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• 1.
Univ Mediterranea Reggio Calabria, Italy.
Linköping University, Department of Physics, Chemistry and Biology, Biology. Linköping University, Faculty of Science & Engineering. USDA ARS, WV 25430 USA. Univ Catania, Italy. Univ Mediterranea Reggio Calabria, Italy.
Metabarcoding: A powerful tool to investigate microbial communities and shape future plant protection strategies2018In: Biological control (Print), ISSN 1049-9644, E-ISSN 1090-2112, Vol. 120Article in journal (Refereed)

Microorganisms are the main drivers shaping the functioning and equilibrium of all ecosystems, contributing to nutrient cycling, primary production, litter decomposition, and multi-trophic interactions. Knowledge about the microbial assemblies in specific ecological niches is integral to understanding the assemblages interact and function the function, and becomes essential when the microbiota intersects with human activities, such as protecting crops against pests and diseases. Metabarcoding has proven to be a valuable tool and has been widely used for characterizing the microbial diversity of different environments and has been utilized in many research endeavors. Here we summarize the current status of metabarcoding technologies, the advantages and challenges in utilizing this technique, and how this pioneer approach is being applied to studying plant diseases and pests, with a focus on plant protection and biological control. Current and future developments in this technology will foster a more comprehensive understanding of microbial ecology, and the development of new, innovative pest control strategies.

• 2.
SLU, Umeå, Sweden .
FOI, Umeå, Sweden . Linköping University, Department of Computer and Information Science, Statistics. Linköping University, The Institute of Technology. SLU, Umeå, Sweden .
Maternal heterozygosity and progeny fitness association in an inbred Scots pine population2013In: Genetica, ISSN 0016-6707, E-ISSN 1573-6857, Vol. 141, no 1-3, p. 41-50Article in journal (Refereed)

Associations between heterozygosity and fitness traits have typically been investigated in populations characterized by low levels of inbreeding. We investigated the associations between standardized multilocus heterozygosity (stMLH) in mother trees (obtained from12 nuclear microsatellite markers) and five fitness traits measured in progenies from an inbred Scots pine population. The traits studied were proportion of sound seed, mean seed weight, germination rate, mean family height of one-year old seedlings under greenhouse conditions (GH) and mean family height of three-year old seedlings under field conditions (FH). The relatively high average inbreeding coefficient (F) in the population under study corresponds to a mixture of trees with different levels of co-ancestry, potentially resulting from a recent bottleneck. We used both frequentist and Bayesian methods of polynomial regression to investigate the presence of linear and non-linear relations between stMLH and each of the fitness traits. No significant associations were found for any of the traits except for GH, which displayed negative linear effect with stMLH. Negative HFC for GH could potentially be explained by the effect of heterosis caused by mating of two inbred mother trees (Lippman and Zamir 2006), or outbreeding depression at the most heterozygote trees and its negative impact on the fitness of the progeny, while their simultaneous action is also possible (Lynch. 1991). However,since this effect wasn’t detected for FH, we cannot either rule out that the greenhouse conditions introduce artificial effects that disappear under more realistic field conditions.

• 3.
Linköping University, The Department of Physics, Chemistry and Biology.
In-vitro studier av inflammatorisk svar från helblod och neutrofila granulocyter vid aktivering med nanopartiklar2007Independent thesis Basic level (degree of Bachelor), 10 points / 15 hpStudent thesis

Syftet med detta arbete var att studera inflammatoriska effekter i mänskligt helblod och neutrofila granulocyter exponerade och stimulerade av nanopartiklar av gadoliniumoxid. Projektet utreder den toxiska potentialen hos nanopartiklar med olika kemiska och morfologiska egenskaper.

Studier har visat att varken ofunktionaliserade eller funktionaliserade nanopartiklarna ger aggregation i blodet. Syreradikalproduktionen ökar däremot. Resultaten av studier i helblod visar att stimulering med spädnings serier av funktionaliserade nanopartiklar ger mindre frisättning av syreradikaler än spädnings serier med ofunktionaliserade nanopartiklar. Detta bekräftas med studier av morfologiska skillnader i neutrofila granulocyter som var stimulerade med olika typer av nanopartiklar. Detta gjordes med hjälp av fluorescensmikroskopi. Resultaten från studierna tyder på att funktionaliserade nanopartiklar är mindre inflammatoriska än ofunktionaliserade nanopartiklar.

• 4.
Institute of Experimental Botany, Centre of the Region Hana for Biotechnological and Agricultural Research, Olomouc, Czech Republic.
Institute of Experimental Botany, Centre of the Region Hana for Biotechnological and Agricultural Research, Olomouc, Czech Republic. Institute of Experimental Botany, Centre of the Region Hana for Biotechnological and Agricultural Research, Olomouc, Czech Republic. Institute of Experimental Botany, Centre of the Region Hana for Biotechnological and Agricultural Research, Olomouc, Czech Republic. Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences. Institute for Bioinformatics and Systems Biology, Helmholtz Center Munich, Neuherberg, Germany. Department of Gene Technology, Tallinn University of Technology, Akadeemia tee 15, Tallinn 19086, Estonia. Department of Gene Technology, Tallinn University of Technology, Akadeemia tee 15, Tallinn 19086, Estonia. Institute of Experimental Botany, Centre of the Region Hana for Biotechnological and Agricultural Research, Olomouc, Czech Republic. Institute of Experimental Botany, Centre of the Region Hana for Biotechnological and Agricultural Research, Olomouc, Czech Republic. Diversity Arrays Technology Pty Ltd, Yarralumla, ACT2600, Australia. Department of Gene Technology, Tallinn University of Technology, Akadeemia tee 15, Tallinn 19086, Estonia. Institute of Experimental Botany, Centre of the Region Hana for Biotechnological and Agricultural Research, Olomouc, Czech Republic. Institute of Experimental Botany, Centre of the Region Hana for Biotechnological and Agricultural Research, Olomouc, Czech Republic.
The in silico identification and characterization of a bread wheat/Triticum militinae introgression line: Characterization of alien introgression in wheat2017In: Plant Biotechnology Journal, ISSN 1467-7644, E-ISSN 1467-7652, Vol. 15, no 2, p. 249-256Article in journal (Refereed)

The capacity of the bread wheat (Triticum aestivum) genome to tolerate introgression from related genomes can be exploited for wheat improvement. A resistance to powdery mildew expressed by a derivative of the cross bread wheat cv. Tähti ⨯ T. militinae (Tm) is known to be due to the incorporation of a Tm segment into the long arm of chromosome 4A. Here, a newly developed in silico method termed RICh (rearrangement identification and characterization) has been applied to characterize the introgression. A virtual gene order, assembled using the GenomeZipper approach, was obtained for the native copy of chromosome 4A; it incorporated 570 4A DArTseq markers to produce a zipper comprising 2,132 loci. A comparison between the native and introgressed forms of the 4AL chromosome arm showed that the introgressed region is located at the distal part of the arm. The Tm segment, derived from chromosome 7G, harbors 131 homoeologs out of the 357 genes present on the corresponding region of Chinese Spring 4AL. The estimated number of Tm genes transferred along with the disease resistance gene was 169. Characterizing the introgression's position, gene content and internal gene order should facilitate not only gene isolation, but may also be informative with respect to chromatin structure and behavior studies.

• 5.
Department of Biology, Middle Tennessee State University, USA.
Department of Biology, Middle Tennessee State University, USA. Department of Biology, Middle Tennessee State University, USA. Linköping University, Department of Physics, Chemistry and Biology, Ecology. Linköping University, The Institute of Technology.
Forest Composition and Structure onGlade-forming Limestones in Middle Tennessee2012In: Castanea, ISSN 0008-7475, Vol. 77, no 4, p. 335-347Article in journal (Refereed)

Within a successional context, the vegetation associated with the cedar gladeecosystem in middle Tennessee develops from bare limestone bedrock to subclimax redcedar,preclimax oak-hickory, and climax mixed hardwood forests. Studies on the composition andstructure of forests associated with cedar glade–forming limestones (Lebanon, Ridley) are rare.We sampled the canopy and understory of six forest stands in middle Tennessee on theselimestones. Observed number of canopy species was 14–24 across stands; estimated richnesswas greater by 1–3 species (bootstrap) or 3–6 species (first-order jackknife) than observedrichness. With the exception of Ailanthus altissima in one stand, all other canopy species werenative. Juniperus virginiana, Fraxinus americana, Carya ovata, and Quercus muehlenbergii wereprimary canopy components in 4 or 6 stands, and C. glabra, Q. shumardii, Ulmus alata, F.quadrangulata, Q. alba, and Q. velutina in 2–3 stands. When we included stands from apreviously published study (most on the non-glade Carters Limestone) with our data, aprincipal components analysis identified three groups with the axes approximating a moisturebedrockgradient and a time-successional gradient. An examination of regeneration in ourstands predicts that (1) mesophytes and/or fire-sensitive species (Acer saccharum, Fraxinus spp.,Celtis spp.) will increase and (2) xerophytes and/or fire-adapted species (Quercus spp., Caryaspp.) will decrease. Altogether, our results strongly suggest that the oak-hickory stage shown insuccessional outlines of vegetation development associated with the cedar glade ecosystem maynot occur in its current state in the future.

• 6.
Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics. Linköping University, The Institute of Technology.
Charge storage properties of biopolymer electrodes with (sub)tropical lignins2014In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 16, no 45, p. 24681-24684Article in journal (Refereed)

The electrochemical and charge storage properties of different lignins inside biopolymer electrodes were studied and correlated with the chemical variations of the lignins as indicated from the nuclear magnetic resonance (NMR) spectroscopic data. The varying fractions of monolignols were found to correlate with charge storage properties. It was found that as the sinapyl to guaiacyl (S/G) ratio increased both the specific capacitance and charge capacity increased considerably. This indicates that quinones generated on S-units can contribute more to charge storage in the biopolymer electrodes.

• 7.
Linköping University, Department of Physics, Chemistry and Biology, Biology. Linköping University, Faculty of Science & Engineering.
Early domestication?: Phenotypic alterations of Red Junglefowl selected for divergent fear of humans2016Doctoral thesis, comprehensive summary (Other academic)

Domestication is the process through which animals adapt to conditions provided by humans. The domesticated phenotype differs from wild ancestors in a number of traits relating to physiology, morphology and behaviour. One of the most striking differences is the animals’ fear response towards humans, and reduced fear of humans is assumed to have been an early prerequisite for the success of domestication. The early alterations seen in the domesticated phenotype may be traits developed as a correlated selection response due to tameness rather than selected upon one by one.

This thesis summarizes a project where Red Junglefowl were selected for divergent fear of humans during six generations. In every generation, fear response to human was assessed in a standardized test and, according to fear score, the animals were bred for either high fear of humans (H) or low fear of humans (L). The animals were, above that of the standardized selection test, behaviourally phenotyped in different tests in each generation mainly focusing on fear, exploration and social behaviour. In addition to behaviour, the animals were phenotyped for body weight, egg weight, metabolism, feed intake, plumage condition, blood plasma corticosterone and peripheral serotonin. After culling, vital organs and brains were harvested and weighed.

In paper I, we demonstrated that the selection trait has a significant genetic heritability and is genetically correlated with other behavioural responses associated with fearfulness and exploration. In paper II, we concluded that animals from the L strain had better plumage condition, higher weight, laid larger eggs and also generated larger offspring. Furthermore, when tested in a social dominance test with a limited resource, they received less and performed more aggression regardless of whether the restricted source was edible or not. In paper III, we revealed that animals from the L strain had higher basal metabolic rate as chicks, gained more weight in relation to feed intake and were bolder in a Novel Object test. Furthermore, the L males had higher plasma levels of peripheral serotonin, but the corticosterone after a restraint stress test did not differ. In paper IV and V, we concluded the project by comparing brain and organ weights as well as behaviour of the parental generation (P0) with the fifth selected generation (S5). The absolute brain weight as well as the weight specific brain weight were larger in the animals selected on H than in the L-animals. The relative weight of telencephalon was significantly higher in H whereas relative weight of cerebellum was significantly lower. Heart, liver, spleen and testes were all relatively heavier in H animals than in L. Interestingly, the behaviours assessed in P0 and S5 seemed to be rather resilient to the selection with only small differences in S5.

To summarize, the selection on divergent tameness in Red Junglefowl has affected several phenotypic traits associated with the domesticated phenotype. The results of this project indicate that tameness in Red Junglefowl could be an underlying factor driving trait modifications towards the domesticated phenotype.

1. Heritability and Genetic Correlations of Fear-Related Behaviour in Red Jungelfowl -Possible Implications for Early Domestication
Open this publication in new window or tab >>Heritability and Genetic Correlations of Fear-Related Behaviour in Red Jungelfowl -Possible Implications for Early Domestication
2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 4, p. e35162-Article in journal (Refereed) Published
##### Abstract [en]

Domesticated species differ from their wild ancestors in a number of traits, generally referred to as the domesticated phenotype. Reduced fear of humans is assumed to have been an early prerequisite for the successful domestication of virtually all species. We hypothesized that fear of humans is linked to other domestication related traits. For three generations, we selected Red Junglefowl (ancestors of domestic chickens) solely on the reaction in a standardized Fear of Human-test. In this, the birds were exposed for a gradually approaching human, and their behaviour was continuously scored. This generated three groups of animals, high (H), low (L) and intermediate (I) fearful birds. The birds in each generation were additionally tested in a battery of behaviour tests, measuring aspects of fearfulness, exploration, and sociality. The results demonstrate that the variation in fear response of Red Junglefowl towards humans has a significant genetic component and is genetically correlated to behavioural responses in other contexts, of which some are associated with fearfulness and others with exploration. Hence, selection of Red Junglefowl on low fear for humans can be expected to lead to a correlated change of other behavioural traits over generations. It is therefore likely that domestication may have caused an initial suite of behavioural modifications, even without selection on anything besides tameness.

##### National Category
Natural Sciences
##### Identifiers
urn:nbn:se:liu:diva-76833 (URN)10.1371/journal.pone.0035162 (DOI)000305336200026 ()
Available from: 2012-04-20 Created: 2012-04-20 Last updated: 2017-12-07
2. Red Junglefowl (Gallus gallus) selected for low fear of humans are larger, more dominant and produce larger offspring
Open this publication in new window or tab >>Red Junglefowl (Gallus gallus) selected for low fear of humans are larger, more dominant and produce larger offspring
2014 (English)In: animal, ISSN 1751-7311, Vol. 8, no 9, p. 1498-1505Article in journal (Refereed) Published
##### Abstract [en]

Many traits associated with domestication are suggested to have developed as correlated responses to reduced fear of humans. Tameness may have reduced the stress of living in human proximity and improved welfare in captivity. We selected Red Junglefowl (ancestors of all domestic chickens) for four generations on high or low fear towards humans, mimicking an important aspect of the earliest period of domestication, and tested birds from the third and fourth generation in three different social tests. Growth and plumage condition, as well as size of eggs and offspring were also recorded, as indicators of some aspects of welfare. Birds selected for low fear had higher weight, laid larger eggs and generated larger offspring, and had a better plumage condition. In a social dominance test they also performed more aggressive behaviour and received less of the same, regardless of whether the restricted resource was feed or not. Hence, dominance appeared to increase as a consequence of reduced fear of humans. Furthermore, egg size and the weight of the offspring were larger in the less fearful birds, and plumage condition better, which could be interpreted as the less fearful animals being better adapted to the environment in which they were selected.

##### Place, publisher, year, edition, pages
Cambridge University Press, 2014
##### Keyword
Red Junglefowl, domestication, fearfulness, selection, social behaviour
##### National Category
Biological Sciences
##### Identifiers
urn:nbn:se:liu:diva-109499 (URN)10.1017/S1751731114001426 (DOI)000342219000013 ()24910136 (PubMedID)
Available from: 2014-08-20 Created: 2014-08-20 Last updated: 2016-11-17
3. Is domestication driven by reduced fear of humans? Boldness, metabolism and serotonin levels in divergently selected red junglefowl (Gallus gallus)
Open this publication in new window or tab >>Is domestication driven by reduced fear of humans? Boldness, metabolism and serotonin levels in divergently selected red junglefowl (Gallus gallus)
2015 (English)In: Biology Letters, ISSN 1744-9561, E-ISSN 1744-957X, Vol. 11, no 9, article id 20150509Article in journal (Refereed) Published
##### Abstract [en]

Domesticated animals tend to develop a coherent set of phenotypic traits. Tameness could be a central underlying factor driving this, and we therefore selected red junglefowl, ancestors of all domestic chickens, for high or low fear of humans during six generations. We measured basal metabolic rate (BMR), feed efficiency, boldness in a novel object (NO) test, corticosterone reactivity and basal serotonin levels (related to fearfulness) in birds from the fifth and sixth generation of the high- and low-fear lines, respectively (44-48 individuals). Corticosterone response to physical restraint did not differ between selection lines. However, BMR was higher in low-fear birds, as was feed efficiency. Low-fear males had higher plasma levels of serotonin and both low-fear males and females were bolder in an NO test. The results show that many aspects of the domesticated phenotype may have developed as correlated responses to reduced fear of humans, an essential trait for successful domestication.

##### Place, publisher, year, edition, pages
ROYAL SOC, 2015
##### Keyword
genetics; domestication; stress
##### National Category
Zoology Evolutionary Biology
##### Identifiers
urn:nbn:se:liu:diva-123162 (URN)10.1098/rsbl.2015.0509 (DOI)000364772300009 ()26382075 (PubMedID)
##### Note

Funding Agencies|research council Formas; ERC [322206]

Available from: 2015-12-07 Created: 2015-12-04 Last updated: 2017-12-01Bibliographically approved
4. Effects of Divergent Selection for Fear of Humans on Behaviour in Red Junglefowl
Open this publication in new window or tab >>Effects of Divergent Selection for Fear of Humans on Behaviour in Red Junglefowl
2016 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 11, p. 1-12Article in journal (Refereed) Published
##### Abstract [en]

Domestication has caused a range of similar phenotypic changes across taxa, relating to physiology, morphology and behaviour. It has been suggested that this recurring domesticated phenotype may be a result of correlated responses to a central trait, namely increased tameness. We selected Red Junglefowl, the ancestors of domesticated chickens, during five generations for reduced fear of humans. This caused a marked and significant response in tameness, and previous studies have found correlated effects on growth, metabolism, reproduction, and some behaviour not directly selected for. Here, we report the results from a series of behavioural tests carried out on the initial parental generation (P0) and the fifth selected generation (S5), focusing on behaviour not functionally related to tameness, in order to study any correlated effects. Birds were tested for fear of humans, social reinstatement tendency, open field behaviour at two different ages, foraging/exploration, response to a simulated aerial predator attack and tonic immobility. In S5, there were no effects of selection on foraging/exploration or tonic immobility, while in the social reinstatement and open field tests there were significant interactions between selection and sex. In the aerial predator test, there were significant main effects of selection, indicating that fear of humans may represent a general wariness towards predators. In conclusion, we found only small correlated effects on behaviours not related to the tameness trait selected for, in spite of them showing high genetic correlations to fear of humans in a previous study on the same population. This suggests that species-specific behaviour is generally resilient to changes during domestication.

PLOS, 2016
##### National Category
Developmental Biology Genetics
##### Identifiers
urn:nbn:se:liu:diva-132742 (URN)10.1371/journal.pone.0166075 (DOI)000387909300035 ()27851792 (PubMedID)
##### Note

European Research Council [322206]; FORMAS [221-2007-838]; Vetenskapsradet [621-2008-5437]

Available from: 2016-11-22 Created: 2016-11-22 Last updated: 2017-11-29Bibliographically approved
• 8.
Linköping University, Department of Physics, Chemistry and Biology, Biology. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Biology. Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biology. Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biology.
Red Junglefowl (Gallus gallus) selected for low fear of humans are larger, more dominant and produce larger offspring2014In: animal, ISSN 1751-7311, Vol. 8, no 9, p. 1498-1505Article in journal (Refereed)

Many traits associated with domestication are suggested to have developed as correlated responses to reduced fear of humans. Tameness may have reduced the stress of living in human proximity and improved welfare in captivity. We selected Red Junglefowl (ancestors of all domestic chickens) for four generations on high or low fear towards humans, mimicking an important aspect of the earliest period of domestication, and tested birds from the third and fourth generation in three different social tests. Growth and plumage condition, as well as size of eggs and offspring were also recorded, as indicators of some aspects of welfare. Birds selected for low fear had higher weight, laid larger eggs and generated larger offspring, and had a better plumage condition. In a social dominance test they also performed more aggressive behaviour and received less of the same, regardless of whether the restricted resource was feed or not. Hence, dominance appeared to increase as a consequence of reduced fear of humans. Furthermore, egg size and the weight of the offspring were larger in the less fearful birds, and plumage condition better, which could be interpreted as the less fearful animals being better adapted to the environment in which they were selected.

• 9.
Linköping University, Department of Physics, Chemistry and Biology, Biology. Linköping University, Faculty of Science & Engineering.
Linköping University, Department of Physics, Chemistry and Biology, Biology. Linköping University, Faculty of Science & Engineering. Linköping University, Department of Physics, Chemistry and Biology, Biology. Linköping University, Faculty of Science & Engineering.
Brain size is reduced by selectionfor tameness in Red Junglefowl–correlated effects in vital organs2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 3306Article in journal (Refereed)

During domestication animals have undergone changes in size of brain and other vital organs. We hypothesize that this could be a correlated effect to increased tameness. Red Junglefowl (ancestors of domestic chickens) were selected for divergent levels of fear of humans for five generations. The parental (P0) and the fifth selected generation (S5) were culled when 48–54 weeks old and the brains were weighed before being divided into telencephalon, cerebellum, mid brain and optic lobes. Each single brain part as well as the liver, spleen, heart and testicles were also weighed. Brains of S5 birds with high fear scores (S5 high) were heavier both in absolute terms and when corrected for body weight. The relative weight of telencephalon (% of brain weight) was significantly higher in S5 high and relative weight of cerebellum was lower. Heart, liver, testes and spleen were all relatively heavier (% of body weight) in S5 high. Hence, selection for tameness has changed the size of the brain and other vital organs in this population and may have driven the domesticated phenotype as a correlated response.

• 10.
Linköping University, Department of Physics, Chemistry and Biology, Biology. Linköping University, Faculty of Science & Engineering.
Linköping University, Department of Physics, Chemistry and Biology, Biology. Linköping University, Faculty of Science & Engineering.
Effects of Divergent Selection for Fear of Humans on Behaviour in Red Junglefowl2016In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 11, p. 1-12Article in journal (Refereed)

Domestication has caused a range of similar phenotypic changes across taxa, relating to physiology, morphology and behaviour. It has been suggested that this recurring domesticated phenotype may be a result of correlated responses to a central trait, namely increased tameness. We selected Red Junglefowl, the ancestors of domesticated chickens, during five generations for reduced fear of humans. This caused a marked and significant response in tameness, and previous studies have found correlated effects on growth, metabolism, reproduction, and some behaviour not directly selected for. Here, we report the results from a series of behavioural tests carried out on the initial parental generation (P0) and the fifth selected generation (S5), focusing on behaviour not functionally related to tameness, in order to study any correlated effects. Birds were tested for fear of humans, social reinstatement tendency, open field behaviour at two different ages, foraging/exploration, response to a simulated aerial predator attack and tonic immobility. In S5, there were no effects of selection on foraging/exploration or tonic immobility, while in the social reinstatement and open field tests there were significant interactions between selection and sex. In the aerial predator test, there were significant main effects of selection, indicating that fear of humans may represent a general wariness towards predators. In conclusion, we found only small correlated effects on behaviours not related to the tameness trait selected for, in spite of them showing high genetic correlations to fear of humans in a previous study on the same population. This suggests that species-specific behaviour is generally resilient to changes during domestication.

• 11.
Linköping University, Department of Physics, Chemistry and Biology, Biology. Linköping University, Faculty of Science & Engineering.
Linköping University, Department of Physics, Chemistry and Biology, Biology. Linköping University, Department of Physics, Chemistry and Biology, Biology. Linköping University, Faculty of Science & Engineering. Linköping University, Department of Physics, Chemistry and Biology, Biology. Linköping University, Faculty of Science & Engineering.
Is domestication driven by reduced fear of humans? Boldness, metabolism and serotonin levels in divergently selected red junglefowl (Gallus gallus)2015In: Biology Letters, ISSN 1744-9561, E-ISSN 1744-957X, Vol. 11, no 9, article id 20150509Article in journal (Refereed)

Domesticated animals tend to develop a coherent set of phenotypic traits. Tameness could be a central underlying factor driving this, and we therefore selected red junglefowl, ancestors of all domestic chickens, for high or low fear of humans during six generations. We measured basal metabolic rate (BMR), feed efficiency, boldness in a novel object (NO) test, corticosterone reactivity and basal serotonin levels (related to fearfulness) in birds from the fifth and sixth generation of the high- and low-fear lines, respectively (44-48 individuals). Corticosterone response to physical restraint did not differ between selection lines. However, BMR was higher in low-fear birds, as was feed efficiency. Low-fear males had higher plasma levels of serotonin and both low-fear males and females were bolder in an NO test. The results show that many aspects of the domesticated phenotype may have developed as correlated responses to reduced fear of humans, an essential trait for successful domestication.

• 12.
University of Calif San Diego, CA 92093 USA; University of Calif San Diego, CA 92093 USA.
Case Western Reserve University, OH 44116 USA. University of Calif San Diego, CA 92093 USA; University of Calif San Diego, CA 92093 USA; US FDA, MD USA. CIC bioGUNE, Spain. University of Calif San Diego, CA 92093 USA. University of Calif San Diego, CA 92093 USA; CIC bioGUNE, Spain; Ikerbasque, Spain. Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering. Case Western Reserve University, OH 44116 USA. University of Calif San Diego, CA 92093 USA; University of Calif San Diego, CA 92093 USA; University of Calif Davis, CA 95616 USA.
Post-translational modifications in PrP expand the conformational diversity of prions in vivo2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 43295Article in journal (Refereed)

Misfolded prion protein aggregates (PrPSc) show remarkable structural diversity and are associated with highly variable disease phenotypes. Similarly, other proteins, including amyloid-beta, tau, alpha-synuclein, and serum amyloid A, misfold into distinct conformers linked to different clinical diseases through poorly understood mechanisms. Here we use mice expressing glycophosphatidylinositol (GPI)anchorless prion protein, PrPC, together with hydrogen-deuterium exchange coupled with mass spectrometry (HXMS) and a battery of biochemical and biophysical tools to investigate how posttranslational modifications impact the aggregated prion protein properties and disease phenotype. Four GPI-anchorless prion strains caused a nearly identical clinical and pathological disease phenotype, yet maintained their structural diversity in the anchorless state. HXMS studies revealed that GPIanchorless PrPSc is characterized by substantially higher protection against hydrogen/deuterium exchange in the C-terminal region near the N-glycan sites, suggesting this region had become more ordered in the anchorless state. For one strain, passage of GPI-anchorless prions into wild type mice led to the emergence of a novel strain with a unique biochemical and phenotypic signature. For the new strain, histidine hydrogen-deuterium mass spectrometry revealed altered packing arrangements of beta-sheets that encompass residues 139 and 186 of PrPSc. These findings show how variation in posttranslational modifications may explain the emergence of new protein conformations in vivo and also provide a basis for understanding how the misfolded protein structure impacts the disease.

• 13.
Totalförsvarets Forskningsinstitut, FOI, Stockholm, Sweden.
Swedish National Forensic Centre (NFC), Linköping, Sweden. Totalförsvarets Forskningsinstitut, FOI, Stockholm, Sweden.
Chemometrics comes to court: evidence evaluation of chem–bio threat agent attacks2015In: Journal of Chemometrics, ISSN 0886-9383, E-ISSN 1099-128X, Vol. 29, no 5, p. 267-276Article in journal (Refereed)

Forensic statistics is a well-established scientific field whose purpose is to statistically analyze evidence in order to support legal decisions. It traditionally relies on methods that assume small numbers of independent variables and multiple samples. Unfortunately, such methods are less applicable when dealing with highly correlated multivariate data sets such as those generated by emerging high throughput analytical technologies. Chemometrics is a field that has a wealth of methods for the analysis of such complex data sets, so it would be desirable to combine the two fields in order to identify best practices for forensic statistics in the future. This paper provides a brief introduction to forensic statistics and describes how chemometrics could be integrated with its established methods to improve the evaluation of evidence in court.

The paper describes how statistics and chemometrics can be integrated, by analyzing a previous know forensic data set composed of bacterial communities from fingerprints. The presented strategy can be applied in cases where chemical and biological threat agents have been illegally disposed.

• 14.
Linköping University, Department of Physics, Chemistry and Biology, Biology.
Welfare in zoo kept felids: A study of resource usage2016Independent thesis Advanced level (degree of Master (Two Years)), 40 credits / 60 HE creditsStudent thesis

Due to a large number of felid species being endangered they are subjects of conservation projects both in situ and ex situ. Keeping felids in zoos are problematic with stereotypic behaviours such as pacing and reproduction difficulties often occurring. The aim of this study was to review research and zoo husbandry knowledge about which resources are most important for the welfare of zoo kept felids, and in addition perform behavioural observations in seven felid species in four Swedish zoos to try to find an order of priority of resources. Observations were performed during opening hours in 36 sessions per species and zoo. The results showed that studies of felid resource usage are missing. Zoo husbandry practice is probably based mainly on traditions and anecdotal knowledge. The observations showed that except for minor differences felids behave similarly regardless of species but the use of resources varies. Small felid species seems to be hiding rather than pacing as a way of coping. Elevated resources and areas as well as numerous hiding places are important to felids but many factors might affect the choice of resting places. Therefore it is important to provide the felids with multiple choices. It is also important to evaluate both species and individuals when designing enclosures and providing resources. More multi-institutional studies with large number of individuals of all zoo kept felid species are needed to gather knowledge about felids needs and preferences of resources.

• 15.
Hannover Medical Sch, Germany.
University of Malaya, Malaysia; University of Malaya, Malaysia; School Basic Appl Science, India. University of Malaya, Malaysia. University of Malaya, Malaysia. Hannover Medical Sch, Germany. Hannover Medical Sch, Germany. Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences. Hannover Medical Sch, Germany. University of Malaya, Malaysia; University of Malaya, Malaysia. University of Malaya, Malaysia; University of Malaya, Malaysia.
Negative Checkpoint Regulatory Molecule 2B4 (CD244) Upregulation Is Associated with Invariant Natural Killer T Cell Alterations and Human Immunodeficiency Virus Disease Progression2017In: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 8, article id 338Article in journal (Refereed)

The CD1d-restricted invariant natural killer T (iNKT) cells are implicated in innate immune responses against human immunodeficiency virus (HIV). However, the determinants of cellular dysfunction across the iNKT cells subsets are seldom defined in HIV disease. Herein, we provide evidence for the involvement of the negative checkpoint regulator (NCR) 2B4 in iNKT cell alteration in a well-defined cohort of HIV-seropositive anti-retroviral therapy (ART) naive, ART-treated, and elite controllers (ECs). We report on exaggerated 2B4 expression on iNKT cells of HIV-infected treatment-naive individuals. In sharp contrast to CD4-iNKT cells, 2B4 expression was significantly higher on CD4+ iNKT cell subset. Notably, an increased level of 2B4 on iNKT cells was strongly correlated with parameters associated with HIV disease progression. Further, iNKT cells from ARTnaive individuals were defective in their ability to produce intracellular IFN-gamma Together, our results suggest that the levels of 2B4 expression and the downstream co-inhibitory signaling events may contribute to impaired iNKT cell responses.

• 16.
Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
Lactate Dehydrogenase and Citrate Synthase activity in cardiac and skeletal muscle of lowland and highland tinamous2013Independent thesis Basic level (degree of Bachelor), 10,5 credits / 16 HE creditsStudent thesis

Tinamous (Tinamidae) have the smallest heart in relation to body mass compared to any other flying bird today (Bishop 1997). This means that heart size is likely to restrict aerobic metabolism. Tinamous inhabit areas from sea level to 4800 m a.s.l., which means that the high altitude living species, Nothoprocta ornata (NO), is exposed to hypoxia. In this study the activity of the two metabolic enzymes Lactate Dehydrogenase (LDH) and Citrate Synthase (CS) was measured and the ratio between the enzyme activities calculated to examine if the small heart of the tinamous affects their aerobic/anaerobic metabolism. The activity of the two enzymes was measured in the heart and the gastrocnemius muscle in the three species Nothoprocta ornata (NO), Nothoprocta perdicaria (NP) and Gallus gallus (GG). CS activity was significantly higher in the heart compared to the skeletal muscle and LDH activity was significant higher in the skeletal muscle than in the heart in all three species. The LDH/CS ratio was significantly higher in NO’s skeletal muscle than in chickens but there was no significant difference between species in the heart. The higher ratio in NO´s muscle could be a sign of a higher anaerobic metabolism that is used in the muscles to compensate for the small heart NO have. In conclusion, the Tinamous

• 17.
Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics. Linköping University, Faculty of Science & Engineering.
University of Basque Country, Spain. Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics. Linköping University, Faculty of Science & Engineering. Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics. Linköping University, Faculty of Science & Engineering. Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics. Linköping University, Faculty of Science & Engineering. University of Basque Country, Spain; Ikerbasque, Spain. Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics. Linköping University, Faculty of Science & Engineering.
High performance PEDOT/lignin biopolymer composites for electrochemical supercapacitors2016In: Journal of Materials Chemistry A, ISSN 2050-7488, Vol. 4, no 5, p. 1838-1847Article in journal (Refereed)

Developing sustainable organic electrode materials for energy storage applications is an urgent task. We present a promising candidate based on the use of lignin, the second most abundant biopolymer in nature. This polymer is combined with a conducting polymer, where lignin as a polyanion can behave both as a dopant and surfactant. The synthesis of PEDOT/Lig biocomposites by both oxidative chemical and electrochemical polymerization of EDOT in the presence of lignin sulfonate is presented. The characterization of PEDOT/Lig was performed by UV-Vis-NIR spectroscopy, FTIR infrared spectroscopy, thermogravimetric analysis, scanning electron microscopy, cyclic voltammetry and galvanostatic charge-discharge. PEDOT doped with lignin doubles the specific capacitance (170.4 F g(-1)) compared to reference PEDOT electrodes (80.4 F g(-1)). The enhanced energy storage performance is a consequence of the additional pseudocapacitance generated by the quinone moieties in lignin, which give rise to faradaic reactions. Furthermore PEDOT/Lig is a highly stable biocomposite, retaining about 83% of its electroactivity after 1000 charge/discharge cycles. These results illustrate that the redox doping strategy is a facile and straightforward approach to improve the electroactive performance of PEDOT.

• 18.
Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
Is spring burning a viable management tool for species-rich grasslands?2012Independent thesis Advanced level (degree of Master (Two Years)), 80 credits / 120 HE creditsStudent thesis

Semi- natural grasslands are species-rich and also one of the most threatened biotopes in Europe. The area of these grasslands has declined and grassland vegetation is threatened as a result of lack of management and land use change. Appropriate management is therefore required to maintain the conservation values and high species richness of semi- natural grasslands. Traditional management, that is, grazing or annual mowing is expensive, which motivates evaluation of alternative cheaper methods of management. Burning is less costly and therefore I evaluated burning along with the conventional methods. The study addressed the main question: is burning an option to mowing and grazing? I searched the literature for available studies suitable for metaanalysis, but located only detailed reports from a series of eleven Swedish long-term field trials. In addition, I collected data in the only one of these trials still running. To facilitate metaanalysis, l used different indicator systems of classification of grassland plants then calculating the odds for a random record being an indicator after one, eight, fourteen, twenty-eight and thirty-nine spring burns. The results show an increasing proportion of grassland indicators of good management in the mowed and grazed plots compared with the burnt plots, indicating a general negative effect of burning on grassland plants compared with mowing and grazing. Hence, burning is not an appropriate long-term management method if the aim is to maintain vegetation diversity in semi-natural grassland.

• 19.
Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba..
Interfaculty Institute for Biochemistry, University of Tübingen, Germany; BioApplications Enterprises, Winnipeg, MB, Canada. Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba.. Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba.. Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba.. Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba.. Department of Human Anatomy and Cell Sciences, and Manitoba Institute of Child Health, Winnipeg, Canada. Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba.. Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba.. Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba.. Department of Neurobiology, International Center of Neurological Restoration, CIREN, Havana, Cuba.
Prolonged Survival and expression of neural markers by bone marrow-derived stem cells transplanted into brain lesions2009In: Medical Science Monitor, ISSN 1234-1010, E-ISSN 1643-3750, Vol. 15, no 2, p. BR47-BR54Article in journal (Refereed)

BACKGROUND: Bone marrow-derived stem cell transplantation is a potentially viable therapeutic option for the treatment of neurodegenerative disease. MATERIAL/METHODS: We have isolated bone marrow stem cells by standard method. We then evaluated the survival of rats' bone marrow mononuclear cells implanted in rats' brain. The cells were extracted from rats' femurs, and marked for monitoring purposes by adenoviral transduction with Green Fluorescent Protein (GFP). Labeled cells were implanted within the area of rats' striatum lesions that were induced a month earlier employing quinolinic acid-based method. The implants were phenotyped by monitoring CD34; CD38; CD45 and CD90 expression. Bone marrow stromal cells were extracted from rats' femurs and cultivated until monolayer bone marrow stromal cells were obtained. The ability of bone marrow stromal cells to express NGF and GDNF was evaluated by RT-PCR. RESULTS: Implanted cells survived for at least one month after transplantation and dispersed from the area of injection towards corpus callosum and brain cortex. Interestingly, passaged rat bone marrow stromal cells expressed NGF and GDNF mRNA. CONCLUSIONS: The bone marrow cells could be successfully transplanted to the brain either for the purpose of trans-differentiation, or for the expression of desired growth factors.

• 20.
Linköping University, Department of Physics, Chemistry and Biology.
Linköping University, Department of Physics, Chemistry and Biology. Linköping University, Department of Physics, Chemistry and Biology. Linköping University, Department of Physics, Chemistry and Biology. Linköping University, Department of Physics, Chemistry and Biology. Linköping University, Department of Physics, Chemistry and Biology. Linköping University, Department of Physics, Chemistry and Biology.
Method Development for Determining the Stability of Heat Stable Proteins Combined with Biophysical Characterization of Human Calmodulin and the Disease Associated Variant D130G2016Independent thesis Basic level (degree of Bachelor), 10,5 credits / 16 HE creditsStudent thesis

Calmodulin is a highly conserved calcium ion binding protein expressed in all eukaryotic species. The 149 amino acid residues in the primary structure are organized in seven α helices with the highly flexible central α helix connecting the two non-cooperative domains of calmodulin. Each domain contains two EF-hand motifs to which calcium ions bind in a cooperative manner, hence the binding of four calcium ions saturate one calmodulin molecule. In the cardiovascular area calmodulin is involved in the activation of cardiac muscle contraction, and mutations that arise in the genetic sequence of the protein often have severe consequences. One such consequential mutation that can arise brings about the replacement of the highly conserved aspartic acid with glycine at position 130 in the amino acid sequence. In this research, the thermal and chemical stability within the C domain of the D130G variant of human calmodulin was investigated using a new method only requiring circular dichroism spectroscopic measurements. Affinity studies within the C domain of the D130G variant of human calmodulin were performed using fluorescence spectroscopy, and the ligands chosen for this purpose were trifluoperazine and p- HTMI. All analytical experiments were performed with the C domain of wild type human calmodulin as a reference. From the new method, it was concluded that the C domain of the D130G variant of human calmodulin has a slightly decreased stability in terms of Tm and Cm values compared to the C domain of wild type human calmodulin. The affinity analyses indicated that neither trifluoperazine nor p-HTMI discriminates between the C domain of the D130G variant of human calmodulin and the C domain of wild type human calmodulin in terms of dissociation constants. The pivotal outcome from this research is that the new method is applicable for determination of the stability parameters Tm and Cm of heat stable proteins.

• 21.
Manitoba Institute of Cell Biology, and Department of Biochemistry and Medical Genetics, Univ. Manitoba, Winnipeg, Canada.
Manitoba Institute of Cell Biology, and Department of Biochemistry and Medical Genetics, Univ. Manitoba, Winnipeg, Canada. Cancer Care Manitoba, Manitoba Institute of Cell Biology, University of Manitoba,. Cancer Care Manitoba, Manitoba Institute of Cell Biology, University of Manitoba,. Cancer Care Manitoba, Manitoba Institute of Cell Biology, University of Manitoba,. Department of Human Anatomy and Cell Science, University of Manitoba, Winnipeg, Canada.
Selected technologies to control genes and their products for experimental and clinical purposes2007In: Archivum Immunologiae et Therapiae Experimentalis, ISSN 0004-069X, Vol. 55, no 3, p. 139-149Article, review/survey (Refereed)

"On-demand" regulation of gene expression is a powerful tool to elucidate the functions of proteins and biologically-active RNAs. We describe here three different approaches to the regulation of expression or activity of genes or proteins. Promoter-based regulation of gene expression was among the most rapidly developing techniques in the 1980s and 1990s. Here we provide basic information and also some characteristics of the metallothionein-promoter-based system, the tet-off system, Muristerone-A-regulated expression through the ecdysone response element, RheoSwitch (R), coumermycin/novobiocin-regulated gene expression, chemical dimerizer-based promoter activation systems, the "Dual Drug Control" system, "constitutive androstane receptor"-based regulation of gene expression, and RU486/mifepristone-driven regulation of promoter activity. A large part of the review concentrates on the principles and usage of various RNA interference techniques (RNAi: siRNA, shRNA, and miRNA-based methods). Finally, the last part of the review deals with historically the oldest, but still widely used, methods of temperature-dependent regulation of enzymatic activity or protein stability (temperature-sensitive mutants). Due to space limitations we do not describe in detail but just mention the tet-regulated systems and also fusion-protein-based regulation of protein activity, such as estrogen-receptor fusion proteins. The information provided below is aimed to assist researchers in choosing the most appropriate method for the planned development of experimental systems with regulated expression or activity of studied proteins.

• 22.
Cancer Care Manitoba, Manitoba Institute of Cell Biology, University of Manitoba.
Cancer Care Manitoba, Manitoba Institute of Cell Biology, University of Manitoba; Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Canada . Cancer Care Manitoba, Manitoba Institute of Cell Biology, University of Manitoba. Cancer Care Manitoba, Manitoba Institute of Cell Biology, University of Manitoba. Department of Radiooncology, University of Erlangen, Erlangen, Germany . Manitoba Institute of Cell Biology, Cancer Care Manitoba; Manitoba Institute of Child Health; Department of Biochemistry and Medical Genetics; Department of Human Anatomy and Cell Science, University Manitoba, Winnipeg, Canada, .
Selected technologies to control genes and their products for experimental and clinical purposes2007In: Archivum Immunologiae et Therapiae Experimentalis, ISSN 0004-069X, E-ISSN 1661-4917, Vol. 55, no 3, p. 139-149Article in journal (Refereed)

"On-demand" regulation of gene expression is a powerful tool to elucidate the functions of proteins and biologically-active RNAs. We describe here three different approaches to the regulation of expression or activity of genes or proteins. Promoter-based regulation of gene expression was among the most rapidly developing techniques in the 1980s and 1990s. Here we provide basic information and also some characteristics of the metallothionein-promoter-based system, the tet-off system, Muristerone-A-regulated expression through the ecdysone response element, RheoSwitch (R), coumermycin/novobiocin-regulated gene expression, chemical dimerizer-based promoter activation systems, the "Dual Drug Control" system, "constitutive androstane receptor"-based regulation of gene expression, and RU486/mifepristone-driven regulation of promoter activity. A large part of the review concentrates on the principles and usage of various RNA interference techniques (RNAi: siRNA, shRNA, and miRNA-based methods). Finally, the last part of the review deals with historically the oldest, but still widely used, methods of temperature-dependent regulation of enzymatic activity or protein stability (temperature-sensitive mutants). Due to space limitations we do not describe in detail but just mention the tet-regulated systems and also fusion-protein-based regulation of protein activity, such as estrogen-receptor fusion proteins. The information provided below is aimed to assist researchers in choosing the most appropriate method for the planned development of experimental systems with regulated expression or activity of studied proteins.

• 23.
Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden. The Bioinformatics Centre, Department of Biology, University of Copenhagen, Copenhagen, Denmark. Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
Shotgun analysis of the marine mussel Mytilus edulis hemolymph proteome and mapping the innate immunity elements.2015In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 15, no 23-24, p. 4021-4029Article in journal (Refereed)

The marine mussel innate immunity provides protection to pathogen invasion and inflammation.In this regard, the mussel hemolymph takes a main role in the animal innate response.Despite the importance of this body fluid in determining the physiological condition of theanimal, little is known about the molecular mechanisms underlying the cellular and humoralresponses. In this work, we have applied aMS (nano-LC-MS/MS) strategy integrating genomicand transcriptomic data with the aim to: (i) identify the main protein functional groups thatcharacterize hemolymph and (ii) to map the elements of innate immunity in the marine musselMytilus edulis hemolymph proteome. After sample analysis and first protein identificationbased onMS/MS data comparison, proteins with unknown functions were annotated with blastusing public database (nrNCBI) information. Overall 595 hemolymph proteins were identifiedwith high confidence and annotated. These proteins encompass primary cellular metabolicprocesses: energy production and metabolism of biomolecules, as well as processes related tooxidative stress defence, xenobiotic detoxification, drug metabolism, and immune response.A group of proteins was identified with putative immune effector, receptor, and signalingfunctions in M. edulis. Data are available via ProteomeXchange with identifier PXD001951(http://proteomecentral.proteomexchange.org/dataset/PXD001951).

• 24.
Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics .
Unsupervised hidden Markov model for automatic analysis of expressed sequence tags2011Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis

This thesis provides an in-depth analyze of expressed sequence tags (EST) that represent pieces of eukaryotic mRNA by using unsupervised hidden Markov model (HMM). ESTs are short nucleotide sequences that are used primarily for rapid identificationof new genes with potential coding regions (CDS). ESTs are made by sequencing on double-stranded cDNA and the synthesizedESTs are stored in digital form, usually in FASTA format. Since sequencing is often randomized and that parts of mRNA contain non-coding regions, some ESTs will not represent CDS.It is desired to remove these unwanted ESTs if the purpose is to identifygenes associated with CDS. Application of stochastic HMM allow identification of region contents in a EST. Softwares like ESTScanuse HMM in which a training of the HMM is done by supervised learning with annotated data. However, because there are not always annotated data at hand this thesis focus on the ability to train an HMM with unsupervised learning on data containing ESTs, both with and without CDS. But the data used for training is not annotated, i.e. the regions that an EST consists of are unknown. In this thesis a new HMM is introduced where the parameters of the HMM are in focus so that they are reasonablyconsistent with biologically important regionsof an mRNA such as the Kozak sequence, poly(A)-signals and poly(A)-tails to guide the training and decoding correctly with ESTs to proper statesin the HMM. Transition probabilities in the HMMhas been adapted so that it represents the mean length and distribution of the different regions in mRNA. Testing of the HMM's specificity and sensitivityhave been performed via BLAST by blasting each EST and compare the BLAST results with the HMM prediction results.A regression analysis test shows that the length of ESTs used when training the HMM is significantly important, the longer the better. The final resultsshows that it is possible to train an HMM with unsupervised machine learning but to be comparable to supervised machine learning as ESTScan, further expansion of the HMM is necessary such as frame-shift correction of ESTs byimproving the HMM's ability to choose correctly positioned start codons or nucleotides. Usually the false positive results are because of incorrectly positioned start codons leadingto too short CDS lengths. Since no frame-shift correction is implemented, short predicted CDS lengths are not acceptable and is hence not counted as coding regionsduring prediction. However, when there is a lack of supervised models then unsupervised HMM is a potential replacement with stable performance and able to be adapted forany eukaryotic organism.

• 25.
University of Liverpool, UK.
University of Liverpool, UK. University of Liverpool, UK. University of Liverpool, UK. University of Liverpool, UK. University of Liverpool, UK. University of Liverpool, UK. University of Liverpool, UK. Safety Assessment, AstraZeneca, Gartuna, Södertälje, Sweden. University of Liverpool, UK. University of Liverpool, UK. University of Liverpool, UK. University of Liverpool, UK. University of Liverpool, UK.
In silico analysis of HLA associations with drug-induced liver injury: use of a HLA-genotyped DNA archive from healthy volunteers2012In: Genome Medicine, ISSN 1756-994X, E-ISSN 1756-994X, Vol. 4, no 6, article id 51Article in journal (Refereed)

BACKGROUND: Drug-induced liver injury (DILI) is one of the most common adverse reactions leading to product withdrawal post-marketing. Recently, genome-wide association studies have identified a number of human leukocyte antigen (HLA) alleles associated with DILI; however, the cellular and chemical mechanisms are not fully understood.

METHODS: To study these mechanisms, we established an HLA-typed cell archive from 400 healthy volunteers. In addition, we utilized HLA genotype data from more than four million individuals from publicly accessible repositories such as the Allele Frequency Net Database, Major Histocompatibility Complex Database and Immune Epitope Database to study the HLA alleles associated with DILI. We utilized novel in silico strategies to examine HLA haplotype relationships among the alleles associated with DILI by using bioinformatics tools such as NetMHCpan, PyPop, GraphViz, PHYLIP and TreeView.

RESULTS: We demonstrated that many of the alleles that have been associated with liver injury induced by structurally diverse drugs (flucloxacillin, co-amoxiclav, ximelagatran, lapatinib, lumiracoxib) reside on common HLA haplotypes, which were present in populations of diverse ethnicity.

CONCLUSIONS: Our bioinformatic analysis indicates that there may be a connection between the different HLA alleles associated with DILI caused by therapeutically and structurally different drugs, possibly through peptide binding of one of the HLA alleles that defines the causal haplotype. Further functional work, together with next-generation sequencing techniques, will be needed to define the causal alleles associated with DILI.

• 26.
Mevalonate Cascade Inhibition by Simvastatin Induces the Intrinsic Apoptosis Pathway via Depletion of Isoprenoids in Tumor Cells2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 44841Article in journal (Refereed)

The mevalonate (MEV) cascade is responsible for cholesterol biosynthesis and the formation of the intermediate metabolites geranylgeranylpyrophosphate (GGPP) and farnesylpyrophosphate (FPP) used in the prenylation of proteins. Here we show that the MEV cascade inhibitor simvastatin induced significant cell death in a wide range of human tumor cell lines, including glioblastoma, astrocytoma, neuroblastoma, lung adenocarcinoma, and breast cancer. Simvastatin induced apoptotic cell death via the intrinsic apoptotic pathway. In all cancer cell types tested, simvastatin-induced cell death was not rescued by cholesterol, but was dependent on GGPP-and FPP-depletion. We confirmed that simvastatin caused the translocation of the small Rho GTPases RhoA, Cdc42, and Rac1/2/3 from cell membranes to the cytosol in U251 (glioblastoma), A549 (lung adenocarcinoma) and MDA-MB231( breast cancer). Simvastatin-induced Rho-GTP loading significantly increased in U251 cells which were reversed with MEV, FPP, GGPP. In contrast, simvastatin did not change Rho-GTP loading in A549 and MDA-MB-231. Inhibition of geranylgeranyltransferase I by GGTi-298, but not farnesyltransferase by FTi-277, induced significant cell death in U251, A549, and MDA-MB-231. These results indicate that MEV cascade inhibition by simvastatin induced the intrinsic apoptosis pathway via inhibition of Rho family prenylation and depletion of GGPP, in a variety of different human cancer cell lines.

• 27.
Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Department of Clinical Pathology and Clinical Genetics.
Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Department of Clinical Pathology and Clinical Genetics. Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Department of Clinical Pathology and Clinical Genetics. University of Southern Denmark, Denmark. Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Department of Clinical Pathology and Clinical Genetics. Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Department of Clinical Pathology and Clinical Genetics. Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Department of Clinical Pathology and Clinical Genetics.
Genome-Wide Association Study to Identify Genes Related to Renal Mercury Concentrations in Mice2016In: Journal of Environmental Health Perspectives, ISSN 0091-6765, E-ISSN 1552-9924, Vol. 124, no 7, p. 920-926Article in journal (Refereed)

BACKGROUND: Following human mercury (Hg) exposure, the metal accumulates in considerable concentrations in kidney, liver, and brain. Although the toxicokinetics of Hg have been studied extensively, factors responsible for interindividual variation in humans are largely unknown. Differences in accumulation of renal Hg between inbred mouse strains suggest a genetic interstrain variation regulating retention or/and excretion of Hg. A. SW, DBA/2 and BALB/C mouse strains accumulate higher amounts of Hg than B10.S.

OBJECTIVES: We aimed to find candidate genes associated with regulation of renal Hg concentrations.

METHODS: A. SW, B10.S and their F1 and F2 offspring were exposed for 6 weeks to 2.0 mg Hg/L drinking water. Genotyping with microsatellites was conducted on 84 F2 mice for genome-wide scanning with ion pair reverse-phase high-performance liquid chromatography (IP RP HPLC). Quantitative trait loci (QTL) were established. Denaturing HPLC was used to detect single nucleotide polymorphisms for haplotyping and fine mapping in 184 and 32 F2 mice, respectively. Candidate genes (Pprc1, Btrc and Nfkb2) verified by fine mapping and QTL were further investigated by real-time polymerase chain reaction. Genes enhanced by Pprc1 (Nrf1 and Nrf2) were included for gene expression analysis.

RESULTS: Renal Hg concentrations differed significantly between A. SW and B10. S mice and between males and females within each strain. QTL analysis showed a peak logarithm of odds ratio score 5.78 on chromosome 19 (p = 0.002). Haplotype and fine mapping associated the Hg accumulation with Pprc1, which encodes PGC-1-related coactivator (PRC), a coactivator for proteins involved in detoxification. Pprc1 and two genes coactivated by Pprc1 (Nrf1 and Nrf2) had significantly lower gene expression in the A. SW strain than in the B10. S strain.

CONCLUSIONS: This study supports Pprc1 as a key regulator for renal Hg excretion.

• 28.
Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115 USA.
Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115 USA. Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115 USA. Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115 USA.
Specification of Neuropeptide Cell Identity by the Integration of Retrograde BMP Signaling and a Combinatorial Transcription Factor Code2003In: Cell, ISSN 0092-8674, E-ISSN 1097-4172, Vol. 113, no 1, p. 73-86Article in journal (Refereed)

Individual neurons express only one or a few of the many identified neurotransmitters and neuropeptides, but the molecular mechanisms controlling their selection are poorly understood. In the Drosophila ventral nerve cord, the six Tv neurons express the neuropeptide gene FMRFamide. Each Tv neuron resides within a neuronal cell group specified by the LIM-homeodomain gene apterous. We find that the zinc-finger gene squeeze acts in Tv cells to promote their unique axon pathfinding to a peripheral target. There, the BMP ligand Glass bottom boat activates the Wishful thinking receptor, initiating a retrograde BMP signal in the Tv neuron. This signal acts together with apterous and squeeze to activate FMRFamide expression. Reconstituting this "code," by combined BMP activation and apterous/squeeze misexpression, triggers ectopic FMRFamide expression in peptidergic neurons. Thus, an intrinsic transcription factor code integrates with an extrinsic retrograde signal to select a specific neuropeptide identity within peptidergic cells.

• 29.
Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
Variation in proactive - reactive personality types in the red junglefowl2013Independent thesis Basic level (degree of Bachelor), 10,5 credits / 16 HE creditsStudent thesis

It has been shown in many species that individuals exhibit consistent differences in behaviour over time and/or across situations. These differences in behaviour are called personality. One way to categorise personality types typically used for rodents, is along a proactive-reactive gradient, which describes how individuals cope with stressful challenges. Proactive individuals pay less attention to their environment, form routines easily and take longer to adapt when routines are broken compared to reactive individuals. Avian species have to date rarely been described along this gradient, thus the generality of this description across species is unclear. The present study has investigated variation in proactivity-reactivity in red junglefowl chicks (Gallus gallus). To observe the chicks’ coping styles, a proactive-reactive test was conducted where the chicks were trained to form a routine, which was then broken. Their behavioural response to this was recorded and used as a measure for proactivity-reactivity. The behavioural response was then linked to individual behavioural variation in additional personality assays. Individuals that were more vigilant in the proactive-reactive test often uttered stress calls and took longer to complete the test. In contrast, individuals that walked more and did not utter stress calls had a shorter time to complete the test. These findings can be used to describe proactive red junglefowl chicks; those that are more stressed when routines are broken, compared to calmer reactive individuals. I found no difference in routine formation between proactive and reactive red junglefowl chicks, suggesting that what describes proactive and reactive individuals may vary across species.

• 30.
Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
Protein Misfolding in Human Diseases2009Doctoral thesis, comprehensive summary (Other academic)

There are several diseases well known that are due to aberrant protein folding. These types of diseases can be divided into three main categories:

1. Loss-of-function diseases
2. Gain-of-toxic-function diseases
3. Infectious misfolding diseases

Most loss-of-function diseases are caused by aberrant folding of important proteins. These proteins often misfold due to inherited mutations. The rare disease marble brain disease (MBD) also known as carbonic anhydrase II deficiency syndrome (CADS) can manifest in carriers of point mutations in the human carbonic anhydrase II (HCA II) gene. We have over the past 10-15 years studied the folding, misfolding and aggregation of the enzyme human carbonic anhydrase II. In summary our HCA II folding studies have shown that the protein folds via an intermediate of molten-globule type, which lacks enzyme activity and the molten globule state of HCA II is prone to aggregation. One mutation associated with MBD entails the His107Tyr (H107Y) substitution. We have demonstrated that the H107Y mutation is a remarkably destabilizing mutation influencing the folding behavior of HCA II. A mutational survey of position H107 and a neighboring conserved position E117 has been performed entailing the mutants H107A, H107F, H107N, E117A and the double mutants H107A/E117A and H107N/E117A. All mutants were severely destabilized versus GuHCl and heat denaturation. Thermal denaturation and GuHCl phase diagram and ANS analyses showed that the mutants shifted HCA II towards populating ensembles of intermediates of molten globule type under physiological conditions. The enormously destabilizing effects of the H107Y mutation is not due to loss of specific interactions of H107 with residue E117, instead it is caused by long range sterical destabilizing effects of the bulky tyrosine residue. We also showed that the folding equilibrium can be shifted towards the native state by binding of the small-molecule drug acetazolamide, and we present a small molecule inhibitor assessment with select sulfonamide inhibitors of varying potency to investigate the effectiveness of these molecules to inhibit the misfolding of HCA II H107Y. We also demonstrate that high concentration of the activator compound L-His increases the enzyme activity of the mutant but without stabilizing the folded protein.

The infectious misfolding diseases is the smallest group of misfolding diseases. The only protein known to have the ability to be infectious is the prion protein. The human prion diseases Kuru, Gerstmann-Sträussler-Scheinker disease (GSS) and variant Creutzfeldt-Jakob are characterized by depositions of amyloid plaque from misfolded prion protein (HuPrP) in various regions of the brain depending on disease. Amyloidogenesis of HuPrP is hence strongly correlated with prion disease.

Our results show that amyloid formation of recHuPrP90-231 can be achieved starting from the native protein under gentle conditions without addition of denaturant or altered pH. The process is efficiently catalyzed by addition of preformed recHuPrP90-231 amyloid seeds. It is plausible that amyloid seeding reflect the mechanism of transmissibility of prion diseases. Elucidating the mechanism of PrP amyloidogenesis is therefore of interest for strategic prevention of prion infection.

1. Unfolding a folding disease: folding, misfolding and aggregation of the marble brain syndrome-associated mutant H107Y of human carbonic anhydrase II
Open this publication in new window or tab >>Unfolding a folding disease: folding, misfolding and aggregation of the marble brain syndrome-associated mutant H107Y of human carbonic anhydrase II
2004 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 342, no 2, p. 619-633Article in journal (Refereed) Published
##### Abstract [en]

Most loss-of-function diseases are caused by aberrant folding of important proteins. These proteins often misfold due to mutations. The disease marble brain syndrome (MBS), known also as carbonic anhydrase II deficiency syndrome (CADS), can manifest in carriers of point mutations in the human carbonic anhydrase II (HCA II) gene. One mutation associated with MBS entails the His107Tyr substitution. Here, we demonstrate that this mutation is a remarkably destabilizing folding mutation. The loss-of-function is clearly a folding defect, since the mutant shows 64% of CO2 hydration activity compared to that of the wild-type at low temperature where the mutant is folded. On the contrary, its stability towards thermal and guanidine hydrochloride (GuHCl) denaturation is highly compromised. Using activity assays, CD, fluorescence, NMR, cross-linking, aggregation measurements and molecular modeling, we have mapped the properties of this remarkable mutant. Loss of enzymatic activity had a midpoint temperature of denaturation (Tm) of 16 °C for the mutant compared to 55 °C for the wild-type protein. GuHCl-denaturation (at 4 °C) showed that the native state of the mutant was destabilized by 9.2 kcal/mol. The mutant unfolds through at least two equilibrium intermediates; one novel intermediate that we have termed the molten globule light state and, after further denaturation, the classical molten globule state is populated. Under physiological conditions (neutral pH; 37 °C), the His107Tyr mutant will populate the molten globule light state, likely due to novel interactions between Tyr107 and the surroundings of the critical residue Ser29 that destabilize the native conformation. This intermediate binds the hydrophobic dye 8-anilino-1-naphthalene sulfonic acid (ANS) but not as strong as the molten globule state, and near-UV CD reveals the presence of significant tertiary structure. Notably, this intermediate is not as prone to aggregation as the classical molten globule. As a proof of concept for an intervention strategy with small molecules, we showed that binding of the CA inhibitor acetazolamide increases the stability of the native state of the mutant by 2.9 kcal/mol in accordance with its strong affinity. Acetazolamide shifts the Tm to 34 °C that protects from misfolding and will enable a substantial fraction of the enzyme pool to survive physiological conditions.

##### Place, publisher, year, edition, pages
Oxford: Elsevier, 2004
##### Keyword
Misfolding, loss-of-function, aggregation, molten globule, misfolding inhibitor
##### National Category
Biochemistry and Molecular Biology
##### Identifiers
urn:nbn:se:liu:diva-21072 (URN)10.1016/j.jmb.2004.07.024 (DOI)
Available from: 2009-09-28 Created: 2009-09-28 Last updated: 2018-04-25Bibliographically approved
2. Thermodynamic interrogation of a folding disease. Mutant mapping of position 107 in human carbonic anhydrase II linked to marble brain disease.
Open this publication in new window or tab >>Thermodynamic interrogation of a folding disease. Mutant mapping of position 107 in human carbonic anhydrase II linked to marble brain disease.
2008 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, no 5, p. 1288-1298Article in journal (Refereed) Published
##### Abstract [en]

Marble brain disease (MBD) also known as Guibaud−Vainsel syndrome is caused by autosomal recessive mutations in the human carbonic anhydrase II (HCA II) gene. HCA II is a 259 amino acid single domain enzyme and is dominated by a 10-stranded β-sheet. One mutation associated with MBD entails the H107Y substitution where H107 is a highly conserved residue in the carbonic anhydrase protein family. We have previously demonstrated that the H107Y mutation is a remarkably destabilizing folding mutation [Almstedt et al. (2004) J. Mol. Biol. 342, 619−633]. Here, the exceptional destabilization by the H107Y mutation has been further investigated. A mutational survey of position H107 and a neighboring conserved position E117 has been performed entailing the mutants H107A, H107F, H107N, E117A and the double mutants H107A/E117A and H107N/E117A. All mutants were severely destabilized versus GuHCl and heat denaturation. Thermal denaturation and GuHCl phase diagram and ANS analyses showed that the mutants shifted HCA II toward populating ensembles of intermediates of molten globule type under physiological conditions. The native state stability of the mutants was in the following order:  wt > H107N > E117A > H107A > H107F > H107Y > H107N/E117A > H107A/E117A. In conclusion:  (i) H107N is least destabilizing likely due to compensatory H-bonding ability of the introduced Asn residue. (ii) Double mutant cycles surprisingly reveal additive destabilization of H107N and E117A showing that H107 and E117 are independently stabilizing the folded protein. (iii) H107Y and H107F are exceptionally destabilizing due to bulkiness of the side chains whereas H107A is more accommodating, indicating long-range destabilizing effects of the natural pathogenic H107Y mutation.

##### Place, publisher, year, edition, pages
Washington: ACS, 2008
##### Keyword
mutant mapping, carbonic anhydrase, misfolding, loss-of-function, molten globule
##### National Category
Biochemistry and Molecular Biology
##### Identifiers
urn:nbn:se:liu:diva-21056 (URN)10.1021/bi701720p (DOI)
Available from: 2009-09-28 Created: 2009-09-28 Last updated: 2018-04-25Bibliographically approved
3. Small-Molecule Suppression of Misfolding of Mutated Human Carbonic Anhydrase II Linked to Marble Brain Disease
Open this publication in new window or tab >>Small-Molecule Suppression of Misfolding of Mutated Human Carbonic Anhydrase II Linked to Marble Brain Disease
2009 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 23, p. 5358-5364Article in journal (Refereed) Published
##### Abstract [en]

Carbonic anhydrase II deficiency syndrome or Marble brain disease (MBD) is caused by autosomal recessive mutations in the human carbonic anhydrase II (HCA II) gene. Here we report a small-molecule stabilization study of the exceptionally destabilized HCA II mutant H107Y employing inhibitors based on p-aminobenzoyisulfonamide compounds and 1,3,4-thiadiazolylsulfonamides as well as amino acid activators. Protein stability assays showed a significant stabilization by the aromatic sulfonamide inhibitors when present at 10 mu M concentration, providing shifts of the midpoint of thermal denaturation between 10 degrees C and 16 degrees C and increasing the free energies of denaturation 0.5-3.0 kcal/mol as deduced from GuHCl denaturation. This study could be used as a starting point for the design of small-molecule folding modulators and possibly autoactivatable molecules for suppression of misfolding of destabilized HCA II mutants.

##### National Category
Natural Sciences
##### Identifiers
urn:nbn:se:liu:diva-19548 (URN)10.1021/bi900128e (DOI)
##### Note
On the day of the defence date tha status of this articel was In Manuscript.Available from: 2009-06-29 Created: 2009-06-26 Last updated: 2018-04-25Bibliographically approved
4. Amyloid fibrils of human prion protein are spun and woven from morphologically disordered aggregates
Open this publication in new window or tab >>Amyloid fibrils of human prion protein are spun and woven from morphologically disordered aggregates
2009 (English)In: Prion, ISSN 1933-6896, Vol. 3, no 4, p. 224-235Article in journal (Refereed) Published
##### Abstract [en]

Propagation and infectivity of prions in human prionopathies are likely associated with conversion of the mainly α-helical human prion protein, HuPrP, into an aggregated form with amyloid-like properties. Previous reports on efficient conversion of recombinant HuPrP have used mild to harsh denaturing conditions to generate amyloid fibrils in vitro. Herein we report on the in vitro conversion of four forms of truncated HuPrP (sequences 90-231 and 121-231 with and without an N-terminal hexa histidine tag) into amyloid-like fibrils within a few hours by using a protocol (phosphate buffered saline solutions at neutral pH with intense agitation) close to physiological conditions. The conversion process monitored by thioflavin T, ThT, revealed a three stage process with lag, growth and equilibrium phases. Seeding with preformed fibrils shortened the lag phase demonstrating the classic nucleated polymerization mechanism for the reaction. Interestingly, comparing thioflavin T kinetics with solubility and turbidity kinetics it was found that the protein initially formed non-thioflavionophilic, morphologically disordered aggregates that over time matured into amyloid fibrils. By transmission electron microscopy and by fluorescence microscopy of aggregates stained with luminescent conjugated polythiophenes (LCPs); we demonstrated that HuPrP undergoes a conformational conversion where spun and woven fibrils protruded from morphologically disordered aggregates. The initial aggregation functioned as a kinetic trap that decelerated nucleation into a fibrillation competent nucleus, but at the same time without aggregation there was no onset of amyloid fibril formation. The agitation, which was necessary for fibril formation to be induced, transiently exposes the protein to the air-water interface suggests a hitherto largely unexplored denaturing environment for prion conversion.

##### Place, publisher, year, edition, pages
Austin: Landes Bioscience Journals, 2009
##### National Category
Natural Sciences
##### Identifiers
urn:nbn:se:liu:diva-21064 (URN)10.4161/pri.3.4.10112 (DOI)000280061100009 ()
Available from: 2009-09-28 Created: 2009-09-28 Last updated: 2018-04-25
• 31.
Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
Unfolding a folding disease: folding, misfolding and aggregation of the marble brain syndrome-associated mutant H107Y of human carbonic anhydrase II2004In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 342, no 2, p. 619-633Article in journal (Refereed)

Most loss-of-function diseases are caused by aberrant folding of important proteins. These proteins often misfold due to mutations. The disease marble brain syndrome (MBS), known also as carbonic anhydrase II deficiency syndrome (CADS), can manifest in carriers of point mutations in the human carbonic anhydrase II (HCA II) gene. One mutation associated with MBS entails the His107Tyr substitution. Here, we demonstrate that this mutation is a remarkably destabilizing folding mutation. The loss-of-function is clearly a folding defect, since the mutant shows 64% of CO2 hydration activity compared to that of the wild-type at low temperature where the mutant is folded. On the contrary, its stability towards thermal and guanidine hydrochloride (GuHCl) denaturation is highly compromised. Using activity assays, CD, fluorescence, NMR, cross-linking, aggregation measurements and molecular modeling, we have mapped the properties of this remarkable mutant. Loss of enzymatic activity had a midpoint temperature of denaturation (Tm) of 16 °C for the mutant compared to 55 °C for the wild-type protein. GuHCl-denaturation (at 4 °C) showed that the native state of the mutant was destabilized by 9.2 kcal/mol. The mutant unfolds through at least two equilibrium intermediates; one novel intermediate that we have termed the molten globule light state and, after further denaturation, the classical molten globule state is populated. Under physiological conditions (neutral pH; 37 °C), the His107Tyr mutant will populate the molten globule light state, likely due to novel interactions between Tyr107 and the surroundings of the critical residue Ser29 that destabilize the native conformation. This intermediate binds the hydrophobic dye 8-anilino-1-naphthalene sulfonic acid (ANS) but not as strong as the molten globule state, and near-UV CD reveals the presence of significant tertiary structure. Notably, this intermediate is not as prone to aggregation as the classical molten globule. As a proof of concept for an intervention strategy with small molecules, we showed that binding of the CA inhibitor acetazolamide increases the stability of the native state of the mutant by 2.9 kcal/mol in accordance with its strong affinity. Acetazolamide shifts the Tm to 34 °C that protects from misfolding and will enable a substantial fraction of the enzyme pool to survive physiological conditions.

• 32.
Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
Thermodynamic interrogation of a folding disease. Mutant mapping of position 107 in human carbonic anhydrase II linked to marble brain disease.2008In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, no 5, p. 1288-1298Article in journal (Refereed)

Marble brain disease (MBD) also known as Guibaud−Vainsel syndrome is caused by autosomal recessive mutations in the human carbonic anhydrase II (HCA II) gene. HCA II is a 259 amino acid single domain enzyme and is dominated by a 10-stranded β-sheet. One mutation associated with MBD entails the H107Y substitution where H107 is a highly conserved residue in the carbonic anhydrase protein family. We have previously demonstrated that the H107Y mutation is a remarkably destabilizing folding mutation [Almstedt et al. (2004) J. Mol. Biol. 342, 619−633]. Here, the exceptional destabilization by the H107Y mutation has been further investigated. A mutational survey of position H107 and a neighboring conserved position E117 has been performed entailing the mutants H107A, H107F, H107N, E117A and the double mutants H107A/E117A and H107N/E117A. All mutants were severely destabilized versus GuHCl and heat denaturation. Thermal denaturation and GuHCl phase diagram and ANS analyses showed that the mutants shifted HCA II toward populating ensembles of intermediates of molten globule type under physiological conditions. The native state stability of the mutants was in the following order:  wt > H107N > E117A > H107A > H107F > H107Y > H107N/E117A > H107A/E117A. In conclusion:  (i) H107N is least destabilizing likely due to compensatory H-bonding ability of the introduced Asn residue. (ii) Double mutant cycles surprisingly reveal additive destabilization of H107N and E117A showing that H107 and E117 are independently stabilizing the folded protein. (iii) H107Y and H107F are exceptionally destabilizing due to bulkiness of the side chains whereas H107A is more accommodating, indicating long-range destabilizing effects of the natural pathogenic H107Y mutation.

• 33.
Laboratory of Stem Cells and Neurogeneration, Institute of Anatomy, Histology and Pathology, Facultu of Medicine and Center for INterdiciplinary Studies on the Nervous System (CISNe), Universitad Austral de Chile; Institute for Molecular Regenerative MEdicine and Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus University, Salzburg, Austira. Linköping University, Department of Physics, Chemistry and Biology, Biology. Linköping University, Faculty of Science & Engineering.
Bisphenol-A and metabolic diseases: epigenetic developmental and transgenerational basis2016In: Environmental Epigenetics, ISSN 2058-5888, Vol. 2, no 3, p. 1-10Article in journal (Refereed)

Exposure to environmental toxicants is now accepted as a factor contributing to the increasing incidence of obesity and metabolic diseases around the world. Such environmental compounds are known as ‘obesogens’. Among them, bisphenol-A (BPA) is the most widespread and ubiquitous compound affecting humans and animals. Laboratory animal work has provided conclusive evidence that early-life exposure to BPA is particularly effective in predisposing individuals to weight gain. Embryonic exposure to BPA is reported to generate metabolic disturbances later in life, such as obesity and diabetes. When BPA administration is combined with a high-fat diet, there is an exacerbation in the development of metabolic disorders. Remarkably, upon BPA exposure of gestating females, metabolic disturbances have been found both in the offspring and later in life in the mothers themselves. When considering the metabolic effects generated by an early developmental exposure to BPA, one of the questions that arises is the role of precursor cells in the etiology of metabolic disorders. Current evidence shows that BPA and other endocrine disruptors have the ability to alter fat tissue development and growth by affecting the capacity to generate functional adipocytes, as well as their rate of differentiation to specific cell types. Epigenetic mechanisms seem to be involved in the BPA-induced effects related to obesity, as they have been described in both in vitro and in vivo models. Moreover, recent reports also show that developmental exposure to BPA generates abnormalities that can be transmitted to future generations, in a process called as transgenerational epigenetic inheritance.

• 34.
Linköping University, Department of Behavioural Sciences and Learning. Linköping University, Department of Physics, Chemistry and Biology.
Höns återhämtning efter stress samt domesticeringens effekter: En biologisk studie med ett lärarperspektiv2011Independent thesis Basic level (degree of Bachelor), 10 credits / 15 HE creditsStudent thesis

The biological study has shown that behaviour of laying hens has changed through domestication and the hens have adapted to live with humans. They have developed less fear of humans and more resistance to stress. In this study hens’ recovery after a stressful experience has been studied. A comparison between the breeds White Leghorn and red junglefowl has been made to see how the domestication has affected the recovery. The questions of this study are therefore: How long does it take for hens’ to recover their natural behaviour, not stressrelated, after a stressful experience? Are there any differences in recovery between the different breeds White Leghorn and red junglefowl? The results did not show a specific time for recovery, but a distinct recovery was noticed in Relaxed Behaviour, Comfort Behaviour, Perch, Vocalisation and Stand Alert. This beacause of the behaviours differed significantly with time. Also significant differences existed between the breeds, both in behaviours that showed recovery and in behavious that did not. A conclusion of what breed that recovered fastest was difficult to make. This because of the differences of behaviour showed different tendencies in recovery. The differences in recovery between the sexes showed tendencies that the males recovered fastest. The domestication has also been studied from a teacherperspective. The study showed that domestication can be a part of biologyeducation in the fields of evolution and genetics in the grades 7-9.

• 35.
Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
Individuell tillväxt och substratval hos en lokalt differentierad population av Asellus aquaticus2013Independent thesis Basic level (degree of Bachelor), 10,5 credits / 16 HE creditsStudent thesis

Local differentiation may occur during a short period of time and is part of the formation of new species. The isopod Asellus aquaticus is an example of a species in which local adaptation has occurred during a short period of time. An establishment of stonewort (Chara spp.) vegetation in Lake Tåkern (in the 2000) resulted in two different Asellus ecotypes; a lighter pigmented, smaller one that lives among stoneworts grazing periphytic algae, and a darker, larger ecotype that feeds on decaying leaves in reed (Phragmites australis vegetation. The purpose of this study was to examine whether there are differences in growth between ecotypes, depending on whether the food was periphytic algae or leaves, and to study the choice of substrates between the two food types. For the study, animals from both habitats were brought in from Lake Tåkern to the laboratory. I found that both the reed and the stonewort animals grew better when feeding on periphytic algae than on leaves, but that there was no difference in weight gain between the two ecotypes. There was no indication that the animals preferred any of the substrates. Results suggest that despite earlier noted differences in behavior, size and pigmentation (which differentiation had brought), there is no evidence that Asellus aquaticus has adapted to feed on plant matter prevailing in their original habitat. The animals grew better when the food was algae maybe due to that the algae, as previous studies show, are easier assimilated, whereas Asellus has to eat a larger amount of leaves to reach the same energy intake.

• 36.
Linköping University, Department of Physics, Chemistry and Biology, Biology. Linköping University, Faculty of Science & Engineering.
Ecophysiology methods: Refining the old, validating the new and developing for the future2016In: Comparative Biochemistry and Physiology A, ISSN 1095-6433, E-ISSN 1531-4332, Vol. 202Article in journal (Other academic)

n/a

• 37.
Linköping University, Department of Physics, Chemistry and Biology, Biology. Linköping University, Faculty of Science & Engineering.
Linköping University, Department of Physics, Chemistry and Biology, Biology. Linköping University, Faculty of Science & Engineering. University of Mayor San Andres, Bolivia. Tinamou Chile SL, Chile. University of Mayor San Andres, Bolivia.
Aerobic performance in tinamous is limited by their small heart. A novel hypothesis in the evolution of avian flight2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 15964Article in journal (Refereed)

Some biomechanical studies from fossil specimens suggest that sustained flapping flight of birds could have appeared in their Mesozoic ancestors. We challenge this idea because a suitable musculoskeletal anatomy is not the only requirement for sustained flapping flight. We propose the "heart to fly" hypothesis that states that sustained flapping flight in modern birds required an enlargement of the heart for the aerobic performance of the flight muscles and test it experimentally by studying tinamous, the living birds with the smallest hearts. The small ventricular size of tinamous reduces cardiac output without limiting perfusion pressures, but when challenged to fly, the heart is unable to support aerobic metabolism (quick exhaustion, larger lactates and post-exercise oxygen consumption and compromised thermoregulation). At the same time, cardiac growth shows a crocodilian-like pattern and is correlated with differential gene expression in MAPK kinases. We integrate this physiological evidence in a new evolutionary scenario in which the ground-up, short and not sustained flapping flight displayed by tinamous represents an intermediate step in the evolution of the aerobic sustained flapping flight of modern birds.

• 38.
University of Leon, Facultad de Ciencias Biologicas y Ambientales, Spain.
Mejora y evolución de los protocolos de congelación de eyaculados de oso pardo (Ursus arctos)2012Doctoral thesis, comprehensive summary (Other academic)

La situación crítica del oso pardo (Ursus arctos) en España plantea la necesidad de aplicar diferentes estrategias de conservación, dentro de las cuales tiene especial relevancia la creación de un banco de recursos genéticos. La eficacia de esta herramienta depende, entre otros factores, de la adaptación específica de los protocolos de congelación espermática ya existentes y que permitan el almacenamiento eficaz de los espermatozoides del oso pardo en un banco de germoplasma. Este trabajo nos permite concluir la utilidad de la selección entre ciclos con el fin de mejorar la calidad de los espermatozoides sometidos a recongelación

• 39.
ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain. ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain. Cabárceno Park, Cantabria, Spain. ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, León, Spain. ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, León, Spain. ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
Quality of frozen-thawed semen in brown bear is not affected by timing of glycerol addition.2011In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 75, no 8, p. 1561-1565Article in journal (Refereed)

We have tested several freezing protocols for brown bear semen, modifying the time when glycerol was added (before and after cooling to 5 °C). No differences were found among protocols, indicating a good tolerance of brown bear semen to glycerol. This finding indicates that freezing protocols for brown bear semen could be modified to fit practical solutions which would facilitate preparation of the seminal samples in the field with the addition of glycerol at ambient temperature.

• 40.
Linköping University, Department of Clinical and Experimental Medicine, Division of Children's and Women's health. Linköping University, Faculty of Medicine and Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Division of Children's and Women's health. Linköping University, Faculty of Medicine and Health Sciences.
Exogenous Individual Lecithin-Phospholipids (Phosphatidylcholine and Phosphatidylglycerol) Cannot Prevent the Oxidative Stress Imposed by Cryopreservation of Boar Sperm.2017In: Journal of veterinary medicine and surgery, ISSN 2574-2868, Vol. 1, no 1Article in journal (Refereed)

Objective: Despite the use of high proportions of the chemically undefined lipoprotein/phospholipid-rich egg-yolk in extenders, boar sperm are highly sensitive to cooling, which induces ROS generation and disrupts the plasma membrane.

Here, we studied whether replacement of hen egg-yolk by commercially defined lecithin phospholipids, derived from egg (LPGE: phosphatidyl glycerol, LPCE: phosphatidyl choline) or soybean (LPCS: phosphatidyl choline), could individually ameliorate such oxidative effects during cryopreservation of ejaculated (sperm rich fraction, SRF) or of cauda-epididymal sperm, retrieved post-mortem from the same males.

Methods: A conventional extender (lactose buffer, with 20% egg-yolk, 0.5% OEP and 3% glycerol) was used as control. Cryodamage was assessed as loss of sperm motility, membrane and acrosome intactness, early membrane destabilization changes, mitochondrial potential, superoxide and ROS production, to finally determine lipid peroxidation (LPO) using specific probes.

Results and conclusion: In general, the exogenous phospholipids assayed were unable of maintaining neither sperm motility nor viability post-thaw compared to controls, owing to increased ROS production and lipid peroxidation. In our study, mitochondrial superoxide production resulted in very high levels for all groups, whereas both ROS production and lipid peroxidation were reduced in the control group, containing emulsified hen egg yolk. Further studies using various dosage and combination of LPCS should be followed for their eventual protective effect.

Keywords: Cryodamage; Sperm; Boar; Mitochondrial activation; Mitochondrial superoxide; ROS production; Lipid peroxidation

• 41.
ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain. ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain. ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, Campus de Vegazana, León Spain. ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, Campus de Vegazana, León Spain. Cabárceno Park, Cantabria, Spain. ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain. ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, Campus de Vegazana, León Spain.
The antioxidant effects of soybean lecithin- or low-density lipoprotein-based extenders for the cryopreservation of brown-bear (Ursus arctos) spermatozoa.2013In: Reproduction, Fertility and Development, ISSN 1031-3613, E-ISSN 1448-5990, Vol. 25, no 8, p. 1185-1193Article in journal (Refereed)

Egg yolk low-density lipoproteins (LDL) and soybean lecithin were evaluated as replacements for egg yolk in extenders used for the cryopreservation of brown-bear spermatozoa. The motility, viability and acrosomal status of post-thawed spermatozoa were analysed, and an egg-yolk extender was used as a control. The total antioxidant capacity of these extenders was tested. Soybean lecithin showed an effect that was dependent on the soybean concentration (2%, 3.5% and 5%) and source (Type A: 24% L-α-phosphatidylcholine, and Type B: 14-23% L-α-phosphatidylcholine). Only semen cryopreserved with 5% Type A soybean exhibited a sperm motility similar to that of semen cryopreserved in egg-yolk-based extender after thawing, although the sperm viability and acrosome status were not as high. Semen frozen in an extender containing LDL (10-15%) exhibited improved sperm viability in comparison with the control, but sperm motility was lower. The LDL-based extender exhibited a higher anti-oxidant activity than the egg-yolk extender and soy lecithin-based extenders. The extenders with higher anti-oxidant activity showed improvements in frozen sperm viability but lower semen motility. These results indicate that soybean lecithin did not have the same protective effect as egg yolk during the freezing of brown-bear spermatozoa but suggest that LDL (10-15%) could be a useful substitute for egg yolk in these extenders.

• 42.
ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain .
ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain. Cabárceno Park, Cantabria, Spain. ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, León, Spain . The Academic Unit of Reproductive and Developmental Medicine, University of Sheffield, Sheffield, UK. The Academic Unit of Reproductive and Developmental Medicine, University of Sheffield, Sheffield, UK. ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, León, Spain . ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
The addition of heat shock protein HSPA8 to cryoprotective media improves the survival of brown bear (Ursus arctos) spermatozoa during chilling and after cryopreservation.2013In: Theriogenology, ISSN 0093-691X, E-ISSN 1879-3231, Vol. 79, no 3, p. 541-550Article in journal (Refereed)

The Cantabrian brown bear survives as a small remnant population in northern Spain and semen cryopreservation for future artificial insemination is one of the measures being implemented for conservation of this species. As part of this program we investigated the value of adding heat shock protein A8 (HSPA8) to media (N-[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid-TRIS-fructose with 20% egg yolk) used for chilling and cryopreserving the spermatozoa. Semen samples from eight brown bears were obtained by electroejaculation during the breeding season. In experiment 1, we tested three concentrations of HSPA8 (0.5, 1, and 5 μg/mL) to determine whether sperm motility (computer assisted sperm analysis system) and sperm survival could be improved during refrigeration (5 °C) up to 48 hours. Results showed that sperm viability (test with propidium iodide) was improved by the addition of 0.5 and 5 μg/mL HSPA8. In experiment 2, HSPA8 was added to the cryopreservation media (6% final glycerol concentration) before the freezing process. Though there were no differences in sperm viability immediately after thawing (analyses to 0 hours), plasma membrane permeability (test with YO-PRO-1) was significantly lower by the presence of HSPA8 (1 μg/mL) and acrosomal damage (test with peanut agglutinin-fluorescein isothiocyanate conjugate) was reduced by higher concentrations of HSPA8 (1 and 5 μg/mL) (analyses after thermal stress test incubating over 2 hours to 37 °C). In experiment 3, results of a simple progression test carried out through artificial mucus (hyaluronic acid 4 mg/mL) showed a significant decrease in the total number of sperm able to swim a distance of 0.5 to 2 cm through a capillary tube for all HSPA8-based extenders. Nevertheless, the distance traveled by the vanguard spermatozoa, which represent a highly motile subpopulation, was restored by the inclusion of 1 and 5 μg/mL HSPA8 in the cryopreservation media. Thus, the HSPA8 addition to extender improves the quality of brown bear (Ursus arctos) sperm during chilling (viability) and after cryopreservation (number of sperm with damaged acrosomes and "apoptotic-like" changes).

• 43.
ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain. ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain. ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, León, Spain. Cabárceno Park, Cabárceno, Cantabria, Spain. SaBio IREC (CSIC-UCLM-JCCM), Albacete, Spain. ITRA-ULE, INDEGSAL, University of León, León, Spain; Molecular Biology (Cell Biology), University of León, León, Spain. ITRA-ULE, INDEGSAL, University of León, León, Spain; Animal Reproduction and Obstetrics, University of León, León, Spain.
Brown bear sperm double freezing: Effect of elapsed time and use of PureSperm(®) gradient between freeze-thaw cycles.2013In: Cryobiology, ISSN 0011-2240, E-ISSN 1090-2392, Vol. 67, no 3, p. 339-346Article in journal (Refereed)

The use of sexed spermatozoa has great potential to captive population management in endangered wildlife. The problem is that the sex-sorting facility is a long distance from the semen collection place and to overcome this difficulty two freeze-thaw cycles may be necessary. In this study, effects of refreezing on brown bear electroejaculated spermatozoa were analyzed. We carried out two experiments: (1) to assess the effects of the two freezing-thawing cycles on sperm quality and to analyze three different elapsed times between freezing-thawing cycles (30, 90 and 180 min), and (2) to analyze the use of PureSperm between freezing-thawing cycles to select a more motile and viable sperm subpopulation which better survived first freezing. The motility, viability and undamaged acrosomes were significantly reduced after the second thawing respect to first thawing into each elapsed time group, but the elapsed times did not significantly affect the viability and acrosome status although motility was damaged. Our results with the PureSperm gradient showed higher values of viability in freezability of select sample (pellet) respect to the rest of the groups and it also showed a significant decrease in the number of acrosome damaged. In summary, the double freezing of bear semen selected by gradient centrifugation is qualitatively efficient, and thus could be useful to carry out a sex-sorting of frozen-thawed bear spermatozoa before to send the cryopreserved sample to a biobank. Given the low recovery of spermatozoa after applying a selection gradient, further studies will be needed to increase the recovery rate without damaging of the cell quality.

• 44.
Lund University, Sweden .
Chalmers University of Technology, Göteborg, Sweden. Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics. Linköping University, The Institute of Technology. Lund University, Sweden . Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics. Linköping University, The Institute of Technology. Chalmers University of Technology, Göteborg, Sweden. Lund University, Sweden . Lund University, Sweden . Lund University, Sweden .
Charge Carrier Dynamics of Polymer: Fullerene Blends: From Geminate to Non-Geminate Recombination2014In: Advanced Energy Materials, ISSN 1614-6832, Vol. 4, no 8, article id 1301706Article in journal (Refereed)

The charge carrier dynamics of a new polymer-fullerene blend are examined on the femtosecond to the millisecond time scale. The full time range is globally fitted using a chemical reaction rate model that includes all key processes, charge generation, energy transfer, charge separation, and recombination, over the full 12 orders of magnitude in time and a factor of 33 in light intensity. Particular attention is paid to the charge recombination processes and it is found that they are highly material specific. Comparison of the dynamics to those of a previously studied polymer: fullerene blend reveals that while for one blend the recombination dynamics are mainly controlled by geminate recombination, the charge recombination in the presently studied polymer: fullerene blend are entirely controlled by non-geminate electron-hole recombination. Carrier density dependence of the non-geminate recombination rate is analyzed and a correlated disorder model of site energies is proposed to explain the observed dependency.

• 45.
University of Sydney, Australia.
Linköping University, Department of Physics, Chemistry and Biology, Theoretical Biology. Linköping University, The Institute of Technology. University of Sydney, Australia. University of Sydney, Australia.
Egg incubation effects generate positive correlations between size, speed and learning ability in young lizards2014In: Animal Cognition, ISSN 1435-9448, E-ISSN 1435-9456, Vol. 17, no 2, p. 337-347Article in journal (Refereed)

Previous studies have suggested that body size and locomotor performance are targets of Darwinian selection in reptiles. However, much of the variation in these traits may derive from phenotypically plastic responses to incubation temperature, rather than from underlying genetic variation. Intriguingly, incubation temperature may also influence cognitive traits such as learning ability. Therefore, we might expect correlations between a reptiles size, locomotor speed and learning ability either due to selection on all of these traits or due to environmental effects during egg incubation. In the present study, we incubated lizard eggs (Scincidae: Bassiana duperreyi) under hot and cold thermal regimes and then assessed differences in hatchling body size, running speed and learning ability. We measured learning ability using a Y-maze and a food reward. We found high correlations between size, speed and learning ability, using two different metrics to quantify learning (time to solution, and directness of route), and showed that environmental effects (incubation temperature) cause these correlations. If widespread, such correlations challenge any simple interpretation of fitness advantages due to body size or speed within a population; for example, survivors may be larger and faster than nonsurvivors because of differences in learning ability, not because of their size or speed.

• 46.
Kolmården Wildlife Park.
Filosofiska institutionen, Stockholms universitet. Linköping University, Department of Culture and Communication, Language and Culture. Linköping University, Faculty of Arts and Sciences. Kungliga Tekniska Högskolan. Carnegie Investment Bank, Swedden.
A proposal to use distributional models to analyse dolphin vocalisation2017In: Proceedings of the 1st International Workshop on Vocal Interactivity in-and-between Humans, Animals and Robots, VIHAR 2017 / [ed] Angela Dassow, Ricard Marxer & Roger K. Moore, 2017, p. 31-32Conference paper (Refereed)

This paper gives a brief introduction to the starting points of an experimental project to study dolphin communicative behaviour using distributional semantics, with methods implemented for the large scale study of human language.

• 47.
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
Structural insights into protein-protein interactions governing regulation in transcription initiation and ubiquitination2015Doctoral thesis, comprehensive summary (Other academic)

Virtually every aspect of the cellular processes in eukaryotes requires that the interactions between protein molecules are well coordinated in different regulatory pathways. Any protein dysfunction involved in these regulatory pathways might lead to various pathological conditions. Understanding the structural and functional peculiarities of these proteins molecular machineries will help in formulating structure-based drug design.

The first regulatory process studied here is the RNA polymerase-II mediated transcription of the eukaryotic protein-coding genes to produce mRNAs. This process requires the formation of the ‘transcription initiation’ by the assembly of Pre-Initiation Complex (PIC) formation at a core promoter region. Regulation at this initiation level is a key mechanism for the control of gene expression that governs cellular growth and differentiation. The transcription Factor IID (TFIID) is a conserved multiprotein general transcription factor with an essential role in  nucleating the PIC formation, composed of TATA Binding Protein (TBP) and about 14 TBP Associated Factors (TAFs). The here presented crystal structure (1.97Å) of TBP bound to TAND1 and TAND2 domains from TAF1 reveals a detailed molecular pattern of interactions involving both transcriptionally activating and repressing regions in TBP, thereby uncovering central principles for anchoring of TBP-binding motifs. Together with NMR and cellular analysis, this work provides the structural basis of competitive binding with TFIIA to modulate TBP in promoter recognition.

In eukaryotes, another fundamental mechanism in the regulation of cellular physiology is the posttranslational modification of substrate proteins by ubiquitin, termed ‘ubiquitination’. Important actors in this mechanism are the ubiquitin-ligases (E3s) that culminate the transfer of ubiquitin to the substrate and govern the specificity of this system. One E3 ligase in particular, TRIM21, defines a subgroup of the Tripartite Motif (TRIM) family, which belongs to the major RING-type of E3 ubiquitin ligases, and plays an important role in pathogenesis of autoimmunity by mediating ubiquitination of transcription factors. The crystal structure (2.86Å) of the RING domain from TRIM21 in complex with UBE2E1, an E2 conjugating enzyme, together with the NMR and SAXS analysis as well as biochemical functional analysis, reveals the molecular basis for the dynamic binding interfaces. The TRIM21 mode of ubiquitin recognition and activation for catalytic transfer of ubiquitin can be modeled onto the entire TRIM family.

Finally, we explored the concepts of conformational selection in proteins as a possible key component for protein-mediated transcriptional regulation. In this framework, MexR, a bacterial repressor of the MexAB-OprM efflux pump, and its mutant Arg21Trp were studied as an example for proteins presenting different conformations. The residue Arg21Trp mutation is clinically identified to cause of Multi-Drug Resistant (MDR) by attenuated DNA binding, and leads to the overexpression of the MexAB-OprM efflux pump. With the crystal structure (2.19Å) of MexR mutant Arg21Trp, in combination with MD-simulations and SAXS for both wild-type and mutant, we could unravel the atomic details of the wild-type conformations consisting in subsets of populations of DNA bound and unbound forms. Remarkably, the mutant Arg21Trp stabilize the DNA unbound state and shifts MexR in a pre-existing equilibrium, from a repressed to a derepressed state.

Taken together, these studies substantially broaden our knowledge at a molecular level in protein interactions that are involved in transcriptional regulation and ubiquitination, studied by a carefully selected combination of complementary structural methods spanning different resolutions and time scales.

1. High-resolution structure of TBP with TAF1 reveals anchoring patterns in transcriptional regulation
Open this publication in new window or tab >>High-resolution structure of TBP with TAF1 reveals anchoring patterns in transcriptional regulation
2013 (English)In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 20, no 8, p. 1008-+Article in journal (Refereed) Published
##### Abstract [en]

The general transcription factor TFIID provides a regulatory platform for transcription initiation. Here we present the crystal structure (1.97 angstrom) and NMR analysis of yeast TAF1 N-terminal domains TAND1 and TAND2 bound to yeast TBP, together with mutational data. We find that yeast TAF1-TAND1, which in itself acts as a transcriptional activator, binds TBPs concave DNA-binding surface by presenting similar anchor residues to TBP as does Mot1 but from a distinct structural scaffold. Furthermore, we show how TAF1-TAND2 uses an aromatic and acidic anchoring pattern to bind a conserved TBP surface groove traversing the basic helix region, and we find highly similar TBP-binding motifs also presented by the structurally distinct TFIIA, Mot1 and Brf1 proteins. Our identification of these anchoring patterns, which can be easily disrupted or enhanced, provides insight into the competitive multiprotein TBP interplay critical to transcriptional regulation.

##### Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 75 VARICK ST, 9TH FLR, NEW YORK, NY 10013-1917 USA, 2013
##### National Category
Engineering and Technology
##### Identifiers
urn:nbn:se:liu:diva-96977 (URN)10.1038/nsmb.2611 (DOI)000322715300016 ()
##### Note

Funding Agencies|Swedish Research Council|621-2011-6028621-2012-5250621-2012-5136|VINNOVA|P32045-1|Swedish Cancer Foundation|11 0681|Swedish Child Cancer Foundation|PROJ09/092|Forum Scientium Award||Canadian Institutes for Health Research|MT-13611|Japan Society for the Promotion of Science|23370077|Knut and Alice Wallenberg foundation||Canada Research Chair||

Available from: 2013-09-05 Created: 2013-09-02 Last updated: 2017-12-06
2. Mutation-Induced Population Shift in the MexR Conformational Ensemble Disengages DNA Binding: A Novel Mechanism for MarR Family Derepression
Open this publication in new window or tab >>Mutation-Induced Population Shift in the MexR Conformational Ensemble Disengages DNA Binding: A Novel Mechanism for MarR Family Derepression
2016 (English)In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 24, no 8, p. 1311-1321Article in journal (Refereed) Published
##### Abstract [en]

MexR is a repressor of the MexAB-OprM multidrug efflux pump operon of Pseudomonas aeruginosa, where DNA-binding impairing mutations lead to multidrug resistance (MDR). Surprisingly, the crystal structure of an MDR-conferring MexR mutant R21W (2.19 angstrom) presented here is closely similar to wildtype MexR. However, our extended analysis, by molecular dynamics and small-angle X-ray scattering, reveals that the mutation stabilizes a ground state that is deficient of DNA binding and is shared by both mutant and wild-type MexR, whereas the DNA-binding state is only transiently reached by the more flexible wild-type MexR. This population shift in the conformational ensemble is effected by mutation-induced allosteric coupling of contact networks that are independent in the wild-type protein. We propose that the MexR-R21W mutant mimics derepression by small-molecule binding to MarR proteins, and that the described allosteric model based on population shifts may also apply to other MarR family members.

##### Place, publisher, year, edition, pages
CELL PRESS, 2016
##### National Category
Structural Biology
##### Identifiers
urn:nbn:se:liu:diva-131908 (URN)10.1016/j.str.2016.06.008 (DOI)000383244600012 ()27427478 (PubMedID)
##### Note

Funding Agencies|European Communitys Seventh Framework Program (FP7) under BioStruct-X [283570]; Swedish e-Science Research Center; Swedish Research Council; Tage Erlander Visiting Professor grant.

The original status of this article was Manuscript and the titel was Population shift disengages DNA binding in a multidrug resistance MexR mutant.

Available from: 2016-10-13 Created: 2016-10-11 Last updated: 2017-11-29
3. Anti-Ro52 Autoantibodies from Patients with Sjögren's Syndrome Inhibit the Ro52 E3 Ligase Activity by Blocking the E3/E2 Interface
Open this publication in new window or tab >>Anti-Ro52 Autoantibodies from Patients with Sjögren's Syndrome Inhibit the Ro52 E3 Ligase Activity by Blocking the E3/E2 Interface
2011 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 42, p. 36478-36491Article in journal (Refereed) Published
##### Abstract [en]

Ro52 (TRIM21) is an E3 ligase of the tripartite motif family that negatively regulates proinflammatory cytokine production by ubiquitinating transcription factors of the interferon regulatory factor family. Autoantibodies to Ro52 are present in patients with lupus and Sjögren's syndrome, but it is not known if these autoantibodies affect the function of Ro52. To address this question, the requirements for Ro52 E3 ligase activity were first analyzed in detail. Scanning a panel of E2 ubiquitin-conjugating enzymes, we found that UBE2D1–4 and UBE2E1–2 supported the E3 ligase activity of Ro52 and that the E3 ligase activity of Ro52 was dependent on its RING domain. We also found that the N-terminal extensions in the class III E2 enzymes affected their interaction with Ro52. Although the N-terminal extension in UBE2E3 made this E2 enzyme unable to function together with Ro52, the N-terminal extensions in UBE2E1 and UBE2E2 allowed for a functional interaction with Ro52. Anti-Ro52-positive patient sera and affinity-purified anti-RING domain autoantibodies inhibited the E3 activity of Ro52 in ubiquitination assays. Using NMR, limited proteolysis, ELISA, and Ro52 mutants, we mapped the interactions between Ro52, UBE2E1, and anti-Ro52 autoantibodies. We found that anti-Ro52 autoantibodies inhibited the E3 ligase activity of Ro52 by sterically blocking the E2/E3 interaction between Ro52 and UBE2E1. Our data suggest that anti-Ro52 autoantibodies binding the RING domain of Ro52 may be actively involved in the pathogenesis of rheumatic autoimmune disease by inhibiting Ro52-mediated ubiquitination.

##### Place, publisher, year, edition, pages
American Society for Biochemistry and Molecular Biology, 2011
##### National Category
Natural Sciences
##### Identifiers
urn:nbn:se:liu:diva-53170 (URN)10.1074/jbc.M111.241786 (DOI)000296538300033 ()
##### Note

Funding agencies|Swedish Research Council||Swedish Foundation for Strategic Research||VINNOVA||CeNano||Swedish Cancer Society||Karolinska Institutet||Linkoping University||King Gustaf Vs 80-Year Foundation||Heart-Lung Foundation||Stockholm County Council||Gustafsson Foundation||Soderberg Foundation||National Cancer Institute of Canada||Swedish Rheumatism Association||Wallenberg Foundation||

Available from: 2010-01-18 Created: 2010-01-18 Last updated: 2017-12-12Bibliographically approved
4. Structure of a TRIM21 - UBE2El complex reveals the specificity of E2 and ubiquitin recognition by TRIM E3 RINGs
Open this publication in new window or tab >>Structure of a TRIM21 - UBE2El complex reveals the specificity of E2 and ubiquitin recognition by TRIM E3 RINGs
##### Abstract [en]

TRIM21, a RlNG-containing E3 ubiquitin-ligase of the TRIM  $\tiny(\underline{tri}partite \ \underline{m}otif)$ protein family, is a major autoantigen in SLE and Sjögren's syndrome as well as a modifier of interferon regulatory factors, thereby regulating innate immune signalling. We herein report the 2.86 Å crystal structure ofhuman TRIM211-91 comprising the RING domain (residues 16-55), in complex with the human E2 conjugating UBE2El enzyme (also denoted UbcH6). The crystal structure, joint with analysis by NMR and SAXS as well as structure-directed mutations and functional assays provides a detailed view of the specificity-determining contacts that support specific E2 recognition in the TRIM family. A detailed comparison of our structure with known E2 bound ubiquitin complexes, supported by biochemical analyses, reveals the molecular basis for TRIM21 interactions with donor ubiquitin that activates catalytic ubiquitin transfer. Finally, our structure convincingly demonstrates the placement of the Ub-targeted Lys61 of the adjacent TRIM21$\small^,$1- 91 close to the catalytically active UBE2El cysteine, and how the Lys61 amide is activated fora nucleophilic attack by hydrogen-bondeffected deshielding by conserved acidic residues at the E2 active site. In all, our structural findings provide molecular details ofthe selectivity involved in TRIM21 interactions with its cognate UBE2E1 enzyme and how TRIM21 positions ubiquitin in a catalytic conformation for ubiquitin transfer, and presents a snapshot of the Ub ligation step on a specific target residue of TRIM211-91 as an auto-ubiquitinated pseudo-substrate at high concentration. Increased structural and functional understanding of the TRIM mediated ubiquitination will aid development ofnovel therapeutic approaches in the entire TRIM family ofproteins.

##### National Category
Chemical Sciences Biochemistry and Molecular Biology
##### Identifiers
urn:nbn:se:liu:diva-122466 (URN)
Available from: 2015-11-03 Created: 2015-11-03 Last updated: 2015-11-13Bibliographically approved
• 48.
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
Rheumatology Unit, Department of Medicine, Center for Molecular Medicine L8:04, Karolinska Institutet, Stockholm, Sweden. Rheumatology Unit, Department of Medicine, Center for Molecular Medicine L8:04, Karolinska Institutet, Stockholm, Sweden. European Molecular Biology Laboratory, Grenoble Outstation, Grenoble, France. Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering. School of Molecular Bioscience, The University of Sydney, New South Wales, Australia. Rheumatology Unit, Department of Medicine, Center for Molecular Medicine L8:04, Karolinska Institutet, Stockholm, Sweden. Department of Medical Biochemistry and Biophysics, Protein Science Facility, Karolinska Institutet, Stockholm, Sweden. Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
Structure of a TRIM21 - UBE2El complex reveals the specificity of E2 and ubiquitin recognition by TRIM E3 RINGsManuscript (preprint) (Other academic)

TRIM21, a RlNG-containing E3 ubiquitin-ligase of the TRIM  $\tiny(\underline{tri}partite \ \underline{m}otif)$ protein family, is a major autoantigen in SLE and Sjögren's syndrome as well as a modifier of interferon regulatory factors, thereby regulating innate immune signalling. We herein report the 2.86 Å crystal structure ofhuman TRIM211-91 comprising the RING domain (residues 16-55), in complex with the human E2 conjugating UBE2El enzyme (also denoted UbcH6). The crystal structure, joint with analysis by NMR and SAXS as well as structure-directed mutations and functional assays provides a detailed view of the specificity-determining contacts that support specific E2 recognition in the TRIM family. A detailed comparison of our structure with known E2 bound ubiquitin complexes, supported by biochemical analyses, reveals the molecular basis for TRIM21 interactions with donor ubiquitin that activates catalytic ubiquitin transfer. Finally, our structure convincingly demonstrates the placement of the Ub-targeted Lys61 of the adjacent TRIM21$\small^,$1- 91 close to the catalytically active UBE2El cysteine, and how the Lys61 amide is activated fora nucleophilic attack by hydrogen-bondeffected deshielding by conserved acidic residues at the E2 active site. In all, our structural findings provide molecular details ofthe selectivity involved in TRIM21 interactions with its cognate UBE2E1 enzyme and how TRIM21 positions ubiquitin in a catalytic conformation for ubiquitin transfer, and presents a snapshot of the Ub ligation step on a specific target residue of TRIM211-91 as an auto-ubiquitinated pseudo-substrate at high concentration. Increased structural and functional understanding of the TRIM mediated ubiquitination will aid development ofnovel therapeutic approaches in the entire TRIM family ofproteins.

• 49.
Linköping University, Department of Physics, Chemistry and Biology. Linköping University, Faculty of Science & Engineering.
Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics. Linköping University, Faculty of Science & Engineering. Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering. Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering. University of Sydney, Australia. Karolinska Institute, Sweden. Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics. Linköping University, Faculty of Science & Engineering. Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
Mutation-Induced Population Shift in the MexR Conformational Ensemble Disengages DNA Binding: A Novel Mechanism for MarR Family Derepression2016In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 24, no 8, p. 1311-1321Article in journal (Refereed)

MexR is a repressor of the MexAB-OprM multidrug efflux pump operon of Pseudomonas aeruginosa, where DNA-binding impairing mutations lead to multidrug resistance (MDR). Surprisingly, the crystal structure of an MDR-conferring MexR mutant R21W (2.19 angstrom) presented here is closely similar to wildtype MexR. However, our extended analysis, by molecular dynamics and small-angle X-ray scattering, reveals that the mutation stabilizes a ground state that is deficient of DNA binding and is shared by both mutant and wild-type MexR, whereas the DNA-binding state is only transiently reached by the more flexible wild-type MexR. This population shift in the conformational ensemble is effected by mutation-induced allosteric coupling of contact networks that are independent in the wild-type protein. We propose that the MexR-R21W mutant mimics derepression by small-molecule binding to MarR proteins, and that the described allosteric model based on population shifts may also apply to other MarR family members.

• 50.
Centre for Speech Technology, KTH, Stockholm, Sweden.
Linköping University, Department of Computer and Information Science, NLPLAB - Natural Language Processing Laboratory. Linköping University, The Institute of Technology. Forschungsinstitut Alexander Koenig, Bonn, Germany. Antelope Park, Gweru, Zimbabwe.
An acoustic analysis of lion roars. II: Vocal tract characteristics2011In: Proceedings from Fonetik 2011, Quarterly Progress and Status Report TMH-QPSR, Volume 51, 2011, 2011, p. 5-8Conference paper (Other academic)

This paper makes the first attempt to perform an acoustic-to-articulatory inversion of a lion (Panthera leo) roar. The main problems that one encounters in attempting this, is the fact that little is known about the dimensions of the vocal tract, other than a general range of vocal tract lengths. Precious little is also known about the articulation strategies that are adopted by the lion while roaring. The approach used here is to iterate between possible values of vocal tract lengths and vocal tract configurations. Since there seems to be a distinct articulatory changes during the process of a roar, we find a smooth path that minimizes the error function between a recorded roar and the simulated roar using a variable length articulatory model.

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