Background

It has previously been shown in an uncontrolled study that the IgE response to vaccine antigens is downregulated by co‐vaccination with cellular Bordetella pertussis vaccine.

Methods

In the present study, we compared in a controlled trial the humoral immune response to diphtheria toxoid (D) and tetanus toxoid (T) in relation to co‐vaccinated cellular or acellular B pertussis vaccine. IgE, IgG4, and IgG to D and T were analyzed at 2, 7, and 12 months of age in sera of children vaccinated with D and T (DT, N = 68), cellular (DTPw, N = 68), 2‐ or 5‐component acellular B pertussis vaccine (DTPa2, N = 64; DTPa5, N = 65).

Results

One month after vaccination, D‐IgE was detected in 10% sera of DTPw‐vaccinated children, whereas vaccination in the absence of whole‐cell pertussis resulted in 50%‐60% IgE positivity. Six months after vaccination, the IgE antibody levels were found to be more persistent than the IgG antibodies. These diphtheria findings were mirrored by those for tetanus. Only minor differences between vaccine groups were found with regard to D‐IgG and T‐IgG. No immediate‐type allergic reactions were observed.

Conclusion

Cellular (but not acellular) B pertussis vaccine downregulates IgE to co‐vaccinated antigens in infants. We assume that the absence of immediate‐type allergic reactions is due to the high levels of IgG antibodies competing with IgE antibodies.

Molecular diagnostics for sepsis is still a challenge due to the presence of compounds that interfere with gene amplification and bacteria at concentrations lower than the limit of detection (LOD). Here, we report on the development of a 3D printed modular microfluidic device (3Dpm mu FD) that preconcentrates bacteria of interest in whole blood and purifies their genomic DNA (gDNA). It is composed of a W-shaped microchannel and a conical microchamber. Bacteria of interest are magnetically captured from blood in the device with antibody conjugated magnetic nanoparticles (Ab-MNPs) at 5 mL/min in the W-shaped microchannel, while purified gDNA of the preconcentrated bacteria is obtained with magnetic silica beads (MSBs) at 2 mL/min in the conical microchamber. The conical microchamber was designed to be connected to the microchannel after the capturing process using a 3D-printed rotary valve to minimize the exposure of the MSBs to interfering compounds in blood. The pretreatment process of spiked blood (2.5 mL) can be effectively completed within about 50 min. With the 3Dpm mu FD, the LOD for the target microorganism Escherichia coli O157:H7 measured by both polymerase chain reaction (PCR) with electrophoresis and quantitative PCR was 10 colony forming unit (CFU) per mL of whole blood. The results suggest that our method lowers the LOD of molecular diagnostics for pathogens in blood by providing bacterial gDNA at high purity and concentration.

Non-consumptive predator effects have been shown to influence a wide range of behavioural, life history and morphological traits. Extra-pair reproduction is widespread among socially monogamous birds and may incur predation costs. Consequently, altered rates of extra-pair reproduction are expected in circumstances characterized by increased adult perceived predation risk. In addition, extra-pair reproduction is expected to be most affected for birds with phenotypes that generally increase predation risk (such as more active individuals). In two consecutive years, perceived predation risk was manipulated for great tits Parus major breeding in 12 nest-box plots by broadcasting sounds of their main predator (European sparrowhawk Accipiter nisus; six plots). As a control treatment, sounds of a sympatric, avian non-predator species were broadcast (Eurasian blackbird Turdus merula; six plots). Levels of extra-pair paternity did not differ between plots with different predation risk treatments. Males that moved more in a novel environment (more active or faster exploring) tended to have offspring with fewer partners, but this effect did not vary with predation risk treatment. From an adaptive viewpoint, predation costs associated with extra-pair reproduction may be small and may not outweigh the benefits of extra-pair behaviour. Research on a broader range of taxa with different mating strategies is now needed to confirm the generality of our findings.

The adaptive evolution of timing of breeding (a component of phenology) in response to environmental change requires individual variation in phenotypic plasticity for selection to act upon. A major question is what processes generate this variation. Here we apply multi-year manipulations of perceived predation levels (PPL) in an avian predator-prey system, identifying phenotypic plasticity in phenology as a key component of alternative behavioral strategies with equal fitness payoffs. We show that under low-PPL, faster (versus slower) exploring birds breed late (versus early); the pattern is reversed under high-PPL, with breeding synchrony decreasing in conjunction. Timing of breeding affects reproductive success, yet behavioral types have equal fitness. The existence of alternative behavioral strategies thus explains variation in phenology and plasticity in reproductive behavior, which has implications for evolution in response to anthropogenic change.

Among-individual behavioral differences (i.e. animal personality) are commonly observed across taxa, although the underlying, causal mechanisms of such differences are poorly understood. Animal personality has been correlated with physiological functions as well as fitness-related traits. Variation in many aspects of monoamine systems, such as metabolite levels and gene polymorphisms, has been linked to behavioral variation. Therefore, here we experimentally investigated the potential role of monoamines in explaining individual variation in personality, using two common pharmaceuticals that respectively alter the levels of serotonin and dopamine in the brain: fluoxetine and ropinirole. We exposed three-spined sticklebacks, a species that shows animal personality, to either chemical alone or to a combination of the two chemicals, for 18 days. During the experiment, fish were assayed at four time points for the following personality traits: exploration, boldness, aggression and sociability. To quantify brain gene expression on short- and longer-term scales, fish were sampled at two time points. Our results show that monoamine manipulations influence fish behavior. Specifically, fish exposed to either fluoxetine or ropinirole were significantly bolder, and fish exposed to the two chemicals together tended to be bolder than control fish. Our monoamine manipulations did not alter the gene expression of monoamine or stress-associated neurotransmitter genes, but control, untreated fish showed covariation between gene expression and behavior. Specifically, exploration and boldness were predicted by genes in the dopaminergic, serotonergic and stress pathways, and sociability was predicted by genes in the dopaminergic and stress pathways. These results add further support to the links between monoaminergic systems and personality, and show that exposure to monoamines can causally alter animal personality.

Among-individual behavioral differences (i.e. animal personality) are commonly observed across taxa, although the underlying, causal mechanisms of such differences are poorly understood. Animal personality has been implicated in correlations with physiological functions as well as affecting fitness-related traits. Variation in many aspects of monoamine systems, such as metabolite levels and gene polymorphisms, has been linked to behavioral variation. Therefore, here we investigated the potential role of monoamines in explaining individual variation in personality, using two common pharmaceuticals that respectively alter the levels of serotonin and dopamine in the brain: fluoxetine and ropinirole. We exposed three- spined sticklebacks, a species that shows animal personality, to either chemical alone or to a combination of the two chemicals, for 18 days. During the experiment, fish were assayed at four time points for the following personality traits: exploration, boldness, aggression and sociability. To quantify brain gene expression on short- and longer-term scales, fish were sampled at two time points. Our results show that monoamine manipulations influence fish behavior. Specifically, fish exposed to either fluoxetine or ropinirole were significantly bolder, and fish exposed to the two chemicals together tended to be bolder than control fish. Our monoamine manipulations did not alter the gene expression of monoamine or stress-associated neurotransmitter genes, but control, untreated fish showed covariation between gene expression and behavior. Specifically, exploration and boldness were predicted by genes in the dopaminergic, serotonergic and stress pathways, and sociability was predicted by genes in the dopaminergic and stress pathways. These results add further support to the links between monoaminergic systems and personality, and show that exposure to monoamines can causally alter animal personality.

1. The causes of individual variation in behavior are often not well understood, and potential underlying mechanisms include both intrinsic and extrinsic factors, such as early environmental, physiological, and genetic differences.
2. In an exploratory laboratory study, we raised three-spined sticklebacks (Gasterosteus aculeatus) under 4 different environmental conditions (simulated predator environment, complex environment, variable social environment, and control). We investigated how these manipulations related to behavior, brain physiology and gene expression later in life, with focus on brain dopamine and serotonin levels, turnover rates, and gene expression.
3. The different rearing environments influenced behavior and gene expression, but did not alter monoamine levels or metabolites. Specifically, compared to control fish, fish exposed to a simulated predator environment tended to be less aggressive, more exploratory, and more neophobic; and fish raised in both complex and variable social environments tended to be less neophobic. Exposure to a simulated predator environment tended to lower expression of dopamine receptor DRD4A, a complex environment increased expression of dopamine receptor DRD1B, while a variable social environment tended to increase serotonin receptor 5-HTR2B and increased serotonin transporter SLC6A4A expression. Despite both behavior and gene expression varying with early environment, there was no evidence that gene expression mediated the relationship between early environment and behavior.
4. Our results confirm that environmental conditions early in life can affect phenotypic variation. However, the mechanistic pathway of the monoaminergic systems translating early environmental variation into observed behavioral responses was not detected.

The causes of individual variation in behavior are often not well understood, and potential underlying mechanisms include both intrinsic and extrinsic factors, such as early environmental, physiological, and genetic differences. In an exploratory laboratory study, we raised three-spined sticklebacks <i>(Gasterosteus aculeatus)</i> under 4 different environmental conditions (simulated predator environment, complex environment, variable social environment, and control). We investigated how these manipulations related to behavior, brain physiology, and gene expression later in life, with focus on brain dopamine and serotonin levels, turnover rates, and gene expression. The different rearing environments influenced behavior and gene expression, but did not alter monoamine levels or metabolites. Specifically, compared to control fish, fish exposed to a simulated predator environment tended to be less aggressive, more exploratory, and more neophobic; and fish raised in both complex and variable social environments tended to be less neophobic. Exposure to a simulated predator environment tended to lower expression of dopamine receptor DRD4A, a complex environment increased expression of dopamine receptor DRD1B, while a variable social environment tended to increase serotonin receptor 5-HTR2B and serotonin transporter SLC6A4A expression. Despite both behavior and gene expression varying with early environment, there was no evidence that gene expression mediated the relationship between early environment and behavior. Our results confirm that environmental conditions early in life can affect phenotypic variation. However, the mechanistic pathway of the monoaminergic systems translating early environmental variation into observed behavioral responses was not detected.

Animal personality has been described in a range of species with ecological and evolutionary consequences. Factors shaping and maintaining variation in personality are not fully understood, but monoaminergic systems are consistently linked to personality variation. We experimentally explored how personality was influenced by alterations in two key monoamine systems: dopamine and serotonin. This was done using ropinirole and fluoxetine, two common human pharmaceuticals. Using the Mediterranean field cricket (Gryllus bimaculatus), we focused on the personality traits activity, exploration, and aggression, with confirmed repeatability in our study. Dopamine manipulations explained little variation in the personality traits investigated, while serotonin manipulation reduced both activity and aggression. Due to limited previous research, we created a dose-response curve for ropinirole, ranging from concentrations measured in surface waters to human therapeutic doses. No ropinirole dose level strongly influenced cricket personality, suggesting our results did not come from a dose mismatch. Our results indicate that the serotonergic system explains more variation in personality than manipulations of the dopaminergic system. Additionally, they suggest that monoamine systems differ across taxa, and confirm the importance of the mode of action of pharmaceuticals in determining their effects on behaviour.

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.

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.

Progress in synthesis technologies and advances in fundamental understanding of materials with low dimensionality has led to the birth of a new scientific field, nanoscience, and to strong expectations of multiple applications of nanomaterials. The physical properties of small particles are unique, bridging the gap between atoms and molecules, on one side, and bulk materials on the other side. The work presented in this thesis investigates the potential of using magnetic nanoparticles as the next generation of contrast agents for biomedical imaging. The focus is on gadolinium-based nanoparticles and cellular activity including the uptake, morphology and production of reactive oxygen species.

Gd ion complexes, like Gd chelates, are used today in the clinic, world-wide. However, there is a need for novel agents, with improved contrast capabilities and increased biocompatibility. One avenue in their design is based on crystalline nanoparticles. It allows to reduce the total number of Gd ions needed for an examination. This can be done by nanotechnology, which allows one to improve and fine tune the physico- chemical properties on the nanomaterial in use, and to increase the number of Gd atoms at a specific site that interact with protons and thereby locally increase the signal. In the present work, synthesis, purification and surface modification of crystalline Gd2O3-based nanoparticles have been performed. The nanoparticles are selected on the basis of their physical properties, that is they show enhanced magnetic properties and therefore may be of high potential interest for applications as contrast agents.

The main synthesis method of Gd₂O₃ nanoparticles in this work was the modified “polyol” route, followed by purification of as-synthesized DEG-Gd2O3 nanoparticles suspensions. In most cases the purification step involved dialysis of the nanoparticle samples. In this thesis, organosilane were chosen as an exchange agent for further functionalization. Moreover, several paths have been explored for modification of the nanoparticles, including Tb³⁺ doping and capping with sorbitol.

Biocompatibility of the newly designed nanoparticles is a prerequisite for their use in medical applications. Its evaluation is a complex process involving a wide range of biological phenomena. A promising path adopted in this work is to study of nanoparticle interactions with isolated blood cells. In this way one could screen nanomaterial prior to animal studies.

The primary cell type considered in the thesis are polymorphonuclear neutrophils (PMN) which represent a type of the cells of human blood belonging to the granulocyte family of leukocytes. PMNs act as the first defense of the immune system against invading pathogens, which makes them valuable for studies of biocompatibility of newly synthesized nanoparticles. In addition, an immortalized murine alveolar macrophage cell line (MH-S), THP-1 cell line, and Ba/F3 murine bone marrow-derived cell line were considered to investigate the optimization of the cell uptake and to examine the potential of new intracellular contrast agent for magnetic resonance imaging.

In paper I, the nanoparticles were investigated in a cellular system, as potential probes for visualization and targeting intended for bioimaging applications. The production of reactive oxygen species (ROS) by means of luminol-dependent chemiluminescence from human neutrophils was studied in presence of Gd₂O₃ nanoparticles. In paper II, a new design of functionalized ultra-small rare earth-based nanoparticles was reported. The synthesis was done using polyol method followed by PEGylation, and dialysis. Supersmall gadolinium oxide (DEG-Gd2O3) nanoparticles, in the range of 3-5 nm were obtained and carefully characterized. Neutrophil activation after exposure to this nanomaterial was studied by means of fluorescence microscopy. In paper III, cell labeling with Gd₂O₃ nanoparticles in hematopoietic cells was monitored by magnetic resonance imaging (MRI). In paper IV, ultra-small gadolinium oxide nanoparticles doped with terbium ions were synthesized as a potentially bifunctional material with both fluorescent and magnetic contrast agent properties. Paramagnetic behavior was studied. MRI contrast enhancement was received, and the luminescent/ fluorescent property of the particles was attributable to the Tb³⁺ ion located on the crystal lattice of the Gd₂O₃ host. Fluorescent labeling of living cells was obtained. In manuscript V, neutrophil granulocytes were investigated with rapid cell signaling communicative processes in time frame of minutes, and their response to cerium-oxide based nanoparticles were monitored using capacitive sensors based on Lab-on-a-chip technology. This showed the potential of label free method used to measure oxidative stress of neutrophil granulocytes. In manuscript VI, investigations of cell-(DEGGd₂O₃) nanoparticle interactions were carried out. Plain (DEG-Gd₂O₃) nanoparticles, (DEG-Gd₂O₃) nanoparticles in presence of sorbitol and (DEG-Gd₂O₃) nanoparticles capped with sorbitol were studied. Relaxation studies and measurements of the reactive oxygen species production by neutrophils were based on chemiluminescence. Cell morphology was evaluated as a parameter of the nanoparticle induced inflammatory response by means of the fluorescence microscopy.

The thesis demonstrates high potential of novel Gd₂O₃-based nanoparticles for development of the next generation contrast agents, that is to find biocompatible compounds with high relaxivity that can be detected at lower doses, and in the future enable targeting to provide great local contrast.

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.

I experimenten undersöktes cellresponsen hos blodceller exponerade med ofunktionaliserade och funktionaliserade nanopartiklarna. Effekterna av funktionaliserade och ofunktionaliserade nanopartiklarna på aggregation och syreradikalproduktion i helblod och hos neutrofila granulocyter studerades med hjälp av lumi-aggregometri.

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.

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.

Background and purpose: To determine the prevalence of vertebral hemangiomas (VHs), establish a new classification of VHs based on their MRI-signal pattern, and study their natural history. Methods: MRI of 1000 consecutive patients who underwent at least two MRI with an interval of at least 3 years. Growth rate and change of MRI-signal pattern during the follow-up period were the parameters included in studying the natural history of VHs. Results: The prevalence of VHs was 41%. VHs were classified as type I-IV with fat-rich VHs (type I), constituted 79% of all VHs. VHs were more common among females 43 degrees/o versus males 39%, p = .22. The most affected vertebra was L1. Occurrence rates for cervical (1%), thoracic (7%), and lumbar spine (10%) differed significantly (p &lt; .001). The prevalence of VHs increased with age regardless of gender or spinal part involved (p &lt; .001). Only 26% of VHs changed their size and 4 degrees/o changed their signal during the average follow-up of 7 years. All VHs were slowly growing lesions (average expected growth of &lt;3 mm/10 years). No significant difference between growth rate of VHs type I (0.25 mm/year) and other types of VHs. None of the VHs that were initially reported as "metastases cannot be rule out" showed alarming change in signal or size. Conclusions: VH can be classified into four types based on their MRI-signal pattern. Regardless of their type, VHs are slowly growing lesions. The presence of typical morphological pattern should enable radiologists to confidently differentiate them from vertebral metastases.

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.

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.

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.

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.

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.

Domestication of animals offers unique possibilities to study evolutionary changes caused by similar selection pressures across a range of species. Animals from separate genera tend to develop a suite of phenotypic alterations referred to as "the domesticated phenotype". This involves changes in appearance, including loss of pigmentation, and alterations in body size and proportions. Furthermore, effects on reproduction and behaviour are typical. It is hypothesized that this recurring phenotype may be secondary effects of the increased tameness that is an inevitable first step in the domestication of any species. We first provide a general overview of observations and experiments from different species and then review in more detail a project attempting to recreate the initial domestication of chickens. Starting from an outbred population of Red Junglefowl, ancestors of all modem chickens, divergent lines were selected based on scores in a standardized fear-of-human test applied to all birds at 12 weeks of age. Up to the eighth selected generation, observations have been made on correlated effects of this selection on various phenotypes. The fear score had a significant heritability and was genetically correlated to several other behavioural traits. Furthermore, low-fear birds were larger at hatch, grew faster, laid larger eggs, had a modified metabolism and increased feed efficiency, had modified social behaviour and reduced brain size. Selection affected gene expression and DNA-methylation in the brains, but the genetic and epigenetic effects were not specifically associated with stress pathways. Further research should be focused on unraveling the genetic and epigenetic mechanisms underlying the correlated side-effects of reduced fear of humans.

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.

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.

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.

BackgroundThe immunomodulatory capacity of breast milk may partially be mediated by microRNAs (miRNA), small RNA molecules that regulate gene expression on a post-transcriptional level and are hypothesized to be involved in modulation of immunological pathways. Here, we evaluate the expression of immune-related miRNAs in breast milk after pre- and postnatal supplementation with Limosilactobacillus reuteri and omega-3 (omega-3) polyunsaturated fatty acids (PUFAs), and the association to infant regulatory T cell (Treg) frequencies. MethodsOne-hundred and twenty women included in a double-blind, randomized, placebo-controlled allergy intervention trial received L. reuteri and/or omega-3 PUFAs daily from gestational week 20. Using Taqman qPCR, 24 miRNAs were analyzed from breast milk obtained at birth (colostrum) and after 3 months (mature milk) of lactation. The proportion of activated and resting Treg cells were analyzed in infant blood using flow cytometry at 6, 12, and 24 months. ResultsRelative expression changed significantly over the lactation period for most of the miRNAs; however, the expression was not significantly influenced by any of the supplements. Colostrum miR-181a-3p correlated with resting Treg cell frequencies at 6 months. Colostrum miR-148a-3p and let-7d-3p correlated with the frequencies of activated Treg cells at 24 months, as did mature milk miR-181a-3p and miR-181c-3p. ConclusionMaternal supplementation with L. reuteri and omega-3 PUFAs did not significantly affect the relative miRNA expression in breast milk. Interestingly, some of the miRNAs correlate with Treg subpopulations in the breastfed children, supporting the hypothesis that breast milk miRNAs could be important in infant immune regulation. Trial registrationClinicalTrials.gov-ID: NCT01542970.

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.

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.

Carbapenem-resistant bacteria are a threat to public health globally and have been found in the environment as well as the clinic. Some bacterial clones are associated with carbapenem resistance genes, such as Escherichia coli sequence type 38 (ST38) and the carbapenemase gene bla(OXA-48). Carbapenem-resistant Enterobacteriaceae (CRE) are a global threat to human health and are increasingly being isolated from nonclinical settings. OXA-48-producing Escherichia coli sequence type 38 (ST38) is the most frequently reported CRE type in wild birds and has been detected in gulls or storks in North America, Europe, Asia, and Africa. The epidemiology and evolution of CRE in wildlife and human niches, however, remains unclear. We compared wild bird origin E. coli ST38 genome sequences generated by our research group and publicly available genomic data derived from other hosts and environments to (i) understand the frequency of intercontinental dispersal of E. coli ST38 clones isolated from wild birds, (ii) more thoroughly measure the genomic relatedness of carbapenem-resistant isolates from gulls sampled in Turkey and Alaska, USA, using long-read whole-genome sequencing and assess the spatial dissemination of this clone among different hosts, and (iii) determine whether ST38 isolates from humans, environmental water, and wild birds have different core or accessory genomes (e.g., antimicrobial resistance genes, virulence genes, plasmids) which might elucidate bacterial or gene exchange among niches. Our results suggest that E. coli ST38 strains, including those resistant to carbapenems, are exchanged between humans and wild birds, rather than separately maintained populations within each niche. Furthermore, despite close genetic similarity among OXA-48-producing E. coli ST38 clones from gulls in Alaska and Turkey, intercontinental dispersal of ST38 clones among wild birds is uncommon. Interventions to mitigate the dissemination of antimicrobial resistance throughout the environment (e.g., as exemplified by the acquisition of carbapenem resistance by birds) may be warranted.IMPORTANCE Carbapenem-resistant bacteria are a threat to public health globally and have been found in the environment as well as the clinic. Some bacterial clones are associated with carbapenem resistance genes, such as Escherichia coli sequence type 38 (ST38) and the carbapenemase gene bla(OXA-48). This is the most frequently reported carbapenem-resistant clone in wild birds, though it was unclear if it circulated within wild bird populations or was exchanged among other niches. The results from this study suggest that E. coli ST38 strains, including those resistant to carbapenems, are frequently exchanged among wild birds, humans, and the environment. Carbapenem-resistant E. coli ST38 clones in wild birds are likely acquired from the local environment and do not constitute an independent dissemination pathway within wild bird populations. Management actions aimed at preventing the environmental dissemination and acquisition of antimicrobial resistance by wild birds may be warranted.

Gulls (Larus spp.) have frequently been reported to carry Escherichia coli exhibiting antimicrobial resistance (AMR E. coli); however, the pathways governing the acquisition and dispersal of such bacteria are not well described. We equipped 17 landfill-foraging gulls with satellite transmitters and collected gull faecal samples longitudinally from four locations on the Kenai Peninsula, Alaska to assess: (a) gull attendance and transitions between sites, (b) spatiotemporal prevalence of faecally shed AMR E. coli, and (c) genomic relatedness of AMR E. coli isolates among sites. We also sampled Pacific salmon (Oncorhynchus spp.) harvested as part of personal-use dipnet fisheries at two sites to assess potential contamination with AMR E. coli. Among our study sites, marked gulls most commonly occupied the lower Kenai River (61% of site locations) followed by the Soldotna landfill (11%), lower Kasilof River (5%) and upper Kenai River (amp;lt;1%). Gulls primarily moved between the Soldotna landfill and the lower Kenai River (94% of transitions among sites), which were also the two locations with the highest prevalence of AMR E. coli. There was relatively high spatial and temporal variability in AMR E. coli prevalence in gull faeces and there was no evidence of contamination on salmon harvested in personal-use fisheries. We identified E. coli sequence types and AMR genes of clinical importance, with some isolates possessing genes associated with resistance to as many as eight antibiotic classes. Our findings suggest that gulls acquire AMR E. coli at habitats with anthropogenic inputs and subsequent movements may represent pathways through which AMR is dispersed.

We tested extended-spectrum beta-lactamase producing bacteria from wild gulls (Larus spp.) sampled in 2009 for the presence of mcr-1. We report the detection of mcr-1 and describe genome characteristics of four Escherichia coli and one Klebsiella pneumoniae isolate from Spain and Portugal that also exhibited colistin resistance. Results represent the earliest evidence for colistin-resistant bacteria in European wildlife.

Here, we report the first detection of carbapenemase-producing Escherichia coli in Alaska and in wildlife in the United States. Wild bird (gull) feces sampled at three locations in Southcentral Alaska yielded isolates that harbored plasmidencoded bla(kpc-2), or chromosomally encoded bla(OXA-48) and genes associated with antimicrobial resistance to up to eight antibiotic classes.

Anthropogenic inputs into the environment may serve as sources of antimicrobial resistant bacteria and alter the ecology and population dynamics of synanthropic wild animals by providing supplemental forage. In this study, we used a combination of phenotypic and genomic approaches to characterize antimicrobial resistant indicator bacteria, animal telemetry to describe host movement patterns, and a novel modeling approach to combine information from these diverse data streams to investigate the acquisition and long-distance dispersal of antimicrobial resistant bacteria by landfill-foraging gulls. Our results provide evidence that gulls acquire antimicrobial resistant bacteria from anthropogenic sources, which they may subsequently disperse across and between continents via migratory movements. Furthermore, we introduce a flexible modeling framework to estimate the relative dispersal risk of antimicrobial resistant bacteria in western North America and adjacent areas within East Asia, which may be adapted to provide information on the risk of dissemination of other organisms and pathogens maintained by wildlife through space and time. Published by Elsevier B.V.

Carbapenem resistant Enterobacteriaceae (CRE) are a threat to public health globally, yet the role of the environment in the epidemiology of CRE remains elusive. Given that wild birds can acquire CRE, likely from foraging in anthropogenically impacted areas, and may aid in the maintenance and dissemination of CRE in the environment, a spatiotemporal comparison of isolates from different regions and timepoints may be useful for elucidating epidemiological information. Thus, we characterized the genomic diversity of CRE from fecal samples opportunistically collected from gulls (Larus spp.) inhabiting Alaska (USA), Chile, Spain, Turkey, and Ukraine and from black kites (Milvus migrans) sampled in Pakistan and assessed evidence for spatiotemporal patterns of dissemination. Within and among sampling locations, a high diversity of carbapenemases was found, including Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-beta-lactamase (NDM), oxacillinase (OXA), and Verona integron Metallo beta-lactamase (VIM). Although the majority of genomic comparisons among samples did not provide evidence for spatial dissemination, we did find strong evidence for dissemination among Alaska, Spain, and Turkey. We also found strong evidence for temporal dissemination among samples collected in Alaska and Pakistan, though the majority of CRE clones were transitory and were not repeatedly detected among locations where samples were collected longitudinally. Carbapenemase-producing hypervirulent K. pneumoniae was isolated from gulls in Spain and Ukraine and some isolates harbored antimicrobial resistance genes conferring resistance to up to 10 different antibiotic classes, including colistin. Our results are consistent with local acquisition of CRE by wild birds with spatial dissemination influenced by intermediary transmission routes, likely involving humans. Furthermore, our results support the premise that anthropogenicallyassociated wild birds may be good sentinels for understanding the burden of clinically-relevant antimicrobial resistance in the local human population.

Due to global warming, our planet will experience more extreme weather conditions. Plants can protect themselves against these abiotic stress conditions with their stress response, which includes alternative splicing of certain genes. Alternative splicing is a post-transcriptional process where a single gene gives rise to different mRNAs, which in turn produces different proteins. In plants, this is usually done by intron retention. One type of protein that may be involved in this stress response are the non-specific lipid transfer proteins (LTPs). Indeed, evidence of intron retention has been found in the LTP genes in the liverwort Marchantia polymorpha, called MpLTPd. To investigate whether this alternative splicing is caused by abiotic stress or not, I subjected the moss to two different types of stress trials, drought and cold, and compared the general expression of the intron in MpLTPd2 and MpLTPd3 from the stressed samples to samples from a moss grown under normal conditions. I found that the expression of the intron did change in the stressed moss, but none of the differences were significant. This suggests that alterative splicing in MpLTPd2 and MpLTPd3 is not caused by cold and drought and that the intron-containing protein plays no role in the protection of M. polymorpha against abiotic stress.

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.

There is little knowledge about olfactory learning in primates, even though primates are known to use olfaction in several behaviors including food selection and territorial defense. Therefore I assessed the olfactory learning and memory performance in five adult spider monkeys (Ateles geoffroyi) using a food-rewarded two-choice olfactory discrimination paradigm. The spider monkeys acquired the initial odor discrimination in 530-1102 trials and in a series of intramodal transfer tasks they needed 30-510 trials to reach the learning criterion. There was a significant negative correlation between the number of trials needed to reach the learning criterion and the number of transfer tasks completed. Thus, as a group, the animals displayed olfactory learning set formation. The number of trials that the spider monkeys needed in initial olfactory learning was comparable to that of other primate species tested previously but higher compared to that of other mammals such as dogs and rats. The learning speed of the spider monkeys in intramodal transfer tasks was similar to that of other mammals tested, suggesting that primates are less prepared to use olfactory cues in the initial solving of a problem but that once they learn the concept, their learning speed with novel odor discrimination problems is not generally slower than that of non-primate mammals. All spider monkeys tested reached the learning criterion in the memory tasks straight on the first testing day, that is: within 30 trials, suggesting similar long-term odor memory capabilities in spider monkeys and other mammals such as dogs, mice and rats.

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

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.

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.

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.

The productivity of marine ecosystems and the services they provide to humans are largely dependent on complex interactions between prey and predators. These are embedded in a diverse network of trophic interactions, resulting in a cascade of events following perturbations such as species extinction. The sheer scale of oceans, however, precludes the characterization of marine feeding networks through de novo sampling. This effort ought instead to rely on a combination of extensive data and inference. Here we investigate how the distribution of trophic interactions at the global scale shapes the marine fish food web structure. We hypothesize that the heterogeneous distribution of species ranges in biogeographic regions should concentrate interactions in the warmest areas and within species groups. We find that the inferred global metaweb of marine fish-that is, all possible potential feeding links between co-occurring species-is highly connected geographically with a low degree of spatial modularity. Metrics of network structure correlate with sea surface temperature and tend to peak towards the tropics. In contrast to open-water communities, coastal food webs have greater interaction redundancy, which may confer robustness to species extinction. Our results suggest that marine ecosystems are connected yet display some resistance to perturbations because of high robustness at most locations.

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 T_m and C_m 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 T_m and C_m of heat stable proteins. 

"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.

"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.

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).

The modern clear-cutting practice in forestry has had a major impact on biodiversity in the forest. Many organisms that depend on structures linked to older forests are declining in number and are now endangered. Today, it is debated whether alternative forestry methods are better at preserving the structure of the forest and the biological diversity. In this literature study, studies were compiled that have compared how different forestry methods have affected biodiversity. The results showed that continuous cover forestry to a greater extent retains the structures of an older forest and had the least impact on the species associated with older, continuous forest. For some of the most sensitive species, however, all felling has a negative impact. Clear-cutting is what changes the species composition the most, and species that depend on continuity forests are often replaced by generalists and species that prefer open habitats. The studies show that greater nature conservation considerations in clear-cutting can sometimes reduce the impact on species composition. All kinds of forestry methods reduce the amount and degrade the quality of dead wood in the forest. The studies show that several alternative forestry methods have a greater potential to preserve or create dead wood compared to clear-cutting. Results also show that there are several suitable methods that can be used in forestry. In order to achieve and maintain a high level of biodiversity at landscape level, a mixture of different forestry methods seems optimal.

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.

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.

African populations are underrepresented in medical genomics studies. For the Somali population, there is virtually no information on genomic markers with significance to precision medicine. Here, we analyzed nearly 900,000 genomic markers in samples collected from 95 unrelated individuals in the North Eastern Somalia. ADMIXTURE program for estimation of individual ancestries revealed a homogenous Somali population. Principal component analysis with PLINK software showed approximately 60% East African and 40% West Eurasian genes in the Somali population, with a close relation to the Cushitic and Semitic speaking Ethiopian populations. We report the unique features of human leukocyte antigens (HLA) in the Somali population, which seem to differentiate from all other neighboring regions compared. Current study identified high prevalence of the diabetes type 1 (T1D) predisposing HLA DR-DQ haplotypes in Somalia. This finding may explain the increased T1D risk observed among Somali children. In addition, ethnic Somalis were found to host the highest frequencies observed thus far for several pharmacogenetic variants, including UGT1A4*2. In conclusion, we report that the Somali population displays genetic traits of significance to health and disease. The Somali dataset is publicly available and will add more information to the few genomic datasets available for African populations.

Fragment-screening by X-ray crystallography (XFS) is an expensive and low throughput fragment drug discovery screening method, and it requires a lot of optimization for each protein target. The advantages with this screening method are that it is very sensitive, it directly gives the three-dimensional structure of the protein-fragment complexes, and false positives are rarely obtained. The aim of this project was to help Sprint Bioscience assess if the advantages with XFS outweigh the disadvantages, and if this method should be used as a complement to their differential scanning fluorimetry (DSF) screening method.

An XFS campaign was run using the oncoprotein vaccinia related kinase 1 (VRK1) as a target protein to evaluate this screening method. During the development of the XFS campaign, a diverse fragment library was created which consisted of 298 fragments that were all soluble in DMSO at 1 M concentration. The crystallization of the protein VRK1 was also optimized in this project to get a robust, high throughput crystallization set up which generated crystals that diffracted at higher resolution than 2.0 Å when they were not soaked with fragments. The soaking protocol was also optimized in order to reduce both the steps during the screening procedure and mechanical stress caused to the crystals during handling. Lastly, the created fragment library was used in screening VRK1 at 87.5 mM concentration with XFS.

23 fragment hits could be obtained from the X-ray crystallography screening campaign, and the mean resolution of the crystal structures of the protein-fragment complexes was 1.87Å. 11 of the 23 fragment hits were not identified as hits when they were screened against VRK1 using DSF. XFS was deemed as a suitable and efficient screening method to complement DSF since the hit rate was high and fragments hits could be obtained with this method that could not be obtained with DSF. However, in order to use this screening method a lot of time needs to be spent in optimizing the crystal system so it becomes suitable for fragment screening. Sprint Bioscience would therefore need to evaluate the cost/benefit ratio of using this screening method for each new project.