Duplicated genes are present in the teleost fish lineage owing to a whole-genome duplication (WGD) event that occured ~ 230-400 million years ago. In the duplication-degeneration-complementation (DDC) model, partitioning of ancestral functions (subfunctionalization) and acquisition of novel functions (neofunctionalization) have been proposed as principal processes for the retention of duplicated genes in the genome. The DDC model was tested by analyzing the differential tissue-specific distribution of transcripts for the duplicated fatty acid-binding protein 10 (fabp10) genes in embryos, larvae and adult zebrafish (Danio rerio). The distribution of zebrafish fabp10a and fabp10b transcripts show a strikingly different tissue-specific pattern leading us to suggest that the zebrafish fabp10 duplicates had been retained in the genome owing to neofunctionalization. In another experiment to test the DDC model, transcriptional regulation of duplicated fabp genes was analyzed in zebrafish fed clofibrate, a peroxisome proliferator-activated receptor (PPAR) agonist. Clofibrate increased the steady-state level of both the duplicated copies of fabp1a/fabp1b.1, and fabp7a/fabp7b mRNA and heteronuclear RNA (hnRNA), but in different tissues of zebrafish. The steady-state level of fabp10a and fabp11a mRNA and hnRNA was elevated in liver of zebrafish, but not for fabp10b and fabp11b. We also investigated the effect of dietary fatty acids (FAs) and clofibrate on the transcriptional regulation of single copy fabp genes, fabp2, fabp3 and fabp6 in zebrafish. The steady-state level of fabp2 transcripts increased in intestine, while fabp3 mRNA increased in liver of zebrafish fed diets differing in FA content. In zebrafish fed clofibrate, fabp3 mRNA in intestine, and fabp6 mRNA in intestine and heart, was elevated. Whether the regulation of fabp gene transcription by clofibrate is controlled either directly or indirectly, the regulatory elements in the zebrafish fabp genes have diverged markedly since the WGD event, thereby supporting the DDC model.

TiPARP is a PARP-like mART that is induced by and negatively regulates AHR transactivation. Despite these insights, not much is known about TiPARP. This study aimed to characterize the regulation of TiPARP by AHR, PDGFR, and ERα, and investigate potential receptor interplay. Gene expression studies revealed that coactivation of AHR and PDGFR can enhance TiPARP expression after 3 h relative to activation of either receptor pathway alone. Gene expression and ChIP studies demonstrated that while co-activation of AHR and ER enhanced AHR, ARNT, and ERα recruitment to the regulatory region of TiPARP, TiPARP mRNA levels were not potentiated by co-activation relative to activation of either pathway. Dissection of the 5’ regulatory region of TiPARP using reporter gene assays revealed that a putative AHRE cluster and an ERE half-site were functional. Lastly, overexpression of TiPARP with an estrogen-responsive reporter revealed that TiPARP can repress ERα signalling and requires its catalytic activity.

No description available.

L’Ostéoarthrose (OA) est une maladie articulaire entrainant une dégénérescence du cartilage et une ossification de l’os sous-chondral. Elle touche un Canadien sur 10 et pourtant l’origine de cette pathologie est encore inconnue. Dans le cadre de ce projet, la contribution de deux facteurs de transcription, NFAT1 et PITX1, dans la régulation transcriptionnelle du promoteur d’IHH a été examiné compte tenu de l’implication potentielle de la voie hedgehog (Hh) et de ces facteurs dans la pathogenèse de l’OA. La voie de signalisation Hh régule la croissance et la différenciation des chondrocytes. Indian hedgehog (IHH), l’un des trois membres de la famille Hh, contrôle leur prolifération et leur différenciation. / Osteoarthritis (OA) is the most common joint disorder and is characterized by cartilage degradation and endochondral ossification. One in every ten Canadians is affected, yet its aetiopathogenesis remains unknown. In this present study, two new regulators of the IHH promoter, NFAT1 and PITX1, were studied. The downregulation of IHH expression by these factors could contribute to the OA pathogenesis. The Hedgehog (Hh) signaling pathway regulates chondrocyte growth and differentiation in the growth plate. Indian hedgehog (IHH), one of its members, stimulates chondrocyte proliferation and osteoblast differentiation. IHH is essential in skeletogenesis, osteoblastogenesis and cartilage growth.

Cellular messenger RNAs (mRNAs) exist almost exclusively in the context of ribonucleoprotein complexes (RNPs), which are largely responsible for the coordinated regulation of mRNA fate, and in particular, the post-transcriptional regulation of mRNA stability and translation. RNA- binding proteins, antisense RNAs, and microRNAs represent three major classes of post- transcriptional regulatory factors that interact with target mRNAs. Significantly, these interactions are dynamically regulated under both basal and stress conditions, such as hypoxia. Given the prominent contributions of post-transcriptional regulation to overall gene expression, a more comprehensive understanding of the underlying mechanisms is required. In this thesis, we present exciting new evidence for the functional importance of post- transcriptional gene regulation, especially in the vascular endothelium. Firstly, we show that the formation of hnRNP E1-containing RNPs contributes significantly to the remarkable basal stability of endothelial nitric oxide synthase (eNOS) mRNAs in endothelial cells by protecting them from inhibitory post-transcriptional forces. However, hypoxia impairs such RNP formation through hnRNP E1 serine phosphorylation and nuclear localization. Together, these mechanisms contribute significantly to decreased eNOS expression and activity in chronic hypoxia. ii Secondly, we reveal an important functional relationship between the microRNA pathway and the HIF-mediated cellular hypoxic response. Specifically, the down-regulation of Dicer and an important number of Dicer-dependent microRNAs in chronic hypoxia represents an important adaptive mechanism that serves to maintain the cellular hypoxic response through HIF-α- and microRNA-dependent mechanisms, with significant implications for the development of RNAi- based therapies. Finally, we provide evidence that the up-regulation of specific microRNAs in acute hypoxia is a potentially important mechanism that serves to suppress global translation initiation in order to conserve energy and ensure cellular survival. Collectively, the findings presented in this thesis provide important new mechanistic insight into the post-transcriptional regulation of eNOS, as well as the functional integration of the microRNA and the cellular hypoxic response pathways.

Cellular messenger RNAs (mRNAs) exist almost exclusively in the context of ribonucleoprotein complexes (RNPs), which are largely responsible for the coordinated regulation of mRNA fate, and in particular, the post-transcriptional regulation of mRNA stability and translation. RNA- binding proteins, antisense RNAs, and microRNAs represent three major classes of post- transcriptional regulatory factors that interact with target mRNAs. Significantly, these interactions are dynamically regulated under both basal and stress conditions, such as hypoxia. Given the prominent contributions of post-transcriptional regulation to overall gene expression, a more comprehensive understanding of the underlying mechanisms is required. In this thesis, we present exciting new evidence for the functional importance of post- transcriptional gene regulation, especially in the vascular endothelium. Firstly, we show that the formation of hnRNP E1-containing RNPs contributes significantly to the remarkable basal stability of endothelial nitric oxide synthase (eNOS) mRNAs in endothelial cells by protecting them from inhibitory post-transcriptional forces. However, hypoxia impairs such RNP formation through hnRNP E1 serine phosphorylation and nuclear localization. Together, these mechanisms contribute significantly to decreased eNOS expression and activity in chronic hypoxia. ii Secondly, we reveal an important functional relationship between the microRNA pathway and the HIF-mediated cellular hypoxic response. Specifically, the down-regulation of Dicer and an important number of Dicer-dependent microRNAs in chronic hypoxia represents an important adaptive mechanism that serves to maintain the cellular hypoxic response through HIF-α- and microRNA-dependent mechanisms, with significant implications for the development of RNAi- based therapies. Finally, we provide evidence that the up-regulation of specific microRNAs in acute hypoxia is a potentially important mechanism that serves to suppress global translation initiation in order to conserve energy and ensure cellular survival. Collectively, the findings presented in this thesis provide important new mechanistic insight into the post-transcriptional regulation of eNOS, as well as the functional integration of the microRNA and the cellular hypoxic response pathways.

TiPARP is a PARP-like mART that is induced by and negatively regulates AHR transactivation. Despite these insights, not much is known about TiPARP. This study aimed to characterize the regulation of TiPARP by AHR, PDGFR, and ERα, and investigate potential receptor interplay. Gene expression studies revealed that coactivation of AHR and PDGFR can enhance TiPARP expression after 3 h relative to activation of either receptor pathway alone. Gene expression and ChIP studies demonstrated that while co-activation of AHR and ER enhanced AHR, ARNT, and ERα recruitment to the regulatory region of TiPARP, TiPARP mRNA levels were not potentiated by co-activation relative to activation of either pathway. Dissection of the 5’ regulatory region of TiPARP using reporter gene assays revealed that a putative AHRE cluster and an ERE half-site were functional. Lastly, overexpression of TiPARP with an estrogen-responsive reporter revealed that TiPARP can repress ERα signalling and requires its catalytic activity.

Borrelia burgdorferi s. l. is a spirochete which causes the multisystemic disorder Lyme disease. As the borreliae lack toxin production, the pathogenicity is thought to involve, at least in part, molecules from the outer surface. Most Lyme disease Borrelia strains express two major outer surface lipoproteins, OspA (31 kD) and OspB (34 kD), on their surface. However, some strains lack the expression of OspA and OspB, but express a smaller 21 to 25 kD OspC protein instead. This thesis focuses on the importance of these proteins in the pathogenesis of Lyme disease. Biochemical and immunochemical studies of the OspA and OspB proteins from strains of various geographic origins show considerable differences in the apparent molecular weights and in their reactivities to monoclonal antibodies. The cloning and sequencing of the ospAB opérons from strains of different origins has demonstrated that the heterogeneity is found also at the DNA level Comparison of the ospAB sequences allows the classification of the strains into three types, which coincide with the recent species designations, B. burgdorferi sensu stricto, B. afzelii and B. garinii The genes are located on a linear plasmid about 50 kb in size, and are cotranscribed as a single message. The expression of the osp operon in different strains was studied by Western blot and Northern blot analysis. The ospAB operon of strains expressing varying amounts of the Osp proteins was cloned and sequenced. The DNA sequence was found to be &gt;99% identical. The regulation appears to be primarily at the transcriptional level. In patients who have received incomplete treatment, B. burgdorferi have been isolated several years after the onset of the disease. As mentioned above, the ospAB loci of different strains show considerable heterogeneity, and it has been speculated that the spirochetes evade the host’s immune system by antigenic variation of the Osp proteins. In a mouse model system it was shown that no variation of the osp genes occurs over the course of an infection, and that other escape mechanisms must be used. The OspB proteins in particular have been shown to be very heterogeneous in different isolates. The MAb 84C recognizes a wide variety of B. burgdorferi strains, and the binding epitope was mapped to a conserved region in the carboxyl terminus of the OspB protein with putative structural and/or functional importance. It is well known that antibodies can kill bacteria in the presence of complement and phagocytes. Some antibodies seem to have a bactericidal effect by themselves. H6831 is a monoclonal antibody recognizing the OspB protein of some B. burgdorferi strains. The bactericidal action of univalent FAb fragments from H6831 was further characterized, and the binding epitope was mapped to a very heterogeneous region of the carboxyl end of the OspB protein. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1994, härtill 5 uppsatser</p> / digitalisering@umu

Carcinoma growth requires a supportive tumor stroma. The concept of reciprocal interactions between tumor and stromal cells has become widely acknowledged and the connective tissue activation seen in the malignant process has been likened to that of a healing wound. Little is, however, known about the specific characteristics of these interactions, distinguishing them from the interplay occurring between epithelial and stromal cells in wound healing. In order to study differences in the humoral effects of malignant and benign epithelial cells on fibroblasts, we used an in vitro coculture model with human oral squamous cell carcinoma cells (SCC) or normal oral keratinocytes (NOK) on one side of a semi-permeable membrane and fibroblasts seeded in gels on the other. Pro-collagens α1(I) and α1(III) were more downregulated in NOK cocultures compared to SCC cocultures. IL-1α was identified as a major keratinocyte-derived soluble factor behind the effects observed. We concluded that SCC are less antifibrotic compared to NOK. There was also a differential expression among enzymes involved in ECM turnover. The urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) were both upregulated by NOK, but not by SCC. Here, rIL-1ra caused further upregulation of PAI-1. Global gene expression in fibroblasts was assessed using Affymetrix™ arrays. In total, 82 transcripts were considered differentially expressed; 52 were up- and 30 were downregulated in SCC compared to NOK cocultures. Among the differentially expressed genes there was an enrichment of genes related to collagens and to a nonspecific, innate-type response. The innate response marker pentraxin (PTX3) was upregulated by keratinocyte-derrived IL-1α in both NOK and SCC cocultures. We observed a considerably higher IL-1α / IL-1ra quotient in SCC cocultures, however, while PTX3 mRNA upregulation was higher in SCC cocultures, there was no difference in the level of PTX3 secreted protein. Taken together, we concluded that NOK and SCC regulate genes important for ECM composition and for the innate immune-response differentially. IL-1α was identified as one important mediator of the observed effects. In general, SCC appeared to be more profibrotic in their effects on fibroblasts.

The vertebrate Dlx gene family consists of multiple convergently transcribed bigene clusters and encodes a group of homeodomain-containing transcription factors crucial for the development of forebrain, branchial arches, sensory organs and limbs. At least four cis-regulatory elements (CREs) are responsible for Dlx expression in the forebrain: URE2 and I12b in the Dlx1/Dlx2 (zebrafish dlx1a/dlx2a) locus, and, I56i and I56ii in the Dlx5/Dlx6 (zebrafish dlx5a/dlx6a) locus. Here, we first show that unlike the other three enhancers, mouse I56ii CRE targets a group of GABAergic projection neurons expressing striatal markers Meis2 and Islet1. Meis2 and Islet1 proteins can activate reporter gene transcription via the I56ii CRE, suggesting that they may be potential upstream regulators of Dlx genes in vivo. To determine whether there exists a dlx-mediated regulatory pathway during zebrafish GABAergic neuron formation, we establish two independent lines of transgenic fish in which the GFP reporter gene is controlled by a 1.4kb dlx5a/dlx6a intergenic sequence (encompassing zebrafish I56i and I56ii) and a 1.1kb fragment containing only I56i CRE, respectively. Our observations reveal that dlx5a/dlx6a regulatory elements exhibit a fairly specific activity in the zebrafish forebrain and may be essential for GABAergic neuron generation, while I56i and I56ii are likely to play distinct roles in modulating this process in different subpopulations of cells. Disruption of dlx1a/dlx2a or dlx5a/dlx6a function leads to a marked decrease of enhancer activity in the diencephalon and midbrain as well as a comparatively lesser extent of reduction in the telencephalon. In order to define the specific contribution of various individual CREs to overall Dlx regulation, we also generate a mutant mouse model in which I12b CRE is selectively deleted. Despite that mice homozygous for I12b loss develop normally and harbor no overt morphological defects in the forebrain, targeted deletion of this enhancer results in a significant reduction of Dlx1/Dlx2 transcript levels and seemingly perturbs cell proliferation in the subpallial telencephalon, particularly in the ventricular and subventricular zones of ganglionic eminences. Taken together, these data illustrate a complex and dynamic Dlx regulation in the early developing forebrain through the implications of multiple Dlx CREs with overlapping and diverse functions.