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There is much observational evidence to suggest that patients with chronic kidney disease are biologically 'older' than their unaffected peers. This is most obviously seen with cardiovascular disease: young patients on haemodialysis have a relative risk of cardiovascular mortality similar to that of people over 50 years their senior in the general population. Moreover, there are striking analogies between the effects of physiological ageing and uraemia on the structure and function of the heart and vasculature. Despite this, little work has been published looking at whether these similarities are reflected at a molecular and cellular level. Two processes implicated in ageing are autophagy and senescence. There is much inferred evidence that these processes are affected by chronic kidney disease. The aim of this work was to investigate whether autophagy and senescence are indeed altered in the uraemic heart, whether these processes might be linked, and whether the findings of these enquiries might suggest their involvement in the pathogenesis of the prematurely aged cardiac phenotype. An in vitro model of the uraemic myocardium was created using rat cardiac myoblast cells exposed to the uraemic toxin indoxyl sulphate, and in vivo models using adenine-diet and subtotal (5/6) nephrectomy rodents. Autophagy was assayed using immunoblotting, PCR array, immunohistochemistry and fluorescence microscopy, and senescence by immunoblotting and as part of an ageing-dedicated PCR array. Though not achieving statistical significance, markers of autophagy activity appeared to be increased in rat cardiac myoblast cells exposed to indoxyl sulfate, and in cardiac tissue from adenine-diet rats. Interestingly markers of autophagy activity were significantly increased in hepatic tissue from subtotal nephrectomy rats. PCR of RNA purified from cardiac tissue from adenine-diet rats demonstrated an expression of ageing-related genes analogous to that in physiological ageing. Though limited by numbers, these findings present evidence to suggest that autophagy may be upregulated as a protective mechanism in the progeroid uraemic heart, a situation possibly comparable to that in physiological ageing. Changes in cardiac autophagy and ageing in uraemia present new avenues for translational research into pathological ageing in chronic kidney disease.

Senescence is a highly efficient mechanism that provides an irreversible barrier to cell cycle progression to prevent undesired proliferation. However, under pathological circumstances, senescence can adversely affect organ function, viability and regeneration. In the context of biliary disease, we hypothesize that senescence is initiated in the bile ducts and spreads to the liver parenchyma, impairing the liver’s regenerative capacity and aggravating the condition. We have developed a mouse model of biliary senescence, based on the conditional deletion of Mdm2 in bile ducts, that mimics clinical features of biliary disease. Using this model, we studied the underlying mechanisms that characterize biliary disease, and established an essential role of TGFβ in paracrine senescence-associated regeneration. Lastly, we disrupted TGFβ signalling to therapeutically rescue this phenotype in our model of biliary senescence. These results reveal the detrimental role of senescence in biliary disease, and a TGFβ- dependent mechanism for dissemination of senescence from the biliary epithelium to the parenchyma, impairing liver function. Finally, we have identified TGFβ signalling disruption as a potential therapeutic target to limit senescence-dependent aggravation in human cholangiopathies.

Hepatitis C virus (HCV) infection is a leading cause of cirrhosis and hepatocellular carcinoma. The degree of fibrosis progression and treatment-related outcomes are critically dependent on the age of the infected individual. Progressive ageing is associated with a decline in the efficacy of adaptive immune system function. T-lymphocytes from aged subjects demonstrate multiple phenotypic and functional changes, including telomere shortening. Short telomeres are associated with poor proliferative capacity, pro-inflammatory responses and increased mortality in clinical studies. This research aimed to study telomere length changes in T-lymphocytes in chronic HCV infection and its relationship to clinical endpoints. Further, the intracellular signalling changes in T-lymphocytes with short telomeres were studied in subjects with chronic HCV. Short CD4+ T-lymphocyte telomeres were associated with the presence of severe hepatic fibrosis independent of other known factors. Telomere length was associated with blood markers of hepatic damage and dysfunction as well as histological markers of inflammation and fibrosis. Further, on prospective follow-up, short CD4+ telomere length at enrolment predicted progression to clinical endpoints of hepatic decompensation, development of hepatocellular carcinoma and death. Short CD4+ telomere length predicted a failure to respond to anti-viral treatment for HCV infection. Unexpectedly, subjects with non-viraemic HCV had short CD8+ telomere length. Liver biopsy tissue from a cohort of subjects with non-viraemic HCV was studied and demonstrated significant inflammation or fibrosis in most. To study the IFN-α signalling pathway in cells with short telomeres, I utilised the phospho-histone γ-H2AX, a downstream signal from short telomeres. CD8+ T-lymphocytes expressing γ-H2AX had the form and function of cells with end-stage differentiation. γ-H2AX+ cells had a pro-inflammatory cytokine secretion profile with high expression of IFN-γ and low IL-2. Further γ-H2AX+ cells were unable to respond to exogenous IFN-α by phosphorylating Stat1. This failure was attributable to a post-receptor defect. T-lymphocyte telomere length changes in HCV may underpin the effect of age on clinical and treatment-related outcome. Short telomeres are associated with intracellular signalling defects which may explain the failure to respond to anti-viral therapy.

Cellular senescence represents status, when the cells cease to divide and remain in permanent cell cycle arrest. Senescence is considered to be an active response of the cell to various extrinsic and intrinsic types of stress such as certain oncogene activation, exposing to several cytokines or drugs and damaged and/or uncapped telomeres. Senescent cells are characterised by extensive modification of gene expression, flattened and enlargement of cellular body. Hypothetically, altered gene expression may lead also to increase of certain surface proteins expression. Such protein can be L1 cell adhesive molecule (L1CAM), which is expressed heterogeneously within the population. This Thesis describes current knowledge of cellular senescence, the mechanism, which may result in establishment of senescence phenotype, and also the characteristic markers of senescence. Thesis also puts together the heterogeneity of L1CAM expression in A375 senescent cells with oxygen consumption rate and extracellular acidification rate performed by Seahorse XFe24 metabolic analyser. Therefore, ells were sorted according to their levels of expressing L1CAM onto low and high L1CAM expressing subpopulations. Obtained data show potential correlation between the rate of L1CAM expression in A375 cells and the metabolic rate. Key...

In addition to the canonical full-length p53 (FLp53), the TP53 gene produces twelve protein isoforms through alternative RNA splicing or initiation of transcription and translation. Two of<br>these isoforms, D133p53a and p53b, have been identified as endogenous regulators of cellular senescence. Cellular senescence is a durable cell cycle arrest that inhibits the continued replication of aged and DNA-damaged cells. This process is a critical mechanism of tumor suppression that<br>prevents initiation and malignant progression and has been leveraged to treat cancers including glioblastoma. However, removal of senescent cells by macrophages is needed to restore tissue homeostasis. This process is impacted by a variety of factors. For example, senescent cells accumulate in aged individuals and can promote chronic inflammation and disease through the senescence-associated secretory phenotype (SASP). As the global population ages, it will become more critical to understand the function of cellular senescence in disease. Targeting senescent cells, either through elimination (senolysis) or reprogramming, may have potential therapeutic value in individuals with a high senescent cell burden. Aged or DNA-damaged cells adopt a senescence-associated p53 isoform profile characterized by reduced expression of D133p53a and increased expression of p53b. Critically, restoration of D133p53a rescues cells from senescence and enhances DNA repair. Targeting p53<br>isoforms may represent a mechanism by which cells can be reprogrammed. A thorough understanding of the contexts in which senescent cells maintain beneficial or harmful roles is<br>critical to developing senescence therapeutics in cancer and aging.

Vancouver Island, British Columbia, is at the northern extent of natural climate zones conducive for grape growing, making vineyards susceptible to any changing weather patterns and temperature extremes. Grapevine monitoring is an important aspect of the viticulture industry, and remote sensing technologies are a powerful aid in reporting vegetation information for better vineyard management practices. However, the understanding of vine spectral responses as viewed by optical sensors has to be developed further, and was undertaken in this study. Chlorophyll pigments drive photosynthesis, a biochemical process in plants, which contributes to physiological performance and productivity, making it an appropriate leaf characteristic for detailed examination. This study aimed to develop a thorough understanding of the relationship between (i) leaf-level spectral reflectance and transmittance properties and (ii) pigment concentrations, via ground-based sampling. This was achieved through the examination of two ground campaign tools, as well as current spectral data processing techniques and workflow methods. A spectrometer and SPAD chlorophyll meter collected nondestructive measurements during leaf senescence and grape harvest, and wet chemical extraction methods determined chlorophyll content (expressed in terms of unit leaf area and leaf fresh weight). Reflectance indices,first order derivative indices, and a continuum removal approach were used to generate eighteen reflectance-based attributes. This study performed a series of chlorophyll estimation models through iterative ordinary least square regression, followed by two methods of model validation. Performance metrics indicated strong models with high explanatory power; the continuum removed depth normalized total area metric was presented as the optimal nondestructive attribute for accurate chlorophyll estimation for leaf level field campaigns (R2 = 0.93). Chlorophyll expressed in units of fresh weight yielded more consistent models than in units of leaf area. The chlorophyll meter also presented compelling results (R2 ≥ 0.78), and both sensors were determined to be appropriate for field validation campaigns for this vineyard study. / Graduate

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Master of Science / Biochemistry and Molecular Biophysics Interdepartmental Program / Anna Zolkiewska / Cellular senescence is a state of irreversible cell cycle arrest in response to non-lethal stress. In cancer cells, senescence can be induced by chemotherapy, radiation, or signals from the tumor microenvironment, such as transforming growth factor β (TGFβ). Senescent cells are metabolically active and have altered gene expression compared to their non-senescent counterparts. Senescent cells release a wide variety of factors, including extracellular domains of transmembrane proteins that require proteolytic cleavage by specific proteases. ADAMs (A Disintegrin and Metalloprotease domain-containing proteins) are enzymes that cleave many transmembrane proteins, such as growth factor precursors or adhesion molecules, and thus may act as sheddases in senescent cells. Here, we investigate ADAM expression levels during TGFβ- induced cellular senescence. SUM149PT and SUM102PT breast cancer cells were incubated with TGFβ, followed by treatment with high doses of paclitaxel to remove actively proliferating, non-senescent cells. Induction of cellular senescence was examined by evaluating changes in cell size and granularity, and by β-galactosidase staining. ADAM mRNA levels were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Among several ADAMs tested, ADAM12 mRNA was significantly upregulated in senescent cells. In addition, we demonstrated that ADAM12 knock-down leads to decreased activation of epidermal growth factor receptor (EGFR), an important modulator of cancer cell growth, survival, and metastasis. This effect of ADAM12 knock-down was likely due to a diminished release of soluble EGF or EGF-like ligands from cells. Since senescent cells often release increased amounts of these ligands, ADAM12 may modulate the senescence secretome in senescent breast cancer cells.

Radiotherapy continues to be a primary modality in the treatment of cancer. DNA damage induced by radiation can promote apoptosis as well as both autophagy and senescence, where autophagy and senescence can theoretically function to prolong tumor survival. A primary aim of this work was to investigate the hypothesis that autophagy and/or senescence could be permissive for DNA repair, thereby facilitating tumor cell recovery from radiation-induced growth arrest and/or cell death. In addition, studies were designed to elucidate the involvement of autophagy and senescence in radiation sensitization by PARP inhibitors and the re-emergence of a proliferating tumor cell population. In the context of this work, the relationship between radiation-induced autophagy and senescence was also determined. Studies were performed using DNA repair proficient HCT116 colon carcinoma cells and a repair deficient Ligase IV (-/-) isogenic cell line. Irradiation promoted a parallel induction of autophagy and senescence that was strongly correlated with the extent of persistent H2AX phosphorylation in both cell lines; however inhibition of autophagy failed to suppress senescence, indicating that the two responses were dissociable. Irradiation resulted in a transient arrest in the HCT116 cells while arrest was prolonged in the Ligase IV (-/-) cells; however, both cell lines ultimately recovered proliferative function, which may reflect maintenance of DNA repair capacity. The PARP inhibitors (Olaparib) and (Niraparib) increased the extent of persistent DNA damage induced by radiation as well as the extent of both autophagy and senescence; neither cell line underwent significant apoptosis by radiation alone or in the presence of the PARP inhibitors. Inhibition of autophagy failed to attenuate radiation sensitization, indicating that autophagy was not involved in the action of the PARP inhibitors. As with radiation alone, despite sensitization by PARP inhibition, proliferative recovery was evident within a period of 10-20 days. While inhibition of DNA repair via PARP inhibition may initially sensitize tumor cells to radiation via the promotion of senescence, this strategy does not appear to interfere with proliferative recovery, which could ultimately contribute to disease recurrence.