<p>Adenovirus type 5 is able to oncogenically transform cells growing in culture. These transformed cells show different degrees of transformation. The interest of my studies was to establish the presence and characterize the structure of virus DNA in adenovirus 5 transformed cell lines and to look for a possible correlation between the integrated viral sequences and the phenotype of the cells. For this purpose, cell lines transformed by wild type (virions or DNA) and by host range mutants (virions) were analyzed for their viral DNA content and for their transformed phenotype.</p> <p>More than the left 8% was always present in virion transformed cells. Cells transformed by host range mutants of complementation Group I generally contained a larger fraction of the genome than did their counterparts transformed by wild type virus. In some host range transformed cells, virtually the entire viral DNA molecule was found colinearly integrated.</p> <p>In the case of the cells transformed by wild type DNA or virions, it was not possible to correlate any particular phenotype with a specific integration pattern. The same pattern was found in the tumorigenic derivatives of a non oncogenic cell line (293) as in the parental line. Studies with cells transformed by Group I host range mutants showed that partial or complete integration of viral DNA into transformed cells was always associated with the production of some viral proteins and with the induction of a partially transformed, non oncogenic phenotype.</p> <p>An interesting finding was the limited number of inserts in cell lines isolated from uncloned populations within a small number of passages after transformation. This suggested the possibility of a limited number of sites available for transformation or alternatively, that a few cells rapidly overgrew the rest. In an attempt to answer this question and to obtain more information on the integration process, DNA was extracted from semipermissive rat cells at different times shortly after infection. Adenovirus 5 wild type and host range mutants from Groups I and II were used in individual experiments. The extracted DNA was alnalyzed for the presence and state of viral DNA. New forms of intracellular viral DNA, which might be intermediates for integration, were found. A fragment having the size of both ends of the viral DNA joined together, and which hybridizes with both ends when these are used separately as probes, was detected in Southern blots after digestion of rat cell DNA with different restriction enzymes. This structure was detected earlier after infection with host range 1 (Group I), than after infection/with host range 6 (Group II) or with wild type, and was found also in Hela cells and in two rat cell lines after infection with host range J or wild type. Studies followed to determine the nature of this new arranged viral DNA provided evidence for covalent head to tail joining and for the formation of circular Ad5 DNA molecules.</p> / Doctor of Philosophy (PhD)

<p>Biochemical and structural abnormalities have been reported in postmortem brain tissue from patients with mood disorders. Studies of the molecular pharmacology of drugs used in the treatment of mood disorders have led to a reinterpretation of earlier models of neuropathology in these diseases. Noradrenergic and serotonergic hypotheses have been expanded to include postsynaptic intracellular signal transduction pathways, regulation of gene expression, and synaptic plasticity. Because much of this evidence was obtained from postmortem brain, the experiments in this study also used postmortem tissue to examine the neuropathology of mood disorders. Human postmortem brain tissue was obtained from the Stanley Consortium Neuropathology Foundation and consisted of pieces of temporal and occipital cortex, and slices of prefrontal cortex and hippocampus from patients with bipolar affective disorder (BD), major depressive disorder (MDD), schizophrenia (SCZ) and controls (N = 15 per group). Components of the cAMP system were examined, as were potential target transcription and neurotrophic factors. Morphological consequences arising from altered cAMP signalling such as sprouting and DNA fragmentation were also investigated. There was a trend towards blunted temporal cortex adenylyl cyclase activity in subjects with mood disorders, and decreased occipital cortex Gas in lithium-treated BD subjects. Temporal cortex CREB level~ were decreased in antidepressant-untreated MDD subjects compared to controls, and normal in those subjects treated by antidepressants at the time of death. Temporal cortex CREB levels were decreased in BD patients treated with anticonvulsants relative to those not treated by anticonvulsants at the time of death. When assessing the effect of suicide on cAMP signalling, subjects who died as a result of suicide had lower temporal cortex CREB levels and CRE-binding than subjects who died of other causes. This effect was most evident in the MDD group. In hippocampus, BDNF levels were increased in subjects treated by antidepressants compared to subjects not treated by these medications at the time of death. This finding was more pronounced in the MDD group. Hippocampal studies also showed that BD subjects had increased mossy fibre staining relative to controls and other diagnoses, suggesting increased sprouting of the dentate gyrus axons. These postmortem findings are consistent with recent animal and cell models of antidepressant and mood stabilizer pharmacology. The changes in post-receptor signalling and hippocampal sprouting are also consistent with current conceptualizations of the neurobiology of mood disorders. These studies support the use of postmortem brain tissue as a clinically relevant research model, and add to the growing literature elucidating the pathophysiology of mood disorders and the molecular pharmacology of their treatments.</p> / Doctor of Philosophy (PhD)

<p>The epidemic rise in the prevalence of allergy and asthma, primarily in the "developed" world, has impelled prolific nodes of inquiry into the epidemiology, aetiology, immunology and management of these syndromes. While studies in human patients have revolutionized pharmacological treatment of asthma and allergy, and have also intimated some of the environmental agents and conditions conducive to disease expression in susceptible populations, only animal models of asthma afford the experimental flexibility upon which detailed in vivo analysis of immunology and pathogenesis depends. Because asthma arises through airway mucosal contact with allergens, chemical pollutants and/ or infectious agents, an authentic animal model of asthma should preserve the airway as the interface of incipient contact with antigen and, by extension, as the immune microenvironment that conditions allergic sensitization. This heuristic is particularly relevant when considering questions about the immunomodulatory effects of local, anti-inflammatory intervention. The research documented in this thesis investigates several immunomodulatory concepts- including pharmacological intervention (Chapter 2), costimulatory molecule blockade (Chapter 3) and chemokinetic manipulation of cell trafficking (Chapters 4 and 5)-in a murine model of airway mucosal sensitization to an innocuous antigen. The salient message informed by these studies is that the outcome of an immune-inflammatory response is very much a reflection of the airway microenvironment in which the immune system initially processes antigen. Of substantive clinical interest, these data indicate that the efficacy of acute, therapeutic intervention must be reconciled with the status of the antigen-specific response once treatment has ceased.</p> / Doctor of Philosophy (PhD)

<p>Experimental and clinical evidence implicate the β-integrin subunit and its associated intracellular effector ILK in the initiation and progression of human breast cancer. Roles for these proteins in promoting the oncogenic process, however, have not been demonstrated in vivo. As a result, experiments were designed to test the tumour-promoting properties of β-integrin and ILK in the mammary glands of transgenic mice. The first of these experiments involved the targeted ablation of a conditional allele of β-integrin in a transgenic mouse model of human breast cancer. Using this approach, it was found that the expression of β-integrin is required for oncogeneinduced transformation of the mammary gland epithelium in vivo. This requirement for β1-integrin expression was observed during both the initiation of tumourigenesis, as well as for maintaining the growth of established tumours. In addition, a block in tumour cell proliferation following ablation of β1-integrin expression was found to be associated with the suppression of F AK autophosphorylation, providing a molecular mechanism underlying the requirement for β1-integrin expression during tumourigenesis. The second experiment was designed to test the oncogenic properties of ILK, through the establishment of transgenic mice overexpressing ILK in the mammary gland epithelium. Following the induction of a hyperplastic mammary gland phenotype, these mice developed solid mammary tumours showing evidence of epithelial-to-mesenchymal transition, confirming that ILK overexpression can contribute to mammary tumourigenesis in vivo. Since expression of both β-integrin and ILK have been reported in samples of aggressive human tumours, these results may have clinical significance to the treatment of human breast malignancies.</p> / Doctor of Philosophy (PhD)

<p>A variety of short oligoribonucleotide sequences were synthesized using the phosphotriester chemical synthesis developed in Neilson's laboratory. These sequences were designed to incorporate a variety of features that would aid in the study of perturbations of helix structure and stability. Variable temperature proton nuclear magnetic resonance (NMR) spectroscopy was used in this study and provides a powerful technique for the study of nucleic acid conformations and for the investigation of the effects of the mispairing and single stranded regions caused by the helix imperfections introduced.</p> <p>The assignment procedure for NMR spectra was improved through the study of a series of related sequences. This study determined the effects that the addition of a nucleotide to the terminus of a sequence or the insertion of a nucleotide into the middle of a sequence would have on the chemical shifts from the rest of the sequence.</p> <p>A series of self-complementary pentaribonucleotides, with a central non-base paired opposition (AGXCU, where X ≡ A, G, C or U), was studied to determine the effects of small loops on duplex stability. In contrast to earlier results, these pentamers formed stable duplexes when X ≡ A or C, although the duplex Tm's were significantly, reduced. These sequences also provided the opportunity to study the sequence effects of adjacent internal G.C base pairs on duplex stability when the middle base pair was A·U, G·C or G.U. Comparison with earlier results using corresponding A·U base pair neighbours, demonstrated the enhanced stabilization of G·C base pairs.</p> <p>The effects of terminal non-base paired (dangling) adenosines were more closely investigated and found to contribute an average of 11°c to the thermal stability of the duplex formed. This study also demonstrated that 5'-dangling adenosines contribute less to overall stability than do 3'-dangling adenosines. However, this effect did display some sequence dependence.</p> <p>The triribonucleotide GpCpA was the first trimer shown to form a stable RNA duplex (Tm = 33°C). The duplex consisted of two G·C base pairs and two 3'-dangling adenosines and had a stability equal to that of the tetramer duplex UpGpCpA with four Watson-Crick base pairs.</p> <p>The influence of base stacking on duplex formation was studied and it was discovered that the direction of base stacking had a definite influence on helix stability. Stacking in the 5'→3' direction was more favourable to duplex formation than stacking in the 3'→5' direction.</p> <p>Lastly, the significance of invariant adenosines at position 14 and 21, in the D-stem of tRNA, was investigated through a model study that suggested the adenosines contributed to D-stem stability.</p> <p>Most of the results presented in this thesis have been published or accepted for publication in:</p> <p>1. Jeremy R. Everett, Donald W. Hughes, Russell A. Bell, Dirk Alkema, Thomas Neilson and Paul J. Romaniuk. "Nearest-Neighbour and Next-Nearest-Neighbour Effects in the Proton NMR Spectra of the Oligoribonucleotides ApGpX and CpApX." (1980) Biopolymers 19, 557.</p> <p>2. Thomas Neilson, Paul J. Romaniuk, Dirk Alkema, Donald W. Hughes, Jeremy R. Everett and Russell A. Bell. "The Effects of Base Sequence on the Stability of Short Ribonucleic Acid Duplexes." (1980) Nucleic Acids Res. Sym. Series No. 7, 293.</p> <p>3. Dirk Alkema, R.A. Bell, P.A. Hader and T. Neilson. "Triplet GpCpA Forms a Stable RNA Duplex." (1981) J. Am. Chem. Soc. 103, 2866.</p> <p>4. Russell A. Bell, Jeremy R. Everett, Donald W. Hughes, Dirk Alkema, Paul Hader, Thomas Neilson and Paul J. Romaniuk. "Nearest-Neighbour and Next-Nearest-Neighbour Effects in the Proton NMR Spectra of the Oligoribonucleotides ApXpG, CpXpG, CpApXpUpG, ApGpXpC and ApGpXpCpU." (1981) Biopolymers 20, 1383.</p> <p>5. Dirk Alkema, Russell A. Bell, Paul A. Hader and Thomas Neilson. "Short RNA duplex Stability: Contribution from non-base paired residues to the direction of stacking." (1981) in "Biomolecular Stereodynamics" R.H. Sarma, ed., Adenine Press, Elmsford, NY., p. 417.</p> <p>6. Dirk Alkema, Russell A. Bell, Paul A. Hader and Thomas Neilson. "Invariant Adenosine Residues Stabilize tRNA D-steins." (1982) Accepted for publication in FEBS Letters.</p> <p>7. Dirk Alkema, Paul A. Hader, Russell A. Bell and Thomas Neilson. "Effects of Flanking G·U· Base Pairs on Internal Watson-Crick, G·U and Non-Bonded Base Pairs Within a Short RNA Duplex." (1982) Accepted for publication in-Biochemistry.</p> <p>Two additional publications will appear shortly:</p> <p>1. Paul A. Hader, Thomas Neilson, Dirk Alkema, Eric C. Kofoid and M.C. Ganoza. "Sequencing of Short RNA Oligomers by Proton Nuclear Magnetic Resonance." (1982) Accepted for publication in FEBS Letters.</p> <p>2. Paul A. Hader, Dirk Alkema, Russell A. Bell and Thomas Neilson. "Parameters for Proton Chemical Shift Prediction in Oligoribonucleotides." (1982) J. Chem. Comm. (in press).</p> / Doctor of Philosophy (PhD)

<p>Overexpression of the Neu/ErbB-2 receptor tyrosine kinase is implicated in the genesis of human breast cancer. Previous studies have observed elevated levels of endogenous ErbB3 protein in Neu-induced tumors. Although it has been suggested that the aberrant co-expression of Neu and ErbB3 may play a critical role in the induction of human breast tumors, the biological significance and the molecular mechanism of ErbB3 regulation during Neu-mediated tumorigenesis remains unclear. The results of this thesis demonstrate that the ability of ErbB3 to be constitutively phosphorylated and to regulate cell cycle progression through the activation of the PI-3K/mTOR pathway fully depends on the activity of Neu. Both PI-3K and mTOR in turn are involved in sustaining high levels of ErbB3 protein by increasing its stability. A mutant of ErbB3 unable to recruit and activate PI-3K (ErbB3-6F), blocks Neu-induced transformation, disrupts the mTOR/4EBP1 pathway and leads to apoptotic cell death. This strongly suggests that ErbB3 's role in these tumors is to provide cell survival signals by recruiting the p85 regulatory unit of PI-3K and coupling overexpressed Neu to the PI-3K/mTOR/4EBPl pathway. This thesis further demonstrates that the mTOR inhibitor rapamycin, delays the onset and inhibits the growth of Neu-induced mammary tumors in transgenic mice. The rapamycin-induced tumor inhibition correlates with downregulation of ErbB3, S6K, 4EBP1 and cyelin D1. I also show that the ErbB3-6F mutant exhibits antitumor activity in vivo by preventing tumor growth and facilitating tumor regression. Therefore, the antitumor activity of the rapamycin analogue, CCI-779 in human breast cancer may be mediated, in part, through the inactivation of ErbB3 thereby inhibiting the critical cell survival signals necessary for transformation. Taken together these results strongly suggest that Neu requires ErbB3 to drive mammary transformation and that the ErbB2/ErbB3 heterodimer may function as an oncogenic unit to recruit distinct yet complimentary signaling pathways that cooperate during mammary tumor progression. The results of this thesis provide a molecular basis for the selective targeting of the ErbB3/PI-3K/mTOR signaling pathway in the treatment of HER2-mediated human breast malignancies in which HER3 is overexpressed.</p> / Doctor of Philosophy (PhD)

<p>Embryonic studies clearly demonstrate that MyoD and Myf5 are required for specification of the myogenic lineage. Moreover, MyoD plays an essential role in initiating expression of myogenin and inducing differentiation. However, the factors and mechanisms which temporally regulate expression of myogenic genes are not fully understood. Furthermore, the controversy of whether cell cycle withdrawal precedes induction of MyoD transcriptional activity, or MyoD activity induces cell cycle withdrawal, still exist. In this thesis, I demonstrate that although MyoD transcriptional activity is regulated by cell cycle regulators, such as pRb, cdk4 and cyclin D 1, these molecules are functioning in a cell cycle-independent manner. In addition, MyoD regulates cell cycle progression, and more specifically S-phase entry, by regulating the stability of cyclin E. Moreover, MyoD regulates expression of cyclin Dl indirectly by modulating activation of NF-lCB. Importantly, I demonstrate how the E-protein, HEB, regulates myogenic gene expression in an isoform-specific, MRF-specific, and promoterspecific manner. In the absence of HEB, MyoD is unable to effectively induce expression of myogenin, and differentiation is inhibited. Therefore, ubiquitously expressed factors, such as pRb and HEB, are able to regulate the restricted pattern of gene expression in myogenic differentiation.</p> / Doctor of Philosophy (PhD)

<p>Six studies (Studies 1-6) were performed to gain insight into selected physiologic (metabolic, cardiorespiratory, thermoregulatory), electromyographic, and biomechanical implications of the high energy cost of walking in children and adolescents with mild cerebral palsy (CP). Controls (CON) were also tested in Studies 3 and 4. Studies 1 and 2 examined issues related to habituation to treadmill walking. The purpose of Study 1 was to determine if after one, 12-15-minute treadmill walking practice session: i) metabolic and cardiorespiratory responses during walking are affected by repeated walking bouts on different days, and ii) if these responses are different at different speeds. After 12-15 minutes of treadmill walking practice, subjects walked on the treadmill (3-minute bouts) at 60, 75, 90% of the fastest walking speed (FWS), on three different days. From Day 1 to Day 3, net ventilation ('VE) and net heart rate (HR) at 90% FWS decreased by 3.6 l'minute⁻¹ and 8 beats·minute⁻¹, respectively. There were no differences between Day 1 and Day 2 or Day 1 and Day 3 for any other metabolic or cardiorespiratory variable at any speed. Between-day reliability of most metabolic and cardiorespiratory responses was ~ 0.95. Since there were no Day 1 to Day 3 differences in metabolic variables, Day 1 to Day 3 decreases at 90% FWS in net HR may reflect reduced emotional stress over time and decreases in net VE, an uncoupling of oxygen uptake (V02) and VE. Despite between-day differences, it appears that reliable metabolic and cardiorespiratory data may be obtained in these subjects after one, 12-15-minute treadmill walking practice session. In Study 2 the subjects practiced walking on the treadmill as in Study 1 and, on a different day, they then walked once on the treadmill for three minutes, at 90% FWS. In this case, the purpose of the study was to determine: i) minute-by-minute differences in lower limb antagonist muscle co-activation and stride length during a 3-minute treadmill walk following 12-15 minutes of treadmill walking practice, and ii) if the minute-by-minute pattern of co-activation is affected by site (thigh or lower leg) and lower limb dominance. During the treadmill walk, non-dominant thigh co-activation decreased between minute 1 and a) minute 2 (6%), b) minute 3 (7.2%). Co-activation for the dominant lower leg decreased between minute 1 and minute 3 (11.3%). Non-dominant thigh coactivation was on average 27.3% higher than for the dominant thigh, independent of time. Thigh co-activation was on average 27.7% higher than for the lower leg, independent of dominance or time. Stride length increased between minute 1 and minute 3 by 2.1 %. These data suggest that 12-15 minutes of treadmill walking practice may be sufficient time to obtain stable co-activation and stable stride length by minute 2 of a fast treadmill walk. The data also suggest that dominance and site affect the magnitude of co-activation. The purpose of Studies 3 and 4 was to determine if children and adolescents with mild spastic CP differ from CON in their thermoregulatory responses during exercise in the heat, where such exercise would have the same oxygen (O2) cost for both groups (Study 3) and where such exercise would have a higher O2 cost for those with CP compared to CON (Study 4). Each subject with CP was individually matched to a CON. The CP subject and their CON-match arm-cranked (Study 3) or walked on the treadmill (Study 4) at the same intensity for three, 10-minute bouts in 35 °C, 50% relative humidity. In Study 3, there were no CP-CON differences in \/02 or in thermal strain. In Study 4, V02, body temperatures, and HR were higher in the CP group compared to CON (V02 was 40% higher, rectal temperature was 0.4 °C higher). Those with CP demonstrated greater thermal strain than CON during treadmill walking where they required more metabolic energy, and thus produced more metabolic heat than CON, but not during arm-cranking where their V02 was matched and heat production was therefore similar between the groups. The primary purposes of Studies 5 and 6 were to determine whether there was a relationship between the subjects' level of habitual physical activity (PA) and their 02 cost of walking (Study 5) or their biomechanical walking economy (Study 6). In both studies subjects walked on the treadmill at the same speeds and with the same amount of practice as in Study 1. HR (Study 5) and movement (Study 6) were also monitored over 2 weekdays and 1 weekend day. In Study 5 habitual PA (derived from monitored HR) was related (r = -.70 to -.84) to net V02 at 60 and 75% FWS, to net V02 m-1 , averaged across the three speeds, and to % peak \/02 at all three speeds. PA was not related to net \/02 at 90% FWS. In Study 6, biomechanical walking economy, as measured by the biomechanical economy quotient (SEQ), at 60, 75 or 90% FWS explained about half of the intersubject variance in PA as measured by accelerometer movement counts. A similar relationship was found between SEQ and accelerometer movement counts at or above the 80th and 90th percentile for both the total minutes d-1, and the number of 5-minute bouts d-1. The data from Studies 5 and 6 suggest that PA in these subjects may be related to their walking economy.</p> / Doctor of Philosophy (PhD)

<p>Intestinal epithelia contribute to gut homeostasis by acting as a selectively permeable barrier and establishing a driving force for water movement through vectorial ion transport. These processes are affected by cytokines. As TGFß levels can be increased in gut inflammation, a situation where epithelial function is often altered. the aim of this thesis was to d.efine the effeets of TGFß on intestinal epithelial barrier and ion transport function. The first study characterized the kinetics and signal transduction pathway behind the novel observation that intestinal epithelial monolayers treated with TGFß display reduced stimulated secretory responses. The mechanisms involved in this process were further delineated in a second study where it was determined TGFp treatment causes a down-regulation and altered sub-cellular localization of the main apical chloride channel (CFTR). The final study defined the mechanism behind TGFp-induced epithelial barrier enhancement in terms of signal transduction pathways and regulation of proteins involved in maintaining a physiologically "tight" barrier. Furthermore, it was determined that TGFß treatment protects against barrier damage caused by pathogenic bacteria, and the mechanisms behind this have been revealed. The first two manuscripts present a substantial body of evidence on the kinetics and mechanisms of TGFß-induced diminished epithelial ion transport. In addition to furthering understanding of cytokine regulation of epithelial function, these data are particularly relevant to the field of enteric disease where water balance is often perturbed. In the tinal manuscript, the protective role of TGFß on epithelial barrier function is illustrated by its ability to preserve banier function from damage caused by the pathogenic bacteria. enterohemonhagic E. coli (EHEC) 0157:H7. Having determined mechanisms underlying ephhelial banier enhancement and protection by TGF~, potential therapeutic targets have been revealed that might be strategic in treating individuals with conditions of increased banier permeability. such as inflammatory bowel disease relapses and EHEC infection.</p> / Doctor of Philosophy (PhD)

<p>The development of synchrony is characteristic of uterine muscle at the end of pregnancy. The coordinated contractile activity of the smooth muscle is effective in delivering the fetuses. A structural change occurs late in gestation, and specialized sites of intercellular communication, gap junctions, appear between the uterine smooth muscle cells. The objectives of this thesis were to characterize the time course of gap junction formation, investigate some factors that govern their appearance, and examine the possible significance of gap junction formation in the myometrium. Electrophysiological methods were used to evaluate the hypothesis that gap junction formation at parturition resulted in improved electrical communication between cells. Improved coupling might allow the synchronization of uterine activity.</p> <p>Gap junctions were shown by quantitative thin section electron microscopy to be present between uterine smooth muscle cells immediately prior to, during, and for a short time following parturition in the rat. Several experiments involving ovariectomy revealed that the stimulus for gap junction formation was systemic in nature. Bilateral ovariectomy of midterm pregnant rats resulted in premature termination of pregnancy and gap junction formation, both of which were blocked by hormone administration. These and other results suggest that progesterone withdrawal may regulate gap junction formation in rat myometrium, but many factors, such as estrogen and prostaglandins, may also be involved.</p> <p>Uterine smooth muscle is a functional electrical syncytium, and properties of electric current flow through the muscle yield indirect estimates of cell-to-cell coupling between cells. Impedance analysis showed that the specific resistance of the cytoplasm of myometrial cells was constant from before term to delivery, but the junctional resistance decreased. Shortly post partum the junctional resistance increased. Cable analysis confirmed that the internal resistance of myometrium was lower at parturition. Thus, improved cell-to-cell communication was associated with a demonstrated increase in gap junction contact between cells. These results are consistent with the hypothesis that gap junction formation at the end of gestation results in improved electrical coupling of uterine smooth muscle cells.</p> / Doctor of Philosophy (PhD)