http://hdl.handle.net/1956/929 Search the Channel search http://bora.uib.no/simple-search http://hdl.handle.net/1956/2323 Title: Comprehensive Analysis of Exported Proteins from Mycobacterium tuberculosis H37Rv <br/> <br/>Authors: Målen, Hiwa; Berven, Frode S.; Fladmark, Kari E.; Wiker, Harald G. <br/> <br/>Abstract: Proteins secreted by Mycobacterium tuberculosis play an essential role in the pathogenesis of tuberculosis. The culture filtrates of M. tuberculosis H37Rv made by Sadamu Nagai (Japan), are considerably enriched for secreted proteins compared to other culture filtrates. Complementary approaches were used to identify the secreted proteins in these culture filtrates: (i) 2-DE combined with MALDI-TOF MS and (ii) LC coupled MS/MS. Peptides derived from a total of 257 proteins were identified of which 144 were identified by more than one peptide. Several members of the immunologically important early secretory antigenic target-6 (ESAT-6) family of proteins were found to be major components. The majority of the identified proteins, 159 (62%), were predicted to be exported through the general secretory pathway. We experimentally verified that the signal peptides, which mediate translocation through the cell membrane, had been removed in 41 of the identified proteins, and in 35 of those, there was an AXA motif N-terminally to the cleavage site, showing that this motif is important for the recognition and cleavage of signal peptides in mycobacteria. A large fraction of the secreted proteins were unknown, suggesting that we have mapped an unexplored part of the exported proteome of M. tuberculosis. <br/> <br/>Description: Original published version is available at: http://dx.doi.org/10.1002/pmic.200600853 http://hdl.handle.net/1956/2219 Title: Uterine cervical neoplasia : aspects of biology and pathology <br/> <br/>Authors: Bertelsen, Bjørn I. http://hdl.handle.net/1956/2214 Title: Time-dependent clock gene expression in mouse and human stem and progenitor cells <br/> <br/>Authors: Tsinkalovsky, Oleg <br/> <br/>Abstract: Time is a fundamental part of all biological processes. During the whole process of evolution, living cells and organisms had to adapt to cyclic variations in the environment. In particular, the light and temperature conditions varied with day and night and with the seasons. The cellular functions and behavior in organisms were regulated according to their daily needs and provision of optimal conditions for survival. Time functions in cells are of two kinds. One is the cyclic variation, where the same events come back with regular intervals. Thereby, cellular adaptation occurs in a cyclic manner. The second way is longitudinal time regulation. From conception on, the development of organs occur sequentially and strictly coordinated in time until a mature fetus is ready for delivery. The periods of gestation, infant, growth and maturity, as well as aging, are strictly regulated in all higher organisms. Total life span is also rather constant within each species, although there are differences of many fold between short-lived and long-lived species. Thus, all the different longitudinal time periods in the life within a species are strictly coordinated to each other and are in concordance with total life span. Time regulation in single cells occurs at shorter intervals, both with cyclic and noncyclic variations. The oscillations may be down to a few seconds or even parts of seconds, such as nerve pulses. Heartbeat in higher organisms usually occurs as a rhythm of about one second or less, respiratory rhythm is slower, while the rhythm of the blood pressure is according to day and night. As a result, many types of time regulation and adaptation are occurring simultaneously in the same organism. Until a few years ago, the study of such time keeping was largely confined to the observation of phenomena from the outside and with mainly a descriptive approach. This has been replaced by a deeper understanding of the underlying biology and regulatory mechanisms at the genetic level (for recent review see Koukkari and Sothern, 2006b). This thesis deals with the hematopoietic system in mice and in men, a highly adaptable tissue with a high cell turnover and many different functions that cover all parts of the body. It is therefore no wonder that time keeping is an important part of the regulatory circuits. The main emphasis of this thesis is on the elucidation of local clock functions in cells of different stage of maturation, and discussion on how the molecular clock in bone marrow (BM) is unique compared to other tissues, as well as different between species. http://hdl.handle.net/1956/2065 Title: In Situ Expression of Cytokines and Cellular Phenotypes in the Lungs of Mice with Slowly Progressive Primary Tuberculosis <br/> <br/>Authors: Mustafa, T.; Phyu, S.; Nilsen, Rune; Jonsson, R.; Bjune, G. <br/> <br/>Abstract: The cellular phenotypes and the expression of cytokines were studied in the lungs of mice, using immunohistochemistry, during different phases of slowly progressive primary murine tuberculosis infection. During the first phase the small focal lesions in healthy mice contained predominantly interleukin-2 (IL-2)- expressing cells. A small number of tumour necrosis factor-a (TNF-a)-, monocyte chemoattractant protein-1 (MCP-1)- and IL-10-expressing cells were also present. IL-4-expressing cells were not detected. During the second phase the mice became unwell, but the bacterial counts and the size of focal lesions stabilized. IL-4- expressing cells appeared. The IL-10-, TNF-a- and MCP-1-expressing cells increased in number. On progression to phase three, the mice became seriously unwell and died rapidly. The inflammation spread to <80% of the lung parenchyma. There was a marked increase in the number of IL-10-expressing cells. Expression of other cytokines was similar to that observed in the second phase. In the lesions, 3±6% of the macrophages (Mf) containing mycobacterial antigens expressed high levels of IL-10 and TNF-a. The absolute numbers of CD3-, CD4- and CD11b-expressing cells in the lesions increased with the progression of infection. The numbers of CD8+ cells were reduced in the last phase of infection. The kinetics of T-lymphocyte subsets and the pattern of cytokine expression changed with the type and degree of tissue injury. The small number of Mf with a heavy load of mycobacterial antigens may be the cause of this disturbance in cytokine balance, thus leading to progression of inflammation. http://hdl.handle.net/1956/2063 Title: Detection and molecular characterization of herpes simplex virus from Tanzania and Norway <br/> <br/>Authors: Kasubi, Mabula Joseph http://hdl.handle.net/1956/1926 Title: Polymorphisms of the XRCC1, XRCC3 and XPD genes and risk of colorectal adenoma and carcinoma, in a Norwegian cohort : a case control study <br/> <br/>Authors: Skjelbred, Camilla Furu; Sæbø, Mona; Wallin, Håkan; Nexø, Bjørn Andersen; Hagen, Per Christian; Lothe, Inger Marie Bowitz; Aase, Steinar; Johnson, Egil; Hansteen, Inger-Lise; Vogel, Ulla; Kure, Elin H. <br/> <br/>Abstract: Background: Genetic polymorphisms in DNA repair genes may influence individual variation in DNA repair capacity, which may be associated with risk of developing cancer. For colorectal cancer the importance of mutations in mismatch repair genes has been extensively documented. Less is known about other DNA repair pathways in colorectal carcinogenesis. In this study we have focused on the XRCC1, XRCC3 and XPD genes, involved in base excision repair, homologous recombinational repair and nucleotide excision repair, respectively. Methods: We used a case-control study design (157 carcinomas, 983 adenomas and 399 controls) to test the association between five polymorphisms in these DNA repair genes (XRCC1 Arg194Trp, Arg280His, Arg399Gln, XRCC3 Thr241Met and XPD Lys751Gln), and risk of colorectal adenomas and carcinomas in a Norwegian cohort. Odds ratio (OR) and 95% confidence interval (95% CI) were estimated by binary logistic regression model adjusting for age, gender, cigarette smoking and alcohol consumption. Results: The XRCC1 280His allele was associated with an increased risk of adenomas (OR 2.30, 95% CI 1.19–4.46). The XRCC1 399Gln allele was associated with a reduction of risk of high-risk adenomas (OR 0.62, 95% CI 0.41–0.96). Carriers of the variant XPD 751Gln allele had an increased risk of low-risk adenomas (OR 1.40, 95% CI 1.03–1.89), while no association was found with risk of carcinomas. Conclusion: Our results suggest an increased risk for advanced colorectal neoplasia in individuals with the XRCC1 Arg280His polymorphism and a reduced risk associated with the XRCC1 Arg399Gln polymorphism. Interestingly, individuals with the XPD Lys751Gln polymorphism had an increased risk of low-risk adenomas. This may suggest a role in regression of adenomas. http://hdl.handle.net/1956/1293 Title: Ectopic germinal center formation in Sjögren’s syndrome: Significance of lymphoid organization <br/> <br/>Authors: Jonsson, Malin Viktoria <br/> <br/>Abstract: Sjögren’s syndrom (SS) is an autoimmune, chronic inflammatory disorder predominantly affecting the salivary and lacrimal glands. The overall aim of this study was to determine clinicopathological features in human and murine disease with regard to organization of ectopic lymphoid tissue, and to explore possible strategies for detection of patients at increased risk for extra-glandular manifestations. Mononuclear cell infiltrates in the shape of germinal centers (GC) were observed in the salivary glands of approximately 1/4th of patients. Phenotypic markers for GC components such as T and B cells, proliferating cells, follicular dendritic cells and plasma cells confirmed the ectopic GC formation. The pattern and distribution of homing and retentive chemokines CXCL12, CXCL13 and CCL21, and adhesion molecules/integrin pairs ICAM/LFA and VCAM/VLA, was described in various lymphoid organizations in minor salivary glands. In addition, local autoantibody production was detected and correlated with serum levels. Focal infiltrates and GC could be observed within the same gland, and were separated by altered B and T cell ratios, higher degree of proliferation and the localization of plasma cells in the periphery of infiltrates. Serum levels of BAFF and APRIL were elevated in pSS, and were in part linked to focus score, elevated serum IgG and autoantibody levels. In a large cohort of pSS, ectopic GC were also associated with higher focus scores, lower mean un-stimulated salivary secretion, Ro/SSA and La/SSB autoantibodies, elevated RF-titres and increased serum IgG. Not all morphological GC could be confirmed by CD21/CD23/CD35 labeling, but clinical features remained comparable. E-cadherin, an adhesion molecule important for epithelial tissue integrity, was investigated in minor salivary glands. E-cadherin is the ligand of integrin αEβ7/CD103 and lymphocytes expressing this integrin were increased in SS compared to non-SS. E-cadherin+ infiltrating cells were identified as CD68+ macrophages. Serum levels of sE-cadherin were increased in pSS compared to healthy blood donors and most likely mirror the chronic inflammatory state. The non-obese diabetic (NOD) mouse is an animal model of SS. We observed significant changes in inflammation between 8 and 17 weeks of age, while hyposalivation was first observed between 17 and 24 weeks. In 1/3rd of mice older than 17 weeks, proliferating cells were observed in the focal infiltrates. Significant differences were detected in serum cytokine levels of IL-2, IL-5 and GM-CSF, and IL-4 and TNF-α in saliva. Salivary secretion correlated with IL-4, IFN-γ and TNF-α levels in saliva of NOD mice, but not with inflammatory changes in the salivary glands. Focal sialadenitis preceded hyposalivation, which occurred without a significant change in inflammation in NOD mice. Proliferating inflammatory cells indicate contribution of local factors in progression of SS-like disease. In conclusion, ectopic germinal centers occur in a sub-group of patients with SS and are characterized by autoantibody production, progressive disease and increased serum IgG. It remains unclear whether the observed GC are a result of long-standing inflammation or indeed have functional properties and thus play an active role in the pathogenesis. A future challenge will be to identify and target trafficking molecules which drive the chronic inflammation in SS, without affecting migration and function of leukocytes required for protective immunity. <br/> <br/>Description: This thesis is based on the following papers, which will be referred to in the text by their Roman numerals: I. Salomonsson S, Jonsson MV, Skarstein K, Hjälmström P, Wahren-Herlenius M, Jonsson R. Cellular basis of ectopic germinal center formation and autoantibody production in the target organ of patients with Sjögren’s syndrome. Arthritis & Rheumatism 2003;48(11):3187-201. II. Jonsson MV, Szodoray P, Jellestad S, Jonsson R, Skarstein K. Association between circulating levels of the novel TNF family members APRIL and BAFF and lymphoid organization in primary Sjögren’s syndrome. Journal of Clinical Immunology, 2005;25(3):189-201. III. Jonsson MV, Skarstein K, Jonsson R, Brun JG. Germinal centers in primary Sjögren’s syndrome indicate a certain clinical immunological phenotype. Submitted. IV. Jonsson MV, Salomonsson S, Gunnvor Øijordsbakken, Skarstein K. Elevated serum levels of soluble E-cadherin in patients with primary Sjögren’s syndrome. Scandinavian Journal of Immunology, 2005;62(6):552-9. V. Jonsson MV, Delaleu N, Brokstad KA, Berggreen E, Skarstein K. Impaired salivary gland function in NOD mice – association with changes in cytokine profile but not with salivary gland histopathology. Arthritis & Rheumatism, in press. http://hdl.handle.net/1956/1139 Title: Functional analysis of tilS homologues in Bacteria and Archaea <br/> <br/>Authors: Stavrum, Ruth <br/> <br/>Abstract: Out of all of the microorganisms sequenced so far about one third of the genes have unknown function. Several studies have shown that information on evolutionary relationships between unknown genes can aid in the prediction of the function of these genes. As a result of all this new information, new methods of identification have been established which sort genes based on sequence similarity between both paralogues and orthologues genes. The Clusters of Orthologous Groups (COG) is one such method, which group genes on the basis of sequence similarity where all groups containing at least three proteins from distant genomes are assumed to belong to the same orthologous group. At the start of this project all, but one of the COGs containing universal genes had been assigned a function. In the last group, COG0037, the genes had been suggested to be ATPases based on conserved motifs. In September, 2003 an article was published where the function of one of the Escherichia coli members in COG0037, TilS (previously known as YaeN), had been determined and TilS was shown to be an RNA modification enzyme. Based on this knowledge it was decided to analyse TilS orthologues for similar function. In this work four TilS homologues; AF1595 and AF1321 from Archaeoglobus fulgidus, Sso0586 from Sulfolobus solfataricus and YdaO from E. coli were expressed in Escherichia coli, and tested for solubility. Out of the four proteins only one, YdaO proved to be soluble. This protein was purified by affinity chromatography and analysed further and shown to exhibit ATPase activity and ability to autophosphorylate. An attempt to verify whether this protein is expressed under normal conditions was unsuccessful. An attempt to determine whether the gene coding for YdaO could be used as a signature gene for E. coli was also unsuccessful.