Ataxia-telangiectasia mutated kinase (ATM), a serine/threonine kinase, plays a role in DNA damage repair, redox sensing, and metabolism. In the heart, ATM contributes significantly in the myocardial infarction (MI)-induced cardiac remodeling with effects on fibrosis, hypertrophy, apoptosis and inflammation. This study investigates the role of ATM deficiency in 14 weeks Western-type diet (WD)-induced cardiac outcomes prior to and 1-day post-MI in a sex-specific manner using wild-type (WT) and ATM heterozygous knockout (hKO) mice. In male mice, ATM deficiency induced rapid body weight gain and preload-associated dysfunction, while WT mice displayed afterload-associated dysfunction 14 weeks post-WD. Myocyte apoptosis and hypertrophy were higher in hKO-WD versus WT-WD. WD increased fibrosis, and expression of collagen-1α1, MMP-2 and MMP-9 only in WT-WD. AMPK activation was higher, while activation of mTOR and NF-kB was lower in hKO-WD versus hKO-NC. Serum levels of IL-12(p70), eotaxin, IFN-γ, MIP-1α, and MIP-1β were higher in hKO-WD versus WT-WD. Conversely, female hKO-WD mice exhibited an attenuation of weight gain and maintenance of heart function. Cholesterol, triglyceride, and glucose levels were higher in female hKO-WD. WD-induced apoptosis and Bax expression were lower in hKO-WD vs WT-WD. Collagen-1α1 expression was higher in hKO-WD vs WT-WD. MMP-2 and MMP-9 expression increased only in WT-WD. MI decreased cardiac function in both male and female mice versus their WD counterparts. The cardioprotective effects of ATM deficiency in terms of heart function were abolished in female mice 1 day post-MI. MI led to a similar infarct size and increase in apoptosis in the two WD-MI groups of both sexes. These data suggest that – 1) ATM deficiency associates with systolic and preload associated diastolic dysfunction, and exacerbates apoptosis, hypertrophy, and fibrosis in male mice in response to WD; 2) In female mice, ATM deficiency plays a cardioprotective role with preserved systolic function and decreased apoptosis in response to WD; 3) the sex-specific cardioprotective effects of ATM deficiency in females were abolished 1day post-MI. Thus, ATM deficiency affects cardiac structure and function in a sex-specific manner in response to WD and early post-MI.

For many years, adipose tissue (AT) was thought to be a tissue primarily responsible for cushioning and insulating organs. However, significant advances in knowledge have shown that AT is necessary for maintaining an optimal metabolic balance through paracrine and endocrine mechanisms. Because AT dysfunction is related with illnesses such as obesity and diabetes, it is vital to understand the mechanisms behind these pathologies to restore metabolic health. Beige AT is a unique form of fat that generates heat through uncoupling protein 1 (UCP1), has a dense neurovascular network, and is associated with enhanced metabolic health. Hence, particular emphasis has been made on unraveling the processes behind thermogenic activation and maintenance, as increasing thermogenic activity offers considerable potential for treating metabolic disorders. Activation of beige AT is dependent on norepinephrine release from sympathetic neurons upon physiological cues such as cold exposure. Studies have revealed a major role of monoamine oxidase a (MAOA)-mediated norepinephrine clearance in the maintenance of thermogenic AT. However, major limitations are still present with regards to the mechanisms of neurotransmitter clearance and their role in thermogenic regulation. The initial objective of this thesis is to evaluate the effect of human white and thermogenic adipocytes on the formation of a neurovascular network in order to maintain thermogenesis and whether MAOA plays a direct role in thermogenic control. We demonstrate that implanted human thermogenic adipocytes generate a more vascularized and innervated AT than non-thermogenic adipocytes. Additional findings revealed that MAOA is expressed in human adipocytes and that inhibiting MAOA promotes thermogenesis. The second objective of this thesis is to determine if hAdipoGel (hAG) - a decellularized AT matrix – enhances mesenchymal stem cell (MSC) proliferation and differentiation, as well as human adipocyte engraftment in vivo. We show that MSC can proliferate in hAG and differentiate effectively into white and thermogenic adipocytes. Additionally, when white adipocytes are implanted with hAG, they differentiate into a fully functioning fat graft capable of integrating with the host. Understanding the thermogenic processes of human AT, in combination with the use of a suitable decellularized matrix, can aid in the development of therapeutic treatments that boost thermogenic activity and hence metabolic health.

Disruption of cilia proteins results in a range of disorders called ciliopathies. However, the mechanism by which cilia dysfunction contributes to disease is not well understood. Intraflagellar transport (IFT) proteins are required for ciliogenesis. They carry ciliary cargo along the microtubule axoneme while riding microtubule motors. Interestingly, IFT proteins localize to spindle poles in non-ciliated, mitotic cells, suggesting a mitotic function for IFT proteins. Based on their role in cilia, we hypothesized that IFT proteins regulate microtubule-based transport during mitotic spindle assembly. Biochemical investigation revealed that in mitotic cells IFT88, IFT57, IFT52, and IFT20 interact with dynein1, a microtubule motor required for spindle pole maturation. Furthermore, IFT88 co-localizes with dynein1 and its mitotic cargo during spindle assembly, suggesting a role for IFT88 in regulating dynein-mediated transport to spindle poles. Based on these results we analyzed spindle poles after IFT protein depletion and found that IFT88 depletion disrupted EB1, γ-tubulin, and astral microtubule arrays at spindle poles. Unlike IFT88, depletion of IFT57, IFT52, or IFT20 did not disrupt spindle poles. Strikingly, the simultaneous depletion of IFT88 and IFT20 rescued the spindle pole disruption caused by IFT88 depletion alone, suggesting a model in which IFT88 negatively regulates IFT20, and IFT20 negatively regulates microtubulebased transport during mitosis. Our work demonstrates for the first time that IFT proteins function with dynein1 in mitosis, and it also raises the important possibility that mitotic defects caused by IFT protein disruption could contribute to the phenotypes associated with ciliopathies.

Non-alcoholic fatty liver disease (NAFLD) is an increasingly prevalent issue in the modern world, predisposing patients to serious pathology such as cirrhosis and hepatocellular carcinoma. Mitochondrial dysfunction, and in particular, diminished hepatic oxidative phosphorylation (OXPHOS) capacity, have been observed in NAFLD livers, which may participate in NAFLD pathogenesis. To examine the role of OXPHOS in NAFLD, we generated a model of enhanced hepatic OXPHOS using mice with liver-specific transgenic expression of LRPPRC, a protein which activates mitochondrial transcription and augments OXPHOS capacity. When challenged with high-fat feeding, mice with enhanced hepatic OXPHOS were protected from the development of liver steatosis and inflammation, critical components in the pathogenesis of NAFLD. This protection corresponded to increased liver and whole-body insulin sensitivity. Moreover, mice with enhanced hepatic OXPHOS have increased availability of oxidized NAD+, which promotes complete fatty acid oxidation in hepatocytes. Interestingly, mice with enhanced hepatic OXPHOS were also protected from obesogenic effects of long-term high-fat feeding. Consistent with this, enhanced hepatic OXPHOS increased energy expenditure and adipose tissue oxidative gene expression, suggesting a communication between the liver and adipose tissue to promote thermogenesis. Examination of pro-thermogenic molecules revealed altered bile acid composition in livers and serum of LRPPRC transgenic mice. These mice had increased expression of bile acid synthetic enzymes, genes which are induced by NAD+ dependent deacetylase SIRT1 activation of the transcriptional co-regulator PGC-1a. These findings suggest that enhanced hepatic OXPHOS transcriptionally regulates bile acid synthesis and dictates whole-body energy expenditure, culminating in protection from obesity.

Obesity results from expansion of white adipose tissue. The inability of white adipose tissue to adequately store lipids leads to ectopic deposition of lipids in non-adipose tissue that can lead to systemic insulin resistance. It is well known that insulin resistance correlates with inflammation of adipose tissue in obese animals and humans. Decreasing inflammation in the adipose tissue has been proven as a therapeutic strategy for improvement of insulin sensitivity in vivo. Numerous factors secreted by immune cells, including macrophages, have been suggested as regulating adipose tissue insulin sensitivity. In the first part of my thesis, I describe the role of one such factor, CD40 in adipose tissue inflammation. The CD40-CD40L dyad acts as co-stimulation in the interaction of antigen-presenting cells, such as macrophages and dendritic cells, with effector cells, such as T cells, in adaptive immunity. We found that CD40 knockout mice were smaller but surprisingly more insulin resistant and glucose intolerant compared to wild-type mice when fed a high fat diet. Consistent with their metabolic phenotype, knockout mice displayed increased adipose tissue inflammation with infiltration of immune cells including macrophages and T cells. Consistent with increased inflammation, CD40 knockout adipose tissue displayed decreased lipid storage. Deficiency of CD40 also led to increased lipid deposition in liver, which may be due to increased lipid release into circulation from the adipose tissue as well as increased lipid synthesis in the liver. CD40 knockout mice had increased hepatic insulin resistance and increased gluconeogensis despite decreased hepatic inflammation. These findings suggest that CD40 is a novel regulator of adipose tissue inflammation in diet-induced obesity. In the second part of this thesis we examined perivascular adipose tissue and brown adipose tissue for the presence of inflammation. In contrast to visceral adipose tissue, macrophage infiltration was absent in perivascular and brown adipose tissue as defined by reduced F480+ cells by flow cytometry and immunohistochemistry. We also found that perivascular adipose tissue was similar to brown adipose tissue as shown by gross morphology and gene expression pattern. Inflammatory gene expression was not increased in brown or perivascular adipose tissue in obese mice as determined by microarray gene expression analysis. These findings suggest that perivascular adipose tissue is more similar to brown adipose tissue than white adipose tissue and that both perivascular and brown adipose tissue are resistant to inflammation. We conclude that, (1) CD40 protects against adipose tissue inflammation in diet-induced obesity, (2) the CD40 knockout mouse is an interesting model of hepatic steatosis with decreased inflammation and (3) perivascular adipose tissue is almost identical to brown adipose tissue in obese mice and that both are resistant to inflammation.

Mycobacterium tuberculosis stably adapts to respiratory limited environments by entering into a nongrowing but metabolically active state termed quiescence. This state is inherently tolerant to antibiotics due to a reduction in growth and activity of associated biosynthetic pathways. Understanding the physiology of the quiescent state, therefore, may be useful in developing new strategies to improve drug efficiency. Here, we used an established in vitro model of respiratory stress, hypoxia, to induce quiescence. We utilized metabolomic and genetic approaches to identify essential and active pathways associated with nongrowth. Our metabolomic profile of hypoxic M. tuberculosis revealed an increase in several free fatty acids, metabolite intermediates in the oxidative pathway of the tricarboxylic acid (TCA) cycle, as well as, the important chemical messenger, cAMP. In tandem, a high-throughput transposon mutant library screen (TnSeq) revealed that a cAMP-regulated protein acetyltransferase, MtPat, was conditionally essential for survival in the hypoxic state. Via 13C-carbon flux tracing we show an MtPat mutant is deficient in re-routing hypoxic metabolism away from the oxidative TCA cycle and that MtPat is involved in inhibiting fatty-acid catabolism in hypoxia. Additionally, we show that reductive TCA metabolism is required for survival of hypoxia by depletion of an essential TCA enzyme, malate dehydrogenase (Mdh) both in in vitro hypoxia and in vivo mouse infection. Inhibition of Mdh with a novel compound resulted in a significantly greater killing efficiency than the first-line anti-M. tuberculosis drug isoniazid (INH). In conclusion, we show that understanding the physiology of the quiescent state can lead to new drug targets for M. tuberculosis.

Autophagy is a cellular process that delivers cytoplasmic materials for degradation by the lysosomes. Autophagy-related (Atg) genes were identified in yeast genetic screens for vehicle formation under stress conditions, and Atg genes are conserved from yeast to human. When cells or animals are under stress, autophagy is induced and Atg8 (LC3 in mammal) is activated by E1 activating enzyme Atg7. Atg8-containing membranes form and surround cargos, close and mature to become the autophagosomes. Autophagosomes fuse with lysosomes, and cargos are degraded by lysosomal enzymes to sustain cell viability. Therefore, autophagy is most frequently considered to function in cell survival. Whether the Atg gene regulatory pathway that was defined in yeast is utilized for all autophagy in animals, as well as if autophagy could function in a cell death scenario, are less understood. The Drosophila larval digestive tissues, such as the midgut of the intestine and the salivary gland, are no longer required for the adult animal and are degraded during the pupal stage of development. Cells stop growing at the end of larval development, and proper cell growth arrest is required for midgut degradation. Ectopic activation of the PI3K/Akt signaling induces cell growth and inhibits autophagy and midgut degradation. Down regulating PI3K/Akt pathway by Pten mis-expression activates autophagy. In addition, mis-expression of autophagy initiator Atg1 inhibits cell growth and knocking down autophagy restore PI3K/Akt activity. Together, these results indicate that autophagy and growth signaling mutually inhibit each other. Midgut destruction relies on the autophagy gene Atg18, but not caspase activation. The intestine length shortens and the cells undergo programmed cell size reduction, a phenomenon that also requires Atg18, before cell death occurs during midgut destruction. To further investigate whether cell size reduction is cell autonomous and requires other Atg genes, we reduced the function of Atg genes in cell clones using either gene mutations or RNAi knockdowns. Indeed, many Atg genes, including Atg8, are required for autophagy and cell size reduction in a cell autonomous manner. Surprisingly, Atg7 is not required for midgut cell size reduction and autophagy even though this gene is essential for stress-induced autophagy. Therefore, we screened for known E1 enzymes that may function in the midgut, and discovered that Uba1 is required for autophagy, size reduction and clearance of mitochondria. Uba1 does not enzymatically substitute for Atg7, and Ubiquitin phenocopies Uba1, suggesting Uba1 functions through ubiquitination of unidentified molecule(s) to regulate autophagy. In conclusion, this thesis describes: First, autophagy participates in midgut degradation and cell death. Second it reveals a previously un-defined role of Uba1 in autophagy regulation. Third it shows that the Atg genes are not functionally conserved and the requirement of some Atg genes can be context dependent.

Obesity is the top risk factor for the development of type 2 diabetes mellitus in humans. Obese adipose tissue, particularly visceral depots, exhibits an increase in macrophage accumulation and is described as being in a state of chronic low-grade inflammation. It is characterized by the increased expression and secretion of inflammatory cytokines produced by both macrophages and adipocytes, and is associated with the development of insulin resistance. Based on these observations, we investigated the potential role of macrophage infiltration on whole body metabolism, using genetic and diet-induced mouse models of obesity. Using flow cytometry and immunofluorescence imaging we found that a significant percentage of macrophages proliferate locally in adipose tissue of obese mice. Importantly, we identified monocyte chemoattractant protein 1 (MCP-1) as the stimulating factor. We also found that ATMs can be targeted for specific gene silencing using glucan encapsulated siRNA particles (GeRPs). Knockdown of the cytokine osteopontin improved regulation of systemic glucose levels as well as insulin signaling in adipocytes. Conversely, targeting lipoprotein lipase (LPL) abrogated the buffering of lipid spillover from adipose tissue, resulting in increased hepatic glucose output. Finally, silencing of the master regulator of inflammation NF-κB in resident liver macrophages called Kupffer cells significantly improved hepatic insulin signaling. Thus this work demonstrates that macrophages can regulate whole body metabolism.

Proper functioning of endocrine cells is crucial for organismal homeostasis. The underlying mechanisms that fine-tune the amount, and the timing of hormone secretion are not clear. JIP1 / MAPK8IP1 (JNK interacting protein 1) is a scaffold protein that mediates cellular stress response, and is highly expressed in endocrine cells, including insulin secreting b-cells in pancreas islets. However, the role of JIP1 in b-cells is unclear. This study demonstrates that b-cell specific Jip1 ablation results in decreased glucose-induced insulin secretion, without a change in Insulin1 and Insulin2 gene expression. Inhibition of both JIP1-kinesin interaction, and JIP1-JNK interaction by genetic mutations also resulted in decreased insulin secretion, suggesting that JIP1 may mediate insulin vesicle trafficking through interacting with kinesin and JNK. Autophagy is a cellular recycling mechanism and implicated in the b-cell function. Both JIP1 and JNK are proposed to regulate autophagy pathway. However, it is unclear whether JNK plays a role in the promotion or suppression of autophagy. The findings of this study show that JNK is not essential for autophagy induction, but can regulate autophagy in a cell and context specific manner. The results in this thesis implies a mechanism that link cellular trafficking and stress signaling pathways in the regulated hormone secretion. In addition to the known role of JIP1 in metabolism and insulin resistance, this finding may also be relevant to endocrine pathologies.

The nucleolus is a plurifunctional organelle with dynamic protein exchange involved in diverse aspects of cell biology. Additionally, the nucleolus has been shown to have a role in the replication of numerous viruses, which includes HIV-1. Several groups have reported HIV-1 vRNA localization within the nucleolus. Moreover, it has been demonstrated the HIV-1 Rev protein localizes to the nucleolus and interacts with nucleolar proteins, including NPM1. Despite evidence for a nucleolar involvement during replication, a functional link has not been demonstrated. I investigated whether introncontaining vRNAs have a Rev-mediated nucleolar localization step prior to export. Furthermore, I examined whether NPM1 mediates Rev nucleolar localization, participates in Rev function, and/or post-transcriptional events during viral replication. I used coupled RNA fluorescence in situhybridization and indirect immunofluorescence to visualize intron-containing vRNA relative to the nucleolus in the absence or presence of Rev expression. An RNAi-based approach was used to examine the role of NPM1 in Rev function and viral replication in cell lines and primary human macrophages. My research findings support a model for a Rev-independent nucleolar localization step of introncontaining vRNA prior to export. Intriguingly, my results also suggest NPM1 does not participate in Rev nucleolar localization or Rev-mediated vRNA export, as previously proposed. Rather, my findings support a novel role for NPM1, the cytoplasmic localization and utilization of a select class of Rev-dependent vRNAs. Collectively, my findings provide novel insight for a functional role of the nucleolus and NPM1 in HIV-1 replication, which enhances our current understanding of HIV-1 biology.