Pax3 and Pax7 belong to a family of conserved transcription factors that play important and diverse roles in development. In the embryo, they carry out similar roles in neural and somite development, but Pax7 fails to compensate for critical functions of Pax3 in the development of limb musculature. Conversely, in the adult, Pax7 is necessary for the maintenance and survival of muscle satellite cells, whereas Pax3 cannot effectively fulfill these roles in the absence of Pax7. To identify the unique roles of Pax7 in adult muscle cells, we have analyzed global binding of Pax3 and Pax7 by ChIP-Seq. Here, we show that despite highly homologous DNA-binding domains, the majority of binding sites are uniquely recognized by Pax7 and are enriched for homeobox motifs. Genes proximal to conserved, unique Pax7 binding sites cluster into specific functional groups which may reflect the unique biological roles of Pax7. Combining Pax7 binding sites with gene expression data, we describe the regulatory networks directed by Pax7 and show that Pax7 binding is associated with positive gene regulation. Moreover, we show Myf5 is a direct target of Pax7 and identify a novel binding site in the satellite cell control region upstream of Myf5.

The enzyme carbonic anhydrase (CA) contributes to multiple physiological processes by catalysing the reversible hydration of carbon dioxide. However, regulation of CA activity in response to homeostatic challenges remains poorly understood. The objectives of this thesis were to investigate whether CA is transcriptionally regulated by cortisol in fish and whether post-translational modification (PTM) of CA occurs in fish. The results of an in vivo reporter assay used to investigate potential transcriptional regulation of zebrafish, Danio rerio, cytoplasmic CA (CAc) were inconsistent, and it remains unclear whether zebrafish CAc is regulated transcriptionally by cortisol. Phosphorylation of rainbow trout, Oncorhynchus mykiss, CAc was predicted from in silico analysis of the putative amino acid sequence and confirmed by Western analysis of phosphoprotein levels following in vitro incubation of CA, purified from trout gill, under conditions designed to potentiate endogenous kinases. Again using in vitro incubations designed to potentiate endogenous kinases and phosphatases, changes to the phosphorylation state of CAc were found to modulate its enzymatic properties. These findings suggest that CA activity may be regulated by signalling pathways that activate cellular protein kinases, and future work should focus on identifying these pathways.

Serratia marcescens, a member of Enterobacteriaceae family, is a causative agent of nosocomial and opportunistic infections. Numerous reports show that the multidrug resistance among S. marcescens is growing. This organism has high-level intrinsic resistance to a variety of antimicrobial agents, which makes the treatment of infections caused by this bacterium very difficult. The major mechanism for antibiotic resistance, especially to fluoroquinolones, in Gram-negative organisms is the active efflux of the antibiotic molecule mediated by efflux pumps belonging to the Resistance-Nodulation-Cell Division (RND) family. It was previously shown that the SdeAB and SdeXY multidrug efflux pumps are important for conferring the intrinsic drug resistance in S.marcescens. In Escherichia coli, the up-regulation of transcriptional activators, such as MarA, SoxS and Rob, affect transcription of acrAB, tolC and micF. Over-expression of Rob results in increased expression of the E. coli AcrAB-TolC efflux pump and decreases outer membrane permeability through up-regulation of micF, resulting in multidrug, organic solvent and heavy metal resistance. In the present study, we report the identification of a rob gene in S. marcescens which has a 70% identity at the DNA level and 71% identity at the amino acid level to that of E. coli. Moreover, the S. marcescens rob demonstrated similar properties to the E. coli rob including having an effect on expression of outer membrane protein F (OmpF) and over-expression of SdeAB and SdeXY, conferring antibiotic resistance to divergent antibacterial agents and tolerance to organic solvents. We performed rob promoter evaluations using transcriptional fusions to the Green Fluorescence Protein (GFP) in the vector pGlow-TOPO and constructed a rob knock-out using the TargeTron Gene Knockout System. Promoter activity assessment, using the pGlow-TOPO reporter plasmid, showed that rob had higher promoter activity at 37°C than 30°C. In the presence of 2,2’-dipyridyl, rob promoter activity was observed to be slightly increased in the early and mid-log phase by 1.4 and 1.1 fold, respectively. We also showed that sodium decanoate and sodium salicylate can reduce the transcription of rob at 30°C and 37°C. This reduction was observed more potently when rob was exposed to sodium decanoate at 30°C. Minimum inhibitory concentration (MIC) for various antibiotics of the S. marcescens rob knock-out demonstrated a decrease in susceptibility to nalidixic acid, tetracycline, chloramphenicol, ciprofloxacin, norfloxacin, and ofloxacin. Over-expression of rob resulted in an increased resistance by 4, 2, and 2-fold to nalidixic acid, tetracycline and chloramphenicol, respectively. In addition, rob over-production displayed 8, 4, and 4-fold increase in resistance to ciprofloxacin, norfloxacin, ofloxacin, respectively. To discover the role of rob in the efflux mechanism, we performed ethidium bromide accumulation assays on over-expressing and knock-out strains. Organic solvent tolerance assays were carried out using n-hexane to determine if rob is involved in expression of efflux pumps. We found the rob null mutant to be sensitive to n-hexane while the over-expression of rob resulted in resistance to n-hexane. RT-PCR of the rob knock-out strain showed a decrease in expression of micF, ompC, sdeXY, sdeAB and tolC, respectively, and an increase in the expression of ompF. To conclude, we identified a rob homolog in S. marcescens which contributes to resistance to multiple antibiotics and tolerance to organic solvent.

Long terminal repeat (LTR) retrotransposons account for approximately 10% of mouse and 8% of human genomes and may play a role in modifying gene expression. Many species harbor retrotransposon families encompassing both autonomous and non-autonomous members. Specifically, the mouse Early Transposon (ETn) family members lack all retroviral genes but are transcriptionally and retrotranspositionally active, causing over 20 known insertional germline mutations. ETns owe their retrotransposition potential to proteins encoded by structurally intact MusD retrotransposons with whom they share LTRs. ETn elements are transcribed at a much higher level than MusD retrotransposons in embryos and undifferentiated cells, suggesting their evasion of host restriction mechanisms. However, mechanisms responsible for the replicative success of non-autonomous retrotransposon subfamilies over their coding-competent relatives are poorly understood. In the first stage of my research, I analyzed regulatory sequences in an ETn LTR responsible for its high promoter activity in the undifferentiated cell line P19. I found that three GC-boxes that may function as Sp1/Sp3 binding sites act synergistically and are indispensable for undifferentiated cell-specific promoter activity of the LTR. Sp1 binding partners may be responsible for the restricted ETn expression. Moreover, I have shown that unlike many retroviruses, ETn elements possess multiple transcription initiation sites and that they have amplified via intracellular retrotransposition in the P19 teratocarcinoma cell line. In the next step of my research, I performed analysis of epigenetic mechanisms as a means of ERV suppression. Specifically, I showed that in embryonic stem cells, autonomous MusD retrotransposons are epigenetically suppressed to a greater degree than non-autonomous ETn retrotransposons, illustrated by a higher level of DNA methylation and a lower level of active histone modifications. I hypothesize that MusD elements may be silenced by DNA methylation and repressive chromatin spreading into the LTR from the CpG-rich internal retroviral sequence absent in ETn elements. I propose that internal structure largely devoid of high CG content enables ETn elements to evade host-imposed transcriptional repression, contributing to their high mutagenic activity in the mouse germline.

This thesis describes a molecular-based study undertaken to analyse the expression of the RAMOSUS1 (RMS1) and RAMOSUS5 (RMS5) genes in pea (Pisum sativum). Both genes encode carotenoid cleavage dioxygenase (CCD) enzymes that are together proposed to control the synthesis of an inhibitor of bud outgrowth termed SMS (Shoot Multiplication Signal). SMS was recently identified as strigolactone. Expression analyses of RMS1 presented here have built upon earlier experiments which demonstrate it to be a highly regulated transcript. RMS1 mRNA levels are known to be rapidly decreased following removal of the shoot apex but are subsequently restored to that of intact plants by auxin (indole-3-acetic acid or IAA). This regulatory mechanism is retained in all five ramosus mutants tested to date. Together with physiological data, this indicates RMS1, and therefore SMS, are required in IAA-mediated suppression of bud outgrowth. Another significant aspect of RMS1 regulation identified in previous studies involves a graft-transmissible, long-distance feedback signal that moves from shoot to root. This feedback regulation is dependent on the RMS2 gene and enhances RMS1 expression levels. Prior to the cloning of RMS5 and its discovery as a second CCD enzyme in the RMS network, reciprocal grafting studies with the rms mutants indicated RMS5 may act in the same pathway as RMS1 to produce SMS. Multiple studies presented here demonstrate that these two CCD genes are expressed in similar tissues and are regulated by the same signals, specifically IAA and the RMS2-dependent feedback signal. Like RMS1, the RMS5 gene also retains its IAA response in the rms mutants. However, RMS5 is generally less responsive to changes in IAA and RMS2-dependent feedback, as it exhibits smaller fluctuations than RMS1 in its expression levels. Together these findings support a general view that RMS1 is more likely to control a rate-limiting step in SMS synthesis. A previous study indicated that RMS1 expression may be up-regulated by IAA through a posttranscriptional mechanism. This thesis sought to more closely examine the RMS1 and RMS5 IAA response by separately observing the effect of IAA on subsequent transcription. New transcripts, termed heterogenous nuclear RNAs (hnRNAs), were relatively quantified in parallel with existing mRNAs in the steady-state cytoplasmic pool. The experiments conducted here provide further evidence that IAA may act post-transcriptionally to stabilise RMS1 mRNA because the changes in hnRNA are not proportional to the changes in mRNA following IAA-modifying treatments. IAA may still function to induce transcription of RMS1, but this does not appear to be a significant mechanism by which IAA regulates RMS1 expression. In contrast, the IAA induction of RMS5 occurs predominantly via new transcription and RMS5 either lacks or is not as strongly subjected to the IAA-mediated mRNA stabilisation mechanism proposed for RMS1. Initial studies described in this thesis also suggest that IAA could act to regulate the expression of the Arabidopsis orthologues MORE AXILLARY BRANCHING (MAX) genes via a post-transcriptional mechanism. Analyses of MAX hnRNA and mRNA levels in Arabidopsis to date indicate it is the RMS5 orthologue MAX3 which exhibits an IAA response most like RMS1. Additional studies into the regulation of RMS1 and RMS5 presented in this thesis provide further insights into the molecular mechanisms controlling their expression levels. In vitro experiments with the translation inhibitor cycloheximide demonstrate that RMS5 expression levels are increased when protein synthesis is reduced, as previously shown for RMS1. Relative quantification of RMS1 and RMS5 hnRNA levels further demonstrate that the induction by cycloheximide is due primarily to an increase in new transcription, indicating that RMS1 and RMS5 are negatively regulated by a rapidly turned-over transcriptional repressor. Tissue specific effects on RMS1 expression were also observed which are consistent with a protein degradation function of the RMS4 F-box in the shoot. This thesis provides further evidence to suggest that SMS acts in concert with IAA to inhibit the sustained outgrowth of axillary buds. RMS1 and RMS5 expression levels are not regulated by a hypothetical fast decapitation signal which is proposed to cause the initial bud outgrowth occurring prior to decapitation-induced IAA depletion. RMS1, RMS5 and SMS are therefore unlikely to control the initial exit of buds from dormancy to an intermediate transition state. Studies here also suggest that enhanced shoot auxin transport and cytokinin biosynthesis are associated with axillary bud outgrowth because the rms mutants contain elevated shoot expression levels of a gene encoding the auxin efflux carrier PIN1 and two genes controlling cytokinin biosynthesis. Several approaches described in this study were used to characterise the RMS1 and RMS5 proteins. Anti-peptide antibodies were generated against both proteins and the results obtained show that although the antibodies are likely to recognise the full-length proteins, further work is required to effectively detect RMS1 and RMS5 in plant tissues via western blotting. Preliminary in situ immunolocalisation results indicate the RMS1 and RMS5 proteins are localised to the vasculature, consistent with gene expression analyses.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 117-142).

Thesis (Ph.D.). / Written for the Division of Experimental Medicine, Dept. of Medicine. Title from title page of PDF (viewed 2008/03/12). Includes bibliographical references.

Thesis (Ph.D.)--University of South Florida, 2001. / Includes vita. Includes bibliographical references (leaves 80-104). Also available online.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [79]-97).

Orientador: Ângela Maria Victoriano de Campos Soares / Resumo: A paracoccidioidomicose (PCM) é uma micose sistêmica endêmica na América Latina, principalmente no Brasil, Argentina e Venezuela, e que promove um importante impacto na saúde pública. Seu agente etiológico é um fungo termodimórfico pertencente ao gênero Paracoccidioides que compreende o Paracocidioides brasiliensis (Pb) e suas espécies crípticas S1, PS2, PS3 e Paracoccidioides lutzii. As consequências da interação do fungo com as células da resposta imune inata, tais como as células dendríticas (DC), destacando a capacidade destas células para instruir a resposta imune adaptativa, não são totalmente compreendidas. Em estudo anterior, descobrimos que DCs não maturam em resposta ao desafio com Pb. Esta falha foi associada à inibição de PGE2 nas DCs pelo fungo, uma vez que este eicosanóide é um fator importante para a maturação dessas células. Na tentativa de melhor entender este processo e suas conseqüências para a instrução da resposta adaptativa CD4, nós buscamos analisar o perfil transcricional de DCs em resposta ao Pb assim como o de linfócitos CD4+ cocultivados com DCs sensibilizados com o fungo. Para estas análises, nós utilizamos a metodologia de RNA-seq, que permitiu o sequenciamento de alto rendimento e a quantificação sistemática de expressão gênica. Após as análises, os genes que foram regulados positivamente ou negativamente em ambas as células (DCs e CD4) foram listados e as funções das proteínas codificadas por eles, identificadas. A análise geral das proteínas co... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor