<p>  </p> <p>Calcium influx into pancreatic beta-cells is required for proper beta-cell growth and function. While the functional significance of calcium influx into the beta-cells is known, the significance of release of calcium from intracellular stores is less understood. Calcium-induced calcium release (CICR) is a process by which calcium influx into the cell through voltage-gated calcium channels activated release of calcium from intracellular stores. The functional significance of CICR is well understood in cardiac and vascular muscle cells in regard to excitation-contraction coupling. However, the functional significance of CICR in beta-cells in not well understood. </p> <p>To investigate the role of RyR2 in pancreatic beta-cell function, we utilized CRISPR-Cas9 gene editing to delete RyR2 from the rat insulinoma INS-1 cell line. we found that RyR2KO cells displayed an enhanced glucose-stimulated Ca2+ integral (area under the curve; AUC) which was sensitive to inhibition by the IP3R antagonist, xestospongin C. Loss of RyR2 also resulted in a reduction in IRBIT protein levels. Therefore, we deleted IRBIT from INS-1 cells (IRBITKO) and found that IRBITKO cells also displayed an increased Ca2+ AUC in response to glucose stimulation. RyR2 KO and IRBIT KO cells had reduced glucose-stimulated insulin secretion and insulin content. RT-qPCR revealed that <em>INS2</em> transcript levels were reduced in both RyR2KO and IRBITKO. Nuclear localization of AHCY were increase in both the RyR2KO and IRBITKO cells, corresponding with increased levels of insulin gene methylation. Proteomic analysis revealed that deletion of RyR2 or IRBIT resulted in differential regulation of 314 and 137 proteins, respectively, with 41 in common. Our results suggest that RyR2 and IRBIT activity regulate insulin content, insulin secretion, and regulate the proteome in INS-1 cells</p> <p>We next sought to assess the consequences on cellular Ca2+ handling in the absence of RyR2 and IRBIT in INS-1 cells. Store-operated Ca2+ entry (SOCE) stimulated with thapsigargin was reduced in RyR2KO cells compared to controls, but this was not different in IRBITKO cells. STIM1 protein levels were not different between the three cell lines. Basal and carbachol stimulated phospholipase C (PLC) activity was reduced specifically in RyR2KO cells and not IRBITKO cells. However, basal PIP2 levels were elevated in both RyR2KO and IRBITKO cells. Insulin secretion stimulated by tolbutamide was reduced in RyR2KO and IRBITKO cells compared to controls, but this was still potentiated by an EPAC-selective cAMP analog in all three cell lines. Cortical f-actin is known to regulate insulin secretion, and levels were markedly reduced in RyR2KO cells compared to control INS-1 cells. Whole-cell Cav channel current density was reduced in RyR2KO cells compared to controls, and Ba2+ current was significantly reduced by PIP2 depletion preferentially in RyR2KO cells over control INS-1 cells. Action potentials stimulated by 18 mM glucose were more frequent in RyR2KO cells compared to controls, and insensitive to the SK channel inhibitor apamin. Taken together, these results suggest that RyR2 plays a critical role in regulating PLC activity and PIP2 levels via regulation of SOCE. RyR2 also regulates beta-cell electrical activity by controlling Cav current density, via regulation of PIP2 levels, and SK channel activation.</p> <p>Lastly, we investigated the role of PDE subtypes cAMP in INS-1 cells and human islets. We utilized subtype selective inhibitors of PDE1, PDE3 and PDE8 to assess the potential of these PDEs as potential therapeutic targets. We found that PDE1 is the primary subtype in INS-1 cells, whereas PDE3 appears to be required in human pancreatic β-cells by cAMP measurements. PDE1 inhibition potentiated glucose-stimulated to the greatest extent in both INS-1 cells and human islets. PDE1 inhibition potentiated CREB phosphorylation to the greatest extent and was also capable of mitigating lipotoxicity in INS-1 cells. Collectivity, this work highlights the role of cAMP compartmentalized signaling in pancreatic β-cells, and this has drastic effects on pancreatic beta-cell function and survival.</p>

<p>The rapid localized cell death at and around sites of attempted pathogen infection, termed the hypersensitive response (HR), is an immune response mechanism commonly utilized in plants. This cell death limits pathogens from accessing host nutrients which often leads to resistance. The interaction of pathogen signals and host receptors that are required for the HR are well studied; however, the processes that regulate cell death during the HR remain enigmatic. The plant lesion mimic mutants, which form spontaneous lesions and/or undergo autoactive cell death in the absence of infection or stress, are commonly used as model systems to study the HR. Some lesion mimic mutants are caused by autoactive alleles of the resistance genes that recognize pathogen signals and trigger the HR. These mutants have facilitated studies of the HR as they allow the study of the HR without the need to control for pathogen infection. Currently, the etiologies of most maize lesion mimic mutants are unknown. Lesion mimic mutants contain numerous metabolic perturbations, including the increased accumulation of salicylic acid (SA), phenylalanine, and intermediates in heme and chlorophyll biosynthesis and catabolism. Some of these perturbations are dependent on the cause of lesion formation. As such, the accumulation of any of these metabolites in a lesion mutant may infer the etiology of that mutant. This dissertation contains three projects related to the molecular characterization of HR and maize lesion mimic mutants. In the first project (Chapter 2), I compared the metabolite profile of 23 maize lesion mimic mutants. This work identified two major findings that were further explored in the other projects in this dissertation. The first major finding is that four of the 23 mutants have metabolic perturbations that are like those of the known HR lesion mutant, <em>Rp1-D21</em>. In project two (Chapter 3), I molecularly characterize, <em>Lesion10</em>, which is one of the mutants that has HR-like metabolic perturbations. Using genome-wide association studies, I identified a gene candidate that may modify <em>Lesion10</em> phenotypic severity. The second major finding from project one is that SA accumulates to higher than wild-type levels in most of the lesion mutants analyzed. In the third project (Chapter 4), I characterized how SA is synthesized in maize and if SA is necessary or sufficient for the formation of lesions during the HR in maize. Using untargeted metabolite analysis, stable isotope feedings, and enzyme assays, I provide evidence of both known SA biosynthetic pathways in maize and demonstrate that the two pathways are interdependent. In addition, I show that increased accumulation of SA is not required for the HR in maize.</p>

Chromatin regulation is a major aspect of cancer development, progression, and treatment. Several small molecule inhibitors of chromatin regulators are currently used for treatment of certain hematological malignancies. However, there is still opportunity for many more patients to benefit from therapeutic approaches that target chromatin regulation, especially in the context of solid tumors. A critical unmet need is the identification of robust biomarkers that can guide the application of epigenetic inhibitors in a precise and personalized manner. In my dissertation, I aim to address this important knowledge gap by studying how perturbation of chromatin can target metabolic and lineage abnormalities in solid tumors for therapeutic benefit. To do this, I have focused on genetic and pharmacological perturbations of chromatin pathways in two cancer models: (1) lung adenocarcinoma (LUAD) with NRF2 activation and (2) neuroendocrine esophageal carcinoma (NEC). In the study on NRF2-active LUAD, we found that histone deacetylase (HDAC) inhibitors can be repurposed to reprogram the epigenomic and metabolic landscape, which leads to specific and potent anti-tumor effects in the context of NRF2 activation. Specifically, we employed a chromatin-focused genetic screen to identify dependencies on chromatin regulators. The screen revealed an NRF2-specific dependency on class I histone deacetylases. Experiments in mouse and human LUAD cell lines in vitro and in vivo indicated an NRF2-specific sensitivity to the class I HDAC inhibitor Romidepsin. Mechanistically, profiling of histone acetylation and gene expression upon Romidepsin treatment revealed a relative loss of histone H4 acetylation at promoters which was associated with reduced gene expression. Many downregulated genes were more essential for the survival of NRF2 hyperactive cancer cells, including genes involved in glutamine and serine metabolism, c-Myc and several of its targets involved in purine and pyrimidine synthesis. These transcriptional changes had corresponding effects on altering the metabolic pathways that NRF2-active cells selectively require for survival. In the study on neuroendocrine esophageal carcinoma (NEC), we identified a crucial role for epigenetic regulation of lineage fate through transcriptional control of the key epidermal transcription factor p63. This project originated from data from my collaborators that indicates a role for p63 in the suppression of basal-to-neuroendocrine identity transition in the developing esophagus. Consistently, I found that p63 is silenced in NEC through a non-genetic mechanism. Reintroducing p63 isoforms in a human NEC cell line showed that ΔNp63α was sufficient to restore squamous marker expression. An epigenetic drug screen assessing p63 gene expression and subsequent validation experiments revealed that inhibition of EZH2, a histone methyltransferase, induced expression of ΔNp63α and genes related to the squamous identity. Analysis of the chromatin state in the TP63 locus showed that EZH2 inhibition led to a loss histone H3 methylation and a gain of histone H3 acetylation and its reader BRD4. These results support the hypothesis that the squamous identity can be reactivated epigenetically in NEC through de-repression of ΔNp63α as a potential therapeutic strategy. Together, these studies contribute to our understanding of the transcriptional response to chromatin perturbation and show that this can be leveraged to modulate cell metabolism and identity, as well as to achieve therapeutic benefit in new contexts of cancer.

<p dir="ltr">Adipose tissue consists of adipocytes that store energy within lipid droplets and are a central component of lipid metabolism. Mammals contain white, brown and beige adipocytes, which differ in their metabolic roles. White adipocytes store energy, in the form of triglycerides, within lipid droplets and predominantly take on an energy storage role. Brown and beige adipocytes promote energy expenditure and the dissipation of energy as heat through non-shivering thermogenesis. Since energy expenditure combats excess caloric intake and overeating, non-shivering thermogenesis has become heavily researched for its potential therapeutic use in combatting the continued increase in obesity and metabolic disorders worldwide.</p><p dir="ltr">In addition to ATP synthesis, mitochondria are required for a multitude of metabolic processes that maintain cellular homeostasis, including non-shivering thermogenesis. Brown and beige adipocyte mitochondria are specialized to perform non-shivering thermogenesis in response to an environmental stressor like cold exposure. Uncoupling protein 1 (UCP1) is uniquely characteristic of brown and beige adipocyte mitochondria, because it allows oxidative phosphorylation to be uncoupled from ATP synthesis. In order to enhance non-shivering thermogenesis, ongoing molecular characterization of brown adipose tissue (BAT) is being conducted to identify proteins that regulate mitochondrial function and UCP1 activity. In this study, I explored the function of LETM1-domain containing 1 (LETMD1), a novel mitochondrial inner membrane protein with unknown function in BAT. We generated a global (<i>Letmd1</i>^<em>KO</em>) and UCP1+ cell-specific <i>(Letmd1</i>^<em>UKO</em><i>) knockout</i><i> </i>mouse model to study the whole-body and cell-autonomous role of LETMD1 in BAT, respectively. Loss-of-function studies resulted in striking, BAT-specific phenotypic differences, including whitened BAT under thermoneutral, room temperature and cold exposure. Both knockout models were cold intolerant without access to food, and became hypothermic within a few hours of fasted cold exposure. Loss of normal mitochondria structure and cristae arrangement were also evident in knockout BAT, resulting in a decreased number of mitochondria and decreased number of cristae per mitochondrion. Mitochondrial DNA copy number was also significantly decreased in both knockout models. Abnormal mitochondria morphology was supported by increased reactive oxygen species (ROS) accumulation in both knockout models and the visualization of protein aggregates and mitophagy-like morphologies in <i>Letmd1</i>^<em>UKO</em><i> </i><i>mice specifically</i>. TurboID proximity labeling of brown adipocytes revealed enrichment of several respiratory chain complex proteins, mitochondrial ribosome proteins and mitochondrial protein import machinery. Moreover, the aggregation of misfolded nuclear-encoded mitochondrial proteins, including several respiratory chain and mitochondrial ribosome proteins, suggested that LETMD1 facilitates mitochondrial protein import and mitochondrial ribosome assembly, thereby compromising respiratory chain assembly and function during non-shivering thermogenesis. Overall, this study identifies LETMD1 as a novel regulator of brown adipocyte mitochondrial structure and thermogenic function and highlights the requirement of LETMD1 for mitochondrial biogenesis.</p>

Mitochondrial disorders resulting from defects in oxidative phosphorylation are the most common form of inherited metabolic disease. Mutations in the human mitochondrial translation elongation factor GFM1 have recently been shown to cause the lethal pediatric disorder Combined Oxidative Phosphorylation Deficiency Syndrome (COXPD1). Children harboring mutations in GFM1 exhibit severe developmental, metabolic and neurological abnormalities. This work describes the identification and extensive characterization of the first known mutations in iconoclast (ico), the Drosophila orthologue of GFM1. Expression of human GFM1 can rescue ico null mutants, demonstrating functional conservation between the human and fly proteins. While point mutations in ico result in developmental defects and death during embryogenesis, animals null for ico survive until the second or third instar larval stage. These results indicate that in addition to loss-of-function consequences, point mutations in ico appear to produce toxic proteins with antimorphic or neomorphic effects. Consistent with this hypothesis, transgenic expression of a mutant ICO protein is lethal when expressed during development and inhibits growth when expressed in wing discs. In addition, animals with a single copy of an ico point mutation are more sensitive to acute hyperthermic or hypoxic stress. Removal of the positively-charged tail of the protein abolishes the toxic effects of mutant ICO, demonstrating that this domain is necessary for the harmful gain-of-function phenotypes observed in ico point mutants. / Further, expression of GFP-tagged constructs indicates that the C-terminal tail enhances ectopic nuclear localization of mutant ICO, suggesting that mislocalization of the protein may play a role in the antimorphic effects of mutant ICO. Taken together, these results illustrate that in addition to loss-of-function effects, gain-of-function effects can contribute significantly to the pathology caused by mutation in mitochondrial translation elongation factors. / by Catherine F. Trivigno. / Thesis (Ph.D.)--Florida Atlantic University, 2010. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2010. Mode of access: World Wide Web.

Ácidos graxos podem atuar na apoptose ativando e/ou desativando sinais que regulam este processo. Células Jurkat (linhagem de linfócitos-T) foram tratadas com ácido palmítico (PA) (50, 100 e 150µM) e avaliada a ativação de vias de morte e de sobrevivência. O tratamento com PA induziu apoptose pela ativação da via intrínseca de maneira dose-dependente. A exposição destas células ao PA elevou a expressão do receptor de insulina e de GLUT-4, obtendo-se correlação positiva com a apoptose. O mesmo foi observado em linfócitos humanos expostos ao PA e de linfócitos de ratos em jejum. O tratamento das células Jurkat com insulina após exposição ao PA promoveu a ativação da via de sinalização insulínica. O PA aumentou a captação de glicose, mas observou-se diminuição de sua oxidação e o acúmulo de lípides. Assim, a via de sinalização da insulina e o metabolismo de glicose não oxidativo são estimulados como parte de uma coordenada resposta de sobrevivência de linfócitos expostos ao PA em baixas concentrações, mas em altas concentrações ocorre apoptose. / Fatty acids affect apoptosis pathway activating or deactivating signals that regulate this process. Jurkat cells (T-lymphocyte lineage) were treated with sub-lethal concentrations of palmitic acid (PA) (50, 100 e 150µM) and the cell death and survival signaling pathways were investigated. PA induced apoptosis in a dose dependent manner activating the intrinsic pathway. Jurkat cells exposed to PA showed increased insulin receptor and GLUT-4 expression and a positive correlation with apoptosis was obtained. Similar effect was observed in human lymphocytes exposed to PA and lymphocytes from fasted rats. Insulin treatment of Jurkat cells after PA exposure promoted the activation of the insulin signaling pathway. Glucose uptake was increased in the presence of PA, however, its oxidation was diminished and ncreased of lipids accumulation. Therefore, insulin signaling and non oxidative glucose metabolism are stimulated as a part of a coordinated response to survival of lymphocytes exposed to low concentration of PA but in high concentration it induces apoptosis.

Aging is a multifactoral biological process of progressive and deleterious changes partially attributed to a build up of oxidatively damaged biomolecules resulting from attacks by free radicals. Methionine sulfoxide reductases (Msrs) are enzymes that repair oxidized methionine (Met) residues found in proteins. Oxidized Met produces two enantiomers, Met-S-(o) and Met-R-(o), reduced by MsrA and MsrB respectively. Unlike other model organisms, our MsrA null fly mutant did not display increased sensitivity to oxidative stress or shortened lifespan, suggesting that in Drosophila, having either a functional copy of either Msr is sufficient. Here, two Msr mutant types were phenotypically assayed against isogenic controls. Results suggest that only the loss of both MsrA and MsrB produces increased sensitivity to oxidative stress and shortened lifespan, while locomotor defects became more severe with the full Msr knockout fly. / by Kelli Robbins. / Thesis (M.S.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web.

Muitos autores associam as doenças alcoólicas hepáticas às deficiências nutricionais. Por outro lado, trabalhos experimentais estabelecem que a hepatotoxidade alcoólica relaciona-se especialmente à geração de espécies reativas através do sistema microsomal que oxida etanol via citocromo 450, principalmente o CYP2E1. O CYP2E1 hepático tem a capacidade de ativar algumas drogas comumente utilizadas, como o acetaminofeno, em seus metabólitos mais tóxicos e promover carcinogênese. Além. disso, o metabolismo pelo CYP2E1 resulta num aumento na produção de espécies reativas, com diminuição nos sistemas de defesa antioxidantes, estabelecendo o estresse oxidativo. Como a expressão do CYP2E1 é muito influenciada por fatores nutricionais e hormonais, este trabalho descreve os efeitos do tratamento com etanol nos níveis de CYP2E1 e sua relação com alguns parâmetros pró- e antioxidantes, considerando três modelos experimentais diferentes. Ratos machos Sprague Dawley com cerca de três meses de idade receberam ad lib. ração Purina (Purina Ind., Brasil) e, separadamente, uma solução 25 % etanol-20%sacarose durante 1, 2, 3 ou 4 semanas. Os grupos controles foram isocaloricamente pareados aos animais que consumiram etanol, ou receberam quantidades de sacarose equivalentes às calorias recebidas com o consumo de etanol. 18 h antes do sacrifício os animais foram mantidos em abstinência alcoólica, recebendo água e ração ou apenas água ad lib. Os resultados indicam que o consumo de etanol pode ser associado à estabilização do CYP2E1. No entanto, nas nossas condições experimentais, a presença da isoforma não está associada ao estresse oxidativo. Esses resultados indicam que as deficiências nutricionais, especialmente o baixo consumo de carboidratos, são fundamentais na potenciação do estresse oxidativo induzido pelo etanol. / Many authors have attributed alcoholic liver disease to dietary deficiencies. On the other hand, experimental studies have established that alcohol hepatotoxicity is especially related to the generation of oxidant species through its microsomal metabolism via cytochrome P-450, mainly CYP2E1. Liver CYP2E1 has a high capacity to activate some commonly used drugs, such as acetaminophen, to their toxic metabolites, and to promote carcinogenesis. Moreover, metabolism by CYP2E1 results in a significant reactive oxygen species (ROS) release, accompanied by the defense systems decrease against oxidative stress. Since the expression of CYP2E1 is very much influenced by hormonal and nutritional factors, this study describes the effects of ethanol treatment on CYP2E 1 levels and their relationship with some pro and antioxidant parameters considering three experimental models. Male Sprague-Dawley rats were fed ad. lib. for 1, 2, 3 or 4 weeks a commercial diet (Purina Ind., Brazil) plus a 25% ethanol-20% sucrose solution. Control groups were isocalorically pair-feed to the leading ethanol-consuming animals, or received isocaloric amounts of sucrose for pairing only ethanol calories. Eighteen hours before sacrifice ethanol was withdrawal and animals had only free access to tap water or they were offered food and water ad. lib. Results have shown that ethanol administration was associated with CYP2E1 stabilization although under our experimental condition it was not associated with any oxidative stress. These findings indicate that dietary deficiencies, especially low carbohydrate intake are crucial in the potentiation of the ethanol-induced oxidative stress.

A metformina é uma droga utilizada para tratamento da diabetes tipo 2. O PPAR-&alpha; tem um papel central no controle imunometabólico. Portanto, o objetivo do estudo foi avaliar os efeitos imunometabólicos da dieta hiperlipídica (HFD), em camundongos C57BL6 (WT) e knockout para PPAR-&alpha;, tratados com metformina. Métodos: Os animais foram submetidos a uma HFD por 12 semanas, nos últimos dez dias de dieta os animais foram tratados com metformina. A oxidação de palmitato no músculo esquelético, As citocinas, no fígado, no tecido adiposo retroperitoneal, em hepatócitos e macrófagos intraperitoneais foram analisados. Resultados: O tratamento aumentou a oxidação de palmitado no músculo, promoveu um efeito anti-inflamatório no tecido adiposo e reverteu à inflamação dos macrófagos. No fígado e nos hepatócitos, a metformina causou um efeito inflamatório. Conclusão: A inflamação hepática foi induzida pelo tratamento e o efeito principal foi a um potencial aumento na inflamação nos hepatócitos. Os macrófagos tiveram uma resposta anti-inflamatória, assim como o tecido adiposo. / Metformin is a drug used to treatment of type 2 diabetes. PPAR-&alpha; plays a central role in immunometabolic control. Therefore, the aim of the study was to evaluate the effects of imumnometabolics of high fat diet (HFD) in C57BL6 mice (WT) and knockout for PPAR-&alpha; treated with metformin. Methods: The animals were subjected to a HFD for 12 weeks in the last ten days of diet the animals were treated with metformin. The palmitate oxidation in skeletal muscle, cytokines in the liver, in the retroperitoneal adipose tissue, hepatocytes and intraperitoneal macrophages were analyzed. Results: The treatment increased palmitate oxidation in muscle, it has promoted an anti-inflammatory effect in adipose tissue and macrophages to inflammation reversed. In the liver and hepatocytes, metformin caused an inflammatory effect. Conclusion: The liver inflammation was induced, and treatment was a main effect to a potential increase in inflammation in hepatocytes. Macrophages have an anti-inflammatory response, as well as adipose tissue.

Ácidos graxos podem atuar na apoptose ativando e/ou desativando sinais que regulam este processo. Células Jurkat (linhagem de linfócitos-T) foram tratadas com ácido palmítico (PA) (50, 100 e 150µM) e avaliada a ativação de vias de morte e de sobrevivência. O tratamento com PA induziu apoptose pela ativação da via intrínseca de maneira dose-dependente. A exposição destas células ao PA elevou a expressão do receptor de insulina e de GLUT-4, obtendo-se correlação positiva com a apoptose. O mesmo foi observado em linfócitos humanos expostos ao PA e de linfócitos de ratos em jejum. O tratamento das células Jurkat com insulina após exposição ao PA promoveu a ativação da via de sinalização insulínica. O PA aumentou a captação de glicose, mas observou-se diminuição de sua oxidação e o acúmulo de lípides. Assim, a via de sinalização da insulina e o metabolismo de glicose não oxidativo são estimulados como parte de uma coordenada resposta de sobrevivência de linfócitos expostos ao PA em baixas concentrações, mas em altas concentrações ocorre apoptose. / Fatty acids affect apoptosis pathway activating or deactivating signals that regulate this process. Jurkat cells (T-lymphocyte lineage) were treated with sub-lethal concentrations of palmitic acid (PA) (50, 100 e 150µM) and the cell death and survival signaling pathways were investigated. PA induced apoptosis in a dose dependent manner activating the intrinsic pathway. Jurkat cells exposed to PA showed increased insulin receptor and GLUT-4 expression and a positive correlation with apoptosis was obtained. Similar effect was observed in human lymphocytes exposed to PA and lymphocytes from fasted rats. Insulin treatment of Jurkat cells after PA exposure promoted the activation of the insulin signaling pathway. Glucose uptake was increased in the presence of PA, however, its oxidation was diminished and ncreased of lipids accumulation. Therefore, insulin signaling and non oxidative glucose metabolism are stimulated as a part of a coordinated response to survival of lymphocytes exposed to low concentration of PA but in high concentration it induces apoptosis.