<p>Human somatic cells have a finite lifespan. In contrast, since most cancers evolve through expansions of mutant clones with increasingly more transformed phenotypes, tomour cells may exhaust the proliferative potential of normal cells and possibly acquire an unlimited replicative capacity (immortality). Thus, one essential step in tumourigenesis may be the acquisition of an immortal phenotype. The results presented in this thesis suggest that telomeres, the terminal structures that prevent illegitimate recombination and ensure the proper segregation of chromosomes, and telomerase, the enzyme that elongates telomeres de novo, play critical roles in the process of immortalization of transformed cells both in tissue culture and in vivo.</p> <p>In a tissue culture model of transformation we have shown that telomeres shorten as normal cells divided, as previously reported (Harley et al., 1990), and that, consistent with this observation, the cells lacked detectable levels of telomerase activity. Cells that were driven to divide beyond their normal lifespan by transformation with viral oncogenes did not directly acquire telomerase activity; consequently telemeres continued to shorten until a proliferative crisis, characterized by cell death, was reached. At crisis, chromosome ends contained very little telomeric DNA and appeared to be unstable since the frequency of dicentric chromosomes, aberrations that can be formed by the fusion of chromosome ends, increased. These data suggest that the critically short telomeres detected at crisis may no longer be functional, resulting in genomic instability and potentially cell death. Immortal clones which survived crisis maintained short, but stable telomeres and had telomerase activity. Similarly, malignant cells from the advanced stages of different cancers also had short telomeres and were telomerase positive. Moreover, in one cancer analyzed, the telomeres of malignant cells were found to be stably maintained in vitro and in vivo. These data, although correlative in nature, strongly suggest that, in culture as well as in vivo, telomerase must be activated to counter the lethal loss of telomeric DNA if cells are to become immortal.</p> / Doctor of Philosophy (PhD)

<p>One of the possible contributing factors in the development of hypertension may be an accelerated or premature vascular ageing process, because of some similar structural and functional alterations in the vasculature, including an impaired β-adrenoceptor mediated vascular relaxation and an increase in polyploid smooth muscle cells. The primary objective of this study was to investigate the possible relationship between development of polyploidy and the plasma membrane β-adrenoceptor in cultured smooth muscle cells from the thoracic aortae of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) from 3-4-(prehypertensive), 10-12-(developing hypertensive), and 28-30-weeks (establish hypertensive) of age.</p> <p>The major findings from this study are: (i) similar to the in vivo state, cultured smooth muscle cells from different age-groups of WKY and SHR contain a heterogeneous population of mononucleated and multinucleated cells, as well as diploid and polyploid smooth muscle cells; (ii) the expression of both smooth muscle cell polyploidy and β-adrenoceptor density increases with age in both SHR and WKY, however, this increase was significantly accelerated in SHR as compared to WKY, suggesting an accelerated or premature ageing process may be involved in SHR as compared to WKY; (iii) both SHR and WKY express functional smooth muscle cell β-adrenoceptors but many of the β-adrenoceptors expressed on cultured SHR smooth muscle cells are not coupled to adenylate cyclase; (iv) elevation of intracellular cAMP levels either by agonist activation of β-adrenoceptors or by direct activation of adenylate cyclase in cultured smooth muscle cells from 3-4-week old WKY and SHR and 10-12-week old WKY resulted in an increase in polyploid cells; (v) a β-adrenoceptor antagonist only partially inhibited the isoproterenol-stimulated increase in polyploid smooth muscle cells in both SHR and WKY; and, (vi) the development of polyploid SMC via the β-adrenoceptor-Gs-protein-adenylate cyclase-cAMP pathway is more efficient in cells from WKY compared to SHR.</p> <p>From these findings, I conclude that: (i) the vascular β-adrenoceptor mediated signalling pathway plays a role in the development of polyploid smooth muscle cells; and, (ii) additional intracellular pathways, independent of the β-adrenoceptor-cAMP intracellular mediated signalling pathway, are involved in the development of smooth muscle cell polyploidy.</p> <p>Since it is the resistance arteries (which show no significant increase of polyploid smooth muscle cells with age and/or duration of hypertension) and not the elastic large arteries (aorta) which play a significant role in the development of hypertension, these findings have more relevance to the premature ageing process than to the development of hypertension.</p> / Doctor of Philosophy (PhD)

<p>Cytokines are polypeptide hormones that act nonenzymatically to regulate host cell functions. These glycoproteins make up a fourth class of soluble intercellular signalling molecules that also include neurotransmitters, endocrine hormones and autacoids and are believed to play a central role in tissue remodelling in inflammation, infection, and wound repair. Numerous studies have now implicated cytokines to be of critical importance in host defense, and a more complete understanding of their molecular function is essential. What is also evident is that the majority of biological functions assigned to cytokines have been characterized by in vitro systems.</p> <p>In vivo confirmation of these reported biological functions is required and has been attempted. To date, this has been difficult to attain with the available animal models. While studies in transgenic mice have revealed a number of biological activities, they probably do not reflect normal physiological responses, since tissues chronically exposed to a cytokine throughout development may undergo alterations in its effector phenotype. Administration of recombinant protein is also problematic as repeated injections with large doses of purified recombinant protein are usually required to maintain physiologic concentrations due to the short half life of most cytokines in the circulation.</p> <p>To overcome these problems, we have developed an alternative approach to investigate cytokine function in vivo. This approach, which we have defined as a "pseudo transgenic" animal model, uses recombinant adenovirus vectors containing cytokine genes to deliver and transiently overexpress cytokines in vivo in a tissue-directed manner to normal adult animals. Using this vector approach, cytokine expression can be targeted to a tissue in a way that may mimic more normal physiologic responses. In this study, recombinant adenovirus type 5 vectors capable of expressing the murine cytokines interleukin-5 (IL-5), interleukin-6 (IL·6), and RANTES were constructed to investigate the in vivo effects of these cytokines on immune and inflammatory responses.</p> <p>The first vector constructed, Ad5E3mlL6, contained the murine IL-6 gene incorporated into the E3 region of the viral genome and was used to characterize the capacity of recombinant adenovirus vectors for cytokine expression. This vector was very efficient for cytokine expression both in vitro and in vivo. In addition, using an adenovirus vector containing luciferase as a reporter gene, we demonstrated that expression could be targeted in a highly tissue-specific manner dependent upon the route of administration.</p> <p>Since IL-6 was reported to be the major mediator of the acute phase response and intraperitoneal administration of adenovirus vector primarily targeted cytokine expression to the liver and spleen in Balb/c mice, our initial investigation involved intraperitoneal injection of the Ad5E3mlL6 vector into Balb/c mice. This study confirmed in vivo biological roles for IL-6 as a major mediator of the acute phase response and as a B and T cell proliferation factor.</p> <p>We then analyzed the effects of expression of IL-5 and IL-6, alone and in combination, on humoral immune responses in the mucosal tissue of the lung. Both cytokines, produced by T helper type 2 lymphocytes, are critical to the development and differentiation of B lymphocytes and in particular to IgA antibody production in the mucosa-associated lymphoid tissue (MALT) and bronchus-associated lymphoid tissue (BALT). These studies, using Ad5E3mlL5 (a vector expressing murine IL-5 in the E3 region of the virus) in conjunction with Ad5E3mIL6, provided in vivo support for the roles of IL-5 and IL-6 in inducing lung mucosal immune responses. Co-administration of these two vectors in C57BI/6 mice synergistically induced up to a-fold increases in antigen-specific IgA antibody production in the lung.</p> <p>In addition, we studied the in vivo effects of RANTES, a molecule reported to be chemotactic for monocytes, on lung inflammatory responses. A vector, Ad5E3mRANTES, was constructed which contained the murine RANTES cDNA in the E3 region of the virus. This vector, when intratracheally instilled into Sprague Dawley rats, targeted expression to the mucosal tissue and induced the recruitment of monocytes to the lung within 24 hours. These effects were transient and this expression did not result in detectable lasting pathologic changes to the organ. These in vivo findings on RANTES function are consistent with its proposed function as a potent monocyte chemotactic factor.</p> <p>Finally, we applied this newly developed adenoviral technology to immune modulation in an animal model of breast cancer. Mammary tumor cells from transgenic mice expressing the polyoma middle T antigen under the control of the mouse mammary tumor virus promoter were transplanted into syngeneic mice to establish subcutaneous tumors. These tumors were directly injected with control virus or Ad5E1mlL6A + vector, a replication-deficient vector containing the murine IL-6 gene in the E1 region of the Ad5 genome. We found that localized vector-derived expression of IL-6 could attenuate tumor growth.</p> <p>In conclusion, the results of this thesis study demonstrate the practical use for recombinant adenovirus vectors to aid in the investigation of cytokine function in vivo. This demonstration that vector-derived expression of cytokine is high and can be targeted to specific tissues suggests their use as potential therapeutic agents for the modulation of immune responses in the treatment of cancer and other diseases.</p> / Doctor of Philosophy (PhD)

<p>p-155 is a soluble platelet protein, with a reduced subunit mobility of 155 kDa, that was first identified using a monoclonal antibody raised against platelets. This thesis describes the identification and characterization of p-155, including its structure, biosynthesis, cells of origin, and cDNA sequence. Studies of the native p-155 protein indicated that it is comprised of variably sized, disulfide linked multimers of p-155 subunits, ranging in size from a 400 kDa trimer to large multimers, many millions of daltons in size. Based on its massive, multimeric structure, the native p-155 protein was designated as multimerin. Comparisons with other multimeric proteins found in platelets indicated that multimerin is a novel protein and also one of the largest proteins stored in platelets. In addition to platelets, multimerin was also found in endothelial cells. Biosynthetic metabolic labeling studies indicated that multimerin is synthesized by Dami cells (a megakaryocytic cell line), and by endothelial cells. Multimerin is a highly glycosylated protein with complex, N-linked carbohydrate accounting for 1/3 of its molecular mass. Cleveland mapping studies were used to investigate the relationship between the different sized multimerin subunits found in platelets and Dami cells. These studies demonstrated peptide homology, indicating that p-155 and p-170 (a larger but less abundant multimerin subunit found in platelets) originate from p-196, the multimerin precursor protein identified in metabolic labeling studies.</p> <p>Multimerin antibodies were used to screen expression human endothelial cell libraries for multimerin cDNA clones and the most 5' cDNA clone was used to rescreen the library for complete 5' sequence. The complete cDNA sequence for multimerin was then determined. The multimerin cDNA sequence encodes a hydrophilic protein of 1228 amino acids with RGDS, EGF-like, partial EGF-like, and putative coiled-coil domains. In addition, the C-terminal region of multimerin resembles the globular head domain of complement Clq and collagens type VIII and X. These studies establish multimerin as a unique, multimeric platelet and endothelial cell protein. The massive size, GDS motif, and repeating structure of multimerin suggest a possible role for this protein in adhesion.</p> / Doctor of Philosophy (PhD)

<p>This thesis addresses regulatory aspects of the constitutive expression of human antithrombin, a serine-protease inhibitor involved in coagulation and thrombosis. An introductory part describes a 5'partial deletion, 480 nt upstream of the third exon of an antithrombin allele, in a kindred with thrombosis and hereditary deficiency in antithrombin. The lack of expression of the abnormal allele prompted the further characterization of elements regulating transcription in the 5' region of the normal antithrombin gene. Two cis-acting elements were found, both able to promote transcription in HepG2 and Cos I cells. The first promoter, at -150/+68 nt, encompassed the presumed transcriptional start site. The second element, in reverse orientation in regard to the first, was located at +895/+391 nt in the first intervening sequence. Following footprint analysis, it was shown that the 5'upstream promoter interacted with trans-acting factors at -92/-65 nt, -11/+37 nt and -124/-10 nt, in nuclear extracts from hepatic or non-hepatic origin. Several transcription-factors were subsequently identified, which interacted with these three elements either through direct binding or heterodimerization; they were the liver-enriched factors HNF4 and C/EBPα as well as the ubiquitous nuclear hormone receptors COUP-TF1, RXRα, PPARα and TRα. Furthermore, in vivo expression in HepG2, HeLa and BSC40 cells, of HNF4, C/EBPα and RXRα increased the efficiency of the 5'upstream promoter, while expression of COUP-TFl, TRα, HNF3α or β repressed the efficiency of the latter promoter and the activating effects of HNF4. In addition, activation by HNF4 was synergized by co-expression of RXRα in BSC40 and HeLa cells. These results suggest that the interplay of liver-enriched and ubiquitous factors modulates the constitutive expression of the antithrombin gene.</p> / Doctor of Philosophy (PhD)

<p>We were interested in localizing the regionally specific brain responses which underlie the emotion processing strategies of men, women and a clinical population of individuals with emotion processing deficits: autism. In two studies we identified the brain regions involved in the recognition of emotion by measuring changes in regional cerebral blood flow (rCBF) using positron tomography. Study I asked adult men and women to perform face detection, identity matching and emotion matching tasks and compared the distribution of rCBF produced by each task. The recognition of facial emotion by males was associated with activation of the right inferior frontal cortex, whereas in females, activation of the frontal cortices bilaterally, and the right middle temporal and right inferior occipital gyri was identified. Between-group comparisons of the activations associated with facial emotion processing revealed that males showed greater right sided activation of medial frontal and superior occipital regions and less activation of the left inferior frontal gyrus, left fusiform gyrus and right amygdala than did women. Localization of function to these regions is consistent with other research results identifying greater distribution and less lateralization of cognitive functions in females than males. In our second study we presented cross-modal (auditory-visual) gender matching and emotion matching tasks to three groups of adults; men, women, and high functioning men with autism. Compared to the gender matching task, emotion matching was associated with activation of a left inferior frontal region in males, and the right superior temporal gyrus and right anterior cingulate gyrus in females. This pattern was similarly reflected in between-group comparisons, which identified significantly greater activations in a left inferior frontal region for males, and greater anterior cingulate and fusiform activations for females. These results identify sex differences in cross-modal emotion processing. Localization to these regions suggests that females placed greater relative processing emphasis on the auditory-prosodic and motivational qualities of the current experience, whereas males relied more on processes engaged in the construction of an integrated emotional experience and the regulation of responses. Cross-modal emotion matching by adults with autism was associated with activation of Broca's region, and bilateral anterior temporal poles. Between-group comparisons identified significantly greater activation of the right anterior temporal pole and anterior cingulate by the adults with autism, as compared to controls. These findings suggest that the adults with autism placed greater emphasis on processes involved in accessing perceptual knowledge to guide the categorization of emotional stimuli, verbal problem solving, directing attention toward cross-modal information sources and/or assessing the motivational content of stimuli.</p> / Doctor of Philosophy (PhD)

<p>Idiopathic pulmonary fibrosis is a chronic inflammatory disease of the lung characterized by pathologic alterations with fibroblast proliferation and disordered deposition of extracellular matrix resulting in impairment of gas exchange often leading to respiratory failure. In the pulmonary interstitium, the fibroblast and the extracellular matrix products they produce, play a pivotal role in maintaining the structural and functional integrity of the lung.</p> <p>In the study of lung fibrosis and other fibrosing diseases much attention has been directed at events which alter fibroblast activities. In this construct however, the lung fibroblast is viewed merely as a homogeneous target cell altering its behaviour as a result of immune and inflammatory events. Importantly, it has become apparent over the years that (1) fibroblasts are themselves effector cells capable of releasing a variety of growth factors and mediators and (2) that fibroblasts comprise a heterogeneous population, both within and between tissues. Therefore, an alternative, but not a mutually exclusive hypothesis considers the heterogeneous nature of the fibroblast population and the potential contribution of various subpopulations to disease expression.</p> <p>Our first report that fibroblasts derived from chronically activated and inflamed human lung tissue behaved differently than normal fibroblasts suggested that fibroblast populations present in fibrotic lung tissue exhibited accelerated growth rates. In order to further examine the growth characteristics of fibrotic lung fibroblasts, a soft agarose culture system was established in which we examined the ability of lung fibroblast primary lines to form colonies under anchorage-independent growth conditions, possibly equating to aggressive or "transformed" behaviour. Fibroblasts derived from the Iungs of patients with idiopathic pulmonary fibrosis exhibited anchorage-independent growth in soft agarose culture whereas fibroblast lines derived from normal adult tissue did not. These colonies were fibroblast-like according to morphology and immunohistochemical stain characteristics, and the ability to grow under semi-solid growth conditions was maintained by the fibrotic derived cells even after selection and expansion of single colonies. Interestingly, fibroblast cell lines derived from neonatal lung tissue also exhibited the ability to grow as colonies under soft agarose growth conditions suggesting newly differentiated fibroblast populations may be prevalent in fibrotic lung tissue.</p> <p>The effect of various growth and differentiating factors on the modulation of the anchorage-independent colony formation phenotype was examined in vitro. Treatment with various growth factors, including PDGF, FGF, EGF, TGFβ and corticosteroid were able to modify the colony forming abilities of fibrotic and neonatal fibroblast lines. Importantly, none of the above treatments was able to induce fibroblasts derived from normal adult lung tissue to form colonies. The ability of IPF fibroblast lines and neonatal lung fibroblast lines to form colonies under soft agarose growth conditions was inhibited by treatment with retinoids, known differentiating agents, implying the modulation/differentiation of a particular fibroblast phenotype toward a more mature phenotype; one incapable of anchorage-independent growth.</p> <p>Since the ability to form colonies under soft agarose growth conditions by fibrotic fibroblasts appeared to be a stable and disease specific phenotype and since in vitro we were able to successfully modulate this behaviour with retinoids, we examined the effect of orally administered retinoic acid on the modulation of the fibrotic lung response in a rat model of bleomycin induced pulmonary fibrosis. In this study, we report that the in vivo administration of all-trans retinoic acid significantly decreased the number of fibrotic lesions and dramatically altered the pattern of collagen deposition in the bleomycin treated lung.</p> <p>These studies have confirmed and extended our earlier hypothesis concerning the contribution of fibroblast heterogeneity to the pathogenesis of pulmonary fibrosis and suggests avenues of intervention that may lead to alteration of the fibrotic tissue and return to normal lung function.</p> / Doctor of Philosophy (PhD)

<p>This research examines the hypothesis that in rats inflammation of the gastrointestinal tract results in a specific decrease of food intake. A model for examining gastrointestinal inflammation, the trinitrobenzene sulphonic acid model, and its effects on feeding is developed and investigated in the rat. The TNB model results in a predictable and reproducible colonic inflammation that is accompanied by a rapid but transient decrease in food intake. Experiments are presented which: i)investigate if the anorexia is due to nonspecific malaise. ii)investigate, by meal pattern analysis, the profile of food intake displayed by TNB treated animals iii)investigate specifically, which mediators released from the sight of inflammation could be an anorexigenic signal to the central nervous system.</p> <p>To demonstrate that the anorexia is not due to malaise we have shown that the anorexia: is not specific to the TNB model, is accompanied by no decrease in water intake and develops even when animals are maintained on highly palatable or low residue liquid diets. More importantly animals specifically decrease meal size not meal frequency. Decreased meal size with maintenance of meal frequency is consistent with the generation of abnormal satiety signals and not a "general malaise." These findings show that rats are capable of performing the behaviours necessary for food intake, and do not show specific conditioned taste aversions.</p> <p>The hypothesis that the generation of abnormal gastric and/or post-gastric satiety signal is responsible for TNB induced anorexia was also investigated. I demonstrated removal of both gastric and post-gastric satiety signals in a sham feeding preparation resulted in normal intake even in otherwise anorexic animals. I also show that animals with TNB induced colonic inflammation, present with a decreased rate of gastric emptying. These data raise the possibility that abnormal satiety signals, generated as a result of decreased gastric emptying result in decreased meal size and an overall anorexia.</p> <p>Finally, by specifically inhibiting synthesis of leukotrienes and prostaglandins respectively, or by using an interleukin-1 receptor antagonist (rhIL-1ra), I examined the role of these inflammatory mediators in the anorexia seen in the TNB model of colitis in the rat. I demonstrate that inhibition of prostaglandin synthesis and blockage of interleukin-1 receptors particularly in the brain, result in a significant attenuation of the anorexia. I conclude that the full expression of the anorexia observed in TNB treated animals is dependant on the production of prostaglandins and occupation of centrally located interleukin-1 receptors. Thus, here I specifically describe a model suitable for the investigation of the mechanisms of gastrointestinal inflammation induced anorexia and generally a model that may be used to examine how peripherally generated signals can alter behaviour by communication with the central nervous system.</p> / Doctor of Philosophy (Medical Science)

<p>A large number of studies have identified that bronchoconstricting agents have increased potency and effect following inhaled ozone, but the contribution of bronchodilatory mediators to attenuation of the airway hyperresponsiveness observed following inhaled ozone remains unclear. The purpose of this investigation was to identify the contribution of nitric oxide synthase (NOS) and cyclooxygenase (COX) to the regulation of ozone-induced airway hyperresponsiveness. These mediators are derived largely from the airway epithelium and ozone exposure is likely, through epithelial damage, to alter the effect of NOS and COX in the airway. Using an ozone exposed guinea pig model of airway hyperresponsiveness, the in vivo responses to inhaled histamine were compared to responses in sham treated animals. Administration of the NOS inhibitor L-NAME (5120 μg/mL) increased the histamine sensitivity after ozone treatment. The non-selective COX inhibitor indomethacin (10 mg/kg) also increased the histamine sensitivity after ozone treatment. The COX-2 selective inhibitor DFU (1 or 10 mg/kg) caused a 2-fold leftward shift after ozone exposure, similar to that observed with indomethacin. The thromboxane antagonist, SQ 29,548 (1 mg/kg i.p.) attenuated the histamine responsiveness in a time dependent manner. The combination of COX and NOS inhibition produced a histamine-induced airway hyperresponsiveness greater than that observed with either NOS or COX inhibition alone. Pre-administration of dexamethasone inhibited the ozone-induced histamine hyperresponsiveness at all time points. Biochemical measurements of NOS activity identified an increase in NOS enzyme activity following ozone. Bronchoalveolar lavage revealed an inflammatory cell profile that did not correlate to the in vivo airway hyperresponsiveness. In vitro tissue bath experiments identified the presence and biological activity of NOS in the guinea pig trachea. Liquid chromatographic detection of nitric oxide metabolites and Western blot detection of COX-2 and NOS isoforms did not provide reliable data. This thesis demonstrates that histamine airway responsiveness following ozone changes rapidly and that both NOS and COX are upregulated a few hours after ozone exposure and, through functional antagonism, modulate ozone-induced histamine airway hyperresponsiveness.</p> / Doctor of Philosophy (PhD)

<p>Mitochondria play a critical role in the provision of energy to individual cells through the synthesis of ATP. The relationship between mitochondrial energy production and cell cycle progression has been explored in this work with the intention of defining the fuel requirements of the cell cycle engine. Because mitochondrial physiology and cell division proved to be intimately linked, we also examined the feasibility of disrupting mitochondrial properties in order to alter cell cycle dynamics. We show that the rate of utilization of mitochondrially-derived ATP fluctuates throughout the cell cycle and that stages where ATP levels are lowest correspond to stages most sensitive to pharmacologic inhibition of mitochondrial function. These particular transition points comprise theoretical "energetic checkpoints" of cell cycle progression. It is here that the biochemical events that are sensitive to changes in the cellular energy status will determine whether the cell continues through its cycle or pauses until a favourable energetic balance is restored. The increase in the number of cells in the G1 component of the cell cycle that results from antagonizing mitochondrial function is accompanied by an augmented proportion of persistently active Retinoblastoma protein (Rbp). Failure to inactivate this tumour suppressor protein, whose role it is to brake cell cycle progression in response to suboptimal conditions, does not result from an upregulation of the "classical" G1 inhibitory proteins but rather does so secondary to a decrease in the availability of a critical G1 cyclin. This sensitive regulatory protein, cyclin D, is essential for the activation of kinases responsible for the initial phosphorylation of Rbp, which renders it inactive and allows passage out of the G1 component of the cell cycle. This is the first work to report cell cycle-specific periods of increased ATP utilization which correlate with checkpoints through which a cell will not pass if its energetic balance is sufficiently disrupted. We also demonstrate that changes in mitochondrial function elicit changes in the core proteins of the cell cycle machinery suggesting some intracellular link between the energy balance within the cell and the proteins responsible for cell cycle progression. Finally this report confirms previous suggestions that mitochondria represent viable targets for altering the division characteristics of a cell population, particularly in the context of the altered mitochondrial phenotype of many tumour cells.</p> / Doctor of Philosophy (PhD)