<p>The addition of dopamine to homogenates or slice preparations from rat caudate nucleus results in a 100-150% increase in tissue cAMP levels. Since protein kinases are the only known physiological receptors for cAMP, it has been hypothesized that the response of the postsynaptic neurons to dopamine may be mediated in part through the phosphorylation of specific membrane proteins. This study examines the effects of dopamine on cAMP and its associated protein kinase in caudate nucleus.</p> <p>Caudate tissue contains a Type II protein kinase that is activated by cAMP, and that catalyzes the phosphorylation of several synaptic membrane proteins. Dopamine, however, did not appear to enhance the phosphorylation of these proteins either in broken cell or intact preparations, although increases in cAMP could be demonstrated under similar experimental conditions.</p> <p>Destruction of postsynaptic neurons using the neurotoxin kainic acid significantly reduced cAMP-dependent protein kinase activity. Destruction of presynaptic nerve terminals, on the other hand, had no effect on the activity of this enzyme system. However, this procedure was associated with an increase in dopamine receptor sensitivity as measured by an increase in dopamine-mediated turning behaviour. This behavioural response was not abolished by intrastriatal administration of kainic acid, although this technique reduced dopamine-sensitive adenylate cyclase and cAMP-dependent protein phosphorylation by 80-90%.</p> <p>The administration of chronic haloperidol also produced behavioural supersensitivity, as well as increases in the number of postsynaptic dopamine receptors, but this was not associated with comparable increases in the activity of dopamine-sensitive adenylate cyclase, or in cAMP-dependent protein phosphorylation. These studies therefore do not support a role for cAMP or cAMP-dependent protein kinases in the mediation of postsynaptic dopamine receptor supersensitivity.</p> <p>Rat caudate nucleus also contains a number of proteins that are phosphorylated by Ca⁺⁺-dependent protein kinases. Substrates for calmodulin-dependent and independent protein kinases were identified and characterized on the basis of their solubilization properties and response to neuroleptic drugs. Dopamine enhanced the phosphorylation of two of these proteins in rat striatal slices. The effects of dopamine on protein phosphorylation could be distinguished from those of depolarizing agents such as KCl or veratridine, and were not mimicked by 8-bromo-cAMP.</p> <p>The present studies demonstrate that increases in cAMP produced by dopamine have no measurable effect on cAMP-dependent protein phosphorylation. Although these data do not definitely preclude a role for cAMP-dependent protein kinases in the regulation of postsynaptic function, they indicate that the physiological relevance of dopamine-mediated increases in cAMP should be re-examined.</p> / Doctor of Philosophy (PhD)

<p>This work encompasses two primary studies utilizing peripheral quantitative computed tomography (PQCT) in the assessment of bone characteristics at the distal radius. PQCT enables separate analyses of cortical, trabecular and total bone density, content and geometry. Further analysis of the pQCT image with specialized software permits in vivo trabecular structure assessment. The first study is a two-year prospective trial evaluating the effects of five different medication regimens (hormone replacement therapy (HRT), etidronate , alendronate, etidronate plus HRT and alendronate plus HRT) used for the treatment of osteoporosis in 123 postmenopausal women with low bone mass. Results of analyses on baseline data found that trabecular structure indices could significantly discriminate between osteoporotic and osteopenic groups of women. Longitudinal data at the lumbar spine (LS) and femoral neck (FN) for women taking any medication ('Any-Tx') showed similar results to those of large clinical trials. For example, the 'Any-Tx' group gained significant bone mineral density (BMD) over baseline at 2 years at the LS (5.5%, p<0.001) and FN (1.6%, p<0.05). Women on combination therapy gained significantly more BMD than women taking one medication over 2 years at the LS and FN. Data suggests that alendronate plus HRT may produce the largest BMD gains at the LS and FN at 2 years. Longitudinal pQCT data for total, cortical and trabecular compartments at the distal radius in the control group over the 2 years suggest endocortical resorption and trabecular thinning with age. PQCT data in the 'Any-Tx' group show gains in total and cortical bone density and cortical content, but losses in trabecular density, content and area. This suggests that anti-resorptive medications promote endocoritcal apposition and reduce intracortical porosity. The second study evaluated five different clinical measurement tools in the prediction of in vitro failure load at the distal radius and found that cortical content measured by pQCT was a significantly better predictor than ultrasound or digital x-ray radiogrammetry.</p> / Doctor of Philosophy (PhD)

<p>Breast cancer research has focused on a number of key molecular events suspected to play critical roles in the establishment and progression of the human disease. For example, the c-ErbB2 receptor is amplified or overexpressed in approximately 30% of all human breast cancer cases, its overexpression inversely correlating with a positive patient prognosis. In addition, the estrogen receptor is a key marker in assessing patient prognosis, the progression of the disease state, and is by far the most utilized target to treat breast cancer with approximately 100 million hours of clinical experience with the antiestrogen, Tamoxifen. While these two receptor systems play critical roles in breast cancer progression, the activation of downstream signaling pathways and its consequences on cellular function are unclear. Interestingly, the c-Src tyrosine kinase has been identified to play a role in the modulation of both the c-ErbB2/Neu and the estrogen receptor. Here I will describe a role for c-Src in modulating c-ErbB2/Neu receptor activation and its associated ability to transform at the molecular level, as well as a role for c-Src in estrogen receptor mediated mammary gland development. Significantly, the association of c-Src to the c-ErbB2/Neu receptor plays an important role in the direct and specific activation of the receptor. Furthermore, the loss of c-Src within the mammary gland negatively impact its development, this delay being mediated via the estrogen receptor. Taken together, these studies demonstrate the importance of c-Src in two receptor systems that have been heavily implicated in development and disease, the estrogen receptor and the c-ErbB2/Neu. In both cases, c-Src plays a critical role in receptor activation, coordinating downstream events impinging on reproductive development and in mammary gland transformation. The understanding of the molecular crosstalk between these receptor systems within the mammary gland may provide insight into potential translational therapies in clinical research.</p> / Doctor of Philosophy (PhD)

<p>Mammalian cells have the ability to endogenously generate and metabolize hydrogen peroxide (H₂O₂). This H₂O₂ can interact with protein and lipid components of intracellular signal transduction pathways to regulate cell behaviour. It has been demonstrated in our laboratory and by others that many types of cells, including transformed cells, require H₂O₂ for efficient growth in culture. Excess H₂O₂ has long been identified as a source of oxidative stress that can mediate cell growth arrest and death. However, the addition of the H₂O₂ scavenger catalase to culture media or the overexpression of intracellular catalase can block proliferation. This effect is reversed by inactivation of the enzyme or upon co-incubation of cells with H₂O₂ and H₂O₂-generating sources such as glucose oxidase. Thus, at non-toxic levels, H₂O₂ appears to act as a cellular growth factor. The purpose of this work is to help elucidate molecular mechanisms underlying this H₂O₂-regulated aspect of cell physiology. The effects of extracellular H₂O₂ level manipulation upon the activities of the HER-2/Neu receptor tyrosine kinase, mitogen-activated protein kinases (MAPKs), and stress-activated protein kinases (SAPKs) are discussed. The c-Jun-NH₂terminal kinase 1 (JNK1), a member of the SAPK family, is involved in several diverse aspects of cellular functioning including apoptosis and transformation. The JNK1 signal has also been implicated as a cell sensor of redox (reducing/oxidizing) stress. We have observed that the growth-inhibitory effect of both high level H₂O₂ treatment and H₂O₂-scavenging catalase treatment is accompanied by a transient increase in JNK1 activity. To determine the importance of this response in growth regulation, the JKN1 signal wa stably altered in SK-OV-3 human ovarian adenocarcinoma cells by the expression of ectopic JNK1 (HA-JNK1). Levels of HA-JNK1 protein expression correlated with increases in basal c-Jun phosphorylation in a dose-dependent manner. Transient expression of HA-JNK1 potentiated cell growth arrest by catalase activity, however with stable expression a degree of resistance to this response was observed. Resistance was accompanied by a lowered endogenous production of H₂O₂. Transient HA-JNK1 expression also reduced H₂O₂ generation, and this effect was reversed by the JNK inhibitor SP600125. These resultts indicate that the JNK1 stress response contributes to the inhibition of proliferation by catalase treatment, possibly via additional reductions in environmental H₂O₂ caused by a lowered endogenous production. Stable amplification of the JNK1 pathway leads to cellular adaptation to its signal, resulting in a diminished reliance upon basal H₂O₂ levels for efficient growth. These data contribute to the understanding of the mechanisms involved with the overexpression and/or hyperactivity of JNKs observed in certain cancers.</p> / Doctor of Philosophy (PhD)

<p>Background: peritoneal dialysis (PD) is a valuable therapy for end-stage renal disease. Changes in the peritoneal transport properties, including increased solute transport and ultrafiltration (UF) dysfinction are main clinical limitations of PD. The capillary wall of peritoneal blood vessels plays a key role as a barrier to solute transport. Therefore, neovascularization of the peritoneal tissues is a main determinant in the functional changes in the peritoneal membrane. I hypothesize that ultrafiltration of dysfunction is caused by increased peritoneal vascularization with a concomitant increase in glucose transport from peritoneal cavity and rapid loss of the ultrafiltration gradient. Further, I hypothesize that this increased vascular surface area is induced by profibrotic and inflammatory cytokines such as transforming growth factor (TGF) β, interleukin (IL) 1β, and tumour necrosis factor (TNF) α through upregulation of angiogenic cytokines such as vascular endothelial growth factor (VEGF). Methods: I used adenovirus mediated gene transfer of cytokines such as TGFβ1, il-1β, and TNFα to the peritoneal of rats and studied the effects of histology, angiogenesis, gene regulation, solute transport, and UF. I also used an animal model of daily peritoneal exposure to dialysate solution. The angiogenic and fibrogenic response of the peritoneum to this model was detailed, along with the effect of overexpression of angiostatin and decorin by adenovirus mediated gene transfer Results: Adenovirus mediated gene transfer of TGFβ1 led to increased peritoneal fibrosis and angiogenesis which persisted to 28 days. These histologic changes were associated with increased solute transport of glucose and decreased UF. In vitro and in vivo, TGFβ1 appeared to up regulate expression of VEGF. Inflammatory cytokines such as TNFα and IL-1β both induced fibrosis, angiogenesis, and peritoneal membrane dysfunction. The kinetics of the response was quite different. TNFα induced a very transient response with a complete resolution of changes by 21 days after adenovirus infection. The response induced by IL-1β was more sustained. I hypothesize that the strong expression of tissue inhibitor of metalloproteinase after IL-1β treatment may explain the prolonged fibrogenic response after IL-1β. Daily exposure to dialysate also induced a strong fibrogenic and angiogenic response in the peritoneum. The anti-angiogenic agent angiostatin, when overexpressed using adenovirus mediated gene transfer, reduced the vascularization and improved the ultrafiltration dysfunction. Decorin, a proteoglycan that binds and inactivates TGFβ, reduced peritoneal fibrosis, but did not alter the angiogenic response, nor did it improve peritoneal membrane function. Discussion: These experiments have clarified the role of angiogenesis in UF dysfunction. The correlation between blood vessel density and ultrafiltration, along with the improvement in UF after treatment with angiostatin, is compelling evidence that vascularization of the peritoneal membrane causes UF dysfunction. There is a close associate between fibrosis and angiogenesis in the perotineum. Fibrosis appears to be necessary for a prolonged angiogenic response. Further work is required to identify the factors leading to the interaction between fibrosis and angiogenesis. The role of the interstitium and lymphatics in solute transport and UF dysfunction needs to be better defined. Finally, anti-angiogenic therapies need to be studied in these models of peritoneal membrane dysfunction and eventually applied to patients on PD to improve the quality and duration of this therapy.</p> / Doctor of Philosophy (PhD)

<p>Enhanced transepithelial antigen transport in intestine of sensitized rats was previously demonstrated using horseradish peroxidase (HRP) as a model antigen. In senitized rats, transepithelial antigen transport was rapid (< 2 min) and of greater magnitude (three-fold) compared to controls. In my project, the essential components involved in the enhanced antigen transcytosis were investigated using mice with genetic deletions for IL-4 and CD23/FcεII. Increased transepithelial antigen transport was demonstrated in actively sensitized IL-4⁺/⁺ mice, but not IL-4⁻/⁻ mice, in which the phenomenon paralleled the expression of CD23 protein on enterocytes. Passively sensitized mice (both IL-4⁺/⁺ and IL-4⁻/⁻ mice) displayed greater antigen transport after transfer of immune serum unless the serum was first depleted of IgE or IL-4. IL-4 added to cultures of epithelial cells upregulated expression of CD23 mRNA. Finally, this augmented antigen uptake system was inhibited by luminal anti-CD23 and was absent in sensitized CD23⁻/⁻ mice. RT-PCR showed that cultured cells expressed only the b isoform of CD23. Sequencing revealed classical and alternative CD23b transcripts lacking exon 5 (b∆5) or 6 (b∆6), in which all forms were translated into functional IgE receptors. Endocytosis of the b∆5 and b∆6, but not the classical CD23b protein, was observed after binding with saturating anti-CD23, suggesting continuous endocytosis of the alternative forms that agrees with their intracellular localization at steady state. Classical CD23b proteins were expressed on the cell surface and only endocytosed upon antigen-induced IgE cross-linking of the receptor. Taken together, the results demonstrated that IgE/CD23 mediates enhanced transepithelial transport of antigen in sensitized mouse intestine, and that IL-4 plays a major regulatory role. We identified the expression of classical and alternative CD23b transcripts in intestinal epithelial cells, and demonstrated taht the translated proteins display distinct endocytic functions. We concluded that antigen binding to epithelial CD23/IgE facilitates its entry into the body resulting in intestinal anaphylaxis.</p> / Doctor of Philosophy (PhD)

<p>Over 90% of retinal ganglion cells (RGCs) die after intraorbital optic nerve transection close to the eye. Several factor contribute to RGC death including: loss of neurotrophic support, overproduction of free radicals, glutamate-mediated excitotoxicty, activation of pro-apoptotic caspases (3 and 9), and reactive glia. In the present study, the model of optic nerve transection was used to study the neuroprotective mechanisms and effects of ciliary neurotrophic factor on the survival of axotomized retinal ganglion cells. It was demonstrated that axotomized RGCs die by apoptosis since overexpression of Neuronal Inhibitory Apoptosis Protein (NAIP), a potent and selective inhibitor of caspase 3, protected axotomized RGCs that would have otherwise died if untreated. Protection of axotomized RGCs was further enhanced by overexpression of Ciliary Neurotrophic Factor (CNTF) using adenoviral and lentiviral vectors. It was demonstrated that intraocular administration of adenoviral vectors selectively transduced retinal Muller cells, and injection of this vector into the optic nerve stump at the time of optic nerve transection selectively transduced a small percentage of RGCs. Regardless of the route of administration, adenoviral-mediated overexpression of CNTF protected axtomized RGCs. In comparison to adenoviral-mediated delivery, the delivery of CNTF using lentiviral vectors protected greater numbers of axotomized RGCs and for an extended period of time not typically seen using the optic nerve transection model. It was assumed that viral-mediated transfer of CNTF rescued axotmized RGCs by directly activating the high affinity CNTF receptor alpha (CNTFRα) expressed on RGCs. However, CNTF can also protect axotomized RGCs indirectly, by activating the low affinity leukemia inhibitory receptor beta (LIFAβ) expressed on retinal astrocytes and Muller cells. It was demonstrated that viral-mediated overexpression of CNTF resulted in phenotypic changes in retinal glial cells (astrocytes and Muller cells) that may have increased their neuroprotective function. Overexpression of CNTF increased retinal levels of the gap junction protein, connexin 43 (Cx43), the intermediate filament glial fibrillary acidic protein (GFAP), the astrocyte-specific glutamate/aspartate trasporter-1, GLAST-1, and the astrocyte specific enzyme, glutamine synthetase (GS). Taken together, these results suggest that CNTF is capable of protecting axotomized RGCs by directly binding to injured neurons or by modulating surrounding glia. Together, these results suggest that pharmacological interventions that maintain or upregulate CNTF expression may confer a clinically beneficial neuroprotection.</p> / Doctor of Philosophy (PhD)

<p>The selective localization in mucosal tissues of lymphoblasts derived from the mesenteric lymph node (MLN) compared to those from peripheral lymch nodes (PLN axial, brachial and inguinal) was studied using an adoptive transfer model in syngeneic CBA/J mice. The localization of lymphoblasts which had been labelled in vitro with ³H-thymidine or ¹²⁵I-deoxyuridine was assessed using autoradiography or radiocounting.</p> <p>I found that the number of MLN lymphoblasts which localized in the small intestine, lung, Peyer's patches, MLN, PLN, spleen, and liver was directly proportional to the number transferred. This relationship was also present in intestinal epithelium and basal and villus lamina propria and in pulmonary parenchyma, BALT and bronchial epithelium. Even at doses of MLN lymphoblasts which approximated four times the daily output of blasts in thoracic duct lymph, I could not saturate the capacity of these tissues to accommodate MLN blasts, nor was their intra-intestinal distribution altered. Because of this dose relationship it is necessary to control the number of blasts transfered when comparing the localization of lymphoblasts from different organ sources using autoradiography. Thus my dose studies show that, contrary to earlier studies which were not controlled in this manner, MLN blasts do not selectively localize compared to PLN blasts in pulmonary parenchyma. In addition the result suggest that any method of increasing the number of lymphoblasts released from MALT (e.g. by mucosal immunization) or increasing the delivery of these cells to a particular organ (by altering the proportion of the carciac output which it receives) will increase the number of immublasts in a tissue.</p> <p>Although the enumeration of radiolabelled or fluorescent cells in tissue sections has been used extensively in the literature to assess localization, the variation in these results has rarely been stated. I found that this variation can be quite high and is largely due to the probability of detecting a low number of cells in a small volume of tissue.</p> <p>I found that the sex of the recipiegt had no effect on the number of MLN lymphoblasts which localized in the small intestine 24 h after transfer. In contrast, initial experiments showed that lymphoblasts from male donors localized two to three times more frequently than those from female donors in the small intestines of recipients of either sex. However this phenomenon disappeared between September 1980 and August 1981 and neither its initial existence nor its subsequent lops have been explained. I found that the gonadal hormone environment in which the MLN lymphoblasts developed did not influence their capability to localize in the small intestine 24 h after transfer. However, I do not know whether this observation relates to the phenomenon of greater localization of male lymphoblasts because this phenomenon had likely already disappeared when the experiments involving hormonally altered donors were performed.</p> <p>Using autoradiography in a series of experiments analyzing the kinetics of lymphoblast localization, I demonstrated that MLN lymphoblasts selectively localize compared to MLN blasts in the small intestine by 0.5 h after transfer. This suggests that one factor in selective localization is the selective entry of MLN blasts from the vasculature, perhaps mediated by specific receptors on blasts and tissue of localization.</p> <p>The concentration of MLN lymphoblasts was the same in the lamina propria adjacent to the Peyer's patch and distant from the Patch at both 0.5 and 24 h after transfer. This suggests that lymphoblasts extravasate in the lamina propria rather than extravasating in the Peyer's patch and subsequently migrating into the lamina propria. In addition, the distribution of blasts in the basal and villus lamina propria was the same at 0.5 and 24 h after transfer but labelled cells appeared in the intestinal epithelium after 0.5 h.</p> <p>The evidence presented in this thesis suggests that the selective localization of MLN lymphoblasts is mediated the vascular endothelium in the lamina propria, perhaps by specific receptors.</p> / Doctor of Philosophy (PhD)

<p>Around day 8 of mouse embryonic development, yolk sac produces circulating nucleated erythroblasts synthesizing primarily embryonic hemoglobins (Hb E). By day 12 of gestation, fetal liver releases nonnucleated erythrocytes containing adult hemoglobins (Hb A). The objective herein was to examine the cellular mechanism of hemoglobin ontogeny during mouse embryonic development.</p> <p>Embryonic peripheral blood cells from 9-12 day embryos were cultured in either plasma clot or methylcellulose for 6-8 days. Pokeweed mitogen stimulated adult spleen cell conditioned medium (SCM) either with or without erythropoietin (Epo) was added to these cultures. Large erythroid colonies were observed by the sixth day of culture. In another type of experiment embryonic fluid was obtained from exocelomic cavity of day 10 embryos and was added to these cultures with Epo, but without SCM. Erythroid colonies were also observed. In some other experiments, cultures of yolk sac cells from 9 - 10 day embryos with SCM, with or without Epo also gave rise to large erythroid colonies after 6 - 8 days in vitro. Furthermore, such colonies were also detected when fetal hepatic cells of 11 - 13 day embryos were cultured with SCM with or without Epo. Hemoglobin synthesis was analysed by various techniques, i.e., electrophoresis, isoelectric focusing and ion-exchange chromatography. In all cultures, only Hb A synthesis could be detected, although the experiments do not rule out the possibility that minute amount of embryonic hemoglobins were present.</p> <p>In another series of experiments, fragments of embryonic tissues from day 8 embryos were cultured for 6-8 days in methylcellulose with Epo and/or SCM. Hemoglobin present in cultures was examined by the sensitive isoelectric focusing microassay. Cultures of early day 8 tissues produce only Hb E. Cultures of late day 8 embryonic tissues with Epo alone produce primarily Hb E with some Hb A as well. Similar cultures with both Epo and SCM produce both Hb E and Hb A, and the proportion of Hb A synthesized is considerably higher. Cultures of day 10 tissues in the presence of both Epo and SCM produce only adult hemoglobins.</p> <p>These results suggest the following model for the development of murine embryonic erythropoiesis: Hemopoietic progenitor cells capable of giving rise to erythroblasts synthesizing Hb E occur early in embryonic development. This is followed shortly by the appearance of erythroid progenitor cells committed to produce erythroblast progeny synthesizing Hb A. The latter progenitor cells are present in circulation. They seed fetal liver and initiate fetal hepatic erythropoiesis.</p> / Doctor of Philosophy (PhD)

<p>The ets genes encode DNA-binding proteins capable of regulating the expression of target genes that contain ETS sites within their promoter sequences. The ets gene PEA3 is overexpressed in a majority of human breast tumour samples, as well as in the mammary tumours and lung metastases of several transgenic mouse models of this disease. The Pea3 subfamily genes, which include Pea3, Erm, and Er81, are expressed during mouse mammary gland development. However, in mammary tumours induced by the HER2/Neu receptor tyrosine kinase, the Pea3 subfamily genes were upregulated by five-two twenty-fold as compared to normal mammary tissue of wild-type control mice. To understand the role of the PEA3 subfamily proteins in mammary tumorigenesis, transgenic mice were generated which overexpress either PEA3 or a dominant-negative mutant of PEA3 (∆NPEA3En) under the transcritional control of the mouse mammary tumour virus (MMTV) promoter/enhancer. ∆NPEA3En was able to inhibit the function of all three PEA3 subfamily proteins and reduce oncogenic Neu-induced transformation of mouse fibroblasts in cell culture-based assays. ∆NPEA3En was able to delay tumour formation as well as reduce the number and the size of tumours that developed in MMTV-neu/∆NPEA3En bi-transgenic mice as compared to MMTV-neu transgenic female mice. The tumours that did form in bi-transgenic mice generally lacked ∆NPEA3En expression indicating that PEA3 subfamily function is required for tumour progression. The mammary gland of MMTV-PEA3 transgenic female mice exhibited increased ductal branching and side bud formation in virgin mice, and decreased lobuloalveolar differentiation during pregnancy. Surprisingly, MMTV-neu/PEA3 bi-transgenic mice had a longer latency in mammary tumour formation than did MMTV-neu female mice, similar to what was observed in MMTV-neu/∆PEA3 bi-transgenic mice had a longer latency in mammary tumour formation than did MMTV-neu/∆NPEA3En bi-transgenic mice. These results implicate PEA3 subfamily protein function during mammary gland development and in Neu-induced mammary tumorigenesis. Thus, inhibiting PEA3 subfamily protein function, or that of one or more specific target gene products, may complement other current therapies in the treatment of human breast cancer</p> / Doctor of Philosophy (PhD)