High mammographic density, associated with increased tissue stiffness, is a strong risk factor for breast cancer per se . In postmenopausal women there is no differences in the occurrence of ductal carcinoma in situ (DCIS) depending on breast density. Preliminary data suggest that dense breast tissue is associated with a pro -inflammatory microenvironment including infiltrating monocytes. However, the underlying mechanism(s) remains largely unknown. A major roadblock to understanding this risk factor is the lack of relevant in vitro models. A biologically relevant 3D model with tunable stiffness was developed by cross -linking hyaluronic acid. Breast cancer cells were cultured with and without freshly isolated human monocytes. In a unique clinical setting, extracellular proteins were sampled using microdialysis in situ from women with various breast densities. We show that tissue stiffness resembling high mammographic density increases the attachment of monocytes to the cancer cells, increase the expression of adhesion molecules and epithelia-mesenchymal-transition proteins in estrogen receptor (ER) positive breast cancer. Increased tissue stiffness results in increased secretion of similar pro-tumorigenic proteins as those found in human dense breast tissue including inflammatory cytokines, proteases, and growth factors. ER negative breast cancer cells were mostly unaffected suggesting that diverse cancer cell phenotypes may respond differently to tissue stiffness. We introduce a biological relevant model with tunable stiffness that resembles the densities found in normal breast tissue in women. The model will be key for further mechanistic studies. Additionally, our data revealed several pro-tumorigenic pathways that may be exploited for prevention and therapy against breast cancer.

Technologies in image acquisition have developed and often provide image volumes in more than two dimensions. Computer tomography and magnet resonance imaging provide image volumes in three spatial dimensions. The image enhancement methods have developed as well and in this thesis work 3D image enhancement with filter networks is evaluated.

The aims of this work are; to find a method which makes the initial parameter settings in the 3D image enhancement processing easier, to compare 2D and 3D processed image volumes visualized with different visualization techniques and to give an illustration of the benefits with 3D image enhancement processing visualized using these techniques.

The results of this work are;

1. a parameter setting tool that makes the initial parameter setting much easier and

2. an evaluation of 3D image enhancement with filter networks that shows a significant enhanced image quality in 3D processed image volumes with a high noise level compared to the 2D processed volumes. These results are shown in slices, MIP and volume rendering. The differences are even more pronounced if the volume is presented in a different projection than the volume is 2D processed in.

Clinical studies have demonstrated that epidermal pigmentation level can affect cerebral oximetry measurements. To evaluate the robustness of these devices, we have developed a phantom-based test method that includes an epidermis-simulating layer with several melanin concentrations and a 3D-printed cerebrovascular module. Measurements were performed with neonatal, pediatric and adult sensors from two commercial oximeters, where neonatal probes had shorter source-detector separation distances. Referenced blood oxygenation levels ranged from 30 to 90%. Cerebral oximeter outputs exhibited a consistent decrease in saturation level with simulated melanin content; this effect was greatest at low saturation levels, producing a change of up to 15%. Dependence on pigmentation was strongest in a neonatal sensor, possibly due to its high reflectivity. Overall, our findings indicate that a modular channel-array phantom approach can provide a practical tool for assessing the impact of skin pigmentation on cerebral oximeter performance and that modifications to algorithms and/or instrumentation may be needed to mitigate pigmentation bias.

Select prion diseases are characterized by widespread cerebral plaque-like deposits of amyloid fibrils enriched in heparan sulfate (HS), a major extracellular matrix component. HS facilitates fibril formation in vitro, yet how HS impacts fibrillar plaque growth within the brain is unclear. Here we found that prion-bound HS chains are highly sulfated, and that the sulfation is essential for accelerating prion conversion in vitro. Using conditional knockout mice to deplete the HS sulfation enzyme, Ndst1 (N-deacetylase / N-sulfotransferase), from neurons or astrocytes, we then investigated how reducing HS sulfation impacts survival and prion aggregate distribution during a prion infection. Neuronal Ndst1-depleted mice survived longer and showed fewer and smaller parenchymal plaques, shorter fibrils, and increased vascular amyloid, consistent with enhanced aggregate transit toward perivascular drainage channels. The prolonged survival was strain-dependent, only affecting mice infected with extracellular, plaque-forming, but not membrane bound, prions. Live PET imaging revealed rapid clearance of recombinant prion protein monomers into the CSF of mice expressing unsulfated HS, further suggesting that HS sulfate groups hinder transit of extracellular prion protein monomers. Our results directly show how a host cofactor slows the spread of prion protein through the extracellular space and identify an enzyme to target to facilitate aggregate clearance. Prions cause a rapidly progressive neurologic disease and death with no curative treatment available. Prion aggregates accumulate exponentially in the brain of affected individuals triggering neuronal loss and neuroinflammation, yet the molecules that facilitate prion protein aggregation are largely unknown. We have found that prions in the brain preferentially bind to a highly sulfated endogenous polysaccharide, known as heparan sulfate (HS). Here we use genetically modified mice that express poorly sulfated, neuron-derived HS, and infect mice with different prions strains. We find that mice infected with a plaque-forming prion strain show a prolonged survival and fewer plaques compared to controls. We also found that recombinant prion protein was efficiently transported within the interstitial fluid of mice having poorly sulfated HS, suggesting more efficient clearance from the brain. Our study provides insight into how HS retains prion aggregates in the brain to accelerate disease and indicates a specific HS biosynthetic enzyme to target to enhance protein clearance.

Cofactors are essential for driving recombinant prion protein into pathogenic conformers. Polyanions promote prion aggregation in vitro, yet the cofactors that modulate prion assembly in vivo remain largely unknown. Here we report that the endogenous glycosaminoglycan, heparan sulfate (HS), impacts prion propagation kinetics and deposition sites in the brain. Exostosin-1 haploinsufficient (Ext1(+/-)) mice, which produce short HS chains, show a prolonged survival and a redistribution of plaques from the parenchyma to vessels when infected with fibrillar prions, and a modest delay when infected with subfibrillar prions. Notably, the fibrillar, plaque-forming prions are composed of ADAM10-cleaved prion protein lacking a glycosylphosphatidylinositol anchor, indicating that these prions are mobile and assemble extracellularly. By analyzing the prion-bound HS using liquid chromatography-mass spectrometry (LC-MS), we identified the disaccharide signature of HS differentially bound to fibrillar compared to subfibrillar prions, and found approximately 20-fold more HS bound to the fibrils. Finally, LC-MS of prion-bound HS from human patients with familial and sporadic prion disease also showed distinct HS signatures and higher HS levels associated with fibrillar prions. This study provides the first in vivo evidence of an endogenous cofactor that accelerates prion disease progression and enhances parenchymal deposition of ADAM10-cleaved, mobile prions.

Phonocardiographic signals contain bioacoustic information reflecting the operation of the heart. Normally there are two heart sounds, and additional sounds indicate disease. If a third heart sound is present it could be a sign of heart failure whereas a murmur indicates defective valves or an orifice in the septal wall. The primary aim of this thesis is to use signal processing tools to improve the diagnostic value of this information. More specifically, three different methods have been developed:

• A nonlinear change detection method has been applied to automatically detect heart sounds. The first and the second heart sounds can be found using recurrence times of the first kind while the third heart sound can be found using recurrence times of the second kind. Most third heart sound occurrences were detected (98 %), but the amount of false extra detections was rather high (7 % of the heart cycles).

• Heart sounds obscure the interpretation of lung sounds. A new method based on nonlinear prediction has been developed to remove this undesired disturbance. High similarity was obtained when comparing actual lung sounds with lung sounds after removal of heart sounds.

• Analysis methods such as Shannon energy, wavelets and recurrence quantification analysis were used to extract information from the phonocardiographic signal. The most prominent features, determined by a feature selection method, were used to create a new feature set for heart murmur classification. The classification result was 86 % when separating patients with aortic stenosis, mitral insufficiency and physiological murmurs.

The derived methods give reasonable results, and they all provide a step forward in the quest for an intelligent stethoscope, a universal phonocardiography tool able to enhance auscultation by improving sound quality, emphasizing abnormal events in the heart cycle and distinguishing different heart murmurs.

Despite the broad anti-microbial and anti-inflammatory properties of silver nanoparticles (AgNPs), their use in bioengineered corneal replacements or bandage contact lenses has been hindered due to their intense yellow coloration. In this communication, we report the development of a new strategy to pre-stabilize and incorporate AgNPs with different colours into collagen matrices for fabrication of corneal implants and lenses, and assessed their in vitro and in vivo activity.

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.

"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.

Background: A cruciate ligament (CL) injury is a severe injury in soccer. Neuromuscular training programs have a well-documented preventive effect, but there are few studies on the effectiveness of such a program at a national level. The Swedish Knee Control Program (KCP) was found to be effective in preventing CL injuries in youth female soccer players. The KCP was implemented nationwide in Sweden in 2010. Purpose: To evaluate the effectiveness of the Swedish KCP in reducing acute knee injuries in soccer players at a nationwide level. Study Design: Descriptive epidemiology study. Methods: All licensed soccer players in Sweden are covered by the same insurance company. Using this insurance database, around 17,500 acute knee injuries that were reported to the insurance company between 2006 and 2015 were included in the study. By matching the number of licensed soccer players with the number of reported injuries each year, the annual incidence of knee and CL injuries was able to be calculated. To evaluate the spread of the KCP nationally, a questionnaire was sent to all 24 Swedish district football associations (FAs) with questions regarding KCP education. The number of downloads of the KCP mobile application (app) was obtained. Results: The incidence of CL injuries decreased during the study period for both male (from 2.9 to 2.4 per 1000 player-years) and female players (from 4.9 to 3.9 per 1000 player-years). The overall incidence of knee injuries decreased in both male (from 5.6 to 4.6 per 1000 player-years) and female players (from 8.7 to 6.4 per 1000 player-years). Comparing before and after the nationwide implementation of the KCP, there was a decrease in the incidence of CL injuries by 6% (rate ratio [RR], 0.94 [95% CI, 0.89-0.98]) in male players and 13% (RR, 0.87 [95% CI, 0.81-0.92]) in female players and a decrease in the incidence of knee injuries by 8% (RR, 0.92 [95% CI, 0.89-0.96]) and 21% (RR, 0.79 [95% CI, 0.75-0.83]), respectively (P amp;lt; .01 for all). This trend corresponded to a reduction of approximately 100 CL injuries each year in Sweden. A total of 21 of 24 district FAs held organized KCP educational courses during the study period. The percentage of district FAs holding KCP courses was between 46% and 79% each year. There were 101,236 downloads of the KCP app. Conclusion: The KCP can be considered partially implemented nationwide, and the incidence of knee and CL injuries has decreased in both sexes at a nationwide level.

The rapid development of new diagnostic procedures, the mapping of the human genome, progress in mapping genetic polymorphisms, and recent advances in nucleic acid- and protein chip technologies are driving the development of personalized therapies. This breakthrough in medicine is expected to be achieved largely due to the implementation of "lab-on-the-chip" technology capable of performing hundreds, even thousands of biochemical, cellular and genetic tests on a single sample of blood or other body fluid. Focusing on a few disease-specific examples, this review discusses selected technologies and their combinations likely to be incorporated in the "lab-on-the-chip" and to provide rapid and versatile information about specific diseases entities. Focusing on breast cancer and after an overview of single-nucleofide polymorphism (SNP)-screening methodologies, we discuss the diagnostic and prognostic importance of SNPs. Next, using Duchenne muscular dystrophy (DMD) as an example, we provide a brief overview of powerful and innovative integration of traditional immuno-histochemistry techniques with advanced biophysical methods such as NMR-spectroscopy or Fourier-transformed infrared (FT-IR) spectroscopy. A brief overview of the challenges and opportunities provided by protein and aptamer microarrays follows. We conclude by highlighting novel and promising biochemical markers for the development of personalized treatment of cancer and other diseases: serum cytochrome c, cytokeratin-18 and -19 and their proteolytic fragments for the detection and quantitation of malignant tumor mass, tumor cell turn-over, inflammatory processes during hepatitis and Epstein-Barr virus (EBV)-induced hemophagocytic lymphohistiocytosis and apoptotic/necrotic cancer cell death. (c) 2006 Elsevier Ltd. All rights reserved.

Introduction: Measuring testosterone concentrations is of interest both in clinical situations and for research, the latter expanding rapidly during recent years. An increased demand for convenient methods has prompted a number of companies to develop enzyme-linked immunosorbent assay (ELISA) kits to measure testosterone concentrations in saliva. However, the inter-comparability of kits from different manufacturers have yet to be determined. Aim of study: The aim of this study was to compare commercially available ELISA kits from four different manufacturers (Salimetrics, IBL, DRG and Demeditec). Methods: Saliva was collected from 50 participants (25 men and 25 women). Each sample was analysed by the four ELISA kits. Results: The correlations between the ELISA kits from Demeditec, DRG and Salimetrics were moderate to high with r-values amp;gt;.77; however, proportional errors between the methods calls for caution. The ELISA kit from IBL malfunctioned and no results from this kit was obtained. Conclusions: Results from studies using the ELISA kits from Demeditec, DRG and Salimetrics are generally comparable; however, translation using the formulae presented in the current study could increase the accuracy of these comparisons.

Background: The literature on upper extremity injuries in professional soccer players is scarce, and further insight into the onset and cause of these injuries as well as potential differences between goalkeepers and outfield players is important. Purpose: To investigate the epidemiology of hand, wrist, and forearm injuries in male professional soccer players between 2001 and 2019. Study Design: Descriptive epidemiology study. Methods: Between the 2001-2002 and 2018-2019 seasons, 120 European male soccer teams were followed prospectively for a varying number of seasons (558 team-seasons in total). Time-loss injuries and player-exposures to training sessions and matches were recorded on an individual basis in 6754 unique players. Injury incidence was reported as the number of injuries per 1000 player-hours, and between-group differences were analyzed using Z statistics and rate ratios (RRs) with 95% CIs. Between-group differences in layoff time were analyzed. Results: In total, 25,462 injuries were recorded, with 238 (0.9%) of these affecting the hand (71.4%; n = 170), wrist (16.8%; n = 40), and forearm (11.8%; n = 28), producing an incidence of 0.065 injuries per 1000 hours. A majority of the injuries were traumatic with an acute onset (98.7%; n = 235). Fractures were the most common injuries recorded (58.8%; n = 140), often involving the metacarpal bones (25.2%; n = 60) and phalanges (10.1%; n = 24). The injury incidence was significantly higher for goalkeepers (115 injuries; 0.265 per 1000 hours) compared with outfield players (123 injuries; 0.038 per 1000 hours) (RR, 7.0 [95% CI, 5.4-9.0]). Goalkeepers also had a significantly longer mean layoff time than outfield players (23 +/- 27 vs 15 +/- 27 days; P = .016). Conclusion: Injuries to the hand, wrist, and forearm constituted less than 1% of all time-loss injuries in male professional soccer players. Fractures were most common and constituted more than half of all injuries. Goalkeepers had a 7-fold higher incidence and an over 1-week longer mean layoff time compared with outfield players.

The aim of this study was to evaluate in vivo a laser Doppler measurement system in porcine brain tissue during thermal lesioning. A 2-mm monopolar radiofrequency lesioning electrode was equipped with optical fibers in order to monitor the lesioning procedure. Laser Doppler and backscattered light intensity signals were measured along the electrode trajectory and during bilateral lesioning in the central gray (70, 80 and 90°C, n = 14). The time course of the coagulation process could be followed by optical recordings. Two separate groups of tissue were identified from the intensity signals. The changes in the perfusion levels in both groups displayed significant changes (p < 0.05, n = 48) at all temperature settings, while backscattered light intensity was significant for only one group at the different temperatures (p < 0.05, n = 39). These results indicate that optical measurements correlate with lesion development in vivo. The study also indicates that it is possible to follow the lesioning process intra-operatively.

Significance: Spatial frequency domain imaging (SFDI) is a diffuse optical measurement technique that can quantify tissue optical absorption (μa) and reduced scattering (μ0 s) on a pixelby-pixel basis. Measurements of μa at different wavelengths enable the extraction of molar concentrations of tissue chromophores over a wide field, providing a noncontact and label-free means to assess tissue viability, oxygenation, microarchitecture, and molecular content. We present here openSFDI: an open-source guide for building a low-cost, small-footprint, threewavelength SFDI system capable of quantifying μa and μ0 s as well as oxyhemoglobin and deoxyhemoglobin concentrations in biological tissue. The companion website provides a complete parts list along with detailed instructions for assembling the openSFDI system. Aim: We describe the design of openSFDI and report on the accuracy and precision of optical property extractions for three different systems fabricated according to the instructions on the openSFDI website. Approach: Accuracy was assessed by measuring nine tissue-simulating optical phantoms with a physiologically relevant range of μa and μ0 s with the openSFDI systems and a commercial SFDI device. Precision was assessed by repeatedly measuring the same phantom over 1 h. Results: The openSFDI systems had an error of 0 6% in μa and −2 3% in μ0 s, compared to a commercial SFDI system. Bland–Altman analysis revealed the limits of agreement between the two systems to be 0.004 mm−1 for μa and −0.06 to 0.1 mm−1 for μ0 s. The openSFDI system had low drift with an average standard deviation of 0.0007 mm−1 and 0.05 mm−1 in μa and μ0 s, respectively. Conclusion: The openSFDI provides a customizable hardware platform for research groups seeking to utilize SFDI for quantitative diffuse optical imaging.

In the developing Drosophila central nervous system (CNS), neural progenitor (neuroblast [NB]) selection is gated by lateral inhibition, controlled by Notch signaling and proneural genes. However, proneural mutants still generate many NBs, indicating the existence of additional proneural genes. Moreover, recent studies reveal involvement of key epithelial-mesenchymal transition (EMT) genes in NB selection, but the regulatory interplay between Notch signaling and the EMT machinery is unclear. We find that SoxNeuro (SoxB family) and worniu (Snail family) are integrated with the Notch pathway, and constitute the missing proneural genes. Notch signaling, the proneural, SoxNeuro, and worniu genes regulate key EMT genes to orchestrate the NB selection process. Hence, we uncover an expanded lateral inhibition network for NB selection and demonstrate its link to key players in the EMT machinery. The evolutionary conservation of the genes involved suggests that the Notch-SoxB-Snail-EMT network may control neural progenitor selection in many other systems.

Biomaterials are materials that are specifically designed to be in contact with biological systems and have for a long time been used in medicine. Examples of biomaterials range from sophisticated prostheses used for replacing outworn body parts to ordinary contact lenses. Currently it is possible to create biomaterials that can e.g. specifically interact with cells or respond to certain stimuli. Peptides, the shorter version of proteins, are excellent molecules for fabrication of such biomaterials. By following and developing design rules it is possible to obtain peptides that can self-assemble into well-defined nanostructures and biomaterials.

The aim of this thesis is to create ”smart” and tunable biomaterials by molecular self-assembly using dimerizing –helical polypeptides. Two different, but structurally related, polypeptide-systems have been used in this thesis. The EKIV-polypeptide system was developed in this thesis and consists of four 28-residue polypeptides that can be mixed-and-matched to self-assemble into four different coiled coil heterodimers. The dissociation constant of the different heterodimers range from μM to < nM. Due to the large difference in affinities, the polypeptides are prone to thermodynamic social self-sorting. The JR-polypeptide system, on the other hand, consists of several 42-residue de novo designed helix-loop-helix polypeptides that can dimerize into four-helix bundles. In this work, primarily the glutamic acid-rich polypeptide JR2E has been explored as a component in supramolecular materials. Dimerization was induced by exposing the polypeptide to either Zn²⁺, acidic conditions or the complementary polypeptide JR2K.

By conjugating JR2E to hyaluronic acid and the EKIV-polypeptides to star-shaped poly(ethylene glycol), respectively, highly tunable hydrogels that can be self-assembled in a modular fashion have been created. In addition, self-assembly of spherical superstructures has been investigated and were obtained by linking two thiol-modified JR2E polypeptides via a disulfide bridge in the loop region. ŒThe thesis also demonstrates that the polypeptides and the polypeptide-hybrids can be used for encapsulation and release of molecules and nanoparticles. In addition, some of the hydrogels have been explored for 3D cell culture. By using supramolecular interactions combined with bio-orthogonal covalent crosslinking reactions, hydrogels were obtained that enabled facile encapsulation of cells that retained high viability.

The results of the work presented in this thesis show that dimerizing α–helical polypeptides can be used to create modular biomaterials with properties that can be tuned by specific molecular interactions. The modularity and the tunable properties of these smart biomaterials are conceptually very interesting andmake them useful in many emerging biomedical applications, such as 3D cell culture, cell therapy, and drug delivery

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Improving the functionality and reducing revision rates are important driving forces in the development of orthopaedic implants. FibMat is a fibrinogen based matrix developed towards commercialisation by the company Optovent AB. This matrix can be coated on implants and act as a local drug delivery system for bisphosphonates (BPs). BPs are drugs inhibiting bone resorption, and applied with FibMat to improve stability of implants in bone, e.g. when fixing bone fractures. In this thesis, FibMat loaded with BP (FibMat/BP) was coated on stainless-steel screws and titanium screws in order to investigate some technology properties relevant to its clinical applicability. Bone-mimicking materials were used to study scrape-off effect upon insertion. The coagulation properties of fibrinogen as well as the structural properties of BPs were studied after exposure to gamma radiation.

The screws were coated with FibMat and BP (alendronate and 14C-alendronate) using standard coupling techniques. The total amount and distribution of BP after insertion was measured by liquid scintillation and autoradiography. Coagulation assays were performed in order to determine the coagulation properties of fibrinogen, exposed to doses up to 35 kGy, mixed with thrombin. The structural properties of four different BPs (alendronate, pamidronate, zoledronate and ibandronate), exposed to doses up to 35 kGy were analysed by transmission infrared spectroscopy.

The results show that FibMat/BP coating on porous stainless-steel screws is virtually unaffected by insertion into bone materials. The anodised, planar titanium screws are more affected by the insertion process, but an even BP distribution in the cancellous material is indicated. The coagulation assays show that gamma-irradiated fibrinogen has a slower coagulation process compared to non-irradiated fibrinogen and form interrupted network unable to clot. The chemical structures of the BPs seem unaffected by exposure to gamma irradiation. In conclusion, the FibMat/BP is a promising technology for local distribution of BP in conjunction with bone implants.

A combined pH- and pressure-measurement device for oesophageal investigations has been designed using monocrystalline antimony pH electrodes and perfused polyvinyl catheters. The combined device facilitates pressure measurements simultaneously with pH recording, both distal and proximal to the pH electrode. The device is easier to pass through the nose to the oesophagus than the conventional glass pH electrode. pH and pressure measurements in the oesophagus are therefore simplified and valuable information about the function of the region of the lower oesophageal sphincter is added owing to the simultaneous recording of the two parameters.

Using simultaneous manometry and cineradiography, oesophageal evacuation was studied while contrast medium was infused via a catheter. The distal half of the oesophagus could be filled with contrast medium without triggering peristalsis. The hydrostatic pressure necessary to open the lower oesophageal sphincter (LES) was of approximately the same magnitude as the pressure gradient between oesophagus and LES. No significant relaxation of the LES could be observed at the initiation of swallowing. The LES may be looked upon not only as a sphincter preventing reflux but also as a gate which must be forced open by food.

In a clinical study of oesophageal manometry with fluid-filled catheters, both a non-perfused system and a perfused system with a syringe pump have been compared to a system with a low-compliance perfusion pump, which served as a reference. Significantly lower values of motility amplitudes, motility derivatives, and partly of LES pressures, and a time delay of up to 0.5 sec of the amplitude maximum were obtained with the non-perfused system and the system with a syringe pump in comparison to the low-compliance system. Since the oesophageal function can be erroneously evaluated by use of a non-perfused system or a perfused system with a syringe pump, such systems cannot be recommended for clinical use.

Esophageal manometric systems with water-filled catheters have been characterized by the use of model experiments. The examined parameters have been: catheter dimension, catheter compliance, catheter resistance, pump type, pump compliance, and perfusion flow. Accurate static pressure measurements have been obtained for perfused systems independently of the investigated parameters. The dynamic characteristics vary with catheter diameter and perfusion flow. For catheters with low diameter, a narrow bandwidth is obtained for the investigated perfusion flows. The results have been expressed in terms of an electric model of the measurement system. Perfusion pumps with low compliance are recommended to improve the dynamic properties of the measurement system.

The long-term properties of commercially available ECG-electrodes were studied by investigating the parameters: polarization potential, electrical impedance, adhesion, and skin reactions during a period of 7 days. As expected, the most stable polarization potentials were obtained for Ag/AgCl electrodes. Certain simple disposable electrodes showed large polarization potential variations. The most stable electrode impedance was obtained for disposable electrodes with stable adhesion and equipped with an electrode cup or similar. Unchanged adhesion and mechanical properties during the test period were shown by the disposable electrodes with a large self-adhesive collar.

Maturation of lymphoid cells is controlled by the action of stage and lineage-restricted transcription factors working in concert with the general transcription and chromatin remodeling machinery to regulate gene expression. To better understand this functional interplay, we used Biotin Identification in human embryonic kidney cells to identify proximity interaction partners for GATA3, TCF7 (TCF1), SPI1, HLF, IKZF1, PAX5, ID1, and ID2. The proximity interaction partners shared among the lineagerestricted transcription factors included ARID1a, a BRG1-associated factor complex component. CUT&RUN analysis revealed that ARID1a shared binding with TCF7 and GATA3 at a substantial number of putative regulatory elements in mouse T cell progenitors. In support of an important function for ARID1a in lymphocyte development, deletion of Aridla in early lymphoid progenitors in mice resulted in a pronounced developmental arrest in early T cell development with a reduction of CD4(+)CD8(+) cells and a 20-fold reduction in thymic cellularity. Exploring gene expression patterns in DN3 cells from Wt and Aridla-deficient mice suggested that the developmental block resided in the DN3a to DN3b transition, indicating a deficiency in beta-selection. Our work highlights the critical importance of functional interactions between stage and lineage-restricted factors and the basic transcription machinery during lymphocyte differentiation.

The high egg-laying capacity of the modern domestic chicken (i.e. White Leghorn, WL) has arisen from the low egg-laying ancestor Red Junglefowl (RJF) via continuous trait selection and breeding. To investigate whether this long-term selection impacted the seminal fluid (SF)-proteome, 2DE electrophoresis-based proteomic analyses and immunoassays were conducted to map SF-proteins/cytokines in RJF, WL and a 9th generation Advanced Intercross Line (AIL) of RJF/WL-L13, including individual SF (n = 4, from each RJF, WL and AIL groups) and pools of the SF from 15 males of each group, analyzed by 2DE to determine their degree of intra-group (AIL, WL, and RJF) variability using Principal Component Analysis (PCA); respectively an inter-breed comparative analysis of intergroup fold change of specific SF protein spots intensity between breeds. The PCA clearly highlighted a clear intra-group similarity among individual roosters as well as a clear inter-group variability (e.g. between RJF, WL and AIL) validating the use of pools to minimize confounding individual variation. Protein expression varied considerably for processes related to sperm motility, nutrition, transport and survival in the female, including signaling towards immunomodulation. The major conserved SF-proteins were serum albumin and ovotransferrin. Aspartate aminotransferase, annexin A5, arginosuccinate synthase, glutathione S-transferase 2 and l-lactate dehydrogenase-A were RJF-specific. Glyceraldehyde-3-phosphate dehydrogenase appeared specific to the WL-SF while angiotensin-converting enzyme, γ-enolase, coagulation factor IX, fibrinogen α-chain, hemoglobin subunit α-D, lysozyme C, phosphoglycerate kinase, Src-substrate protein p85, tubulins and thioredoxin were AIL-specific. The RJF-SF contained fewer immune system process proteins and lower amounts of the anti-inflammatory/immunomodulatory TGF-β2 compared to WL and AIL, which had low levels- or lacked pro-inflammatory CXCL10 compared to RJF. The seminal fluid proteome differs between ancestor and modern chicken, with a clear enrichment of proteins and peptides related to immune-modulation for sperm survival in the female and fertility.

Limited evidence has shed light on how aSYN proteins affect the oligodendrocyte phenotype and pathogenesis in synucleinopathies that include Parkinsons disease (PD) and multiple system atrophy (MSA). Here, we investigated early transcriptomic changes within PD and MSA O4(+) oligodendrocyte lineage cells (OLCs) generated from patient-induced pluripotent stem cells (iPSCs). We found impaired maturation of PD and MSA O4(+) OLCs compared to controls. This phenotype was associated with changes in the human leukocyte antigen (HLA) genes, the immunoproteasome subunit PSMB9, and the complement component C4b for aSYN p.A53T and MSA O4(+) OLCs, but not in SNCA(trip) O4(+) OLCs despite high levels of aSYN assembly formation. Moreover, SNCA overexpression resulted in the development of O4(+) OLCs, whereas exogenous treatment with aSYN species led to significant toxicity. Notably, transcriptome profiling of genes encoding proteins forming Lewy bodies and glial cytoplasmic inclusions revealed clustering of PD aSYN p.A53T O4(+) OLCs with MSA O4(+) OLCs. Our work identifies early phenotypic and pathogenic changes within human PD and MSA O4(+) OLCs.

NIMA-related kinase7 (NEK7) plays a multifunctional role in cell division and NLRP3 inflammasone activation. A typical expression or any mutation in the genetic makeup of NEK7 leads to the development of cancer malignancies and fatal inflammatory disease, i.e., breast cancer, non-small cell lung cancer, gout, rheumatoid arthritis, and liver cirrhosis. Therefore, NEK7 is a promising target for drug development against various cancer malignancies. The combination of drug repurposing and structure-based virtual screening of large libraries of compounds has dramatically improved the development of anticancer drugs. The current study focused on the virtual screening of 1200 benzene sulphonamide derivatives retrieved from the PubChem database by selecting and docking validation of the crystal structure of NEK7 protein (PDB ID: 2WQN). The compounds library was subjected to virtual screening using Auto Dock Vina. The binding energies of screened compounds were compared to standard Dabrafenib. In particular, compound 762 exhibited excellent binding energy of -42.67 kJ/mol, better than Dabrafenib (-33.89 kJ/mol). Selected drug candidates showed a reactive profile that was comparable to standard Dabrafenib. To characterize the stability of protein-ligand complexes, molecular dynamic simulations were performed, providing insight into the molecular interactions. The NEK7-Dabrafenib complex showed stability throughout the simulated trajectory. In addition, binding affinities, pIC50, and ADMET profiles of drug candidates were predicted using deep learning models. Deep learning models predicted the binding affinity of compound 762 best among all derivatives, which supports the findings of virtual screening. These findings suggest that top hits can serve as potential inhibitors of NEK7. Moreover, it is recommended to explore the inhibitory potential of identified hits compounds through in-vitro and in-vivo approaches.

COVID-19 is characterised by systemic immunological perturbations in the human body, which can lead to multi-organ damage. Many of these processes are considered to be mediated by the blood. Therefore, to better understand the systemic host response to SARS-CoV-2 infection, we performed systematic analyses of the circulating, soluble proteins in the blood through global proteomics by mass-spectrometry (MS) proteomics. Here, we show that a large part of the soluble blood proteome is altered in COVID-19, among them elevated levels of interferon-induced and proteasomal proteins. Some proteins that have alternating levels in human cells after a SARS-CoV-2 infection in vitro and in different organs of COVID-19 patients are deregulated in the blood, suggesting shared infection-related changes.The availability of different public proteomic resources on soluble blood proteome alterations leaves uncertainty about the change of a given protein during COVID-19. Hence, we performed a systematic review and meta-analysis of MS global proteomics studies of soluble blood proteomes, including up to 1706 individuals (1039 COVID-19 patients), to provide concluding estimates for the alteration of 1517 soluble blood proteins in COVID-19. Finally, based on the meta-analysis we developed CoViMAPP, an open-access resource for effect sizes of alterations and diagnostic potential of soluble blood proteins in COVID-19, which is publicly available for the research, clinical, and academic community. Baba & ccaron;ic et al. performed systematic analyses of blood proteins in COVID-19 patients through mass-spectrometry proteomics, showing that a large part of the soluble blood proteome is altered. The authors then developed an open-access resource, CoViMAPP, for meta-analysis of MS proteomics studies of COVID-19 patients.

Continuous monitoring of electroencephalography (EEG), with a focus on amplitude-integrated EEG (aEEG), has been used in neonatal intensive care for decades. A number of systems have been suggested for describing and quantifying aEEG patterns. Extensive full-montage EEG monitoring is used in specialised intensive care units. The American Clinical Neurophysiology Society published recommendations for defining and reporting EEG findings in critically ill adults and infants. Swedish neonatologists and clinical neurophysiologists collaborated to optimise simplified neonatal continuous aEEG and EEG recordings based on these American documents. Conclusion: This paper describes the Swedish consensus document produced by those meetings.

In humans, noroviruses (NVs) cause acute epidemic and viral gastroenteritis. NVs do not only infect humans; viruseshave also been found in pigs, cows, sheep, mice and dogs. The focus in this project has been on the murine norovirus(MNV). MNV is a member of the viral family Caliciviridae and it consists of a single-stranded, positive sense RNAgenome. The genome includes three open reading frames (ORFs), ORF1 encodes for a polyprotein that consists of theprecursor to the 6-7 non-structural (NS) proteins. The polyprotein is cleaved by the NS6 protease. The NS6 isresponsible for all the cleaving in ORF1 and that makes it an attractive target for antiviral drugs. The NS6 proteinstructure has been determined at 1.66 Å resolution using X-ray diffraction techniques. Surprisingly, the electrondensity map revealed density for a peptide bound in the active site. The peptide had a length of 7 residues andoriginated from the C-terminus of another chain in an adjacent asymmetric unit. The active site triad was composed ofthe conserved residues; histidine 30, aspargine 54 and cysteine 139, however in the structure the cysteine 139 ismutated to an alanine to inactivate the protease. Activity assays were performed to probe the importance of the residuein position 109 in the β-ribbon located close to the active site. The three full-length constructs with the mutations;I109A, I109S and I109T were found to have less activity than the full-length wt (1-183). A truncated protease, lacking9 residues in the C-terminus, also had less activity. This indicates that the terminal residues are also important foractivity.

The aim of this study was to investigate the potential of creating an antimicrobial bandage composed of antimicrobial peptides and enzymes attached to a polysaccharide material such as a cellulose bandage. This bandage could be used for problematic wounds such as burn wounds which are prevalent to infections. Binding to cellulose was done by utilizing a carbohydrate binding domain (CBD) which was fused to an antimicrobial agent via a linker. The linker between the CBD and agent contains a thrombin site which allows the agents to be released when in contact with blood. The release mechanism of the antimicrobial agents increases the reach of the agents in addition to increasing their activity, as proven in this study. Both the CBD binding capacity and the thrombin cleavage mechanism were proven to be effective. The peptide used in this study was Pln1. The enzymes used were the bacteriophage lysins CHAP and PlyF307, where the latter additionally contains a peptide domain. Results showed that Pln1, CHAP and PlyF307 were highly effective against the gram-negative bacteria, whilst CHAP had the highest activity against gram-positive bacteria. A cellulose bandage was applied to bacterial cultures containing bound agents, simulating a bandage applied to a wound. The bandage without the addition of thrombin was found to be antibacterial, however, it showed a significant increase of antibacterial activity when thrombin was added, releasing the agent and allowing it full activity.

The central nervous system (CNS) consists of an enormous number of cells, and large cellular variance, integrated into an elaborate network. The CNS is the most complex animal organ, and therefore its establishment must be controlled by many different genetic programs. Considering the high level of complexity in the human CNS, addressing issues related to human neurodevelopment represents a major challenge. Since comparative studies have revealed that neurodevelopmental programs are well conserved through evolution, on both the genetic and functional levels, studies on invertebrate neurodevelopmental programs are often translatable to vertebrates. Indeed, the basis of our current knowledge about vertebrate CNS development has been greatly aided by studies on invertebrates, and in particular on the Drosophila melanogaster (fruit fly) model system.

This thesis attempted to identify novel genes regulating neural cell specification and proliferation in the CNS, using the Drosophila model system. Moreover, I aimed to address how those genes govern neural progenitor cells (neuroblasts; NBs) to obtain/maintain their stemness identity and proliferation capacity, and how they drive NBs through temporal windows and series of programmed asymmetric division, which gradually reduces their stemness identity in favor of neural differentiation, resulting in appropriate lineage progression. In the first project, we conducted a forward genetic screen in Drosophila embryos, aimed at isolating genes involved in regulation of neural proliferation and specification, at the single cell resolution. By taking advantage of the restricted expression of the neuropeptide FMRFa in the last-born cell of the NB lineage 5-6T, the Ap4 neuron, we could monitor the entire lineage progression. This screen succeeded in identifying 43 novel genes controlling different aspects of CNS development. One of the genes isolated, Ctr9, displayed extra Ap4/FMRFa neurons. Ctr9 encodes a component of the RNA polymerase II complex Paf1, which is involved in a number of transcriptional processes. The Paf1C, including Ctr9, is highly conserved from yeast to human, and in the past couple of years, its importance for transcription has become increasingly appreciated. However, studies in the Drosophila system have been limited. In the screen, we isolated the first mutant of Drosophila Ctr9 and conducted the first detailed phenotypic study on its function in the Drosophila embryonic CNS. Loss of function of Ctr9 leads to extra NB numbers, higher proliferation ratio and lower expression of neuropeptides. Gene expression analysis identified several other genes regulated by Ctr9, which may explain the Ctr9 mutant phenotypes. In summary, we identified Ctr9 as an essential gene for proper CNS development in Drosophila, and this provides a platform for future study on the Drosophila Paf1C. Another interesting gene isolated in the screen was worniou (wor), a member of the Snail family of transcription factors. In contrast to Ctr9, whichdisplayed additional Ap4/FMRFa neurons, wor mutants displayed a loss of these neurons. Previous studies in our group have identified many genes acting to stop NB lineage progression, but how NBs are pushed to proliferate and generate their lineages was not well known. Since wor may constitute a “driver” of proliferation, we decided to study it further. Also, we identified five other transcription factors acting together with Wor as pro-proliferative in both NBs and their daughter cells. These “drivers” are gradually replaced by the previously identified late-acting “stoppers.” Early and late factors regulate each other and the cell cycle, and thereby orchestrate proper neural lineage progression.

Introduction

This study aimed to investigate circulating biomarkers associated with the risk of developing diabetic peripheral neuropathy (DPN) and nephropathy in type 1 diabetes (T1D).

Materials and methods

Patients with childhood-onset T1D (n = 49, age 38.3 ± 3.8 yrs.) followed prospectively were evaluated after 30 years of diabetes duration. DPN was defined as an abnormality in nerve conduction tests. Matrix metalloproteinase-9 (MMP-9) and its tissue inhibitor TIMP-1, neutrophil gelatinase-associated lipocalin-2 (NGAL), soluble P-selectin (sP-selectin), estimated GFR (eGFR), micro/macroalbuminuria and routine biochemistry were assessed. For comparison, control subjects were included (n = 30, age 37.9 ± 5.5 yrs.).

Results

In all, twenty-five patients (51 %) were diagnosed with DPN, and nine patients (18 %) had nephropathy (five microalbuminuria and four macroalbuminuria). Patients with DPN had higher levels of TIMP-1 (p = 0.036) and sP-selectin (p = 0.005) than controls. Patients with DPN also displayed higher levels of TIMP-1 compared to patients without DPN (p = 0.035). Patients with macroalbuminuria had kidney disease stage 3 with lower eGFR, higher levels of TIMP-1 (p = 0.038), and NGAL (p = 0.002). In all patients, we found only weak negative correlations between eGFR and TIMP-1 (rho = −0.304, p = 0.040) and NGAL (rho = −0.277, p = 0.062, ns), respectively. MMP-9 was higher in patients with microalbuminuria (p = 0.021) compared with normoalbuminuric patients.

Conclusions

Our findings indicate that TIMP-1 and MMP-9, as well as sP-selectin and NGAL, are involved in microvascular complications in T1D. Monitoring and targeting these biomarkers may be a potential strategy for treating diabetic nephropathy and neuropathy.

Extensive research has been devoted to developing methods for assessing core body temperature, and to determine which method is most accurate. A number of wireless dermal thermometers for home use are presently available, but their relation to core body temperature and suitability for use in clinical research has hitherto not been assessed. The current study aimed to evaluate such thermometers by comparing them to the results of a rectal thermometer. Four wireless dermal thermometers for home use (FeverSmart, iThermonitor, Quest Temp Sitter, and Thermochron iButton) were applied to 15 patients during 24 h, and rectal temperature was measured at four occasions. Pearson correlation revealed moderate correlation for the Feversmart (r = 0.75), iThermonitor (r = 0.79), and Thermochron iButton (r = 0.71) systems. The Quest Temp Sitter system malfunctioned repeatedly, and the correlation (r = 0.29) for this method should therefore be assessed with caution. All dermal thermometers rendered lower average temperatures than Terumo c405 (Feversmart -0.70 +/- 0.65 degrees C; iThermonitor -0.77 +/- 0.53 degrees C, Quest Temp Sitter -1.18 +/- 0.66 degrees C, and Thermochron iButton -0.87 +/- 0.65 degrees C). Sensitivity of the dermal thermometers for detecting core temperatures amp;gt;= 38.0 degrees C was low, ranging from 0.33 to 0.6, but improved to 0.60 to 0.80 after adjusting temperatures by the methods average deviation from rectal temperature. The results from the dermal thermometers tested here showed an insufficient correlation to core temperature to be used for core temperature monitoring in clinical research and practice. Unfortunately, other options for non-invasive temperature measurements are few. The two thermometers with the least unsatisfactory performance profile in our evaluations were the Feversmart and iThermonitor systems.

Commentary to: Activation of ATM and Histone H2AX Phosphorylation Induced by Mitoxantrone But Not by Topotecan is Prevented by the Antioxidant N-acetyl-L-Cysteine Xuan Huang, Akira Kurose, Toshiki Tanaka, Frank Traganos, Wei Dai and Zbigniew Darzynkiewicz

This study aimed to evaluate the predictive value and clinical impact of a clinically implemented artificial neural network software model. The software detects intracranial hemorrhage (ICH) from head computed tomography (CT) scans and artificial intelligence (AI)-identified positive cases are then annotated in the work list for early radiologist evaluation. The index test was AI detection by the program Zebra Medical Vision-HealthICH+. Radiologist-confirmed ICH was the reference standard. The study compared whether time benefits from using the AI model led to faster escalation of patient care or surgery within the first 24 h. A total of 2,306 patients were evaluated by the software, and 288 AI-positive cases were included. The AI tool had a positive predictive value of 0.823. There was, however, no significant time reduction when comparing the patients who required escalation of care and those who did not. There was also no significant time reduction in those who required acute surgery compared with those who did not. Among the individual patients with reduced time delay, no cases with evident clinical benefit were identified. Although the clinically implemented AI-based decision support system showed adequate predictive value in identifying ICH, there was no significant clinical benefit for the patients in our setting. While AI-assisted detection of ICH shows great promise from a technical perspective, there remains a need to evaluate the clinical impact and perform external validation across different settings.

In this work dynamic variation of gate bias is used on a gas-sensitive SiC field effect transistor ("GasFET") to optimize its sensitivity and increase its selectivity. Gate bias ramps introduce strong hysteresis in the sensor signal. The shape of this hysteresis is shown to be an appropriate feature both for the discrimination of various gases (ammonia, carbon monoxide, nitrogen monoxide and methane) as well as for different gas concentrations (250 and 500 ppm). The shape is very sensitive to ambient conditions as well as to the bias sweep rate. Thus, the influences of oxygen concentration, relative humidity, sensor temperature and cycle duration, i.e., sweep rate, are investigated and reasons for the observed signal changes, most importantly the existence of at least two different and competing processes taking place simultaneously, are discussed. Furthermore, it is shown that even for very fast cycles, in the range of seconds, the gas-induced shape change in the signal is strong enough to achieve a reliable separation of gases using gate bias cycled operation and linear discriminant analysis (LDA) making this approach suitable for practical application.

Hydrogels of cellulose nanofibrils (CNFs) are promising wound dressing candidates due to their biocompatibility, high water absorption, and transparency. Herein, two different commercially available wood species, softwood and hardwood, were subjected to TEMPO-mediated oxidation to proceed with delignification and oxidation in a one-pot process, and thereafter, nanofibrils were isolated using a high-pressure microfluidizer. Furthermore, transparent nanofibril hydrogel networks were prepared by vacuum filtration. Nanofibril properties and network performance correlated with oxidation were investigated and compared with commercially available TEMPO-oxidized pulp nanofibrils and their networks. Softwood nanofibril hydrogel networks exhibited the best mechanical properties, and in vitro toxicological risk assessment showed no detrimental effect for any of the studied hydrogels on human fibroblast or keratinocyte cells. This study demonstrates a straightforward processing route for direct oxidation of different wood species to obtain nanofibril hydrogels for potential use as wound dressings, with softwood having the most potential.

Recent advances in microfluidic devices put a high demand on small, robust and reliable pumps suitable for high-throughput applications. Here we demonstrate a compact, low-cost, directly attachable (clip-on) electroosmotic pump that couples with standard Luer connectors on a microfluidic device. The pump is easy to make and consists of a porous polycarbonate membrane and poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) electrodes. The soft electrode and membrane materials make it possible to incorporate the pump into a standard syringe filter holder, which in turn can be attached to commercial chips. The pump is less than half the size of the microscope slide used for many commercial lab-on-a-chip devices, meaning that these pumps can be used to control fluid flow in individual reactors in highly parallelized chemistry and biology experiments. Flow rates at various electric current and device dimensions are reported. We demonstrate the feasibility and safety of the pump for biological experiments by exposing endothelial cells to oscillating shear stress (up to 5 dyn/cm2) and by controlling the movement of both micro- and macroparticles, generating steady or oscillatory flow rates up to ± 400 μL/min.

In the work presented this thesis, two different conditions that are classified as protein misfolding diseases: Alzheimer's disease and lysozyme amyloidosis and proteins that could have a beneficial effect in these diseases, have been studied using Drosophila melanogaster, commonly known as the fruit fly. The fruit fly has been used for over 100 years to study and better understand fundamental biological processes. Although the fruit fly, unlike humans, is an invertebrate, many of its central biological mechanisms are very similar to ours. The first transgenic flies were designed in the early 1980s, and since then, the fruit fly has been one of the most widely used model organisms in studies on the effects of over-expressed human proteins in a biological system; one can regard the fly as a living, biological test tube. For  most proteins, it is necessary that they fold into a three-dimensional structure to function properly. But sometimes the folding goes wrong; this may be due to mutations that make the protein unstable and subject to misfolding. A misfolded protein molecule can then aggregate with other misfolded proteins. In Alzheimer's disease, which is the most common form of dementia, protein aggregates are present in the brains of patients. These aggregates are composed of the amyloid-β (Aβ) peptide, a small peptide of around 42 amino acids which is cleaved from the larger, membrane-bound, protein AβPP by two different enzymes, BACE1 and γ-secretase. In the first part of this thesis, two different fly models for Alzheimer’s disease were used: the Aβ fly model, which directly expresses the Aβ peptide, and the AβPP-BACE1 fly model, in which all the components necessary to produce the Aβ peptide in the fly are expressed in the fly central nervous system (CNS). The two different fly models were compared and the results show that a significantly smaller amount of the Aβ peptide is needed to achieve the same, or an even greater, toxic effect in the AβPP-BACE1 model compared to the Aβ model. In the second part of the thesis, these two fly models for Alzheimer’s disease were again used, but now to investigate whether lysozyme, a protein involved in our innate immune system, can counteract the toxic effect of Aβ generated in the fly models. And indeed, lysozyme is able to save the flies from Aβ-induced toxicity. Aβ and lysozyme were found to interact with each other in vivo. The second misfolding disease studied in this thesis is lysozyme amyloidosis. It is a rare, dominantly inherited amyloid disease in which mutant variants of lysozyme give rise to aggregates, weighing up to several kilograms, that accumulate around the kidneys and liver, eventually leading to organ failure. In the third part of this thesis, a fly model for lysozyme amyloidosis was used to study the effect of co-expressing the serum amyloid P component (SAP), a protein that is part of all protein aggregates found within this disease class. SAP is able to rescue the toxicity induced by expressing the mutant variant of lysozyme, F57I, in the fly's CNS. To further investigate how SAP was able to do this, double-expressing lysozyme flies, which exhibit stronger disease phenotypes than those of the single-expressing lysozyme flies previously studied, were used in the fourth part of this thesis. SAP was observed to reduce F57I toxicity and promote F57I to form aggregates with more distinct amyloid characteristics. In conclusion, the work included in this thesis demonstrates that: i) Aβ generated from AβPP processing in the fly CNS results in higher proteotoxicity compared with direct expression of Aβ from the transgene, ii) lysozyme can prevent Aβ proteotoxicity in Drosophila and could thus be a potential therapeutic molecule to treat Alzheimer’s disease and iii) in a Drosophila model of lysozyme amyloidosis, SAP can prevent toxicity from the disease-associated lysozyme variant F57I and promote formation of aggregated lysozyme morphotypes with amyloid properties; this is important to take into account when a reduced level of SAP is considered as a treatment strategy for lysozyme amyloidosis.

The aggregation of the amyloid-beta (A beta) peptide into fibrillar deposits has long been considered the key neuropathological hallmark of Alzheimers disease (AD). A beta peptides are generated from proteolytic processing of the transmembrane A beta precursor protein (A beta PP) via sequential proteolysis through the beta-secretase activity of beta-site A beta PP-cleaving enzyme (BACE1) and by the intramembranous enzyme gamma-secretase. For over a decade, Drosophila melanogaster has been used as a model organism to study AD, and two different approaches have been developed to investigate the toxicity caused by AD-associated gene products in vivo. In one model, the A beta peptide is directly over-expressed fused to a signal peptide, allowing secretion of the peptide into the extracellular space. In the other model, human A beta PP is co-expressed with human BACE1, resulting in production of the A beta peptide through the processing of A beta PP by BACE1 and by endogenous fly gamma-secretase. Here, we performed a parallel study of flies that expressed the A beta(1-42) peptide alone or that co-expressed A beta PP and BACE1. Toxic effects (assessed by eye phenotype, longevity and locomotor assays) and levels of the A beta(1-42), A beta(1-40) and A beta(1-38) peptides were examined. Our data reveal that the toxic effect per amount of detected A beta(1-42) peptide was higher in the flies co-expressing A beta PP and BACE1 than in the A beta(1-42)-expressing flies, and that the co-existence of A beta(1-42) and A beta(1-40) in the flies co-expressing A beta PP and BACE1 could be of significant importance to the neurotoxic effect detected in these flies. Thus, the toxicity detected in these two fly models seems to have different modes of action and is highly dependent on how and where the peptide is generated rather than on the actual level of the A beta(1-42) peptide in the flies. This is important knowledge that needs to be taken into consideration when using Drosophila models to investigate disease mechanisms or therapeutic strategies in AD research.

The human central nervous system (CNS) displays the greatest cellular diversity of any organ system, consisting of billions of neurons, of numerous cell sub-types, interconnected in a vast network. Given this enormous complexity, decoding the genetic programs controlling the multistep process of CNS development remains a major challenge. While great progress has been made with respect to understanding sub-type specification, considerably less is known regarding how the generation of the precise number of each sub-type is controlled.

The aim of this thesis was to gain deeper knowledge into the regulatory programs controlling cell specification and proliferation. To address these questions I have studied the Drosophila embryonic CNS as a model system, to thereby be able to investigate the genetic mechanisms at high resolution. Despite the different size and morphology between the Drosophila and the mammalian CNS, the lineages of their progenitors share similarity. Importantly for this thesis, both species progenitors show elaborate variations in their proliferation modes, either giving rise to daughters that can directly differentiate into neurons or glia (type 0), divide once (type I), or multiple times (type II).

The studies launched off with a comprehensive chemical forward genetic screen, for the very last born cell in the well-studied lineage of progenitor NB5-6T: the Ap4 neuron, which expresses the neuropeptide FMRFa. NB5-6T is a powerful model to use, because it undergoes a programmed type I>0 daughter cell proliferation switch. An FMRF-eGFP transgenic reporter was utilized as readout for successful terminal differentiation of Ap4/FMRFa and thereby proper lineage progression of the ∼20 cells generated. The strongest mutants were mapped to genes with both known and novel essential functions e.g., spatial and temporal patterning, cell cycle control, cell specification and chromatin modification. Subsequently, we focused on some of the genes that showed a loss of function phenotype with an excess of lineage cells. We found that Notch is critical for the type I>0 daughter cell proliferation switch in the NB5-6T lineage and globally as well. When addressing the broader relevance of these findings, and to further decipher the Notch pathway, we discovered that selective groups of E(spl) genes is controlling the switch in a close interplay with four key cell cycle factors: Cyclin E, String, E2F and Dacapo, in most if not all embryonic progenitors. The Notch mediation of the switch is likely to be by direct transcriptional regulation. Furthermore, another gene identified in the screen, sequoia, was investigated. The analysis revealed that sequoia is also controlling the daughter cell switch in the CNS, and this partly through context dependent interactions with the Notch pathway.

Taken together, the findings presented in this thesis demonstrate that daughter cell proliferation switches in Drosophila neural lineages are genetically programmed, and that Notch contributes to the triggering of these events. Given that early embryonic processes is frequently shown to be evolutionary conserved, you can speculate that changeable daughter proliferation programs could be applied to mammals, and contribute to a broader understanding of proliferation processes in humans as well.

Thiopurine S-methyltransferase (TPMT) belongs to the Class I S-adenosylmethionine-dependent methyltransferase (SAM-MT) super family of structurally related proteins. Common to the members of this large protein family is the catalysis of methylation reactions using S-adenosylmethionine (SAM) as a methyl group donor, although SAM-MTs act on a wide range of different substrates and carry out numerous biologically important functions. While the natural function of TPMT is unknown, this enzyme is involved in the metabolism of thiopurines, a class of pharmaceutical substances administered in treatment of immune-related disorders. Specifically, methylation by TPMT inactivates thiopurines and their metabolic intermediates, which reduces the efficacy of clinical treatment and increases the risk of adverse side effects. To further complicate matters, TPMT is a polymorphic enzyme with over 40 naturally occurring variants known to date, most of which exhibit lowered methylation activity towards thiopurines. Consequently, there are individual variations in TPMTmediated thiopurine inactivation, and the administered dose has to be adjusted prior to clinical treatment to avoid harmful side effects.

Although the clinical relevance of TPMT is well established, few studies have investigated the molecular causes of the reduced methylation activity of variant proteins. In this thesis, the results of biophysical characterization of two variant proteins, TPMT*6 (Y180F) and TPMT*8 (R215H), are presented. While the properties of TPMT*8 were indistinguishable from those of the wild-type protein, TPMT*6 was found to be somewhat destabilized. Interestingly, the TPMT*6 amino acid substitution did not affect the functionality or folding pattern of the variant protein. Therefore, the decreased in vivo functionality reported for TPMT*6 is probably caused by increased proteolytic degradation in response to the reduced stability of this protein variant, rather than loss of function.

Also presented herein are novel methodological approaches for studies of TPMT and its variants. Firstly, the advantages of using 8-anilinonaphthalene-1-sulfonic acid (ANS) to probe TPMT tertiary structure and active site integrity are presented. ANS binds exclusively to the native state of TPMT with high affinity (K_D ~ 0.2 μm) and a 1:1 ratio. The stability of TPMT was dramatically increased by binding of ANS, which was shown to co-localize with the structurally similar adenine moiety of the cofactor SAM. Secondly, an enzyme activity assay based on isothermal titration calorimetry (ITC) is presented. Using this approach, the kinetics of 6-MP and 6-TG methylation by TPMT has been characterized.

Inflammation initiates tendon healing and then normally resolves more or less completely. Unresolved inflammation might disturb the remodeling process. We hypothesized that suppression of inflammation during the early remodeling phase by systemic dexamethasone treatment can improve healing. 36 rats underwent Achilles tendon transection and were randomized to dexamethasone or saline on days 0-4 after surgery (early inflammatory phase), and euthanasia day 7. Another 54 rats received injections days 5-9 (early remodeling phase) and were euthanized day 12 for mechanical, histological and flow cytometric evaluation. Dexamethasone treatment days 0-4 reduced the cross-sectional area, peak force and stiffness by day 7 to less than half (p amp;lt; 0.001 for all), while material properties (peak stress and elastic modulus) were not significantly affected. In contrast, dexamethasone treatment days 5-9 increased peak force by 39% (p = 0.002) and stiffness by 58% (p amp;lt; 0.001). The cross-sectional area was reduced by 42% (p amp;lt; 0.001). Peak stress and elastic modulus were more than doubled (p amp;lt; 0.001 for both). Semi-quantitative histology at day 12 showed that late dexamethasone treatment improved collagen alignment, and flow cytometry revealed reduced numbers of CD8a(+) cytotoxic T cells in the tendon callus. These results suggest that downregulation of lingering inflammation during the early remodeling phase can improve healing.

We increase our understanding of augmenting a cellular immune response, by using an HIV-1 protease-derived epitope (PR75−84), and variants thereof, coupled to the C-terminal, of the B subunit of cholera toxin (CTB). Fusion proteins were used for immunizations of HLA-A0201 transgenic C57BL/6 mice. We observed different capacities to elicit a cellular immune response by peptides with additions of five to ten amino acids to the PR epitope. There was a positive correlation between the magnitude of the elicited cellular immune response and the capacity of the fusion protein to bind GM-1. This binding capacity is affected by its ability to form natural pentamers of CTB. Our results suggest that functional CTB pentamers containing a foreign amino acid-modified epitope is a novel way to overcome the limited cellular immunogenicity of minimal peptide antigens. This way of using a functional assay as readout for improved cellular immunogenicity might become highly valuable for difficult immunogens such as short peptides (epitopes).

The cardiac ventricular action potential depends on several voltage-gated ion channels, including Na-V, Ca-V, and K-V channels. Mutations in these channels can cause Long QT Syndrome (LQTS) which increases the risk for ventricular fibrillation and sudden cardiac death. Polyunsaturated fatty acids (PUFAs) have emerged as potential therapeutics for LQTS because they are modulators of voltage-gated ion channels. Here we demonstrate that PUFA analogues vary in their selectivity for human voltage-gated ion channels involved in the ventricular action potential. The effects of specific PUFA analogues range from selective for a specific ion channel to broadly modulating cardiac ion channels from all three families (Na-V, Ca-V, and K-V). In addition, a PUFA analogue selective for the cardiac IKs channel (Kv7.1/KCNE1) is effective in shortening the cardiac action potential in human-induced pluripotent stem cell-derived cardiomyocytes. Our data suggest that PUFA analogues could potentially be developed as therapeutics for LQTS and cardiac arrhythmia.

Rapid DNA instruments have in recent years been developed, enabling analysis of forensic samples with a minimum of human intervention. Initially intended for fast handling of reference samples, such as samples from suspects in booking suites, attention shifted to include crime scene samples. The aim of this study was to determine whether or not the RapidHIT System (IntegenX) is fit for crime scene samples. The first runs gave very poor results, which was found to be due to an incorrect firmware setting leading to no or just minute amounts of amplicons being injected for electrophoresis. After solving this problem, 28 full runs (seven samples each) applying NGM SElect Express were performed comprising various amounts of blood on cotton swabs. Six of the runs failed completely, four due to cartridge leakage and in two runs the PCR mix was not injected. For 155 samples with 1–5ÎŒL blood (volumes for which complete DNA profiles are expected), 119 samples (77%) gave complete DNA profiles. Among the most serious failures were incorrect allele calling and leakage of DNA extract or PCR product. Other general issues were failure to export results, anode motor breakdown and broken capillary array. Due to the encountered problems with software, hardware and cartridges, together with the low success rate, it was decided not to continue towards implementation of the RapidHIT System in casework.

The ultimate cause of cancer related deaths is metastasis. This thesis is about three of the main human cancers; breast, colorectal and pancreatic cancer, that together account for more than 25% of the cancer-related deaths worldwide. The focus of the thesis is the spread of cancer, metastasis, and the aim was to investigate mechanisms that can be of importance for this process. We analyzed patient samples to validate the role of epithelialto-mesenchymal transition in vivo and found regulations of many related factors. However, these changes tend to fluctuate along the metastatic process, something which makes targeting complicated. We, moreover, focused on the influence of the tumor microenvironment for metastatic spread. In pancreatic cancer, the stroma constitutes the main part of many tumors. We analyzed the crosstalk between tumor and stromal cell and focused on the mediating inflammatory factor interleukin-1 (IL-1) and regulation of microRNAs. The results showed that the most commonly mutated factor in pancreatic cancer, KRAS, associates with the expression of IL-1 and subsequent activation of stromal cells. Blocking KRAS signaling together with IL-1 blockage give a more pronounced effect on in vitro proliferation and migration of cancer cells and suggests the use of a combination therapy. The cancer-associated activation of the stroma was found to be related to changes in microRNA expression. microRNA was analyzed separately in epithelial cells and stromal cells after microdissection of matched samples of primary and secondary tumors of breast and colorectal cancers. miR-214 and miR-199a were upregulated in stroma associated with progressive tumors and in pancreatic cancer stroma we could show that their expression alters the activation of stromal cells and thereby the growth and migratory ability of associated pancreatic tumor cells. In  breast and colorectal cancers we found several common microRNAs to be up- or downregulated in line with progression. We could show that one of these candidates, miR-18a, had a prognostic value in metastatic breast cancer. To further develop these studies we analyzed this microRNA in circulating microvesicles, i.e. exosomes, and investigated their role in the preparation of a pre-metastatic niche. MicroRNAs are stable biomarkers in the circulation, especially protected in exosomes, which can moreover specifically deliver their message to recipient cells. These studies facilitate the understanding of metastatic behavior and suggest new targets to stop cancer metastasis.