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Diabetic nephropathy represents the leading cause of end stage renal disease worldwide and requires a kidney transplant or dialysis to survive. The number of patients suffering from diabetes is expected to increase, thus the number of patients with diabetic nephropathy is expected to concomitantly increase. Current treatment for diabetic nephropathy is not sufficient to prevent disease progression in most patients thus there is a need to develop novel therapies to treat diabetic nephropathy. The earliest changes that occur during the pathogenesis of diabetes occur in the glomerulus. The mesangial cells are a subpopulation of cells in the glomerulus that are responsible for coordinating responses with other nearby cell types. Transforming growth factor (TGF)-β1 is a cytokine that mesangial cells secrete, and has been identified as a profibrotic factor during the pathogenesis of diabetic nephropathy. Concerns have been raised in the use of direct anti-TGF-β1 therapy due to adverse events (such as dyspepsia and diarrhea) and lack of efficacy of anti-TGF-β1 monoclonal antibody LY2382770 in patients with diabetic nephropathy. Thus, therapy aimed at modulating TGF-β1 expression or activity may be efficacious in the treatment of diabetic nephropathy while avoiding potential adverse effects. The hypothesis of this thesis is that SREBP-1 and cell surface GRP78 are novel regulators of TGF-β1 signaling in mesangial cells. Our first study aims to define a novel pathway by which SREBP-1 regulates TGF-β1 signaling in kidney mesangial cells. Our results indicate that SREBP-1 regulates the expression of the type I TGF-β1 receptor through its secretion in exosomes. Our second study expands on these findings and aims to determine if inhibition of SREBP in vivo with the inhibitor fatostatin may prevent diabetic nephropathy. Our results indicate that treatment with fatostatin does not prevent diabetic nephropathy, but accentuates kidney injury in non-diabetic mice. Preliminary results from our lab have indicated that under diabetic conditions, GRP78 is upregulated at the cell surface and may contribute to the activation of SREBP-1 in an ER-stress dependent mechanism. Our third study thus aims to characterize the expression of cell surface GRP78 in diabetic conditions, and to determine its pathological relevance in the development of diabetic nephropathy. Our results have established novel pathways by which TGF-β1 signaling is regulated in mesangial cells. This will assist in identification of novel therapeutic targets that may be of use in the treatment of diabetic nephropathy. / Thesis / Doctor of Philosophy (PhD) / Diabetic kidney disease is the leading cause of end stage renal disease and represents an important risk factor for mortality. The goal of this thesis is to understand and describe the pathways and mechanisms that contribute to the development of diabetic kidney disease in order to identify novel therapeutic targets. This thesis has identified the protein sterol regulatory element binding protein (SREBP)-1 and the cell surface presentation of another protein, the 78 kDa glucose regulated protein (GRP78), as contributors to diabetic kidney disease. Furthermore, this thesis has demonstrated that anti-SREBP therapy with the drug fatostatin did not prevent diabetic kidney disease. These studies show that while inhibiting SREBP-1 and cell surface GRP78 may be effective in the treatment of diabetic kidney disease, the drug fatostatin should not be used for treatment.

The lower airways of patients with chronic airway diseases including cystic fibrosis (CF) are colonized by diverse communities of microorganisms. Over-time the airways of some 60% of CF patients become permanently colonized and dominated by Pseudomonas aeruginosa. Chronic infection of P. aeruginosa has been associated with a decline in pulmonary function, worse prognosis, and eventual patient mortality. Although P. aeruginosa evolves within the CF airways resulting in complex populations, the mechanism by which these complex populations contribute to disease progression is not well understood. Here we show diversity among isolates by observed changes in genome sequences of a strain of P. aeruginosa, known as Prairie Epidemic Strain (PES). Using whole genome sequencing and comparative genomics we identified a large core genome across 195 PES isolates from 57 CF patients of the Calgary Adult Cystic Fibrosis Clinic (CACFC) where 88% of the pangenome was categorized as core genes. Single nucleotide polymorphism (SNPs) mutations were shown to be the largest contributor of diversity at the nucleotide level compared to other polymorphism types consisting of 87% of the total polymorphisms present across the 195 PES isolates. CRISPR arrays and mobile elements such as prophage and plasmids demonstrate this strain of P. aeruginosa was stable over 30 years. In a second aim, I show variation in the populations of P. aeruginosa across an exacerbation event further highlighting the complexity of the lung bacterial community. Distinct populations of P. aeruginosa at the onset and resolution of an exacerbation within a single CF patient were identified by SNPs. These results a model where adaptive radiation as well as natural mutations contribute to the heterogeneity and diversification within populations of P. aeruginosa in CF patients. Understanding the evolution and population structure of PES through the identification of important genes and mutations through the clinical course of an exacerbation can aid in identifying new targets for patient treatment of P. aeruginosa in CF. / Thesis / Master of Science (MSc) / Cystic fibrosis is a life-threatening disease characterized by cycles of stability and respiratory illness. Bacterial species within the lungs of these patients are the main contributor to disease progression. I investigated a specific transmissible epidemic strain, Pseudomonas aeruginosa Prairie Epidemic Strain, using a unique collection of samples provided by collaborators at the adult cystic fibrosis clinic in Calgary. Using these samples, I first explored the differences between patients over a period of 34 years. I hypothesized that similar changes in genome sequences will be observed in multiple patients with a possible commonality in disease progression. Second, I explored the role this bacterial pathogen may play in cycles of respiratory illness. I hypothesize that a specific bacterial subpopulation could initiate these cycles and be identified by changes at the genome level. This research provides further knowledge of an epidemic strain of cystic fibrosis.

Chronic kidney disease (CKD) is a major cause of morbidity and mortality, affecting more than 10% of the world’s population. CKD is associated with excessive renal fibrosis, which leads to declining kidney function and eventual kidney failure. In CKD, glomerular mesangial cells (MC), resident fibroblasts and tubular epithelial cells undergo phenotypic activation and transition in response to profibrotic and proinflammatory cytokines such as transforming growth factor β1 (TGFβ1). These activated renal cells excessively produce extracellular matrix (ECM) proteins that replace functional renal tissue and lead to renal fibrosis. Caveolae are small omega-shaped invaginations of the plasma membrane that mediate signaling transduction events. Formation of caveolae require the protein caveolin-1 (cav-1). We have previously shown that the ability of MC to produce matrix proteins is dependent on cav-1 expression. Unfortunately, clinically targeting cav-1 within the kidneys, specifically within MC, is technically challenging and as of yet unfeasible. Thus, to better understand how cav-1 deletion is protective, we carried out a microarray screen comparing cav-1 wild-type (WT) and knockout (KO) MC. Here, we discovered significant up-regulation of a TGFβ superfamily inhibitory protein, follistatin (FST). FST specifically targets and neutralizes activin A (ActA) but not TGFβ1. TGFβ1 and ActA both belong to the TGFβ superfamily of cytokines and growth factors. While TGFβ1 itself is a known key mediator of renal fibrosis, therapies aimed at directly inhibiting TGFβ1 in kidney diseases have not been successful due to opposing profibrotic and anti-inflammatory effects. ActA has been shown to act as a strong profibrotic and proinflammatory agent in various organs, including the lungs and liver. We along with others have observed elevated levels of ActA within the kidneys and serum of mice and humans with CKD. Functionally, ActA has been shown to contribute to ECM production in the kidneys. Hence, we hypothesized that ActA inhibition through FST could prove beneficial in CKD. In this thesis, our first study elucidated a novel molecular pathway by which cav-1 regulates expression of the FST in MC. Our results indicate that FST is negatively regulated by cav-1 through a PI3K/PKC zeta/Sp1 transcriptional pathway. Our second study expands on these findings and tests whether exogenous FST administration protects against the progression of CKD in a surgical mouse model of CKD. Here, we discovered that FST acts as a reactive oxygen species (ROS) scavenger and that exogenous administration of FST protects against the development of CKD through the inhibition of renal fibrosis and oxidative stress. Lastly, our third study determined whether microRNAs (miRNAs) are implicated in post-transcriptionally regulating FST through cav-1 and whether these FST-targeting miRNAs can be utilized therapeutically to protect against the development and progression of CKD. Here, we determined that a FST-targeting miRNA, microRNA299a-5p, is significantly downregulated in cav-1 deficient MC, upregulated in vivo in a mouse model of CKD and that its inhibition, in vitro and in vivo protects against the accumulation of ECM proteins and renal fibrosis. These studies collectively suggest that FST is an effective therapeutic option for the management of CKD. / Thesis / Doctor of Philosophy (PhD) / Chronic kidney disease results from excessive fibrosis (scarring) within the kidneys. The goal of this thesis is to understand the molecular mechanisms involving the regulation of an antifibrotic protein, follistatin, in glomerular mesangial cells and to identify its therapeutic potential in chronic kidney disease. This thesis has identified that follistatin, an endogenous inhibitor of the profibrotic cytokine activin A, is regulated transcriptionally by Sp1 and post-transcriptionally by microRNA299a-5p. Furthermore, this thesis has demonstrated that exogenous recombinant follistatin administration protects against the progression of chronic kidney disease and that microRNA299a-5p targeting may be an alternative approach to block renal fibrosis. These studies collectively show that follistatin is an effective treatment for the management of chronic kidney disease.

The cystic fibrosis transmembrane conductance regulator (CFTR) is one of the most studied membrane protein models because of its clear clinical significance. Mutations within the CFTR gene lead to cystic fibrosis, the most common autosomal recessive genetic disorder in the Caucasian population. CFTR, a large 160 kDa glycoprotein, is a chloride ion channel in the ABC superfamily of transporter proteins. Due to low natural abundance of CFTR and difficulties producing sufficient amounts in heterologous systems, the exact protein function/structure relationship is unknown. Expression of CFTR in E. coli is lethal and mammalian culture systems are expensive and low yielding. However, successful bioproduction of many complex human proteins has been shown in transgenic plants. Our research objective is to develop tobacco as a model system for expressing human CFTR. Constructs of full-length CFTR fused to the 35S double enhanced promoter could not be propagated in E. coli, suggesting that the CFTR product generated by "leaky" expression was detrimental to bacteria. Two strategies were undertaken to address the problem: 1) a plant intron was introduced into CFTR sequence and 2) a more tightly regulated wound-inducible promoter MeGATM was used. Tobacco was transformed with all constructs. CFTR presence was determined by polymerase chain reaction (PCR). Expression and intron splicing was analyzed by reverse transcriptase-PCR. Splicing did not occur presumably due to intron /exon contexts. In tobacco expressing MeGA:CFTR, however, novel high-molecular-weight membrane-associated proteins were immunodetected using anti-CFTR antibodies suggesting that tobacco may be capable of producing human CFTR. / Master of Science

Cystic fibrosis is an autosomal recessive disease caused by mutations in the CFTR gene. A significant number of mutations (~13%) alter the correct splicing of the CFTR gene, causing the transcription of aberrant transcripts resulting in the production of a non-functional CFTR channel. We focus our research on two intronic CF causing mutations, 3272-26A>G and 3849+10kbC>T that create a new acceptor and donor splice site, respectively, generating in the inclusion of intronic portions into the mRNA. We developed a new genome editing approach to permanently correct the abovementioned mutations by means of CRISPR nucleases. We exploited the use of either Streptococcus pyogenes Cas9, SpCas9, or Acidaminococcus sp. BV3L6, AsCas12a, to edit the aberrant splicing sites and restore the production of the correct transcript, avoiding modifications of the CFTR coding sequence. A comparative analysis between SpCas9 and AsCas12a revealed that the use of AsCas12a with a single crRNA efficiently edits the target loci, producing correctly spliced mRNAs in both 3272-26A>G and 3849+10kbC>T mutations. Furthermore, this genetic repair strategy proved to be highly specific, exhibiting a strong discrimination between the mutated and the wild-type allele and no detectable off-target activity with genome-wide analysis. The selected crRNAs were tested in patients derived primary airway cells and intestinal organoids compound heterozygous for the 3272-26A>G or 3849+10kbC>T mutations, that are considered relevant CF models for translational research. The efficient splicing repair and the complete recovery of CFTR channel activity observed confirmed the goodness of the proposed gene editing strategy. These results demonstrated that allele-specific genome editing with AsCas12a can correct aberrant CFTR splicing mutations, paving the way for a permanent splicing correction in genetic diseases.

Obesity and insulin resistance are associated with a constellation of features including hypertension, dyslipidaemia, type 2 diabetes, and premature cardiovascular disease, collectively termed the metabolic syndrome. Non-alcoholic fatty liver disease (NAFLD) represents the hepatic component of this syndrome, incorporating a spectrum of liver disease with increasing morbidity and mortality, from simple steatosis, to non-alcoholic steatohepatitis (or NASH), fibrosis, cirrhosis and ultimately hepatocellular carcinoma. However, factors influencing this progression are incompletely understood. In this thesis I sought to investigate pathways which promote hepatic inflammation and fibrosis by studying two contrasting dietary models of NAFLD in mice in which the risk of hepatic inflammation, insulin resistance and fibrosis differ; namely the methionine and choline deficient diet (MCDD) which induces steatohepatitis, hepatic insulin resistance, and weight loss, and the choline deficient diet (CDD) which may be protected from insulin resistance, and leads to steatosis without inflammation or weight loss. I investigated the possible molecular mechanisms underlying these differences, and whether they influenced progression to hepatic fibrosis induced by carbon tetrachloride (CCl4).