<p>The incidence and prevalence of people suffering from end stage renal disease is increasing. Proteinuria, particularly the presence of albumin in urine is concerning because proteinuria is associated with the progression to end stage renal disease (ESRD). Understanding the mechanisms involved in damaging the glomerular filtration barrier is essential. Transforming growth factor beta (TGFB) is a key cytokine in mediating glomerulosclerosis and proteinuria. Not much is known about the downstream pathways that mediates the renal damage and proteinuria.</p> <p>I hypothesize that TGFB induces proteinuria through podocyte de-differentiation and this occurs through SMAD dependent and independent pathways.</p> <p>Methods: I used adenovirus mediated gene transfer of TGFB1 to rat renal artery to study the effects of TGFB1 on renal structure and functions. To study the importance of SMAD3 in mediating downstream effects of TGFB1 in proteinuria and podocyte effacement, I used an anti-glomerular basement membrane model in SMAD3+/+ and SMAD3-/- mice to induce glomerulonephritis and proteinuria.</p> <p>Results: Transient TGFB1 overexpression via AdTGFB1 induced significant proteinuria, podocyte foot process effacement, nephrin down-regulation, and nephrinuria. The expression of synaptopodin was also significantly down-regulated by TGFB1. TGFB1 increased the expression of the angiopoietin receptor, Tie2, in podocyte cell culture. In cultured podocytes, TGFB1 downregulated the gene and protein expression of both nephrin and synaptopodin. These findings suggest that locally produced TGFB1 can cause podocyte de-differentiation marked by a loss of synaptopodin, nephrin, and foot process effacement; this process is partly regulated by angiopoietins. This process represents a novel pathway that may explain proteinuria in a variety of common renal diseases.</p> <p>Both SMAD3+/+ and SMAD3-/- mice had proteinuria after induction of anti-GBM glomerulonephritis, though to a lesser extent in SMAD3-/- mice. SMAD3-/- and SMAD3+/+ mice developed significant glomerulonephritis with progressive interstitial fibrosis and chronic renal impairment. The SMAD3+/+ mice were found to be more prone to fibrotic changes, interstitial damage and tubular and glomerulosclerosis than the SMAD3-/- mice. This suggests that TGFB1 signals through pathways other than SMAD3 such as those triggered by hypoxia.</p> <p>Conclusion: I have shown that TGFB1 upregulation via AdTGFB1 induces proteinuria through podocyte dedifferentiation and FP effacement. Angiopoietins are essential for TGFB1 mediated podocyte injury. The effects of TGFB are partially mediated through SMAD3 as there is residual podocyte effacement and proteinuria in the SMAD3-/- mice. Hence there are SMAD3 dependent and independent pathways involved in proteinuria.</p> / Doctor of Science (PhD)

The embryonic cell cycle of Xenopus laevis consists of rapid oscillations between S and M phase occurring in the absence of gap phases and their associated regulatory checkpoints. The end of the 12th cell cycle marks the onset of the midblastula transition (MBT) when the cell cycle lengthens establishing gap phases, their associated checkpoint pathways, and the initiation of zygotic transcription. During cell cycles 2-12, cyclins A and B are translated and expressed once per cell cycle until the MBT when their protein levels decrease due to a newly transcribed zygotic factor that leads to the deadenylation and subsequent loss of their mRNAs. In contrast, cyclin E is expressed at a constitutively high level during cell cycles 2-12. Furthermore, cyclin E levels are terminally lost coincident with initiation of the MBT in the continued presence of its adenylated mRNA. Terminal disappearance of cyclin E appears to be maternally directed and is not affected by zygotic transcription, translation, replication, or the nuclear to cytoplasmic ratio. This has led to the hypothesis that cyclin E is part of an autonomous maternally directed timer that determines the timing of the MBT. To investigate this possibility we have used antisense oligonucleotides to knockdown cyclin E and assess the affects on the timing of the MBT. Premature knockdown of cyclin E did not affect the timing of the MBT indicating it is not part of the maternal timer. Furthermore, prior to the MBT cyclin E protein has an unusually long half life. However, despite an increased stability the constitutively high levels of cyclin E require a low level of translation. We have also determined that the stable pattern of adenylation observed for cyclin E1 is specified by three cis-acting elements in its' 3' UTR. Deletion of the NPS, eCPE/ARE3, and ARE2 abolished adenylation. Additionally, a putative stem loop in ARE2 is targeted by ElrA the Xenopus homolog of HuR and a member of the ELAV gene family. Loss of adenylation required disruption of ElrA binding. These findings demonstrate ElrA functions in the correct adenylation of cyclin E1 mRNA.

The innate immune system provides nonspecific defenses against pathogens. Many diseases occur because of malfunctions in the innate immune system. In the present thesis, I have investigated two independent mechanisms of innate immunity in the lung and liver. Both mechanisms involve responses to bacterial infection and/or components of the bacterial wall (lipopolysaccharide). In the first model, I studied the role of submucosal glands in lung innate immunity through the use of tracheal xenograft airways with and without glands. This work provides evidence that submucosal glands are a major source of antibacterials that are critical for maintaining sterile airways. In the second model, I studied host responses to a gram-negative bacterial cell wall component (lipopolysaccharide) and how the liver coordinates cytokine responses that lead to endotoxic shock. This work examined how hepatic induction of NFkappaB and TNFalpha influenced survival in this lethal murine model of endotoxemic shock. My findings suggest that during the course of lethal endotoxic shock, NFkappaB activation has a predominantly pro-inflammatory effect in the liver through the induction of TNFalpha, and that TNFalpha influences the role of NFkappaB as an anti-apoptotic factor in the liver. In conclusion, my thesis suggests that maintaining a homeostatic balance in response to pathogens is an important function of the complex innate immune system.

<p>Bronchial thermoplasty has recently been FDA approved as a novel therapy for use on adults suffering from severe asthma. The procedure uses radiofrequency energy to heat the airways to 65°C for 10 s. This has been shown in dogs to lead to a reduction of airway smooth muscle mass and in humans to improve quality of life and asthma control. Early cellular reactions to this treatment are unclear; as well, there is limited information regarding thermal sensitivity of airway smooth muscle when exposed to extreme temperatures (50-65°C). We examined the cellular impact of bronchial thermoplasty by investigating the response of airway smooth muscle to heat by immersing bovine tracheal strips and bronchial segments in heated Krebs. We confirmed dramatically decreased functionality over the temperature range 50-60°C at 1 h and 24 h in all tissues. TUNEL analysis noted significant cell death in all tissues heated to 65°C and limited cell death in bronchial tissues treated with <55°C. Immunohistochemical analysis showed an effect of temperature on caspase 3 activation in bronchi; tracheal strips demonstrated co-localization of caspase 3 and TUNEL at 55°C but not 65°C. These data suggests that cell death of airway smooth muscle contributes to the cellular effects observed following heating to 65°C; at lower temperatures, cell death may be limited. We conclude that bronchial thermoplasty (heat treatment to 65°C for ~30 seconds) leads to a number of structural and functional changes in the airway smooth muscle, which culminate in marked loss of function and cell death.</p> / Master of Health Sciences (MSc)

<p><strong><em>Purpose</em></strong></p> <p>To examine the potential of the anti-diabetic agent Metformin (MET) to enhance responses of NSCLC to ionizing radiation (IR).</p> <p><strong><em>Experimental Design</em></strong></p> <p>Human NSCLC A549, H1299 and SK-MES cells were treated with IR, MET or the mTOR inhibitor rapamycin and subjected to proliferation, clonogenic, immunoblotting, cell cycle and apoptosis assays. A549 and H1299 cells were grafted into flanks of immunosuppressed mice and treated with MET and/or IR. Tumours were analyzed by immunoblotting and immunohistochemistry.</p> <p><strong><em>Results</em></strong></p> <p>MET(2.5uM-5mM) caused dose-dependent inhibition of proliferation (10-70%)in all lines, inibited clonogenic survival and sensitized cells to IR. In A549 cellsMET caused inhibition of proliferation comparable to rapamycin, stimulated expression and activation of the ATM and AMPK-p53-p21^cip1and inhibited the Akt-mTOR-4-EBP1 pathway.MET caused G1 arrest of cell cycle, enhanced apoptosis and induced sustained DNA repair foci of gH2AX. MET and IR alone inhibited xenograft growth and combined treatment enhanced that further. IR and MET induced sustained enhancement of expression and activity of ATM-AMPK-p53-p21^cip1and inhibitionof Akt-mTOR-4-EBP1 pathways in tumours also. MET reduced expression of angiogenesis and enhanced expression of apoptosis markers in both control and radiated tumours.</p> <p><strong><em>Conclusions</em></strong></p> <p>Clinically achievable(uM) doses ofMET inhibit human NSCLC cell and tumour growth and sensitize them to IR.This is accompanied by desirable modulation of molecular signals, inhibition of angiogenesis and induction of apoptosis. Our results suggest that MET could be a clinically useful adjunct to radiotherapy in NSCLC and support clinical investigation of MET in combination with radiotherapy.</p> / Master of Science (MSc)

<p>Normal cellular energy metabolism is fundamentally altered in cancer cells to facilitate rapid production of new cellular components, thereby enabling uncontrolled cell growth. Specifically, cancer cells rely on glycolysis and alternative pathways such as lipid and glutamine metabolism for energy, while diverting substrates away from oxidative metabolism regardless of the prevalence of oxygen in the microenvironment. This hallmark of cancer cells is referred to as the Warburg effect, the precise regulation of which is poorly understood despite several decades of research. In comparing the global gene expression profiles of ovarian cancer cells to those that overexpress Ets-1, we have revealed that this transcription factor is involved, at least in part, to this cancer-associated metabolic switch. To support the validity of these findings, we have shown that Ets-1 functionally regulates glycolytic dependence in ovarian and breast cancer cells, while concomitantly displaying a decreased capacity for oxidative phosphorylation. Reactive oxygen species are a normal byproduct of metabolism, and are produced excessively in cancer cells leading to oxidative stress. Interestingly, our genomic pathway analyses uncovered enrichments in antioxidant pathways associated with increased Ets-1 expression. Accordingly, we have also observed that Ets-1 regulates increased intracellular glutathione levels, and induces the activity of key antioxidant enzymes under oxidative stress. Sulfasalazine, an agent that restricts cystine uptake, was shown to be effective for decreasing these high glutathione levels during oxidative stress. These results are clinically relevant because high glutathione levels are associated with iii therapeutic resistance in cancer cells. Collectively, the evidence presented has identified a novel role for the transcription factor Ets-1 in the regulation of cancer energy metabolism, as well as the response to oxidative stress. We have also described a mechanism for Ets- 1-mediated therapeutic resistance, suggesting that this transcription factor may be a promising novel target to enhance conventional cancer therapies.</p> / Doctor of Philosophy (Medical Science)

<p>PTEN is a potent tumour suppressor protein. Aggressive and metastatic prostate cancer (PC) is associated with a reduction or loss of PTEN expression. PTEN reduction often occurs without gene mutations, and its downregulation is not fully understood. Herein, we show that PTEN is incorporated in the cargo of exosomes derived from cancer cells, and this is an exclusive characteristic of cancer cells; normal cells do not incorporate PTEN in their exosomes. We found that this process is affected by the expression of oncogenes, with activation of oncogenic molecules leading to increased PTEN incorporation into exosomes. PTEN expressed in exosomes can be transferred to other cells that have a reduction or loss of PTEN expression. The transferred PTEN is active, as cells showed a substantial increase in phosphatase activity upon treatment with PTEN-bearing exosomes. PTEN transferred through exosomes is also competent to confer tumour-suppression activity to acceptor cells. After incubation with PTEN-bearing exosomes, recipient cells exhibited decreased AKT phosphorylation, changes in the expression of cell cycle mediators indicating cell cycle arrest, and decreased proliferation. These data suggest that exosomal PTEN may be able to compensate for PTEN loss in cancer cells, by transferring the active protein to cancer cells where it can then perform its role as a tumour suppressor.</p> / Master of Science (MSc)

<p>There has been a steady increase in the number of patients with chronic kidney disease. The etiology has been linked to excessive fibrosis progression until the kidney function becomes compromised. We are investigating tubulointerstitial fibrosis (TIF) specifically, as it correlates strongly with the decline of renal function.</p> <p>In our study we investigated the role that active SREBP may play in apoptosis and fibrosis in vivo and in vitro. Treating HK-2 cells with TNFα resulted in the cleavage of SREBP and activation of its SRE promoter. By utilizing a number of inhibitors, we found TNFα induced SREBP cleavage through both a caspase independent and dependent manner.</p> <p>We used fatostatin, a SCAP inhibitor, to reduce the amount of active SREBP in animals in the unilateral ureter obstruction (UUO) model. This model is well known for its development of TIF. Fatostatin decreased SREBP-1 and SREBP-2 activation in mice after 7 and 14 days. Fatostatin increased glomerulotubular integrity and proximal tubular mass as evaluated using lectin staining, along with reducing the number of cells undergoing apoptosis as evaluated by TUNEL staining. Using the Masson Trichrome, Picrosirius red and fibronectin staining, we found a reduction of fibrosis. Fatostatin was also found to attenuate the accumulation of infiltrating myofibroblasts and T cells. These results point to a pathological role for SREBP in TIF.</p> / Master of Health Sciences (MSc)

Alcohol use can negatively impact financial, cognitive, and psychiatric aspects of human life. In the brain, alcohol can have many devastating effects. Alcohol is a well-known cytotoxic agent that can cause specific brain pathology in humans; however, the exact biological mechanisms are not well-elucidated. Animal models are invaluable tools to investigate potential novel treatments in a substance abuse model. Mice studies can be used to screen for negative outcomes prior to human trials. We hypothesize that the C1q tumor necrosis factor-related protein, CTRP3, overexpression in mice reduces neuroinflammation from ethanol consumption that has been coupled with a high fat diet when compared to control mice. The CTRP family of proteins are adipokines and CTRP3 specifically influences cell viability, metabolism, and peripheral inflammation levels. Antibody specific immunoblotting is used to probe protein expression changes in neuroinflammatory markers in mouse cerebellum brain tissue in an overexpression mouse model of CTRP3 when compared to high-fat ethanol exposed mice and baseline control mice. The two proteins examined are MAG and GFAP. Myelin associated glycoprotein, or MAG, is a protein expressed by oligodendrocytes that mediate axonal growth and myelin interactions with neurons in the brain. Oligodendrocytes are extremely sensitive to oxidative stress to which cognitive deficits in ethanol exposure is thought to be attributed. Glial fibrillary acidic protein, or GFAP, is a marker of astrocyte reactivity. Astrocytes are cells in the brain that are responsible for environmental stabilization and actively participate in neurotransmission. Currently, GFAP alterations in ethanol-exposed animals are dose and age dependent. We chose to use young adult mice where GFAP reactiveness is increased during chronic ethanol exposure. The proposed studies are essential in determining CTRP3’s relationship to detrimental neuroinflammatory effects of alcohol and high fat diet in mice. The data obtained from these studies will provide compelling evidence for future clinical trials to investigate CTRP3 as a therapeutic agent in people with a high fat diet that use alcohol chronically.

Tuberculosis is the leading cause of death by single infectious disease worldwide; novel antibiotics are needed to continue to treat this disease. To goal of this project is to provide proof-of-principle support for the idea that targeting protein-protein interactions (PPI) is an appropriate course for the discovery of new drugs. This study optimized the M-PFC assay, which allows detection of PPI in Mycobacteria, through the use of stronger promoters and inducible expression of a peptide blocker by riboswitch. To accomplish this, promoter induction studies were used to find stronger promoters for the M-PFC, optimization of the riboswitch as a method for inducible protein expression within this system, and the addition of both elements to the existing version of the M-PFC. This M-PFC targets DosR homodimerization; this process is known to be essential for survival within the host. This study optimizes a system that may be used to screen for drugs that are capable of interrupting this interaction.