The prevalence of type II diabetes is increasing at an alarming rate due to the western world lifestyle. Type II diabetes is characterized by an insulin resistance distinguished by impaired glucose uptake in adipose and muscle tissues. The molecular mechanisms behind the insulin recistance and also the knowledge considering normal insulin signalling in fat cells, especially in humans, are still unclear. Insulin receptor substrate (IRS) is known to be important for medating the insulin-induced signal from the insulin receptor into the cell. We developed and optimized a method for transfection of primary human adipocytes by electroporation. By recombinant expression of proteins, we found a proper IRS to be crucial for both mitogenic and metabolic signalling in human adipocytes. In human, but not rat, primary adipocytes we found IRS1 to be located at the plasma membrane in non-insulin stimulated cells. Insulin stimulation resulted in a two-fold increase of the amount of IRS1 at the plasma membrane in human cells, compared with a 12-fold increase in rat cells. By recombinant expression of IRS1 we found the species difference between human and rat IRS1 to depend on the IRS proteins and not on properties of the host cell. The adipocytes function as an energy store, critical for maintaining the energy balance, and obesity strongly correlates with insulin resistance. The insulin sensitivity of the adipocytes with regard to the size of the cells was examined by separating small and large cells from the same subject. We found no increase of the GLUT4 translocation to the plasma membrane following insulin stimulation in the large cells, whereas there was a two-fold increase in the small cells. This finding supports the idea of a causal relationship between the enlarged fat cells and reduced insulin sensitivity found in obese subjects. The insulin receptor is located and functional in a specific membrane structure, the caveola. The morphology of the caveola and the localization of the caveolar marker proteins caveolin-1 and -2 were examined. Caveolae were shown to be connected to the exterior by a narrow neck. Caveolin was found to be located at the neck region of caveolae, which imply importance of caveolin for maintaining and sequestering caveolae to the plasma membrane. In conclusion, the transfection technique proved to be highly useful for molecular biological studies of insulin signal transduction and morphology in primary adipocytes.

Pancreatic cancer is a devastating disease that leaves patients with a very poor prognosis and few therapeutic options. Many of the treatment options available are the same that have been used for almost 2 decades. There is a dire need for both novel treatments for this disease as well as novel strategies of treatment. This body of work will introduce and provide evidence in support of a novel combination therapy for pancreatic cancer treatment, a novel strategy of modifying currently used chemotherapeutics for pancreatic cancer therapy, and a novel transgenic preclinical mouse model of pancreatic cancer. Sabutoclax, an antagonist of the anti-apoptotic Bcl-2 proteins, and Minocycline, a commonly used antibiotic, show potent synergy when used in combination in both pancreatic cancer cells and in multiple immune-deficient and immune-competent mouse models of pancreatic cancer. Sabutoclax alone is capable of inducing cell cycle arrest and apoptosis in cells and its cytotoxicity is enhanced significantly when combined with Minocycline. This combination results in the loss of Stat3 activation both in vitro and in vivo, which is essential for its toxicity. It also inhibits tumor growth and prolongs survival in the KPC transgenic mouse model of pancreatic cancer. Also presented here are studies that demonstrate efficacy in vivo of modified versions of Gemcitabine and Paclitaxel. These drugs are linked to a peptide that shows specificity for the EphA2 receptor, which is overexpressed on the surface of pancreatic cancer cells and only minimally on normal cells. This peptide results in increased cellular uptake of drug, as it is bypassing its normal mechanism of entry. These normal mechanisms are often dysregulated in cancer, leading to decreased uptake and drug resistance. The use of these modified drugs show significantly increased tumor growth inhibition as compared to the parent drug alone. Finally, we provide data on the characterization of a novel transgenic mouse model of pancreatic cancer. This model, the Pan Met View (PMV) mouse, combines the commonly used KPC transgenic mouse model of pancreatic cancer and a mouse that expresses a Luciferase reporter gene under the control of the cancer-specific promoter, CCN1. Our data shows that double transgenic PMV mice can now be used to follow primary tumor and metastasis development in real time by Bioluminescent imaging (BLI) through disease progression and potentially therapy. This strategy will enhance the use of genetically engineered mouse models (GEMMS) to study cancer initiation and progression with potential to non-invasively monitor therapy. These chapters present novel and exciting data that have the potential to open multiple avenues of translational study and result in significant advances in pancreatic cancer therapy.

Group X secretory phospholipase A2 (GX sPLA2) hydrolyzes membrane phospholipids producing free fatty acids and lysophospholipids. Previous studies from our lab suggest that mice with targeted deletion of GX sPLA2 (GX KO) have increased age-related weight gain due to an increase in overall adiposity. Paradoxically, this increased adiposity is associated with improved age-related glucose intolerance. GX KO mice also demonstrate a reduced inflammatory response to lipopolysaccharide injection. In vitro studies indicate this phenotype may be attributable to blunted macrophage mediated inflammatory responses. Given the role of macrophages in promoting adipose tissue (AT) inflammation and metabolic dysfunction in response to diet-induced obesity, we hypothesized that GX KO mice would be protected from the obesity related metabolic derangements associated with overfeeding. Unexpectedly, GX KO mice were only partially protected from high fat (HFD) diet-induced glucose intolerance and showed no improvement in HFD-induced insulin resistance. Moreover, GX KO mice were not protected against HFD-induced AT inflammation. GX sPLA2 is produced as a proenzyme (pro-GX sPLA2), and propeptide cleavage is required for enzymatic activity. Furin-like proprotein convertases (PCs) have recently been implicated in the proteolytic activation of pro-GX sPLA2; however the identity of individual PCs involved is unclear. Previous findings from our lab have shown that GX sPLA2 is expressed in the adrenals where it regulates glucocorticoid production. GX KO mice have increased plasma corticosterone levels under both basal and ACTH-induced stress conditions. However, how GX sPLA2 is regulated in the adrenals is still uncertain. We hypothesized that PCs may be involved in the proteolytic activation of pro-GX sPLA2 in the adrenals. Here we report the novel findings that the PCs, furin and PCSK6, proteolytically activate pro-GX sPLA2 in Y1 adrenal cells. Furthermore, we demonstrate that PC dependent processing of pro-GX sPLA2 is necessary for GX sPLA2 dependent suppression of steroidogenesis. Finally, we provide evidence that pro-GX sPLA2 processing by PCs is enhanced in response to adrenocorticotropic hormone (ACTH), suggesting a novel mechanism for negatively regulating adrenal steroidogenesis. Cumulatively, these studies provide valuable insight into the function and regulation of GX sPLA2.

The integrity and barrier properties of intestinal epithelium are determined by specialized adhesive structures known as intercellular junctions; composed of adherens junctions (AJs), tight junctions (TJs) and focal adhesions that mediate cell-cell and cell matrix interactions, respectively. These two types of epithelial cell adhesions regulate each other during disruption and restitution of the epithelial barrier. Inflammatory cytokines such as interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) are elevated during intestinal inflammation. The most notable effects of IFNγ and TNFα on intestinal epithelial homeostasis involve disruption of apical junctions and attenuation of cell migration. Although molecular mechanisms underlying these effects remain poorly understood, expressional downregulation of different adhesion proteins may play a major role in the cytokine-dependent disruption of the intestinal epithelial barriers. This thesis is based on the hypothesis that inhibition of the protein translation initiation machinery promotes the disruption of the intestinal epithelial barrier and attenuates epithelial restitution during mucosal inflammation. This study was focused on two eukaryotic translation initiation factors, eIF4G1 and eIF4G2, which play essential roles in the regulation of cap-dependent protein translation. Expression of both translation initiation factors was dramatically downregulated in model intestinal epithelial cell monolayers treated with IFNγ and TNFα in parallel to cytokine-induced disruption of the epithelial barrier. siRNA or shRNA-mediated downregulation of either eIF4G1, or eIF4G2 increased permeability of well-differentiated SK-CO15 intestinal epithelial cell monolayers and decreased expression of different adherens junction and tight junction proteins. Furthermore depletion of these translation initiating factors inhibits different modes of migration (wound healing and transfilter migration) of stem-cell like and well-differentiated intestinal epithelial cells. These findings suggest that eukaryotic translation initiation factors of the eIF4G family play unique roles in regulating integrity and restitution of the intestinal epithelial barrier. Downregulation of these translation initiating factors may mediate disruption of the intestinal epithelial barriers during mucosal inflammation.

Abstract Although great strides have been made over the decades in development and optimization of anti-cancer therapies, even highly effective drugs often fail to completely eliminate tumors. Residual tumor cells can enter into a state of dormancy for prolonged periods of time but eventually are able to regain proliferative capacity and reemerge as chemotherapy-resistant disease. Because recurrent disease is a leading contributor to patient’s mortality, it is paramount to identify strategies for effectively destroying residual tumor cells. Cytotoxic drugs and ionizing radiation are used as standard therapies in a variety of cancers. These modalities induce apoptosis, autophagy and senescence. Senescence is a state of prolonged growth arrest, which cells are able to eventually escape regaining proliferative capacity. Autophagy is generally considered to be a protective mechanism; however, it can take non-protective or even cytotoxic form in response to anti-cancer treatments. Furthermore, chemotherapy or radiation induced autophagy was shown to be a contributor to the immune response against tumor cells. Using a model of Triple Negative Breast Cancer, we were able to show increased immunosurveillance of tumor cells after enhanced autophagy was achieved by combining epigenetic remodeling with chemotherapy. Alternatively, we were able to achieved effective clearance of tumor cells induced into senescence by chemotherapy or radiation by the senolytic drug ABT-263 (Navitoclax). In summary, autophagy and senescence alone or in concert, can be induced by conventional anti-tumor modalities. Those processes can be modulated independently to achieve clearance of residual tumor cells following anti-cancer therapies.

Human High Temperature requirement A2 (HtrA2) also known as Omi, is a serine protease located in the mitochondria with an important function in both cell survival and death. My results show the proteolytic activity of Omi/HtrA2 varies under different conditions. I characterized the optimal condition for Omi/HtrA2 protease activity using an in vitro assay system. Additionally, I identified a new allosteric regulation of Omi/HtrA2 through interaction with a specific substrate, the MUL1 protein. MUL1 is a multifunctional E3 ubiquitin ligase anchored in the outer mitochondrial membrane with domains both inside mitochondria and in the cytoplasm. The data shown here strongly supports the hypothesis that Omi/HtrA2 activity is modulated by a number of different mechanisms. Some of these conditions, such as pH or substrate denaturation might reflect the state of mitochondria under stress. It has been known that Omi/HtrA2 is a stress activated protease, but the mechanism of its regulation has not been fully elucidated. Furthermore, the allosteric regulation of Omi/HtrA2 by specific substrates, can be another mechanism that provides a feedback loop to increase the activity of the enzyme. The findings from this project contribute new information on the mechanisms of activation of Omi/HtrA2 protease. They support the hypothesis that mitochondrial stress might be involved in the regulation of Omi/HtrA2 protease.

<p>Glomerulosclerosis (GS) is the irreversible scarring of glomerular tissue which underlies the development of chronic kidney disease (CKD). Increased intraglomerular capillary pressure (P_gc) is a major contributor to the development of GS and can occur in both hypertensive and diabetic patients. With elevated P_gc, <em>in vitro</em> and <em>in vivo</em> evidence suggest that mesangial cells (MC) experience cyclic stretch and secrete pro-fibrotic factors such as connective tissue growth factor (CTGF) which contributes to GS. The signaling pathways that are activated in response to elevated P_gc and lead to extracellular matrix (ECM) production in MCs are the main focus of this thesis.</p> <p>Previous data demonstrated activation of the Rho GTPase, Rac1, with cyclic stretch in MCs. Furthermore, the most characterized effector of Rac1, p21-activated kinase (PAK), has been observed to have a role in endothelial cells (ECs) exposed to mechanical stress. We thus proposed that the Rac1-PAK signaling pathway is involved in mechanical stress signaling in MCs.</p> <p>Our data demonstrate that Rac1-PAK signaling was activated in response to cyclic stretch and required for stretch-induced CTGF production in MCs. RhoA activation was also regulated by Rac1-PAK signaling, and RhoA/ROCK were observed to mediate CTGF upregulation with stretch. Further investigation on the role of Rac1-PAK signaling and how it regulates CTGF in MCs exposed to stretch, will provide insight into potential therapeutic targets to delay the progression of hypertension-mediated CKD.</p> / Master of Science in Medical Sciences (MSMS)

<p>Megakaryocyte cultures are a strong tool for the in vitro investigation of platelet production in platelet disorders. Peripheral blood derived hematopoietic progenitor cells (PB-HPCs) are the most accessible source of HPCs with high potential to produce mature megakaryocytes in vitro; however, they are present in low numbers making peripheral blood an inefficient source. Additionally, a megakaryocyte culture with an optimized thrombopoietin (TPO) concentration is required which can reliably allow the investigation of suppressive effects of antibodies/plasma from immune thrombocytopenia (ITP) patients. In this study, we developed a megakaryocyte culture with the utilization of human PB-HPCs in an efficient fashion resulting in the production of high purity megakaryocytes in a TPO-dependent manner.</p> <p>The mononuclear fraction was collected from 180 mL of peripheral whole blood and CD34+ cells were isolated by a positive selection yielding the average of 5.5 x 105 ± 2.5 x 105 CD34+ cells (n = 18). Using 96-well tissue-culture plates and seeding 10,000 CD34+ cells/well, the average of 13 experiments in triplicate can be set up utilizing isolated CD34+ in an efficient manner. Capitalizing on a TPO dose-dependent megakaryocyte production experiment, 20 ng/mL was established as the TPO concentration which resulted in the production of mature megakaryocytes without reaching the plateau in megakaryopoiesis response. On day 11 of culture, the expression of megakaryocytic lineage (CD41/61+) and maturation (CD41/61+CD42+) markers peaked at 90.65% and 76.10%. In conclusion, this culture system has broad application for investigation of platelet disorders and drug discovery which can be accessible to all researchers.</p> / Master of Science (MSc)

<p>Huntington’s disease (HD) is a progressive, neurodegenerative disorder that leads to the selective loss of neurons in the striatum and the cerebral cortex. HD is caused by a CAG trinucleotide repeat expansion beyond the normal length in the <em>IT15 </em>(<em>Htt</em>) gene. The CAG stretch codes for an elongated polyglutamine tract within the amino‐terminus of the huntingtin protein. Polyglutamine tracts with lengths exceeding 37 repeats cause HD whereas repeat lengths below do not. This phenomenon has plagued the HD community since the discovery of the gene in 1993. In this thesis, we sought to elucidate the molecular mechanism by which huntingtin becomes toxic at polyglutamine lengths above 37. Using Förster resonance energy transfer (FRET) techniques, we describe an intramolecular proximity between the first 17 residues (N17) and the proline-rich regions, which flank the polyglutamine tract of huntingtin. We report that we can precisely measure differences between the conformations adopted by the huntingtin protein with polyglutamine tracts below and above the pathogenic repeat threshold of 37 repeats. Our data supports the hypothesis that polyglutamine tracts below the pathogenic threshold can act as a flexible hinge allowing the N17 domain to freely fold back upon huntingtin and come into close 3D proximity with the polyproline region. This flexibility is lost in polyglutamine tracts with >37 repeats resulting in a diminished spatial proximity between N17 and the polyproline domain.</p> / Doctor of Philosophy (PhD)

Anti-tumor therapeutic strategies based on combinations of chemotherapeutic agents with a death inducing ligand such as TNF-α Related Apoptosis Inducing Ligand (TRAIL), are directed towards selective and effective cancer cell apoptosis and enhanced therapeutic response. We previously demonstrated that proteasome inhibition sensitizes TRAIL resistant prostate cancer cells to TRAIL-mediated apoptosis via stabilization of the active p18 subunit of initiator caspase-8. The present study investigated the functional link between caspase-8 and the proteasome, by analyzing the impact of caspase-8 ubiquitination and proteasomal degradation on the outcomes of the extrinsic apoptosis pathway in cancer cells. Caspase-8 ubiquitination status was assessed by polyubiquitin immunoprecipitation (IP) and fluorescent microscopy. Apoptosis induction in response to death receptor stimuli or proteasome inhibitor was evaluated using the Annexin V/Propidium iodide staining (PI). To determine the consequences of proteasome inhibition on caspase-8 stability, trafficking, and activity following death receptor activation, we used the TRAIL-resistant human prostate cancer LNCaP cells, and the caspase-8 deficient Neuroblastoma 7 (NB7) cells, as cellular models for reconstituting the non-cleavable mutant forms of caspase-8. Our findings demonstrate that the non-cleavable forms of caspase-8 are capable of inducing apoptosis comparably to wild-type caspase-8 upon treatment with proteasome inhibitor and GST-TRAIL. Furthermore, caspase-8 processing into its active subunits preceded caspase-8 polyubiquitination, implicating caspase-8 processing as a potential regulatory mechanism, rather than a requirement for caspase-8 activation in apoptosis induction. The mechanistic control of caspase-8 by ubiquitination in cancer cells may have significant significance in bypassing mechanisms of therapeutic resistance in human tumors and optimization of anti-cancer treatment strategies in human tumors and optimization of anti-cancer treatment strategies.