Despite very extensive studies on molecular mechanisms of learning and memory formation it is little known about individual variation in the learning skills within a random animal population and about the differences in the brain biochemistry behind this variation. In the present study, we have focused on the expression and distribution of nitric oxide synthase (NOS), one of the molecules implemented in activity-dependent neuroplasticity, in the rat hippocampus, the structure critical for episodic memory in humans and animals. The aim of the present study was to investigate the differences in expression of three different NOS isoforms: neural (n), epithelial (e), and inducible (i), in four hippocampal subregions (CA1, CA3, DG, and hilus) between Wistar rats classified on the basis of their performance in partially baited 12-arm radial maze as &ldquo / good&rdquo / and &ldquo / poor&rdquo / learners. The NOS isoforms were visualized on coronal hippocampal sections using fluorescent immunohistochemistry technique and n- and eNOS images were processed using ImageJ software, while iNOS immunoreactivity (IR) was assessed by counting immunoreactive cells. In this study, overall hippocampal levels of nNOS were significantly higher than those of eNOS and iNOS. The level of n and eNOS was higher in CA1 compared to DG/hilus areas, but lower than that in CA3 region. The expression of iNOS was the highest in CA1 and the lowest in hilus region. nNOS IR was significantly higher in &ldquo / poor&rdquo / than in &ldquo / good&rdquo / learners but only in CA1 region. No significant between-group differences were found in eNOS expression. iNOS expression was higher in &ldquo / poor&rdquo / learners but it did not reach the required significance level.

Dupuytren's contracture (DC) is a fibromatous disease of the palmar fascia of unknown etiology. The present study was undertaken in order to assess pathophysiological mechanisms and consequences. In a cohort study of 2,375 patients operated for DC at the Department of Hand Surgery, Uppsala there was a male: female ratio of 5.9:1. Women had a higher mean age at first operation than men. One-third of the men and one-quarter of the women required repeated surgery. Early age at first operation was associated with recurrent disease. The risk of cancer was determined in 15,212 patients operated on for DC in Sweden. The overall relative risk was increased by 24%. There was a significantly increased risk for buccal, oesophageal, gastric, lung and pancreatic cancers, which indicates that smoking and alcohol abuse are probable risk factors for DC. Furthermore, there was an increased frequency of fibrosarcoma and malignant fibrous histiocytoma, the cause of which is unexplained The causes of death were evaluated in a national cohort of 16,517 patients operated for DC. There was an overall increased mortality (SMR=1.06), inversely related to age and significant for both sexes, in patients under 70 years. The risk estimate was highest for endocrine-, gastrointestinal-, and respiratory diseases, and accidents. There was also an increased SMR for cardiovascular diseases in younger patients more than 10 years after surgery. The most probable mechanism is related to smoking and other lifestyle factors. Outcome after surgery was not related to the immunohistochemical expression of connective tissue activation markers, such as collagen type IV, integrin α5, laminin, smooth muscle α-actin, procollagen type I, and desmin, in surgical specimens in a prospectively investigated group of patients. Furthermore, there were no associations between gender, age at onset of DC, number of operations, heredity, diabetes mellitus, or medication for cardiovascular disease, and the expression of the different markers. The individual characteristics that place a person at high risk are, thus, not obviously related to ongoing connective tissue production at time of surgery or to connective tissue activity in its conventionally used sense.

Onychophorans are closely allied to the arthropods and possess a body organisation more similar to Middle Cambrian fossils than to recent arthropods. This means that onychophorans in some respects can be regarded as a model for the last common ancestor to both the Arthropoda and the Onychophora. This thesis mainly deals with the morphology of the head region of the Onychophora, but developmental investigations of the expression of a key regulatory gene, engrailed, are also carried out. The innervation of the head was found to differ from that reported in earlier investigations. The nerves that support the mouth were found to originate from three different regions of the brain. That innervation pattern suggests that present day onychophorans with a ventrally placed mouth, have evolved from an ancestor with a terminal mouth. Furthermore it was confirmed that the onychophoran structure with the unfortunately chosen term labrum is not homologous to the structure in arthropods that bears this name. Instead it is a muscular outgrowth from the pharynx. The embryological investigations gave further support for an ancestral and terminal mouth. The two most anterior oral lips are first located on the dorso-frontal side of the head, and later migrate to their final position at the ventral side. This phenomenon also explains their somewhat unexpected innervation from the dorsum. It was also established that the eye originates at a position posterior to the antenna. This is reversed compared to the condition in arthropods, were the eye is innervated from the protocerebrum and the first antenna from the dutocerebrum, and implies that the eye and antenna are not serially homologous between the two groups. A structure in the onychophoran head that has gained little attention is the hypocerebral organ, also termed infracerebral organ. It has been suggested as a corpora allata analog by earlier workers. Its ultrastructure was investigated, and great similarities to the corpora allata of the stick insect Carausius morosus were found. However, the lack of innervation of the hypocerebral organ of Onychophora poses a problem since the corpora allata of insects is controlled by nerves. Instead, cellular strands were found that connected the hypocerebral organ with the brain, and it is possible that these strands act as an alternative communication. The expression pattern of the segment polarity gene engrailed was found to be different from that reported in an earlier account of onychophorans. Engrailed was expressed in a subset of developing neurons in the brain anlage and in the ectoderm and mesoderm of the limb buds. The engrailed positive cells in the brain anlage were located in the area were the first commissure will form. This indicates that engrailed might have a function in axon guidance, as has been reported in other organisms. Later embryonic stages showed expression in the neuropile of the brain. There were no indications of this gene acting in determination of segment polarity. This suggests that there may be at least two copies of engrailed in onychophorans.

The field of proteomics has rapidly expanded due to the completion of the human genome sequence. This thesis validates affinity-purified monospecific antibodies of polyclonal origin, for protein profiling in a broad spectrum of normal tissues and cells. Validation of antibodies is crucial for development of reliable binders for target proteins and this thesis evaluates the generation and application of large sets of msAbs in different settings. MsAbs were generated towards recombinant Protein Epitope Signature Tag (PrEST) antigens using a stringent affinity-purification strategy, presented in the first study. The specificity of msAbs was studied using reverse phase protein arrays and immunohistochemistry (IHC), and results presented over 90% success rate in the protein array analysis. In IHC, 81% of the msAbs displayed apparent specific staining in normal tissues. MsAbs were also compared with commercial analogs (cAbs) using IHC and Western blot. Results presented similar outcome between msAbs and cAbs in both applications, although interpretation suggested more extensive IHC staining patterns with msAbs than with monoclonal analogs. For antibody validation, an approach called paired antibodies was presented and involved the generation of two msAbs towards non-overlapping epitopes on the same protein. Similarities in protein detection between paired antibodies were studied using three different antibody-based methods. Similar results were observed in several applications, indicating that this strategy can be a useful tool for studying known and unknown proteins. Given the reliability of msAbs, they were also applied in a study investigating the impact of tissue fixatives on protein detection. The study showed that different fixation mechanisms appeared to affect protein recognition by indicating that aldehyde-based fixation, e.g. induced by neutral buffered formalin, was preferred for tissues used in IHC and non-aldehyde based fixation was applicable for tissues used in protein extraction analysis and Western blotting. Conclusively, validation results suggest that msAbs are reliable affinity binders that can be used as valuable tools for proteome-wide protein profiling in tissues and cells.

Strength training is widely used to increase performance in sports with high physical demands. The use of drugs such as anabolic steroids among athletes is a wellknown phenomenon, and the effects of these drugs on physical performance documented. The studies presented in this thesis focused on the mechanisms of muscle fiber hypertrophy in the vastus lateralis and the trapezius muscles of strength trained elite athletes. The main hypothesis was that the muscle adaptations to strength training and anabolic steroids are muscle specific. Biopsies were obtained from the trapezius and the vastus lateralis from three groups of elite power lifters. Nine used drugs, ten did not and seven had previously used drugs. Six sedentary males served as controls. The biopsies were frozen and cut in serial cross sections. Histological and immunohistochemical staining techniques were used to analyze muscle fiber morphology and pathology. Fiber type distribution, fiber area, myonuclei number and distribution, satellite cell number and proportion of split fibers were counted and compared for the two muscles within and between the groups. The main findings were that: a) Muscle fiber hypertrophy by strength training is further increased by anabolic steroids. b) The number of nuclei per muscle fiber is higher in power lifters using anabolic steroids compared to non-steroids using lifters. c) Among power lifters who have withdrawn from anabolic steroid usage and training for several years, the number of myonuclei, both subsarcolemmal and internal, remains high. d) In active power lifters, anabolic steroids have no further effect on the number of satellite cells per fiber. e) Power lifters have a high proportion of split fibers. High intensity resistance training increases muscle strength and banned substances such as testosterone and anabolic steroids can enhance the training effects. The studies on muscle cell morphology presented in this thesis reveals that anabolic steroids and testosterone increases muscle fiber size and adds more nuclei to the muscle cell. Based on the morphological appearance of muscle sections from doped and nondoped power lifters, we conclude that testosterone and anabolic steroids enhances the hypertrophic effects of training without adding new features. The addition of myonuclei by training and doping appears to be longer lasting in some muscles than in others. The high proportion of split fibers in power lifter is probably due to high mechanical stress. The findings and conclusions in this thesis raise questions regarding relevant suspension times for athletes caught with banned substances in the body.

To construct a complete map of the human proteome landscape is a vital part of the total understanding of the human body. Such a map could enrich the mankind to the extent that many severe diseases could be fully understood and hence could be treated with appropriate methods. In this study, immunohistochemical (IHC) data from ~6000 proteins, 65 cell types in 48 tissues and 47 cell lines has been used to investigate the human proteome regarding protein expression and localization. In order to analyze such a large data set, different statistical methods and algorithms has been applied and by using these tools, interesting features regarding the proteome was found. By using all available IHC data from 65 cell types in 48 tissues, it was found that the amount of tissue specific protein expression was surprisingly small, and the general impression from the analysis is that almost all proteins are present at all times in the cellular environment. Rather than tissue specific protein expression, the localization and minor concentration fluctuations of the proteins in the cell is responsible for molecular interaction and tissue specific cellular behavior. However, if a quarter of all proteins are used to distinguish different tissues types, there are a proportion of proteins that have certain expression profiles, which defines clusters of tissues of the same kind and embryonic origin. The estimation of expression levels using IHC is a labor-intensive method, which suffers from large variation between manual annotators. An automated image software tool was developed to circumvent this problem. The automated image software was shown to be more robust then manual annotators, and the quantification of expressed protein levels of the stained imaged was in the same range as the manual annotations. A more thorough investigation of the stained image estimations made by the automated software revield a significant correlation between the estimated protein expression and the cell size parameters provided by the automated software. To make it feasible to compare protein expression levels across different cell lines, without the cell line size bias, a normalization procedure was implemented and evaluated. It was found that when the normalization procedure was applied to the protein expression data, the correlation between protein expression values and cell size was minimized, and hence comparisons between cell lines regarding protein expression is possible. In addition, using the normalized protein expression data, an analysis to investigate the degree of correlation between mRNA levels and proteins for 1065 gene products was performed. By using two individual microarray data sets for estimation of RNA levels, and normalized protein data measured by the automated software as estimation of the protein levels, a mean correlation of ~0.3 for was found. This result indicates that a significant proportion of the manufactured antibodies, when used in IHC setup, are indeed an accurate measurement of protein expression levels. By using antibodies directed towards human proteins, plasma samples were investigated regarding metabolic dysfunctions. Since plasma is a complex sample, an optimization regarding protocol for quantification of expressed proteins was made. By using certain characteristics within the dataset, and by using a suspension bead microarray, the protocol could be evaluated. Expected characteristics within the dataset were found in the subsequent analysis, which showed that the protocol was functional. Using the same experimental outline will facilitate future applications, e.g. biomarker discovery. / QC 20100728 / Human Proteome Resource

The aim of this thesis was to characterize the expression of the peptides ghrelin and obestatin, as well as other neuroendocrine markers in human normal tissues, in invasive breast cancer and a wide panel of neuroendocrine tumors (NETs). In normal tissues the expression of ghrelin and obestatin was mainly localized to the gastric mucosa, and in lesser extent in the remaining gastrointestinal tract, endocrine pancreas and mammary glands. Double immunofluorescence studies demonstrated that ghrelin and obestatin were co-localized in the same cells displaying the same cytoplasmic distribution. In normal breast tissue, ghrelin, obestatin, adrenomedullin, apelin and vesicular monoamine transporter 2 were specifically demonstrated in the luminal epithelial cells. Consecutive sections indicated that mammary epithelial cells could express several of these peptides. Secretogranin II and III were also detected in breast tissue, but their presence was restricted to the outer layer of myoepithelial cells, whereas chromogranin B immunoreactivity was found in both the epithelial and myoepithelial cells. Ghrelin and obestatin immunoreactivity was seen in invasive breast cancer, where the expression could be correlated to factors associated with prognosis. Furthermore, multivariate analysis indicated that ghrelin expression was a possible independent prognostic factor for prolonged recurrence-free and breast cancer-specific survival. In a panel of NETs and endocrine-related disorders it was revealed that ghrelin and obestatin immunoreactivity was primarily found in tumors originating from the respective normal tissues. The two proteins were detected in only a few cases and only occasional tumor cells were immunoreactive. In conclusion, ghrelin and obestatin are localized in the gastrointestinal tract, endocrine pancreas and mammary glands. This thesis has contributed to our understanding of the distribution of ghrelin and obestatin in both normal tissue and tumor cells. A potential role of ghrelin as a prognostic factor in invasive breast cancer has been identified and should be further explored.

Background: Ambient air pollution is associated with adverse health effects, but the sources and components, which cause these effects is still incompletely understood. The aim of this thesis was to investigate the pulmonary effects of a variety of common air pollutants, including diesel exhaust, biomass smoke, and road tunnel and subway station environments. Healthy non-smoking volunteers were exposed in random order to the specific air pollutants and air/control, during intermittent exercise, followed by bronchoscopy. Methods and results: In study I, exposures were performed with diesel exhaust (DE) generated at transient engine load and air for 1 hour with bronchoscopy at 6 hours post-exposure. Immunohistochemical analyses of bronchial mucosal biopsies showed that DE exposure significantly increased the endothelial adhesion molecule expression of p-selectin and VCAM-1, together with increased bronchoalveolar lavage (BAL) eosinophils. In study II, the subjects were exposed for 1 hour to DE generated during idling with bronchoscopy at 6 hours. The bronchial mucosal biopsies showed significant increases in neutrophils, mast cells and lymphocytes together with bronchial wash neutrophils. Additionally, DE exposure significantly increased the nuclear translocation of the aryl hydrocarbon receptor (AhR) and phosphorylated c-jun in the bronchial epithelium. In contrast, the phase II enzyme NAD(P)H-quinone oxidoreductase 1 (NQO1) decreased after DE. In study III, the 2-hour exposures took place in a road tunnel with bronchoscopy 14 hours later. The road tunnel exposure significantly increased the total numbers of lymphocytes and alveolar macrophages in BAL, whereas NK cell and CD56+/T cell numbers significantly decreased. Additionally, the nuclear expression of phosphorylated c-jun in the bronchial epithelium was significantly increased after road tunnel exposure. In study IV, the subjects were exposed to metal-rich particulate aerosol for 2 hours at a subway station with bronchial biopsy and BAL sampling at 14 hours. The subway exposure significantly increased the concentration of glutathione disulphide (GSSG) in BAL, with no airway inflammatory responses. In contrast, the number of neutrophils in the bronchial mucosa and the nuclear expression of phosphorylated c-jun in the bronchial epithelium tended to decrease after the subway exposure. In study V, the exposure to biomass smoke lasted 3 hours. Bronchoscopy was conducted 24 hours post exposure. The investigated biomass combustion emissions resulted in a significant increase in total glutathione and reduced glutathione in BAL, without any evident acute airway inflammatory responses.     Conclusion: The present thesis presents data from exposures of healthy subjects to a variety of common air pollutants, as compared with an air reference. Oxidative as well as bronchial mucosal and bronchoalveolar responses differed between these air pollutants, with the most pronounced airway effects seen after exposure to diesel exhaust. This may be due to differences in pulmonary deposition, physicochemical characteristics, toxicological pathways and potency. Additional studies will assist in addressing dose-response and time kinetic aspects of the airway responses.

Proteins are essential building blocks in every living cell, and since the complete human genome was sequenced in 2004, researchers have attempted to map the human proteome, which is the functional representation of the genome. One such initiative is the Human Protein Atlas programme (HPA), which generates monospecific antibodies towards all human proteins and uses these for high-throughput tissue profiling on tissue microarrays (TMAs). The results are publically available at the website www.proteinatlas.org. In this thesis, TMAs were used for analysis of expression patterns in various research areas. Different search queries in the HPA were tested and evaluated, and a number of potential biomarkers were identified, e.g. proteins exclusively expressed in islets of Langerhans, but not in exocrine glandular cells or other abdominal organs close to pancreas. The identified candidates were further analyzed on TMAs with pancreatic tissues from normal and diabetic individuals, and colocalization studies with insulin and glucagon revealed that several of the investigated proteins (DGCR2, GBF1, GPR44 and SerpinB10) appeared to be beta cell specific. Moreover, a set of proteins differentially expressed in lung cancer stroma was further analyzed on a clinical lung cancer cohort in the TMA format, and one protein (CD99) was significantly associated with survival. In addition, TMAs with tissue samples from different species were generated, e.g. for mapping of influenza virus attachment in various human and avian tissues. The results showed that the gull influenza virus H16N3 attached to human respiratory tract and eye, suggesting possible transmission of the virus between gull and human. TMAs were also used for analysis of protein expression differences between humans and other primates, and two proteins (TCF3 and SATB2) proved to be significantly differentially expressed on the human lineage at both the protein level and the RNA level.   In conclusion, this thesis exemplifies the potential of the TMA technology, which can be used for analysis of expression patterns in a large variety of research fields, such as biomarker discovery, influenza virus research or further understanding of human evolution.

The myocardial infarction (MI) is one of the leading causes of death in the world today. Accumulated atherosclerotic plaque occluding cardiac blood vessels results in a lack of oxygen supply to parts of the heart, and consequentially the death cardiomyocytes. The damaged area is replaced by scar tissue because of the heart’s insufficient regenerative capability, and the contraction property of the post-MI heart is therefore compromised. The recent findings of an endogenous cardiac progenitor cell (CPC) population gives hope for the establishment of new methods for medical treatments of the post-MI heart. Compared to other stem/progenitor cell sources, the CPCs are committed to a cardiac fate which places them in the forefront of interesting cell sources for regenerative treatments. In this thesis, the distribution of stem cell antigen 1 (Sca-1) positive CPCs in the healthy mouse myocardium, as well as the healthy and post-MI rat left ventricle was determined and compared to the total amount of nuclei. An immunohistochemistry protocol for the detection of Sca-1+ cells was established, and the number of Sca-1+ cells and the total number of nuclei in the different mouse and rat tissue samples were counted using laser scanning cytometry (LSC). The results could conclude a significantly higher distribution of Sca-1+ cells in the mouse atrium compared to the mouse ventricle, and a significantly higher distribution of Sca-1+ cells in the 8 days post-MI rat left ventricle compared to the healthy rat left ventricle. Furthermore, a heterogeneous distribution within the 8 days post-MI rat left ventricle was observed.