The upregulation of the inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production have been implicated in inflammatory pathologies. Although research has revealed that glucocorticoids (GCs) such as dexamethasone and hydrocortisone inhibit iNOS expression and NO production, it remains unclear how these compounds attenuate iNOS expression and function. In response, this thesis has compared the effects of nonselective GCs (i.e., dexamethasone and hydrocortisone) with a selective GC namely, fluticasone propionate (fluticasone) to identify the precise GC actions that regulate the iNOS pathway. Additional investigations were performed to distinguish the GC and non-GC actions using receptor antagonists. Since the effects of GCs on upstream signalling pathways remain vague, further studies were conducted to investigate whether fluticasone regulates the p38 mitogen-activated protein kinases or protein kinase B (Akt) pathways, both of which have been reported to be critical for the induction of iNOS. All experiments were conducted using primary cultures of rat aortic smooth muscle cells (RASMCs). The cells were activated with bacterial LPS (100 μg/mL) and interferon-gamma (IFN-γ, 100 U/mL) to induce iNOS and NO. Nitrite levels in cellular supernatants were quantified by the Griess assay, and expressions of iNOS, phospho-p38 (P-p38), and phospho-Akt (P-Akt) were investigated by western blotting. Dexamethasone (0.1-10.0 μM) inhibited iNOS expression and NO production in a concentration dependent manner that was significant at higher concentrations (0.3-10.0 μM). Hydrocortisone (0.01-10.0 μM) also inhibited iNOS expression and NO production in a concentration dependent manner which was significant at the higher concentrations (0.1-10.0 μM). By contrast, fluticasone (0.1 nM-3.0 μM) inhibited NO production and iNOS expression only partially (~50%), and the effects were significant at 1 nM-3 μM. RU-486 (10 μM), a GC receptor (GCR) blocker, was able to reverse the inhibitions caused by dexamethasone, hydrocortisone, and fluticasone, though eplerenone (0.1-10.0 μM), the mineralocortocoid receptor blocker, had no effect. Fluticasone also inhibited the phosphorylation of p38 and Akt in activated RASMCs. The inhibitions were reversed upon incubation with RU-486 (10 μM) for 1 h prior to the addition of fluticasone. The partial inhibition of iNOS and NO by fluticasone suggests that the actions of dexamethasone and hydrocortisone were not restricted solely to GCR and that other receptors or pathways, if not both, might regulate iNOS and NO in RASMCs. In conclusion, the nonselective GCs (i.e., dexamethasone and hydrocortisone) showed a full inhibition of iNOS expression and function, whereas fluticasone only partially inhibited both processes. The inhibitions were reversed by RU-486, but not eplerenone, which strongly suggests a GC-mediated response to all three compounds investigated. Regarding fluticasone, mechanistic studies revealed that the GC can regulate key signalling pathways associated with the induction of iNOS. More specifically, fluticasone reduced the phosphorylation of p38 and Akt, thereby suggesting that its actions can be mediated by suppressing these kinase pathways, which are widely reported to critically regulate iNOS expression and function.

Epigenetics is the study of heritable biological variation not related to changes in DNA sequence. Epigenetic processes are responsible for establishing and maintaining transcriptional programs that define cell identity. Defects to epigenetic processes have been linked to a host of disorders, including mental retardation, aging, cancer and neurodegenerative diseases. The ability to control and engineer epigenetic systems would be valuable both for the basic study of these critical cellular processes as well as for synthetic biology. Indeed, while synthetic biology has made progress using bottom-up approaches to engineer transcriptional and signaling circuitry, epigenetic systems have remained largely underutilized. The predictive engineering of epigenetic systems could enable new functions to be implemented in synthetic organisms, including programmed phenotypic diversity, memory, reversibility, inheritance, and hysteresis. This thesis broadly focuses on the development of foundational tools and intellectual frameworks for applying synthetic biology to epigenetic regulation in the model eukaryote, Saccharomyces cerevisiae. Epigenetic regulation is mediated by diverse molecular mechanisms: e.g. self-sustaining feedback loops, protein structural templating, modifications to chromatin, and RNA silencing. Here we develop synthetic tools and circuits for controlling epigenetic states through (1) modifications to chromatin and (2) self-templating protein conformations. On the former, the synthetic tools we develop make it possible to study and direct how chromatin regulators operate to produce distinct gene expression programs. On the latter, we focus our studies on yeast prions, which are self-templating protein conformations that act as elements of inheritance, developing synthetic tools for detecting and controlling prion states in yeast cells. This thesis explores the application of synthetic biology to these epigenetic systems through four aims: Aim 1. Development of inducible expression systems for precise temporal expression of epigenetic regulators Aim 2. Construction of a library of chromatin regulators to study and program chromatin-based epigenetic regulation. Aim 3. Development of a genetic tool for quantifying protein aggregation and prion states in high-throughput Aim 4. Dynamics and control of prion switching Our tools and studies enable a deeper functional understanding of epigenetic regulation in cells, and the repurposing of these systems for synthetic biology toward addressing industrial and medical applications. / 2019-10-08T00:00:00Z

I investigated two aspects of in vivo splicing that are poorly understood: spliceosome disassembly and recycling, and proofreading. To this end, I used the auxin-inducible degron (AID) to individually deplete several splicing factors in budding yeast and then I measured the effect on co-transcriptional spliceosome assembly through chromatin immunoprecipitation. In addition, using RNA next-generation sequencing, I measured the frequency of splicing errors following depletion or mutation of the fidelity factor, Prp22. I show that formation of the pre-spliceosome (the first stage of spliceosome assembly) is rapidly inhibited by global defects in late stages of spliceosome assembly. I demonstrate that this is due to the accumulation of arrested spliceosomes that sequester the splicing machinery and, as a result, causes a recycling defect. This suggests that spliceosomes that lack essential splicing factors are not always properly disassembled and recycled in vivo, and warns about potential systematic secondary effects when perturbing single components of the spliceosome. Secondly, I describe the development of a new version of the AID system for budding yeast, called the B-estradiol AID. To the best of my knowledge, an AID system for budding yeast that is fast-acting, tightly-controlled and gratuitous, was lacking until now. Lastly, I show that absence of Prp22 protein, which was previously proposed to play a role in splicing fidelity, correlates with more mistakes in 3’ss selection of many endogenous intron-containing transcripts in vivo. This provides indirect evidence to suggest that Prp22-dependent splicing proofreading is physiologically important. The data from this analysis will be useful in ongoing studies to try to identify common features that could improve our understanding of the mechanism of Prp22’s function in splicing proofreading.

Claritromicina era considerada um antibiótico de escolha para infecções causadas pelo complexo Mycobacterium abscessus, entretando, recentemente falhas no tratamento com este antibiótico têm sido reportadas. A resistência adquirida a claritromicina está relacionada à mutações pontuais que acarretam substituição da adenina na posição 2058 ou na posição 2059 na região do gene rrl que codifica o domínio peptidil transferase do rRNA 23S. Um mecanismo secundário de resistência à claritromicina tem sido descrito como resistência induzida, que é conferida pelo polimorfismo T/C no nucleotídeo 28 do gene erm(41). A resistência adquirida pode ser detectada em até 3 dias de incubação do M. abscessus com a claritromicina enquanto que a resistência induzida requer mais do que 5 dias de incubação. Por outro lado, o uso de marcadores moleculares para detecção de resistência adquirida e induzida no complexo M. abscessus têm sido propostos. O objetivo desse estudo foi avaliar o perfil de suscetibilidade e os marcadores moleculares de resistência à claritromicina no complexo M. abscessus. Um total de 42 isolados de um estudo prévio de vigilância, entre os anos 2007 e 2013 foram utilizados. O perfil de suscetibilidade para a claritromicina foi determinado por microdiluição em caldo com leituras em 3, 5, 7 e 14 dias. Mutações nos genes rrl e erm(41) foram avaliados por PCR com primers específicos e posterior sequenciamento. Resistência à claritromicina, em até 3 dias de incubação, foi observada em 31 dos 42 (73,8%) isolados. Resistência induzida à claritromicina foi observada em 6 de 11 (54,5%) isolados que apresentaram resistência após 5 ou 7 dias de incubação. Todos os isolados com resistência induzida foram M. abscessus subsp. massiliense. Além disso, todos os 28 isolados de M. abscessus subsp. massiliense apresentaram deleção em erm(41). Apenas cinco isolados foram sensíveis à claritromicina após 14 dias de incubação. Nenhum dos 42 isolados apresentaram mutação pontual na região de peptidil transferase do rRNA 23S e todos os isolados apresentaram o polimorfismo T/C no nucleotídeo 28 do gene erm(41). Os dados deste estudo indicam a falta de correlação dos marcadores moleculares com a expressão de resistência à claritromicina. / Infections due to Mycobacterium abscessus complex used to respond to clarithromycin treatment but more recently treatment failure with this antibiotic has been reported. Acquired resistance to clarithromycin is related to substitutions at the adenine either at position 2058 or at position 2059 in a region of the rrl gene encoding to the peptidyltransferase domain of the 23S rRNA. A secondary mechanism related to clarithromycin resistance has been described as an inducible resistance, conferred by T/C polymorphism at the 28th nucleotide in erm(41) gene. Acquired resistance can be detected up to 3 days of incubation of the M. abscessus with the clarithromycin while inducible resistance requires more than 5 days of incubation. Molecular markers to detect acquired and inducible resistance in M. abscessus complex isolates were proposed. This study evaluated the profile of susceptibility and the molecular markers of clarithromycin resistance in M. abscessus complex. A total of 42 isolates from a previous surveillance study (2007 to 2013) were used in this study. The susceptibility profile for clarithromycin was determined by broth microdilution with reads at 3, 5, 7 and 14 days. Mutations in rrl and erm(41) genes were evaluated by PCR with specific primers followed by sequencing. Clarithromycin resistance, up to 3 days of incubation, was observed in 31 of 42 (73.8%) isolates. Inducible clarithromycin resistance was observed in 6 of 11 (54.5%) isolates which presented resistance only after 5 or 7 days of incubation. All isolates with inducible resistance were identified as M. abscessus subsp. massiliense. Moreover, all 28 M. abscessus subsp. massiliense had a deletion in erm(41). Only five isolates proved to be susceptible to clarithromycin after 14 days of incubation. None of the 42 isolates presented a point mutation in the peptidyltransferase region of the 23S rRNA (rrl) and all isolates presented the T/C polymorphism at the 28th nucleotide of the erm(41) gene. The data of this study indicates a lack of correlation of molecular markers of clarithromycin resistance for both acquired and inducible resistance to clarithromycin.

abstract: Biomass synthesis is a competing factor in biological systems geared towards generation of commodity and specialty chemicals, ultimately limiting maximum titer and yield; in this thesis, a widely generalizable, modular approach focused on decoupling biomass synthesis from the production of the phenylalanine in a genetically modified strain of E. coli BW25113 was explored with the use of synthetic trans-encoded small RNA (sRNA) to achieve greater efficiency. The naturally occurring sRNA MicC was used as a scaffold, and combined on a plasmid with a promoter for anhydrous tetracycline (aTc) and a T1/TE terminator. The coding sequence corresponding to the target binding site for fourteen potentially growth-essential gene targets as well as non-essential lacZ was placed in the seed region of the of the sRNA scaffold and transformed into BW25113, effectively generating a unique strain for each gene target. The BW25113 strain corresponding to each gene target was screened in M9 minimal media; decreased optical density and elongated cell morphology changes were observed and quantified in all induced sRNA cases where growth-essential genes were targeted. Six of the strains targeting different aspects of cell division that effectively suppressed growth and resulted in increased cell size were then screened for viability and metabolic activity in a scaled-up shaker flask experiment; all six strains were shown to be viable during stationary phase, and a metabolite analysis showed increased specific glucose consumption rates in induced strains, with unaffected specific glucose consumption rates in uninduced strains. The growth suppression, morphology and metabolic activity of the induced strains in BW25113 was compared to the bacteriostatic additives chloramphenicol, tetracycline, and streptomycin. At this same scale, the sRNA plasmid targeting the gene murA was transformed into BW25113 pINT-GA, a phenylalanine overproducer with the feedback resistant genes aroG and pheA overexpressed. Two induction times were explored during exponential phase, and while the optimal induction time was found to increase titer and yield amongst the BW25113 pINT-GA murA sRNA variant, overall this did not have as great a titer or yield as the BW25113 pINT-GA strain without the sRNA plasmid; this may be a result of the cell filamentation. / Dissertation/Thesis / Masters Thesis Chemical Engineering 2016

Claritromicina era considerada um antibiótico de escolha para infecções causadas pelo complexo Mycobacterium abscessus, entretando, recentemente falhas no tratamento com este antibiótico têm sido reportadas. A resistência adquirida a claritromicina está relacionada à mutações pontuais que acarretam substituição da adenina na posição 2058 ou na posição 2059 na região do gene rrl que codifica o domínio peptidil transferase do rRNA 23S. Um mecanismo secundário de resistência à claritromicina tem sido descrito como resistência induzida, que é conferida pelo polimorfismo T/C no nucleotídeo 28 do gene erm(41). A resistência adquirida pode ser detectada em até 3 dias de incubação do M. abscessus com a claritromicina enquanto que a resistência induzida requer mais do que 5 dias de incubação. Por outro lado, o uso de marcadores moleculares para detecção de resistência adquirida e induzida no complexo M. abscessus têm sido propostos. O objetivo desse estudo foi avaliar o perfil de suscetibilidade e os marcadores moleculares de resistência à claritromicina no complexo M. abscessus. Um total de 42 isolados de um estudo prévio de vigilância, entre os anos 2007 e 2013 foram utilizados. O perfil de suscetibilidade para a claritromicina foi determinado por microdiluição em caldo com leituras em 3, 5, 7 e 14 dias. Mutações nos genes rrl e erm(41) foram avaliados por PCR com primers específicos e posterior sequenciamento. Resistência à claritromicina, em até 3 dias de incubação, foi observada em 31 dos 42 (73,8%) isolados. Resistência induzida à claritromicina foi observada em 6 de 11 (54,5%) isolados que apresentaram resistência após 5 ou 7 dias de incubação. Todos os isolados com resistência induzida foram M. abscessus subsp. massiliense. Além disso, todos os 28 isolados de M. abscessus subsp. massiliense apresentaram deleção em erm(41). Apenas cinco isolados foram sensíveis à claritromicina após 14 dias de incubação. Nenhum dos 42 isolados apresentaram mutação pontual na região de peptidil transferase do rRNA 23S e todos os isolados apresentaram o polimorfismo T/C no nucleotídeo 28 do gene erm(41). Os dados deste estudo indicam a falta de correlação dos marcadores moleculares com a expressão de resistência à claritromicina. / Infections due to Mycobacterium abscessus complex used to respond to clarithromycin treatment but more recently treatment failure with this antibiotic has been reported. Acquired resistance to clarithromycin is related to substitutions at the adenine either at position 2058 or at position 2059 in a region of the rrl gene encoding to the peptidyltransferase domain of the 23S rRNA. A secondary mechanism related to clarithromycin resistance has been described as an inducible resistance, conferred by T/C polymorphism at the 28th nucleotide in erm(41) gene. Acquired resistance can be detected up to 3 days of incubation of the M. abscessus with the clarithromycin while inducible resistance requires more than 5 days of incubation. Molecular markers to detect acquired and inducible resistance in M. abscessus complex isolates were proposed. This study evaluated the profile of susceptibility and the molecular markers of clarithromycin resistance in M. abscessus complex. A total of 42 isolates from a previous surveillance study (2007 to 2013) were used in this study. The susceptibility profile for clarithromycin was determined by broth microdilution with reads at 3, 5, 7 and 14 days. Mutations in rrl and erm(41) genes were evaluated by PCR with specific primers followed by sequencing. Clarithromycin resistance, up to 3 days of incubation, was observed in 31 of 42 (73.8%) isolates. Inducible clarithromycin resistance was observed in 6 of 11 (54.5%) isolates which presented resistance only after 5 or 7 days of incubation. All isolates with inducible resistance were identified as M. abscessus subsp. massiliense. Moreover, all 28 M. abscessus subsp. massiliense had a deletion in erm(41). Only five isolates proved to be susceptible to clarithromycin after 14 days of incubation. None of the 42 isolates presented a point mutation in the peptidyltransferase region of the 23S rRNA (rrl) and all isolates presented the T/C polymorphism at the 28th nucleotide of the erm(41) gene. The data of this study indicates a lack of correlation of molecular markers of clarithromycin resistance for both acquired and inducible resistance to clarithromycin.

Activation of inducible nitric oxide synthase (iNOS) and oxidative stress have been shown to be associated with compromised cardiovascular function in streptozotocin (STZ)-induced type 1 diabetes. The aim of the project is to investigate cardiovascular abnormalities in a rat model of type 2 diabetes (Zucker diabetes fatty or ZDF rats) and two models of metabolic syndrome (fructose-fed rats and Zucker obese rats), and to provide direct evidence linking iNOS and oxidative stress to abnormal cardiovascular function in these disorders. Blood pressure, cardiac contractility, cardiac index, regional flow, vascular resistance and venous tone were measured in diseased as well as normal rats. Biochemical analyses such as activities of iNOS, immunostaining of iNOS and western-blot analysis of iNOS in the heart tissue were carried out. The results showed that cardiac contractile response to dobutamine was compromised in the ZDF rats, and this was associated with increased myocardial protein expression as well as activity of iNOS. The formation of peroxynitrite was increased in the heart tissue of the ZDF rats. Selective inhibition of iNOS by 1400W (N-3-aminomethyl-benzyl-acetamidine) did not alter responses to dobutamine in the control rats, but augmented the contractile effects of dobutamine in the diabetic rats. The regional blood flow was altered in the ZDF rats, and iNOS played a negligible role in regulating regional flow in the ZDF rats. Although venous response to noradrenaline was also altered in the Zucker obese rats, NOS may not be involved in venous tone regulation. Anti-oxidative treatment with N-acetylcysteine inhibited the development of insulin resistance, blood pressure elevation and the increase of 8-isoprostane formation in the fructose-fed rats. We conclude that heart function is compromised and regional blood flow is altered in the ZDF rats. Activation of iNOS plays an important role in suppressing heart dysfunction but does not affect regional blood flow. In Zucker obese rats with metabolic syndrome, iNOS may not be involved in changes of venous function. Oxidative stress is associated with both abnormality of heart dysfunction in type 2 diabetes (by formation of peroxynitrite due to iNOS activation) and development of hypertension and insulin resistance in metabolic syndrome. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

Of the thousands of dividing progenitor cells (PCs) generated daily in the adult brain only a very small proportion survive to become mature neurons through the process of neurogenesis. Identification of the mechanisms that regulate cell death associated with neurogenesis would aid in harnessing the potential therapeutic value of PCs. Apoptosis, or programmed cell death, is suggested to regulate death of PCs in the adult brain as overexpression of B-cell lymphoma 2 (Bcl-2), an anti-apoptotic protein, enhances the survival of new neurons. To directly assess if Bcl-2 is a regulator of apoptosis in PCs, this study examined the outcome of removal of Bcl-2 from the developing PCs in the adult mouse brain. Retroviral mediated gene transfer of Cre into adult floxed Bcl-2 mice eliminated Bcl-2 from developing PCs and resulted in the complete absence of new neurons at 30 days post viral injection. Similarly, Bcl-2 removal through the use of nestin-induced conditional knockout mice resulted in reduced number of mature neurons. The function of Bcl-2 in the PCs was also dependent on Bcl-2-associated X (BAX) protein, as demonstrated by an increase in new neurons formed following viral-mediated removal of Bcl-2 in BAX knockout mice. Together these findings demonstrate that Bcl-2 is an essential regulator of neurogenesis in the adult hippocampus.

One mechanism of plasticity within the adult mammalian brain is the dynamic process of adult neurogenesis that is functionally important in physiological and pathological conditions. During this process, neurons develop from adult neural stem cells (NSCs) via intermediate neural progenitors (NPCs) through several processes including proliferation, survival, differentiation, migration and integration. Despite neurogenesis during development sharing these same processes, there is growing evidence highlighting unique mechanisms that regulate adult versus embryonic neurogenesis. The studies in this thesis test the cell-intrinsic function of genes that have defined roles in embryonic neurogenesis and undefined roles in adult hippocampal neurogenesis using a combination of transgenic inducible mice and in vivo retroviral techniques. The first study examines the microtubule associated protein Doublecortin (DCX), which is transiently expressed by NPCs and is critical for neuronal migration. Our results show that, in the context of adult hippocampal neurogenesis, DCX is not required for the survival or differentiation of the NPCs within the subgranular zone (SGZ). The second study examines the functional role of the autophagy-associated gene 5 (Atg5) which is critical for embryonic neurogenesis and survival. Our findings demonstrate that the intracellular recycling process of autophagy is active throughout maturation of adult hippocampal NPCs and that ablation of Atg5 produces a drastic reduction in NPC survival, without altering the neuronal fate of these cells. The third study examines the requirement of the familial-Alzheimer’s disease associated genes, presenilin 1 and presenilin 2 (PS1 & PS2), which are critical for embryonic NSC maintenance and differentiation. Similar to the findings with DCX, our results demonstrate that presenilins are dispensable for adult neurogenesis. Altogether, these studies add to the growing evidence suggesting differences in the regulation of adult versus embryonic neurogenesis, and highlight autophagy as a novel regulator of survival for adult generated granule neurons in the hippocampus.

Recent advances in cellular reprogramming have enabled the generation of embryoniclike cells from virtually any cell of the body. These inducible pluripotent stem cells (iPSCs) are capable of indefinite self-renewal while maintaining the ability to differentiate into all cell types. Nowhere will this technology have a greater impact than in the ability to generate disease and patient-specific cell lines. Here we explore the capacity of human iPSCs reprogrammed from peripheral blood endothelial progenitor cells lines to differentiate into atrial myocytes for the study of patient specific atrial physiology. Methods and Results: Late outgrowth endothelial progenitor cells (EPCs) cultured from clinical blood samples provided a robust cell source for genetic reprogramming. Transcriptome analysis hinted that EPCs would be comparatively more amenable to pluripotent reprogramming than the traditional dermal fibroblast. After 6 passages, EPCs were transduced with a doxycycline inducible lentivirus system encoding human transcription factors OCT4, SOX2, KLF4 and Nanog to permit differentiation after removal of doxycycline. The high endogenous expression of key pluripotency transcripts enhanced the ease of iPSC generation as demonstrated by the rapid emergence of typical iPSC colonies. Following removal of doxycycline, genetically reprogrammed EPC-iPSC colonies displayed phenotypic characteristics identical to human embryonic stem cells and expressed high levels of the pluripotent markers SSEA-4, TRA1-60 and TRA1-81. After exposure to conditions known to favor atrial identity, EPC- iPSC differentiating into sheets of beating cardiomyocytes that expressed high levels of several atrial-specific expressed genes (CACNA1H, KCNA5, and MYL4). Conclusions: EPCs provide a stable platform for genetic reprogramming into a pluripotent state using a doxycycline conditional expression system that avoids reexpression of oncogenic/pluripotent factors. Human EPC-derived iPSC can be differentiated into functional cardiomyocytes that express characteristic markers of atrial identity.