Keith Walley
Relevant Thesis-Based Degree Programs
Graduate Student Supervision
Doctoral Student Supervision
Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.
Genetic variation contributes to outcome from sepsis. A large number of associations have been observed between genetic variants and sepsis outcome, however, identification of causal single nucleotide polymorphisms (SNPs), or their mechanisms of action, have not been successfully elucidated. The aims of this project are to identify causal variants in two candidate genes and determine whether these variants are involved in the mechanisms leading to altered outcomes in sepsis. The known pathophysiology of sepsis is complex and involves dysregulation of several systemic processes, including the coagulation and inflammatory systems. Based on this knowledge, and known literature on genetic variation in coagulation genes, PROC was chosen as a candidate gene in which to search for causal SNPs. In addition, based on the known role of lipids in sepsis, as well as the already identified causal SNPs in the PCSK9 gene, PCSK9 was selected as a second candidate gene to test the hypothesis that genetic variation in lipid mediators alters outcome in sepsis. Two intronic SNPs were found in the PROC gene (rs2069915 and rs2069916) that are in high linkage disequilibrium and appear to modify untranslated mRNA, leading to lower concentrations of circulating protein C in individuals homozygous for the major alleles of these SNPs. Furthermore, in the PCSK9 gene, an intronic SNP (rs644000) was found that appears to mark known Loss-of-Function and Gain-of-Function coding SNPs, and was associated with outcome in two cohorts of patients with septic shock, and with a reduction of cytokine levels in a subset of these patients. Additionally, using murine genetic Pcsk9 knock-out and pharmacologic inhibition strategies in a murine model of systemic bacteremia, a markedly attenuated global, cardiovascular and inflammatory cytokine response to lipopolysaccharide administration was observed. Furthermore, increased endotoxin clearance was measured after PCSK9 knock-out. Together these results indicate that reduction of PCSK9 activity in both mice and humans reduces the inflammatory response and improves outcome in septic shock.The work presented here furthers the understanding of the role played by non-coding SNPs in protein expression and has implications for a new, potentially personal, drug strategy for sepsis patients in intensive care units.
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Septic shock (sepsis accompanied by cardiovascular failure) is an extreme manifestation ofthe host inflammatory response to severe infection. Tumor necrosis factor alpha (TNFα)super family induced nuclear factor kappa B (NF-κB) signaling pathways play a critical rolein the pathophysiology of the disease. A better understanding of how TNFα induced NF-κBsignaling influences the pathogenesis of septic shock is imperative.NF-κB signaling is activated by a canonical pathway or a non-canonical pathway. Thecanonical NF-κB pathway requires the IκB kinase (IKK) complex comprised of IKKα/β/γ.Activation of the IKK complex in response to inflammatory stimuli, such as TNFα, results inubiquitin-dependent degradation of IκBα or IκBβ, releasing p50 related dimers to thenucleus. In response to TNFα superfamily induced inflammation in the non-canonicalpathway, NF-κB inducing kinase (NIK), a docking molecule, recruits IΚΚα to p100 thusactivating IΚΚα. This phosphorylates p100, which is then degraded, releasing p52containing RelB heterodimers to the nucleus.It has been shown that genetic variation in key inflammatory genes contributes to outcome insepsis. We hypothesized that genetic variation in genes of TNFα super family induced NF-κB signaling would be associated with mortality in septic shock. Specifically, we first testedthe hypothesis that genetic variation within the cytosolic members of the canonical and noncanonicalpathway may be associated with mortality in septic shock. We found that the CCgenotype of NIK rs7222094 is associated with increased mortality and organ dysfunction inseptic shock patients. This is perhaps due to altered regulation of NF-κB pathway genes,including CXCL10. We then tested the hypothesis that genetic variation in genesupregulated by these pathways may be associated with mortality in septic shock. We showedthat the G allele of TNFAIP2 rs8126 is associated with increased mortality and organdysfunction in septic shock patients. We then elucidated a novel biological mechanismwhereby TNFAIP2 is a novel inhibitor of Ras, CREB and NF-κB; TNFAIP2 levels arecontrolled by rs8126; and these by allele differences are reflected in the inhibiton of Ras,CREB and NF-κB.
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Master's Student Supervision
Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.
Acute cardiac dysfunction due to myocardial infarction and septic shock may contribute to hypotension, low cardiac output, or death. A common outcome of these disease states is the induction of inflammation, a major contributor to acute cardiac dysfunction. Acute inflammatory reactions often involve the Toll-Like receptor/NFκB pathway. Toll-Like receptors (TLRs) are members of the innate immune system and are responsible for recognizing foreign Pathogen Associated Molecular Patterns (PAMPs) expressed by infecting organisms. Ligation of cardiomyocyte TLRs by PAMPs initiates an inflammatory response and can reduce cardiomyocyte contractility. Recently, endogenous molecules known as Damage Associated Molecular Patterns (DAMPs) which are released during myocardial infarctions have proven to initiate an inflammatory response similar to PAMPs in inflammatory cells. We hypothesize that DAMPs ligate to TLRs causing a pro-inflammatory response by cardiomyocytes whilst reducing cardiac contractility. Additionally, this pro-inflammatory response can be attenuated by using TLR9 ligand, CpG, in a pre-treatment model. Our first objective was to assay a variety of DAMPs for an effect on the inflammatory and functional responses of cardiomyocytes, and determine which TLRs are involved. We found that the DAMP, HSP70, induces an inflammatory response via TLR2 and the NFkB signalling pathway. Our second objective was to determine if a TLR ligand was capable of activating NFκB signalling inducing a tolerant state without affecting cardiac contractility. We found that pre-treatment with TLR9 ligand (CpG) induced tolerance and down-regulated NFkB signalling and expression of inflammatory markers. In accord with these observations, we found CpG pre-treatment attenuated decreases in cardiomyocyte contractility in an LPS model and coronary artery ligation model of ischemia reperfusion. Microarray analysis identified a group of NFkB pathway inhibitors induced by CpG that may be responsible for the diminished inflammatory response. TNFAIP3 (A20) was identified as a highly regulated suppressor of NFkB in cardiomyocytes. In conclusion, a discrete set of TLRs play a major role in myocardial pro-inflammatory responses caused by DAMPs, PAMPs and ischemic injury that lead to decreased contractility and inflammation. However, using TLR9 ligand CpG pre-emptively has shown to reduce the inflammatory response while maintaining proper cardiac function.
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