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Human cells maintain lipid homeostasis by regulated cleavage of membranebound transcription factors, so-called sterol-regulatory element-binding proteins (SREBPs). The mature forms of SREBP-1 and -2 are transcriptional activators of lipogenic genes controlling cholesterol, fatty acids, and triglyceride biosynthesis and uptake. As the SREBPs play a central role in the regulation of the lipoprotein metabolism, we supposed that specific sequence variations, which correspond to single-nucleotide polymorphisms in these genes, and certain drugs, that influence the expression of SREBP, may result in alterations in plasma lipoprotein concentrations. A syndrome characterized by hypertriglyceridemia, hypercholesterolemia, hyperinsulinemia and lipodystrophy has been found to be associated with antiretroviral treatment (ART) including protease inhibitors. A marker predicting this syndrome has been identified in the gene encoding the sterolregulatory element-binding protein-1c (SREBP-1c), a regulator of triglycerides, cholesterol, insulin and adipocytes. A possible inhibition of SREBP-1c-dependent genes by the protease inhibitor indinavir and its possible reversal by the lipid-lowering drug simvastatin were studied in cell culture. The effects of indinavir and simvastatin on SREBP-1c-dependent genes were compared with the effects of indinavir and simvastatin on SREBP1c-independent genes. In fact, indinavir inhibited the SREBP-1c-dependent genes encoding the lipoprotein lipase and the fatty acid synthase in a dosedependent manner but not the SREBP-1c-independent gene encoding the low-density lipoprotein receptor. Furthermore, simvastatin antagonized the indinavir-induced SREBP-1c-inhibition. Thus, indinavir inhibits important effector genes of the SREBP-1c pathway, which may explain major antiretroviral treatment-related adverse effects. A single-nucleotide polymorphism (3' 322C/G SNP) identified in the sterolregulatory element-binding protein-1c (SREBP-1c) gene was predictive of highly active antiretroviral therapy-related hyperlipoproteinemia. Increases in cholesterol, triglyceride and insulin were less frequently associated with homozygous SREBP-1c-3' 322G (genotype 22) than with heterozygous/homozygous SREBP-1c-3'322C (genotypes 11/12). The differences in messenger RNA conformation can explain the pharmacogenetic basis of these findings. The mRNA stability of both homozygous genotypes of SREBP-1c-3’322C/G was compared in the stably transfected T-REx cell lines using a real-time quantitative polymerase chain reaction method. The mRNA of the SREBP-1c-3’322C isoform (genotype 11) was shown to have a more abundance decay rate than 3’322G isoform (genotype 22). Thus, the sequence variation (3’322C/G SNP) in the coding 3’ end of the gene affects the secondary structure of the SREBP-1c mRNA, influences its degradation rate and, therefore, causes differences in the regulation of SREBP-1c expression. In the process of this thesis, three new splice variants of the human SREBP-1 gene that shared different combinations of the SREBP-1a and -1c exons at the 3’ end of mRNA were identified. The splice variant containing exons 17, 18a and 18c was designated as SREBP-1d, the splice variant containing exons 17, 18a, 18c and 19c was termed as SREBP-1e, and the splice variant containing exons 17, 18c and 20f was named as SREBP-1f. Analysis of tissue distribution showed that the new splice variants SREBP-1e and -1d were ubiquitously found in various human tissues and tumor-derived cells, whereas wild-type SREBP-1c and SREBP-1f transcripts were relatively tissue-specific. This high abundance led us to the hypothesis that splice variants SREBP-1e and SREBP-1d play a more general role in regulating cellular lipid levels as compared to other isoforms. This thesis concludes that the sterol-regulatory element-binding protein (SREBP)-1c is crucial in the metabolic side-effects associated with highly active antiretroviral therapy using protease inhibitors. Moreover, regulation mechanism mediated by the transcription factor SREBP-1 is a model of a complex gene regulation system composed of different related levels: promoter regulation of effector genes, differences in mRNA stability and tissue specific splice variants in different quantities.

BACKGROUND: Early exposure of infants and long-term immunity suggest that colonization with Moraxella catarrhalis is more frequent than is determined by routine culture. We characterized a reservoir of M. catarrhalis in pharyngeal lymphoid tissue. METHODS: Tissue from 40 patients (median age, 7.1 years) undergoing elective tonsillectomy and/or adenoidectomy was analyzed for the presence of M. catarrhalis by culture, real-time DNA and RNA polymerase chain reaction (PCR), immunohistochemical analysis (IHC), and fluorescent in situ hybridization (FISH). Histologic sections were double stained for M. catarrhalis and immune cell markers, to characterize the tissue distribution of the organism. Intracellular bacteria were identified using confocal laser scanning microscopy (CLSM). RESULTS: Twenty-nine (91%) of 32 adenoids and 17 (85%) of 20 tonsils were colonized with M. catarrhalis. Detection rates for culture, DNA PCR, RNA PCR, IHC, and FISH were 7 (13%) of 52, 10 (19%) of 52, 21 (41%) of 51, 30 (61%) of 49, and 42 (88%) of 48, respectively (P

BACKGROUND Moraxella catarrhalis, a major nasopharyngeal pathogen of the human respiratory tract, is exposed to rapid downshifts of environmental temperature when humans breathe cold air. The prevalence of pharyngeal colonization and respiratory tract infections caused by M. catarrhalis is greatest in winter. We investigated how M. catarrhalis uses the physiologic exposure to cold air to regulate pivotal survival systems that may contribute to M. catarrhalis virulence. RESULTS In this study we used the RNA-seq techniques to quantitatively catalogue the transcriptome of M. catarrhalis exposed to a 26 °C cold shock or to continuous growth at 37 °C. Validation of RNA-seq data using quantitative RT-PCR analysis demonstrated the RNA-seq results to be highly reliable. We observed that a 26 °C cold shock induces the expression of genes that in other bacteria have been related to virulence a strong induction was observed for genes involved in high affinity phosphate transport and iron acquisition, indicating that M. catarrhalis makes a better use of both phosphate and iron resources after exposure to cold shock. We detected the induction of genes involved in nitrogen metabolism, as well as several outer membrane proteins, including ompA, m35-like porin and multidrug efflux pump (acrAB) indicating that M. catarrhalis remodels its membrane components in response to downshift of temperature. Furthermore, we demonstrate that a 26 °C cold shock enhances the induction of genes encoding the type IV pili that are essential for natural transformation, and increases the genetic competence of M. catarrhalis, which may facilitate the rapid spread and acquisition of novel virulence-associated genes. CONCLUSION Cold shock at a physiologically relevant temperature of 26 °C induces in M. catarrhalis a complex of adaptive mechanisms that could convey novel pathogenic functions and may contribute to enhanced colonization and virulence.