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Title: EFFECTS OF GHRKO VISCERAL FAT TRANSPLANT OF INSULIN SIGNALING.
Creator: Bennis, Mohammed, Masternak, Michal, University of Central Florida
Abstract / Description: Insulin sensitivity has been positively correlated with a healthy and extended lifespan, while insulin resistance, decreased insulin sensitivity, has been linked to aging and is the main indicative of type 2 diabetes. Growth Hormone Receptor/ Binding Protein Knockout mice (GHRKO), although obese, are characterized by high insulin sensitivity and a prolonged lifespan. Due to the absence of growth hormone receptors (GHR), growth hormone (GH) is unable to activate its downstream pathway....
Show moreInsulin sensitivity has been positively correlated with a healthy and extended lifespan, while insulin resistance, decreased insulin sensitivity, has been linked to aging and is the main indicative of type 2 diabetes. Growth Hormone Receptor/ Binding Protein Knockout mice (GHRKO), although obese, are characterized by high insulin sensitivity and a prolonged lifespan. Due to the absence of growth hormone receptors (GHR), growth hormone (GH) is unable to activate its downstream pathway. Interestingly, the secretory activity of visceral fat in GHRKO mice is altered stimulating insulin sensitivity. In this study, we transplanted normal (N) mice with GHRKO visceral fat pads to determine the role of visceral fat developed with the absence of GH signaling on the insulin-signaling pathway in animals with physiologically normal GH action. We found that the visceral fat transplant (VFT) helped the normal mice gain the beneficial effects of fat developed in the absence of GH and caused improvement of their whole body insulin sensitivity when comparing with sham-operated mice and with mice that received visceral fat from N animals. In presented study, RT-PCR was used to determine the levels of hepatic mRNA expression between three experimental groups including Normal-sham mice (N-S), normal mice transplanted with visceral fat from normal animals (N-N), and normal mice receiving visceral fat from GHRKO mice (N-KO). Additionally, Western Blot and ELISA were used to determine the level of total and phosphorylated proteins. By studying the effect of visceral fat transplant from GHRKO or N mice on the whole body insulin signaling in N male mice, and testing different genes expression and proteins quantification, we can shed light on the mechanism by which white adipose tissue (WAT) regulates whole body insulin sensitivity and longevity as well as understanding the role of WATs in development of diabetes and the process behind insulin resistance.
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Date Issued: 2015
Identifier: CFH0004781, ucf:45372
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFH0004781

Title: THE EFFECTS OF GROWTH HORMONE AND THYROXINE TREATMENT ON THE INSULIN SIGNALING OF FEMALE AMES DWARF MOUSE SKELETAL MUSCLE TISSUE.
Creator: Do, Andrew, Masternak, Michal, University of Central Florida
Abstract / Description: Ames dwarf (df/df) mice are deficient in anterior pituitary hormones: growth hormone (GH), thyroid stimulating hormone (TSH), and prolactin (PRL) due to a spontaneous, homozygous mutation of prop1df gene. These dwarf mice exhibit characteristics such as delayed growth and development coupled with delayed aging, increased lifespan, overall increased insulin sensitivity, as well as resistance to certain diseases and cancers. The mutant mice possess low blood glucose, low serum insulin, and...
Show moreAmes dwarf (df/df) mice are deficient in anterior pituitary hormones: growth hormone (GH), thyroid stimulating hormone (TSH), and prolactin (PRL) due to a spontaneous, homozygous mutation of prop1df gene. These dwarf mice exhibit characteristics such as delayed growth and development coupled with delayed aging, increased lifespan, overall increased insulin sensitivity, as well as resistance to certain diseases and cancers. The mutant mice possess low blood glucose, low serum insulin, and lower body temperature. Their enhanced longevity (about 40-60% longer lifespan than normal mice) is associated with their GH deficiency and disruption in the somatotropic axis (GH/IGF-1 hormonal pathway) as well as increased insulin sensitivity, which is supported by other mutant mouse models for longevity like Snell dwarfs and growth hormone receptor knock-out (GHRKO) mice. When young male Ames dwarf mice were treated with GH replacement therapy, they showed increased body growth to nearly match the normal mouse phenotype. In conjunction to an increase in physical growth, however, GH treatment also decreases the longevity and insulin sensitivity that are characteristic of these mice to levels seen in normal mice. Because of the lack of TSH, they also have undetectable levels of Thyroxine (T4). While T4 treatment didn't increase bodyweight of dwarfs to the same extent as GH treatment, the T4 treated mice retained their enhanced lifespan. Although df/df mice have enhanced whole-body insulin sensitivity, the male skeletal muscle was previously shown to be less responsive to insulin than their liver. In our study we analyzed the insulin signaling pathway in skeletal muscle from female mice after treatment with GH or GH combined with T4. Gene expression and protein expression were investigated in the skeletal muscle of female Ames dwarf mice that were treated with GH or GH and T4 therapy. Real Time Polymerase Chain Reaction (RT-PCR) was used to analyze the expression of mRNA involved with insulin and GH signaling, while western blots were used to analyze protein expression. This project found that female Ames skeletal muscle didn't respond to GH treatment to the same extent as males, and that GH and T4 treatment tends to neutralize the effects seen in GH-only treatment.
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Date Issued: 2013
Identifier: CFH0004483, ucf:45074
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFH0004483

Title: Malondialdehyde (MDA) and Glutathione Peroxidase (GPx) are elevated in Crohns disease-associated with Mycobacterium avium subspecies paratuberculosis (MAP).
Creator: Qasem, Ahmad, Naser, Saleh, Masternak, Michal, Parthasarathy, Sampath, Andl, Claudia, University of Central Florida
Abstract / Description: Inflamed tissue in Crohn's disease (CD) are continuously producing toxic oxygen metabolites leading to cellular injury and apoptosis. Here, we are evaluating the role of Mycobacterium avium subspecies paratuberculosis (MAP) in oxidative stress in CD by evaluation of lipid peroxidation and antioxidant defense activity. Specifically, we measured malondialdehyde (MDA) level and selenium-dependent glutathione peroxidase (GPx) activity in the plasma from patients and cattle infected with MAP. The...
Show moreInflamed tissue in Crohn's disease (CD) are continuously producing toxic oxygen metabolites leading to cellular injury and apoptosis. Here, we are evaluating the role of Mycobacterium avium subspecies paratuberculosis (MAP) in oxidative stress in CD by evaluation of lipid peroxidation and antioxidant defense activity. Specifically, we measured malondialdehyde (MDA) level and selenium-dependent glutathione peroxidase (GPx) activity in the plasma from patients and cattle infected with MAP. The level of MAP antibodies in bovine sera was determined by IDEXX kit whereas detection of MAP DNA was performed by IS900-based nPCR. A total of 42 cattle (21 infected with MAP and 21 healthy controls), 27 CD subjects, 27 of CD-healthy relatives, 66 subjects with various diseases and 34 non-related healthy subjects were investigated. Overall, GPx activity was significantly higher in MAP infected humans (0.80941(&)#177;0.521) versus MAP (-ve) samples (0.42367(&)#177;0.229 units/ml), P(<)0.01. Similarly, the average of GPx activity in cattle infected with MAP was 1.59(&)#177;0.65 units/ml compared to 0.46907(&)#177;0.28 units/ml in healthy cattle (P(<)0.01). Although it was not statistically significant, MDA average level was higher in MAP infected human samples versus MAP (-ve) controls (1.11(&)#177;0.185 nmol/ml versus 0.805(&)#177;0.151 nmol/ml, respectively). Similarly, MDA average level in CD samples that are MAP+ (1.703(&)#177;0.231 nmol/ml) was higher than CD samples that are MAP (-ve) (1.429(&)#177;0.187 nmol/ml). In cattle, MDA average level in MAP infected samples was significantly higher at 3.818(&)#177;0.45 nmol/ml compared to 0.538(&)#177;0.18 nmol/ml in healthy cattle (P(<)0.01). Clearly, the data demonstrated that MAP infection is associated with oxidative stress and resulting in the pathophysiology of worsening of the condition of CD patients.
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Date Issued: 2016
Identifier: CFE0006699, ucf:51906
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006699

Title: Characterization of neural cells derived from reelin-deficient schizophrenic patient iPS cells.
Creator: Roberts, Nicole, Sugaya, Kiminobu, Ebert, Steven, Masternak, Michal, University of Central Florida
Abstract / Description: Reelin is a large, extracellular glycoprotein that binds to several membrane receptors on neural stem cells (HNSCs), neural progenitor cells (NPCs), and neuroblasts of mammals to direct their migration. Previously, our lab established the presence of Reelin increased migration of wild-type fetal-derived HNSC's, both in vitro and in vivo. In addition, we demonstrated that Reelin protein treatment also increases the formation of radial glia via Notch-1 signaling, in vitro. Radial glia are...
Show moreReelin is a large, extracellular glycoprotein that binds to several membrane receptors on neural stem cells (HNSCs), neural progenitor cells (NPCs), and neuroblasts of mammals to direct their migration. Previously, our lab established the presence of Reelin increased migration of wild-type fetal-derived HNSC's, both in vitro and in vivo. In addition, we demonstrated that Reelin protein treatment also increases the formation of radial glia via Notch-1 signaling, in vitro. Radial glia are precursors to NPCs, as well as a scaffold for neuroblast migration during cortical lamination. Reelin has long been associated with Schizophrenia (SZ). Because post-mortem brains are limited to describing the end-point of the disease, heterozygous haplodeficient Reelin knock-out (Reeler) mice are used to model developmental aspects of SZ in vivo. However, SZ is a complex, polyfactoral disease with a myriad of dysfunctional pathways that may have unforeseen effects on Reelin signaling. K. Brennand et al. (2014) reported low Reelin mRNA expression and cellular characteristics mirroring the Reeler mouse in induced pluripotent stem (iPS) cell-derived NPCs and neurons from living SZ patients. Building upon this and our work with stem cells, here we consider Reelin's effects on migration of Reelin-deficient iPS cell-derived NPCs. Reelin treatment of consists of secreted Reelin from transfected human embryonic kidney 293 cells (HEK 293) with the pCRL RELN gene-containing plasmid created by G. D'Arcangelo (1997) and given to us by T. Curran. Using the metric of cellular migration, this is the first time it have been shown that SZ iNPCs are capable of receiving and reacting to extracellular Reelin. Due to our validation of this model, further work using iPS cell-derived neural cells can confidently be used for future disease modeling and drug discovery of Reelin-deficient SZ.
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Date Issued: 2018
Identifier: CFE0007361, ucf:52091
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0007361

Title: Embryonic Stem Cell-Derived Exosomes Inhibit Doxorubicin-Induced Pyroptosis in Cell Culture Models.
Creator: Tavakoli Dargani, Zahra, Singla, Dinender, Masternak, Michal, Siddiqi, Shadab, Steward, Robert, University of Central Florida
Abstract / Description: Doxorubicin (Dox) is a potent chemotherapeutic drug used for the treatment of various cancers. Unfortunately, its use is limited as Dox induces adverse cardiotoxicity (DIC) and muscle toxicity (DIMT), which are mediated through oxidative stress, ER stress, and inflammation. However, it remains unknown whether Dox induces an inflammation mediated cell death, called (")pyroptosis("). The current study is designed to determine whether Dox induces pyroptosis in cardiac and muscle cell culture...
Show moreDoxorubicin (Dox) is a potent chemotherapeutic drug used for the treatment of various cancers. Unfortunately, its use is limited as Dox induces adverse cardiotoxicity (DIC) and muscle toxicity (DIMT), which are mediated through oxidative stress, ER stress, and inflammation. However, it remains unknown whether Dox induces an inflammation mediated cell death, called (")pyroptosis("). The current study is designed to determine whether Dox induces pyroptosis in cardiac and muscle cell culture models. Moreover, the protective effects of embryonic stem cell-derived exosomes (ES-Exos) in inhibiting pyroptosis will also be determined. For this purpose, we designed two different cell culture models using H9c2 cadiomyoblasts and Sol 8 cells. For the DIC model, H9c2 were exposed to Dox to induce pyroptosis and then treated with exosomes. Cells were divided into 4 groups: Control, Dox, Dox+ES-Exos, and Dox+MEF-Exos (negative control). Furthermore, to generate the DIMT model, Sol 8 cells were incubated with Dox+THP-1 conditioned medium (TCM) to induce toxicity and inflammation, which was followed by exosomes treatment. We assigned cells into 5 groups: Control, Dox+TCM, Dox+TCM+ES-Exos, Dox+TCM+MEF-Exos (negative control), and Dox+TCM+ES-Exos+GW4869 compound (exosomes inhibitor, negative control). Our data shows that Dox treatment significantly increased pyroptotic marker expression including TLR-4, NLRP3, caspase-1, IL1-?, Caspase-11, and gasdermin-D as well as increased pro-inflammatory TNF-? and IL-6 expression in H9c2 cells. There was also a significant increase in caspase-1, IL1-?, and IL-18 expression in Dox+TCM treated Sol 8 cells. Conversely, increased pyroptosis and inflammation post-Dox treatment were inhibited by ES-Exos in both culture models. No significant changes observed upon MEF-Exos and GW4869 compound treatments. In conclusion, our data shows Dox induces pyroptosis and inflammation within cardiac and skeletal muscle cells, which can be inhibited following treatment with ES-exosomes. This is a novel study with new mechanistic observations on the pathophysiological role of pyroptosis in Dox-induced cardio and muscle toxicities.
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Date Issued: 2018
Identifier: CFE0007416, ucf:52700
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0007416

Title: Role of Lipid Peroxide Derived Dicarboxylic Acids in Atherosclerotic Calcification.
Creator: Riad, Aladdin, Parthasarathy, Sampath, Altomare, Deborah, Masternak, Michal, Naser, Saleh, University of Central Florida
Abstract / Description: Cardiovascular diseases, including atherosclerosis, are the leading cause of death in the United States. Atherosclerotic lesions are formed by deposition of lipids in the intima of arteries. Upon exposure to oxidative stresses, low-density lipoprotein (LDL) is converted to highly atherogenic oxidized LDL (ox-LDL) particles, contributing to disease development and progression. Advanced disease stages may result in calcification of lesions. This calcification process is important, as it has...
Show moreCardiovascular diseases, including atherosclerosis, are the leading cause of death in the United States. Atherosclerotic lesions are formed by deposition of lipids in the intima of arteries. Upon exposure to oxidative stresses, low-density lipoprotein (LDL) is converted to highly atherogenic oxidized LDL (ox-LDL) particles, contributing to disease development and progression. Advanced disease stages may result in calcification of lesions. This calcification process is important, as it has been shown to be associated with stable plaques that are less prone to rupture. Calcification is present in lipid rich domains of lesions, however neither the composition of the mineralized calcium deposits nor its relationship to lipid peroxidation or the lipid rich atherosclerotic core has previously been identified. This study provides evidence that the lipid peroxide derived dicarboxylic acid (DCA), azelaic acid (AzA) induces calcification in smooth muscle cells, thereby providing the link between calcification and overall plaque burden, and association of calcification with the lipophilic region of the lesion. The potential of lipid peroxide-derived lipophilic DCAs to promote calcification upon exposure to vascular smooth muscle cells was tested. 13-hydroperoxylinoleic acid (HPODE) treatment resulted in the cellular conversion to 9-oxononanoic acid (ONA) and AzA as determined by mass spectrometry analysis. Delivery of AzA via lysophosphatidylcholine (Lyso-PtdCho) micelles induced calcification of human aortic smooth muscle cells (HASMC). AzA was identified in calcified human and mouse atherosclerotic plaques. Calcification of HASMC due to AzA treatment resulted in a less inflammatory and oxidative environment as indicated by genetic expression. These results demonstrate that DCAs may contribute to atherosclerotic calcification thus accounting for the latter's relationship to plaque burden and association with lipids. This study also challenges the dogma that arterial calcification represents the deposition of calcium phosphate and has implications with the development of new therapeutic strategies in treating late stage atherosclerosis.
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Date Issued: 2018
Identifier: CFE0007413, ucf:52730
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0007413

Title: Role of Kruppel-like Factor 8 (KLF8) in Cancer and Cardiomyopathy.
Creator: Lahiri, Satadru, Zhao, Jihe, Parthasarathy, Sampath, Masternak, Michal, Siddiqi, Shadab, University of Central Florida
Abstract / Description: Cancer and cardiovascular diseases are two most fatal diseases causing innumerable death each year. Understanding the mechanisms underlying these diseases is critical for developing proper therapeutic approach. Kr(&)#252;ppel-like factor 8 (KLF8) is a member of Kr(&)#252;ppel-like family transcription factors that is overexpressed in many types of cancers. There is no report on role of KLF8 in cardiovascular diseases to date. KLF8 transcriptionally activates or represses a host of target...
Show moreCancer and cardiovascular diseases are two most fatal diseases causing innumerable death each year. Understanding the mechanisms underlying these diseases is critical for developing proper therapeutic approach. Kr(&)#252;ppel-like factor 8 (KLF8) is a member of Kr(&)#252;ppel-like family transcription factors that is overexpressed in many types of cancers. There is no report on role of KLF8 in cardiovascular diseases to date. KLF8 transcriptionally activates or represses a host of target genes to promote cancer cell proliferation, migration, invasion and epithelial to mesenchymal transition during tumor progression. Studies proposed in this thesis identified a novel posttranslational modification of KLF8 essential for its role in promoting cancer cell migration and discovered a novel function of KLF8 in cardiomyopathy. In our first study, we identified serine 48 (S48) as a novel phosphorylation site on KLF8. Pharmacological and genetic manipulations of various potential kinases further revealed ERK2 as the kinase responsible for this novel phosphorylation. Functional studies indicated that this phosphorylation is crucial for protecting KLF8 protein from degradation in the nucleus and promoting cancer cell migration. Preclinical xenograft models have indicated an important role of KLF8 for tumor progression. To investigate role of KLF8 in spontaneous tumorigenesis better recapitulating pathology in patients, we established the first Cre-regulated conditional KLF8 transgenic mouse model. Upon induction of global expression of the KLF8 transgene, spontaneous mammary and testicular tumors were formed in a small population of the mice by their mid-age, as expected considering the long latency required for tumor progression. Surprisingly, however, nearly 100% of KLF8 the mice died with a significantly enlarged heart, which did not occur to any littermate control mouse. Further characterization of the mice revealed that the global expression of the transgene caused striking systolic dysfunction leading to fatal dilated cardiomyopathy. Importantly, these similar phenotypes were reproduced in heart-specific KLF8 transgenic mice. Cardiovascular disease PCR array identified a number of genes potentially mediating KLF8-induced cardiac pathology. These results identified a previously unimagined function of KLF8 in the heart, shed new light on the mechanisms of cardiac diseases and provide novel preclinical mouse models for future translational research.
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Date Issued: 2016
Identifier: CFE0006692, ucf:51914
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006692

Title: Apolipoprotein-AI Regulates Hepatic VLDL Secretion by Controlling Intracellular VLDL-Trafficking.
Creator: Gurwani, Bhavesh, Siddiqi, Shadab, Masternak, Michal, Naser, Saleh, University of Central Florida
Abstract / Description: Cardiovascular diseases cause 17 million deaths annually, which is estimated to increase to 23 million deaths by the year 2030. One of the major risk factors for the pathogenesis of cardiovascular diseases is increased secretion of very-low density lipoproteins (VLDL) by the liver; however, reduced VLDL-secretion causes fatty liver disease. Synthesis and secretion of VLDL by the liver plays an important role in maintaining overall lipoprotein homeostasis. Assembly of VLDL occurs along with...
Show moreCardiovascular diseases cause 17 million deaths annually, which is estimated to increase to 23 million deaths by the year 2030. One of the major risk factors for the pathogenesis of cardiovascular diseases is increased secretion of very-low density lipoproteins (VLDL) by the liver; however, reduced VLDL-secretion causes fatty liver disease. Synthesis and secretion of VLDL by the liver plays an important role in maintaining overall lipoprotein homeostasis. Assembly of VLDL occurs along with the expression of apolipoproteinB-100 (apoB100) and its lipidation at the endoplasmic reticulum (ER) level. Once formed in the ER lumen, the nascent VLDL is transported to the Golgi for its maturation. In the Golgi compartment, the nascent VLDL acquires apolipoproteinAI (apoAI), more triglycerides, and its apoB100 undergoes phosphorylation and glycosylation. These modifications are necessary for VLDL-exit from the trans-Golgi network (TGN) and this step is mediated by post-Golgi VLDL transport vesicle (PG-VTV). The transport of mature VLDL from the TGN to the plasma membrane (PM) is required for its secretion by the liver but remains to be studied. Our group has shown that the nascent VLDL particles do not contain apoAI, however, VLDL acquires apoAI in the cis-Golgi compartment. Interestingly, apoAI comes off the VLDL as soon as VLDL is secreted into the blood. We hypothesised that apoAI plays an important role in post-TGN VLDL trafficking and thus controls VLDL secretion by the liver. To determine the role of apoAI in the formation of PG-VTV and VLDL secretion, we knocked down apoAI in the hepatocytes using apoAI specific siRNA. The deficiency of apoAI did not have any effect on the expression of apoB100 and other apolipoprotein synthesis that are involved in VLDL synthesis; however, VLDL secretion was significantly reduced. Next, we overexpressed apoAI using plasmid with apoAI gene sequence and checked for the effects in VLDL secretion from the hepatocytes. We observed a significant increase in VLDL secretion from apoAI-overexpressing hepatocytes which is consistent with knockdown results. To determine the role of apoAI in post-TGN trafficking of the mature VLDLs, we isolated sub-cellular organelles from apoAI knockout (apoAI KO) and control mice. Subsequently, we performed in vitro PG-VTV budding assays to assess the effect of apoAI silencing on PG-VTV formation from the TGN. Our results strongly suggest that the deficiency of apoAI increases PG-VTV formation (i.e. TGN-exit of mature VLDL) but significantly reduces VLDL-triglyceride secretion from the hepatocytes. We conclude that apoAI controls VLDL secretion by the liver by regulating post-TGN trafficking of mature VLDL.
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Date Issued: 2016
Identifier: CFE0006685, ucf:51908
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006685

Title: Metabolic Effects of 17a-Estradiol are Growth Hormone Independent and Sex Specific.
Creator: Sidhom, Silvana, Masternak, Michal, Altomare, Deborah, Siddiqi, Shadab, University of Central Florida
Abstract / Description: Aging is a major risk factor for metabolic syndromes and type two diabetes. With growing elderly populations worldwide and increasing incidence of age-related diseases there is a great need to develop pharmacological interventions that would delay aging and protect from age-related diseases. 17-alpha estradiol (17?-E2) is an epimer of the primary female sex hormone estradiol and has been shown to extend lifespan and downregulate markers of age-related metabolic dysfunction in male mice....
Show moreAging is a major risk factor for metabolic syndromes and type two diabetes. With growing elderly populations worldwide and increasing incidence of age-related diseases there is a great need to develop pharmacological interventions that would delay aging and protect from age-related diseases. 17-alpha estradiol (17?-E2) is an epimer of the primary female sex hormone estradiol and has been shown to extend lifespan and downregulate markers of age-related metabolic dysfunction in male mice. Because 17?-E2 does not induce feminization in males it holds potential as a novel therapeutic in humans for age-related metabolic dysfunction. Importantly, we have previously shown that 17?-E2 causes an increase of circulating and hepatic IGF-1 in aged mice, without any changes in GH release in treated animals. Based on this we propose a new hypothesis that 17?-E2 acts through a novel, GH-independent pathway stimulating production of IGF-1 and positively modulating metabolic function in a sex-specific manner. Here we studied 17?-E2 treated long-lived growth hormone receptor knockout (GHRKO) mice, characterized by severely reduced circulating and hepatic IGF-1 due to GH-resistance. We found increases in circulating IGF-1 after treatment in normal and GHRKO male mice, with no effect in female mice, which supports our hypothesis that 17?-E2 induces GH independent IGF-1 production. To determine novel genetic pathways activated by 17?-E2 we performed sequencing of hepatic RNA. Our analysis indicated differential regulation of steroid biosynthesis and insulin signaling pathways. The validation of our sequencing data using qPCR showed significant upregulation of genes involved in insulin action. Importantly, differential regulation of these pathways was present in normal male mice, with no changes in normal females or either male or female GHRKO animals. In summary, this new data supports our hypothesis of a sex-specific effect of 17?-E2 treatment and differing mechanisms of action by which 17?-E2 upregulates IGF-1 independently of GH action.
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Date Issued: 2019
Identifier: CFE0007726, ucf:52424
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0007726

Title: Embryonic Stem Cell Derived Exosomes Enhance Cardiac Stem Cell Differentiation into Heart Cells.
Creator: Hammond, Jamillah, Singla, Dinender, Masternak, Michal, Davidson, Victor, University of Central Florida
Abstract / Description: Transplantation of embryonic stem (ES) cells into the ischemic and infarcted heart has proven to repopulate cardiac cell populations, attenuate structural cardiac remodeling, and rescue cardiac function. Unfortunately, the pluripotency of ES cells increases risk of teratoma formation in vivo. Exosomes, smaller in comparison to ES cells, are cell free carriers of miRNA, proteins, and lipids, and do not suggest risk of teratoma formation. Exosomes have been proposed to mediate and attenuate...
Show moreTransplantation of embryonic stem (ES) cells into the ischemic and infarcted heart has proven to repopulate cardiac cell populations, attenuate structural cardiac remodeling, and rescue cardiac function. Unfortunately, the pluripotency of ES cells increases risk of teratoma formation in vivo. Exosomes, smaller in comparison to ES cells, are cell free carriers of miRNA, proteins, and lipids, and do not suggest risk of teratoma formation. Exosomes have been proposed to mediate and attenuate regeneration following myocardial infarction (MI), however, the role of exosomes derived from ES cells (ES-Exo) in activating resident cardiac stem cells (CSCs) to undergo cardiac differentiation is not established. In the present study, Stem cell antigen 1 positive (Sca-1+ve) CSCs were isolated, incubated with exosomes, and evaluated for differentiation into the major heart cell types in vitro. Observations of in vitro cardiac differentiation were further established in an in vivo model of MI. Ligation of the coronary artery, or a sham surgery was performed in C57BL/6 mice 8-12 weeks of age. Mice were split among four study groups: sham, MI, MI + H9c2-Exo (a cell line control), (&) MI + ES-Exo. ES-Exo were transplanted via intramyocardial (IM) injection immediately following coronary artery ligation. At day 14 (D14), echocardiography was used to evaluate cardiac function. Differentiation into the major heart cells was determined by sarcomeric ?-actin (cardiomyocytes) and smooth muscle ?-actin (vascular smooth muscle cells) immunostaining. Hematoxylin and Eosin and Masson's Trichrome staining assessed cardiomyocyte hypertrophy and fibrosis, respectively. Immunostaining for major heart cellular markers revealed significant activation of resident Sca-1+ve CSCs to undergo cardiac differentiation after ES-Exo treatment. Cardiomyocyte hypertrophy and myocardial fibrosis were significantly increased following coronary artery ligation. Results from histological staining revealed significantly decreased levels of hypertrophy and fibrosis in hearts transplanted with ES-Exo following coronary ligation. In summary, our findings advocate ES-Exo as a viable treatment option to repopulate the myocardium with viable heart cells, attenuate cardiac remodeling, and rescue cardiac function.
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Date Issued: 2018
Identifier: CFE0007188, ucf:52254
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0007188

Title: Alpha-Tocopherol Reduces VLDL Secretion Through Modulation of the VLDL Transport Vesicle.
Creator: Clay, Ryan, Siddiqi, Shadab, Altomare, Deborah, Masternak, Michal, University of Central Florida
Abstract / Description: The liver distributes serum triacylglycerol (TAG) via the very low-density lipoprotein (VLDL), and an increase in VLDL production may result in hyperlipidemia. VLDL synthesis consists of lipidation of Apolipoprotein B100 (ApoB) as it is co- translationally translocated across the endoplasmic reticulum (ER) membrane, and this nascent VLDL particle must undergo subsequent maturation and post-translational modification in the Golgi. The ER-to-Golgi trafficking of VLDL represents the rate...
Show moreThe liver distributes serum triacylglycerol (TAG) via the very low-density lipoprotein (VLDL), and an increase in VLDL production may result in hyperlipidemia. VLDL synthesis consists of lipidation of Apolipoprotein B100 (ApoB) as it is co- translationally translocated across the endoplasmic reticulum (ER) membrane, and this nascent VLDL particle must undergo subsequent maturation and post-translational modification in the Golgi. The ER-to-Golgi trafficking of VLDL represents the rate-limiting step in VLDL secretion and is mediated by the VLDL Transport Vesicle (VTV). Many in vivo studies have indicated that vitamin E (alpha-tocopherol) supplementation protects against atherosclerosis and can reduce hepatic steatosis in nonalcoholic fatty liver disease (NAFLD), but its effects at the molecular level on hepatic lipid metabolism are poorly understood. To investigate the effects of alpha-tocopherol on hepatic VLDL secretion and cellular lipid retention, we performed several experiments in HepG2 (human) and McARH- 7777 (rat) hepatoma cell lines including pulse-chase experiments using 3H-oleic acid (3H- OA), confocal microscopy with BODIPY lipid droplet staining, and an in vitro VTV budding assay. Our results demonstrate a significant reduction of 3H-TAG secretion and ApoB media expression in response to 100 uM alpha-tocopherol, with a corresponding decrease in markers of VTV biogenesis in western blots of whole cell lysates (WCL) and retention of ApoB within the cell, indicating disruption of an early step in VLDL biogenesis. Further evidence indicates an increase in size and lipidation of the VTV and VLDL particle. BODIPY staining as well as 3H-TAG retention in WCLs was also sharply reduced. Overall, these results indicate that alpha-tocopherol reduces VLDL secretion, partially disrupts hepatic VLDL synthesis and VTV biogenesis, increases the lipidation of remaining VLDL particles, and diminishes overall cellular lipid droplet retention.
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Date Issued: 2019
Identifier: CFE0007617, ucf:52538
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0007617

Title: Diabetes Phenotypes in Transgenic Pancreatic Cancer Mouse Models.
Creator: Albury, Toya, Altomare, Deborah, Zhao, Jihe, Masternak, Michal, Khaled, Annette, University of Central Florida
Abstract / Description: Protein Kinase B/AKT, a serine/threonine kinase with three isoforms (AKT1-3), is downstream of phosphatidylinositol 3-kinase (PI3K), and signals through the phosphorylation and subsequent activation or inhibition of downstream substrates, such as mammalian target of rapamycin complex 1 (mTORC1) or glycogen synthase kinase 3 beta (GSK-3?), respectively. The AKT1 isoform is predominantly recognized for regulation of cell survival, growth, and proliferation, due to its constitutive activation in...
Show moreProtein Kinase B/AKT, a serine/threonine kinase with three isoforms (AKT1-3), is downstream of phosphatidylinositol 3-kinase (PI3K), and signals through the phosphorylation and subsequent activation or inhibition of downstream substrates, such as mammalian target of rapamycin complex 1 (mTORC1) or glycogen synthase kinase 3 beta (GSK-3?), respectively. The AKT1 isoform is predominantly recognized for regulation of cell survival, growth, and proliferation, due to its constitutive activation in pancreatic cancers (e.g., islet cell carcinoma and pancreatic adenocarcinoma). The progression of pancreatic ductal adenocarcinoma (PDAC), the most lethal common cancer, is initiated by activation mutations of the KRas oncogene. This leads to additional molecular changes, such as activation of the AKT1 oncogene, which drives PDAC progression and tumor formation. By mating transgenic mice with activation of KRas (Pdx- Cre;LSL-KRasG12D) and mice with activation of AKT1 (Pdx- Tta;TetO-MyrAKT1) we were able to produce mice with two activated oncogenes (AKT1Myr/KRasG12D) for comparative studies. Kaplan-Meier survival curves, histology, and genomic/proteomic analysis were used to characterize the incidence and frequency of histological (e.g. presence of mucin-4 in pancreatic intraepithelial neoplasms) and genetic (e.g. loss of tumor suppressors p16Ink4a and p19Arf) alterations known to commonly occur in human pancreatic cancer, as well as delineate the role of AKT1 in accelerating pancreatic tumor progression and metastasis. We determined that AKT1Myr/KRasG12D mice, unlike other PDAC mouse models, accurately mimic the human PDAC progression molecularly, structurally, and temporally. Interestingly, the AKT1Myr and AKT1Myr/KRasG12D models both exhibit a pre-tumor, diabetic phenotype. While, AKT1 hyperactivation in various cancers has been thoroughly studied, its role in glucose metabolism has been noted, but comparatively overlooked. As early as the 1900s a relationship between diabetes and pancreatic cancer has been proposed. With 80% of PDAC patients suffering from hyperglycemia or diabetes prior to diagnosis, one prevailing theory is that new onset diabetes is an early marker for pancreatic cancer. This is also supported by experimental and clinical studies, such as the resolution of diabetes with tumor removal and the induction of hyperglycemia with the implantation of cancer cell lines. To better understand the role of AKT1 and its hyperactivation in glucose metabolism, AKT1Myr mice were characterized via metabolic (e.g. glucose/insulin tolerance test) and histological (e.g. immunohistochemistry) studies. Beginning at weaning, 3 weeks of age, the glucose intolerant AKT1Myr mice exhibited non-fasted hyperglycemia, which progressed to fasted hyperglycemia by 5 months of age. The glucose intolerance was attributed to a fasted hyperglucagonemia, and hepatic insulin resistance detectable by reduced phosphorylation of the insulin receptor following insulin injection into the inferior vena cava. Additionally, AKT1Myr/KRasG12D mice currently being studied, appear to display a more severe diabetic phenotype, with fasted hyperglycemia noticeable at an earlier age, fasted hyperglucagonemia, polyuria, muscle wasting, and bloating. Treatment of both models with doxycycline diet, to turn-off the transgene, caused attenuation of the non-fasted and fasted hyperglycemia, thus affirming AKT1 hyperactivation as the trigger. These newly revealed roles of AKT1, along with future studies of these mouse models, will better delineate the molecular mechanisms responsible for the individual and joint roles of AKT1 and KRas in pancreatic cancer oncogenesis, the initiation of cancer associated diabetes, and the association of these two diseases.
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Date Issued: 2015
Identifier: CFE0006245, ucf:51081
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006245

Title: Differential Expression Of Proteins Involved In VLDL Trafficking Causes Reduced VLDL Secretion In Male Ames Dwarf Mice.
Creator: Ahmed Moinuddin, Faisal, Siddiqi, Shadab, Masternak, Michal, Naser, Saleh, University of Central Florida
Abstract / Description: Cardiovascular diseases (CVDs) have been recorded as the number one cause of death worldwide, accounting for 32% of total deaths annually. More than two-thirds of all CVD cases are associated with atherosclerosis, which is the accumulation of fats and other substances causing plaque formation in the interior walls of major arteries. This leads to narrowing of the lumen and hardening of the arteries, ultimately resulting in angina, heart attack and/or stroke. Studies have shown that the...
Show moreCardiovascular diseases (CVDs) have been recorded as the number one cause of death worldwide, accounting for 32% of total deaths annually. More than two-thirds of all CVD cases are associated with atherosclerosis, which is the accumulation of fats and other substances causing plaque formation in the interior walls of major arteries. This leads to narrowing of the lumen and hardening of the arteries, ultimately resulting in angina, heart attack and/or stroke. Studies have shown that the pathogenesis of atherosclerosis and associated CVDs is strongly linked to elevated secretion of liver-specific lipoproteins called very-low-density-lipoprotein (VLDL). VLDLs are crucial lipoproteins responsible for transportation of triacylglycerides (TAGs), chemically inert particles that are physiologically significant for their energy storing capacity, from the liver to peripheral tissues. These VLDL particles are synthesized in the lumen of the endoplasmic reticulum (ER) of hepatocytes, transported from the ER to the cis-Golgi in special transport vesicles called VLDL-transport-vesicles (VTVs) and secreted into plasma through a highly regulated secretory pathway. Previous studies from our laboratory have shown that VTV-mediated ER-to-Golgi VLDL trafficking is the rate-limiting step in overall VLDL secretion from hepatocytes into plasma. In this project, we investigated intracellular VLDL trafficking and VLDL secretion in Ames dwarf (Prop1df, df/df) mice, a mutant mouse model homozygous for a recessive mutation at Prop1 gene locus (Prop1df) having deficiency of growth hormone (GH), thyroid stimulating hormone (TSH) and prolactin (PRL). This model is characteristic of prolonged longevity (~50% longer) and improved insulin sensitivity in comparison to their wild-type (N) counterparts. Ames dwarf (df/df) mice have recently been shown to have highly reduced plasma TAG levels, associating them with reduced susceptibility to atherosclerosis and associated CVDs. The underlying mechanism responsible for reduced VLDL secretion in Ames dwarf mice is yet to be characterized. We hypothesize that VTV-mediated trafficking of VLDL is reduced in Ames dwarf mice because of reduced expression of proteins regulating VLDL and VTV formation. To test our hypothesis, we first performed VTV-budding assay using cellular fractions isolated separately from Ames dwarf (df/df) and wild-type (N) mice livers. Our results show a significant (45%) reduction in VTV-budding process in Ames dwarf (df/df) mice compared to wild-type (N). Next we performed 2-dimensional differential gel electrophoresis (2-DIGE) on VTV and whole cell lysate (WCL) samples in order to examine the differences in protein expression and to have highly specific protein separation. ExPASy database was used to analyze protein spots that allowed us in identifying proteins specifically expressed in each of the mouse groups. Employing western blotting, samples (ER, cytosol, VTV and WCL) from both sets of mice were tested for expression levels of VLDL and VTV associated proteins (ApoB100, Sec22b, CideB, MTP, Apo-A1 and Apo-AIV) with ?-actin as the loading control. Significant differences in expression level of these proteins were observed which strongly suggest that the formation of VTV from ER in male Ames dwarf (df/df) mice is reduced compared to wild-type (N). Overall, we conclude that the differential expression of proteins required for VLDL transport causes reduced VLDL secretion in male Ames dwarf (df/df) mice.
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Date Issued: 2015
Identifier: CFE0005916, ucf:50829
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0005916

Title: The contribution of visceral fat to positive insulin signaling in Ames dwarf mice.
Creator: Menon, Vinal, Masternak, Michal, Khaled, Annette, Altomare, Deborah, Estevez, Alvaro, University of Central Florida
Abstract / Description: Ames dwarf (df/df) mice are homozygous for a spontaneous mutation in the prop1 gene due to which there is no development of anterior pituitary cells (-) somatotrophs, lactotrophs and thyrotrophs, leading to a deficiency of growth hormone (GH), prolactin (PRL) and thyrotropin (TSH). They tend to become obese as they age, but still live longer and healthier lives compared to their wild-type littermates, being very insulin sensitive, showing no signs of diabetes and cancer. These mutant mice...
Show moreAmes dwarf (df/df) mice are homozygous for a spontaneous mutation in the prop1 gene due to which there is no development of anterior pituitary cells (-) somatotrophs, lactotrophs and thyrotrophs, leading to a deficiency of growth hormone (GH), prolactin (PRL) and thyrotropin (TSH). They tend to become obese as they age, but still live longer and healthier lives compared to their wild-type littermates, being very insulin sensitive, showing no signs of diabetes and cancer. These mutant mice also have high circulating levels of anti-inflammatory and anti-diabetic adiponectin. Plasma levels of this adipokine usually decrease with an increase in accumulation of visceral fat (VF). We thus believe that VF in df/df mice, developed in the absence of GH signaling, may be functionally different from the same fat depots in normal (N) mice and may be beneficial, rather than detrimental, to the overall health of the animal. We performed surgeries involving removal of VF depots (epididymal and perirenal fat) in both groups of mice and hypothesize that the beneficial effects of visceral fat removal (VFR) will be present exclusively in N mice as VF in df/df mice contributes to enhanced insulin sensitivity by producing decreased levels of pro-inflammatory adipokines like TNF? and IL-6. We found that VFR improved insulin sensitivity only in N mice but not in the df/df mice. This intervention led to an upregulation of certain players of the insulin signaling pathway in the skeletal muscle of N mice only, with no alteration in df/df mice. The subcutaneous fat of df/df mice showed a downregulation of these insulin signaling genes upon VFR. Compared to N mice, epididymal fat of df/df mice (sham-operated) had increased gene expression of some of the players involved in insulin signaling and a decrease in transcript levels of TNFa. Ames dwarf mice had decreased levels of IL-6 protein in EF and in circulation. High circulating levels of adiponectin and decreased levels of IL-6 in circulation could contribute to the high insulin sensitivity observed in the Ames dwarf mice. Understanding the mechanisms responsible for VF having positive effects on insulin signaling in df/df mice would be important for future treatment of obese diabetic patients.
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Date Issued: 2013
Identifier: CFE0004890, ucf:49654
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0004890

Title: Regulation of Extra-Pituitary Prolactin in Monocytes and Macrophages.
Creator: Barrett, Richard, Parthasarathy, Sampath, McKinstry, Karl, Masternak, Michal, Zhao, Jihe, University of Central Florida
Abstract / Description: Recently it has been shown that leukocytes are capable of producing prolactin (PRL). Evidence of extra-pituitary PRL (ePRL) production is so far been limited to primates and is not shared across other mammal species such as mice and rats. While ePRL is characterized as an identical protein to traditional pituitary PRL, it is controlled under an alternative promoter and is thus regulated differently from pituitary PRL. Little is known about what regulates ePRL or its direct role in human...
Show moreRecently it has been shown that leukocytes are capable of producing prolactin (PRL). Evidence of extra-pituitary PRL (ePRL) production is so far been limited to primates and is not shared across other mammal species such as mice and rats. While ePRL is characterized as an identical protein to traditional pituitary PRL, it is controlled under an alternative promoter and is thus regulated differently from pituitary PRL. Little is known about what regulates ePRL or its direct role in human physiology, but given that PRL has well over 300 described functions, it is likely that the autocrine and paracrine effects of this hormone could have far reaching implications in overall physiology. This work takes some of the first steps in understanding how leukocyte ePRL is regulated. Our results show that, adrenergic hormones are one key stimulus in ePRL expression in monocytes/macrophages. This is particularly intriguing considering the opposing role of these two signals in settings such as adipose tissue where adipose tissue macrophages are constantly exposed to pro-lipolytic adrenergic hormones that would in turn stimulate production of an anti-lipolytic hormone, PRL. Further, our work shows that the inflammatory phenotype of the leukocytes influences basal expression of PRL and overall ePRL expression increases significantly as monocytes differentiate into macrophages, as is a common occurrence in adipose tissue. The final portion of our work shows how monocytes/macrophages also respond to preadipocytes directly. These stem cell precursors to mature adipose cells release an unknown factor that stimulates ePRL production in monocytes/macrophages. Analysis of our gene array shows many of the genes stimulated by adipose stem cells alongside PRL are important genes in tissue regeneration and remodeling, a possible role that fits well with known effects of PRL. Understanding such primate specific interactions between the immune system and major metabolic tissues such as adipose fills vital gaps in knowledge that may explain why so many treatments fail when transitioning from mouse models to humans.
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Date Issued: 2018
Identifier: CFE0007309, ucf:52164
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0007309

Title: Novel Cytokine Signaling and Molecular Therapeutic Strategy in Pancreatic Cancer.
Creator: Gitto, Sarah, Altomare, Deborah, Khaled, Annette, Zhao, Jihe, Copik, Alicja, Masternak, Michal, University of Central Florida
Abstract / Description: Pancreatic ductal adenocarcinoma (PDAC) is highly chemo-resistant and has a five year survival rate of (
Show morePancreatic ductal adenocarcinoma (PDAC) is highly chemo-resistant and has a five year survival rate of (<)8%. Risk factors of pancreatic cancer, such as chronic pancreatitis, help to elicit a pro-tumor immune response, and highly fibrotic environment that promotes tumorigenesis. To study how chronic pancreatitis promotes cancer initiation, traditional KRasG12D mice and double mutant Akt1Myr/KrasG12D mice were used to model microenvironment changes. Akt1Myr/KrasG12D mice were more susceptible to chronic tissue damage, accelerated tumor development and metastatic disease. These mice exhibited histological changes consistent with immune cell privilege, where M2 macrophages and non-cytotoxic eosinophils were co-localized with fibrotic regions. IL-5 expression was up regulated in pancreatic cells undergoing acinar to ductal metaplasia and then diminished in advanced lesions. Tumor cells treated with IL-5 exhibit increased migration and activation through STAT5 signaling. Collectively, the results suggest that eosinophils, which are responsive to IL-5, are key mediators in the pancreatic environment subjected to chronic inflammation and injury.Current therapeutics fall short in increasing patient survival. There remains an urgent need for innovative treatments and thus we tested difluoromethylornithine (DFMO) in combination with a novel polyamine transport inhibitor, Trimer44NMe, against Gemcitabine-resistant PDAC cells. Prior clinical failures when targeting polyamine biosynthesis with DFMO monotherapy may be due to tumor escape via an undefined polyamine transport system. In pancreatic tumor cells DFMO alone and with Trimer44NMe significantly reduced PDAC cell viability by inducing apoptosis or cell cycle arrest. In vivo orthotopic PDAC growth with DFMO treatment resulted in decreased c-Myc expression, a readout of polyamine pathway dysfunction. Moreover, dual inhibition significantly prolonged survival of tumor-bearing mice, and increased M1 macrophage infiltration and reduced FoxP3 expression. Collectively, these studies demonstrate that targeting polyamine pathways in PDAC is a promising immunomodulating therapy that increases survival.
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Date Issued: 2017
Identifier: CFE0007283, ucf:52168
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0007283

Title: Development of Molecular Diagnostic Tools for Mycobacterium Species.
Creator: Bengtson, Hillary, Kolpashchikov, Dmitry, Rohde, Kyle, Self, William, Jewett, Travis, Masternak, Michal, University of Central Florida
Abstract / Description: This dissertation focuses on the development of diagnostic tools for mycobacteria using hybridization based technologies including binary deoxyribozyme (BiDz) sensors and microarrays. The genus Mycobacterium, is a diverse group of bacteria containing 150+ species including M. tuberculosis (M.tb) and non-tuberculous mycobacteria (NTM) which exhibit a range of pathogenicity, drug susceptibility and growth characteristics. M. tuberculosis (M.tb) is the causative agent of tuberculosis (TB) and...
Show moreThis dissertation focuses on the development of diagnostic tools for mycobacteria using hybridization based technologies including binary deoxyribozyme (BiDz) sensors and microarrays. The genus Mycobacterium, is a diverse group of bacteria containing 150+ species including M. tuberculosis (M.tb) and non-tuberculous mycobacteria (NTM) which exhibit a range of pathogenicity, drug susceptibility and growth characteristics. M. tuberculosis (M.tb) is the causative agent of tuberculosis (TB) and the leading cause of infectious disease related deaths worldwide. The control of TB is limited by the lack of sensitive and specific diagnostic tools available at the point of care (POC). The studies presented here illustrate the advances in our technology for the detection and differentiation of M.tb and NTM. The use of BiDz sensors enables the selective recognition of DNA/RNA analytes containing single nucleotide polymorphisms associated with species-specific identification, drug susceptibility testing (DST) and strain typing. First, we developed a platform for the detection of M.tb and drug susceptibility using multiplex PCR and BiDz sensors. However, this method relies on the use of expensive instrumentation which is often not available in high TB burden countries. Therefore, additional studies focused on the development of tools for the detection of isothermal amplification products and the direct detection of rRNA. Based on these findings, we also developed an NTM species typing tool using BiDz sensors for species identification in ~1 hour. Despite the advantages of BiDz sensor technology, their use is limited to the detection of a few selected mutations. To address this limitation, we developed a 15-loci multiplex PCR followed by analysis with a custom microarray for high-throughput identification of SNPs. The work presented in this dissertation has the potential to enable the rapid, specific and sensitive identification of mycobacterial species necessary to reduce the diagnostic delay, ensure initiation of effective therapy, and prevent further transmission.
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Date Issued: 2017
Identifier: CFE0006856, ucf:51735
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006856

Title: A major double strand repair pathway and cancer-associated circulating proteins are effecters of epigenetic revision.
Creator: Allen, Brittany, Masternak, Michal, Khaled, Annette, Zhao, Jihe, Muller, Mark, Siddiqi, Shadab, University of Central Florida
Abstract / Description: DNA methylation is a vital epigenetic process that acts as a major control mechanism for gene expression. In addition to its essential role in many normal cellular processes, it is also implicated in a wide variety of disease states and processes including cancer. Along with genetic mutations, aberrant DNA methylation patterns, specifically the inappropriate DNA methylation or demethylation of CpG residues, may activate oncogenes or suppress tumor suppressor genes, respectively. These changes...
Show moreDNA methylation is a vital epigenetic process that acts as a major control mechanism for gene expression. In addition to its essential role in many normal cellular processes, it is also implicated in a wide variety of disease states and processes including cancer. Along with genetic mutations, aberrant DNA methylation patterns, specifically the inappropriate DNA methylation or demethylation of CpG residues, may activate oncogenes or suppress tumor suppressor genes, respectively. These changes can generate or facilitate the progression of tumorigenesis and tend to accumulate throughout the development of cancer. Although they play such a major role in cancer and in other diseases, it remains unclear what causes these epigenetic revisions to occur. This dissertation will focus on uncovering mechanisms that are sources of epigenetic revision, specifically as they relate to cancer. Due to rapid cell division and increased DNA damage, cells are increasingly dependent on DNA repair as they continue on a path of tumorigenic progression. We hypothesize that DNA repair, specifically the repair of DNA double strand breaks (DSB) by Non-Homologous End Joining (NHEJ) may play a role in inappropriate epigenetic revision. Using a GFP reporter system inserted into the genome of HeLa cells, we are able to induce targeted DNA damage that enables the cells, after successfully undergoing NHEJ repair, to express WT GFP. These GFP+ cells were segregated into two expression classes, one with robust expression (Bright) and the other with reduced expression (Dim). Using a DNA hypomethylating drug (AzadC) we were able to demonstrate that the different GFP expression levels was due to differential methylation statuses of CpGs in regions on either side of the break site. Deep sequencing analysis of this area in sorted Bright and Dim populations revealed a collection of different epi-alleles that display patterns of DNA methylation following repair by NHEJ. These patterns differ between Bright and Dim cells which are hypo- and hypermethylated, respectively, and between the post-repair populations and the original, uncut cells. These data suggest that NHEJ repair facilitates a rewrite of the methylation landscape in repaired genes, elucidating one potential source for the altered methylation patterns seen in cancer cells.The Dim cells generated during this study are known to have a hypermethylated GFP gene that is correlated with reduced expression, allowing it to be used as a screening tool for hypomethylating agents. We used this tool to screen the blood serum of patients with head and neck squamous cell carcinoma (HNSCC). We found that the serum from HNSCC patients, but not from healthy individuals, contains some factor that causes hypomethylation in exposed cells. Further, we were able to identify this factor as a protein capable of effecting changes in DNA methylation, gene expression, and miRNA levels in the treated Dim cells. The novel concept presented in this study has immense implications on the study of cancer progression as it evidences circulating proteins, presumably released by cancer cells, which are able to effect gene expression in cells that are distal to the location of the cancer. Further, the fact that these proteins are in circulation makes them a potential target for use in diagnostics. Changes in DNA methylation play a major role in the development of cancer and understanding the mechanisms by which this occurs could provide new therapeutic targets for preventing this process from contributing to tumorigenesis. This dissertation presents potential sources of epigenetic revision in cancer and thus provides answers to a major question that has yet to be answered in the area of cancer research.
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Date Issued: 2017
Identifier: CFE0006555, ucf:51333
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006555

VLDL (2)	+ -
VLDL secretion (2)	+ -
17-alpha Estradiol (1)	+ -
17?-E2 (1)	+ -
AKT (1)	+ -

Aging (1)	+ -
Ames (1)	+ -
Ames dward mice (1)	+ -
Ames dwarf (1)	+ -
ApoB (1)	+ -
Azelaic acid (1)	+ -
Cancer (1)	+ -
Cardiomyopathy (1)	+ -
Cardiotoxicity (1)	+ -
Crohn's disease (1)	+ -
DNA methylation (1)	+ -
DNA repair (1)	+ -
Diabetes Mellitus (1)	+ -
Doxorubicin (1)	+ -
ER (1)	+ -
Embryonic Stem Cell (1)	+ -
Eosinophils (1)	+ -
Epigenetic (1)	+ -
Exosomes (1)	+ -
GH (1)	+ -
GHKRO (1)	+ -
GHRKO mice (1)	+ -
Genetically engineered mouse models (1)	+ -
Glutathione Peroxidase (1)	+ -
Golgi. (1)	+ -

ETD Collection (16)	+ -
Honors Collection (2)	+ -

Masternak, Michal (18)	+ -
University of Central Florida (18)	+ -
Siddiqi, Shadab (7)	+ -
Altomare, Deborah (6)	+ -
Zhao, Jihe (5)	+ -

Khaled, Annette (4)	+ -
Naser, Saleh (4)	+ -
Parthasarathy, Sampath (4)	+ -
Singla, Dinender (2)	+ -
Ahmed Moinuddin, Faisal (1)	+ -
Albury, Toya (1)	+ -
Allen, Brittany (1)	+ -
Andl, Claudia (1)	+ -
Barrett, Richard (1)	+ -
Bengtson, Hillary (1)	+ -
Bennis, Mohammed (1)	+ -
Clay, Ryan (1)	+ -
Copik, Alicja (1)	+ -
Davidson, Victor (1)	+ -
Do, Andrew (1)	+ -
Ebert, Steven (1)	+ -
Estevez, Alvaro (1)	+ -
Gitto, Sarah (1)	+ -
Gurwani, Bhavesh (1)	+ -
Hammond, Jamillah (1)	+ -
Jewett, Travis (1)	+ -
Kolpashchikov, Dmitry (1)	+ -
Lahiri, Satadru (1)	+ -
McKinstry, Karl (1)	+ -
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