<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)

<p>Transection of the optic nerve (axotomy) is a model of central nervous system (CNS) injury. Eighty to ninety percent of retinal ganglion cells (RGCs) die within two weeks after transection of the optic nerve. We demonstrated that the recently discovered neurotrophic factors glial cell-line derived neurotrophic factor (GDNF) and neurturin (NTN) enhance the survival of axotimized RGCs. We also showed that these factors produce additive neurotrophic effects with brain derived neurotrophic factor, suggesting that they act by independent mechamisms. We also demonstrated that the high affinity receptor for GDNF (GFRα-1) is localized to RGCs, Muller glia, and the astrocytes in the retina. GDNF was localized to RGCs, photoreceptors, and pigment epithelial cells, suggesting possible autocrine or paracrine roles for GDNF in the retina. Our results also demonstrate that an indirect neurprotective mechanism of GDNF is the upregulation of the glutamate transporter, GLAST-1, in the retinal glia. This mechanism may protect RGCs from glutamate mediated excitotoxicity. Our findings suggest that multiple neurotrophic factors may be required for the rescue of injured CNS neurons, as suggested by the findings that these factors act independently. We demonstrated that there are increases inducible nitric oxide synthase (iNOS) expression in retinal glial cells, and increases in constitutive (cNOS) expression in RGCs after axotomy, and that increases in NOS expression parallel the timecourse of RGC degeneration. Our results indicate that NO plays a toxic role in promoting RGC death after axotomy, as demonstrated by the findings that NOS inhibition enhances the survival of axotomized RGCs. Furthermore, we examined the neuroprotective effects of the anti-inflammatory cytokines-10 (IL-10), IL-4, and transforming growth factor-β (TGF-β on RGC survival. These cytokines have been shown to inhibit iNOS expression and NO synthesis by glial cells in vitro. Our in vivo studies showed that IL-10 and IL-4 enhanced the survival of axotomized RGCs, and suggest that one of the neuroprotective mechanisms of these cytokines is inhibiting peroxynitrite formation in the retina. These findings suggest that IL-10 and IL-4 inhibit NO synthesis in the retina may inhibit reactivity after optic nerve transection. Our findings point to the possible benefit of IL-10 or IL-4 treating CNS trauma and diseases of the CNS that demonstrate the presence of activated NO synthesizing glial cells.</p> / Doctor of Philosophy (PhD)

<p>Chlamydia pneumoniae is a Gram-negative bacterial pathogen that has evolved to survive completely within the intracellular environment of a host cell. The obligate intracellular lifestyle of this bacterium necessitates an efficient invasion strategy, exemplified by a broad host cell tropism with little propensity for any single cell type and the ability to replicate within both professional phagocytic cells and cells with low phagocytic capacity. After cellular invasion, C. pneumoniae actively evades host immune defenses, establishes a parasitic relationship with the host cell and forms a microcolony by dividing within a non-fusogenic cytoplasmic vacuole called an inclusion. The varied spectrum of host cell pathways modified by C. pneumoniae suggests a complex interaction between the bacteria and the host cell. Identifying the requisite host-pathogen interactions contributing to C.pneumoniae virulence is a major research goal. While the morphological features of the chlamydial development cycle have been described in detail, our understanding of the cellular and molecular events underlying C.pneumoniae invasion and potential mechanisms of disease pathogenesis are preliminary. The work presented in this thesis examines cellular and molecular interactions between C.pneumoniae and various types of host cells during invasion and intracellular growth of chlamydiae. Some of the research questions addressed herein are framed within the context of atherosclerosis and coronary artery disease, with the a posteriori reasoning that a potential microbiologic contribution to human atherosclerosis, specifically due to C.pneumoniae infection, is suggested by several converging lines of investigation. We found that c.pneumoniae invasion of human epithelial cells requires bacterial-induced remodeling of the host acting cytoskeleton and the activation of at least two host cell signal transduction pathways. These host modifications are required for C.pneumoniae uptake but not cellular attachment, suggesting that the C.pneumoniae invasion sequence is biphastic, whereby initial attachment is rapidly followed by activation of host cell signaling and actin polymerization to facilitate uptake. Secondly, we used cDNA array technology to study the endothelial cell transcriptional response to C.pneumoniae infection and found a prominent transactivation of several cytokines, chemokines and smooth muscle cell growth factor genes during early times after infection. Furthermore, using cell culture-based experiments and an established rabbit model of C. pneumoniae-induced artherosclerosis, we showed that C. pneomoniae infection is associated with paracrine activation of smooth muscle cell proliferation and aortic intimal thickening, potentially mediated through platelet-derived growth factor.</p> / Doctor of Philosophy (PhD)

<p>Bipolar disorder, or manic-depressive illness, is a common psychiatric illness affecting 1.5% of the population. It is a chronic illness that requires long-term (often lifetime) pharmacotherapy. The need for lifetime treatment with mood stabilizers (i.e. lithium, valproate and carbmazepine) and high relapse rates suggests that changes in gene expression may be involved in the mechanism of action of these drugs. Using differential display, we identified the 78-kilodalton glucose-regulated protein, GRP78, as being at valporate-regulated gene in both rat brain and cultured cells. GRP78 belongs to a family of resident endoplasmic reticulum proteins, referred to as teh ER stress proteins, which includes GRP94 and calreticulin. These proteins act as both calcium binding proteins and molecular chapterones within the endoplasmic reticulum and when overexpressed protect the cell against cytotoxic insults. In addition to GRP78, we were able to show that GRP94 and calreticulun are also upregulated in a concentration-and-time-dependent manner in response to valporate. This effect was shown to be either drug-or drug class-specific, as treatment with other psychotropic drugs did not elicit a similar response. In patient samples, all three ER stress proteins were increased in postportem temporal cortex from depressed subjects to who died by suicide. These results suggest a possible cytoprotective role for mood stabilizers, since studies have shown that valporate as well as lithium can regulate the expression of proteins with known cytoprotective properties. Indeed, when rat hippocampal neurons were treated for 7 days with the mood stabilizers, lithium, valporate and carbamzepine, a significant reduction in NMDA-mediated cytoplasmic vacuolization could be quantified using transmission electron microscopy. In conclusion, the results of this thesis contribute to a growing number of studies that propose neuroprotective for mood stabilizing drugs, and suggest that valporate might act in part by regulating ER stress proteins.</p> / Doctor of Philosophy (PhD)

<p>The alterations in renal vascular structure and function, and their role in the development and maintenance of hypertension were examined in Kyoto Wistar spontaneously hypertensive rats (SHR) and Wistar Kyoto normotensive controls (WKY). A study of the renal vascular bed in SHR with established hypertension and age matched normotensive WKY indicated that when the isolated kidney was perfused at a variety of flow rates, under maximally relaxed conditions, the renal vascular resistance (RVR) was similar between SHR and WKY. Consistent with the above finding, morphometric measurements of light and electron micrographs indicated that the lumen diameter of relaxed main renal, interlobar, arcuate and interlobular arteries, as well as the preglomerular arterioles was similar in SHR and WKY. The cross-sectional areas of total intima, endothelium, subendothelial space, internal elastic lamina and total adventitia, as well as the volume fraction of axons, nerve sheath cells, fibroblasts, collagen and fluid filled space within the adventitia were only modestly altered in SHR. However, with the exception of the preglomerular arterioles, the media of all the renal arterial classes of SHR exhibited an increase in smooth muscle cell (SMC) cross-sectional area and volume that was produced by SMC hypertrophy and/or hyperplasia, while the extracellular space surrounding the SHCs was increased in both arteriolar and pre-arteriolar vessels.</p> <p>Based on these structural alterations, it was hypothesized that, if the mass of the arterial media is increased, contraction from the adventitial side would tend to push the media of the thicker hypertensive vessel into the lumen to a greater degree than the thinner walled WKY vessel. Under relaxed conditions, the RVR would be expected to be similar between SHR and WKY; however, during contraction RVR should be elevated to a greater degree in SHR than WKY. To test the above hypothesis, pharmacological studies were undertaken. Consistent with the model, at maximal relaxation the RVR was similar in SHR and WKY, while contraction of the renal vascular bed with infused norepinephrine (HE), BaCl₂ angiotensin II, or by stimulating the periarterial nerves produced a larger elevation of RVR in SHR. Aside from a modest increase in HE sensitivity within the renal vasculature of WKY, the contractile sensitivity to the various agents was not altered when SHR and WKY were compared. These studies indicated that the nerve mediated contractile responses within the renal vasculature were mediated by alpha₁ and dopamine receptors, and the proportion of the maximal response attributed to each receptor was similar in SHR and WKY.</p> <p>Similar alterations, but of lesser magnitude, as those present in SHR with established hypertension were found to occur in prehypertensive SHR. The RVR at maximal relaxation was similar to that present in WKY, while the lumen diameter of the main renal, interlobar and cortical arteries was not modified between the two groups. All renal arteries of prehypertensive SHR that were studied exhibited an increase in the cross-sectional area ratio of arterial wall (intima + media) in relation to the lumen, and an increased number of SMC layers within the media. Consistent with the proposed model, when the renal vasculature of prehypertensive SHR was maximally contracted by infusing NE or by stimulating the periarterial nerves (under conditions where the presynaptic uptake of NE was blocked) the amplitude of RVR change was higher in prehypertensive SHR than WKY. As in SHR with established hypertension, the contractile sensitivity of the renal vasculature to NE was modestly increased in WKY.</p> <p>To further test if such alterations occur independently of high blood pressure, hydralazine (an antihypertensive drug that crosses the placental barrier) was fed to female SHR prior to, and during, pregnancy, and subsequently to newborn rats from birth to 21 weeks of age. These animals were compared to similarly treated WKY and nontreated SHR and WKY controls. Treated SHR maintained normal blood pressure throughout the treatment period. The in utero and post-natal normalization of blood pressure in SHR had virtually no effect in altering the renal vascular wall thickness. Within most of the arteries studied, SHR with normalized blood pressure had similar cross-sectional quantities of media and SMC layers as were present in untreated SHR, and greater quantities than that present in either control or treated WKY. The withdrawal of hydralazine from 26 week old in utero and post-natally treated SHR resulted in the re-establishment of hypertension within two days of withdrawal to the levels that were present in control SHR.</p> <p>These results suggest that the thickening of the renal vascular wall in SHR could be of etiological importance in the initiation and maintenance of high blood pressure in SHR, and that such changes are not a secondary modification produced by the elevation of blood pressure.</p> / Doctor of Philosophy (PhD)

<p>Direct assessments of the relative contributions of the major energy releasing pathways in human skeletal muscle during heavy exercise are difficult to obtain due to the invasive measurements required. With an isolated muscle preparation the muscles' environment is carefully controlled and all metabolic measurements are directly obtained. For this reason the isolated perfused rat hindquarter model, previously used to study resting muscle metabolism, was developed to examine the metabolism and performance heavily contracting skeletal muscle.</p> <p>Energy calculations based upon measurements of O₂ uptake (aerobic metabolism), lactate production (anaerobic glycolysis) and CP hydrolysis (alactic anaerobiosis) were made during 20 minutes of repetitive tetanic stimulation. During the initial 5 minutes of stimulation isometric tension production was high but fatigued rapidly and anaerobic involvement in energy production was large (30%), especially in the fast-twitch glycolytic muscle fibers. Muscle glycogenolysis provided the majority of substrate for both anaerobic glycolysis and aerobic metabolism. During the final 15 minutes of stimulation aerobic metabolism dominated (90%) while 60% of peak tension was held, mainly by the fast-twitch oxidative, glycolytic muscle fibers. Glycogen utilization was minimal and intramuscular triacylglycerol became the dominant fuel for oxidative metabolism, contributing 62% of the energy produced.</p> <p>Perfusions with acidotic mediums (metabolic and respiratory) reduced muscle glycogenolysis and lactate accumulation by 35% during the initial 5 minutes of stimulation. The decreased glycolytic flux reduced the availability of carbohydrate substrate for aerobic metabolism and O₂ uptake decreased. The associated reduction in energy release produced an increased rate of tension decay. Total energy release and tension production were also reduced during acidosis in the final 15 minutes of stimulation. The decreased glycolytic flux appeared to be due to an earlier fall in muscle pH during acidosis and subsequent inhibition of key regulatory enzymes such as phosphorylase and phosphofructokinase. However an alternate hypothesis is that acidosis exerted a direct negative effect on the excitation-contraction coupling mechanism, thereby reducing the need for energy production.</p> / Doctor of Philosophy (PhD)

<p>The studies contained in this thesis describe physical, biochemical and immunochemical characterization of human malignant melanoma cell surface antigens defined by a monkey antiserum and a mouse monoclonal antibody (MoAb).</p> <p>The monkey anti-CaCL 73-36 antiserum identified, melanoma-associated surface antigens (MAAs) that were shed from cultured melanoma cells. The shed MAAs appeared to be similar to shed HLA-A,B,C antigens in terms of flotation on KBr, sedimentation in sucrose gradient, and association with B2-microglobulin.</p> <p>The MoAb 140.240, which reacted with a melanoma-specific oncofetal antigen identified an epitope on an 87kd molecule that was integrally associated with melanoma cell plasma membrane. This molecule was a monomeric sialoglycoprotein that originated from a 77kd precursor polypeptide (p77). The precursor was converted to an intermediate 83kd giycopolypeptide (gp83) which in turn was further glycosylated to yield the mature 87kd glycopolypeptide (gp87).</p> <p>Both gp87 and p77 were detectable in the chase medium of melanoma cells metabolically labelled in the presence of tunicamycin (TM), but had molecular weights slightly larger than their cellular counterparts. Comparison of tryptic peptide maps of cellular and shed gp87 showed complete overlapping, except that the shed form contained two additional methionine-containing peptides. Surface radioiodination of TM-treated cells showed that p77 was also expressed on the cell surface.</p> <p>The purification of gp87 from membrane lysate and spent medium of cultured melanoma cells was achieved by a procedure involving ion-exchange, gel filtration, and antibody affinity column chromatography. Compared with the starting materials, the procedure yielded a purification factor of >3,300 and 28.6% recovery for cellular gp87, and a purification factor of >1,200 and 41.5% recovery for shed gp87. On SDS-PAGE, the purified preparations gave a single band of 87kd.</p> <p>Immunochemical analysis of MoAb 96.5 to a 97kd MAA (p97) developed in another laboratory showed that gp87 and p97 were identical molecules, although the epitope recognized by MoAb 140.240 was distinct from that recognized by MoAb 96.5.</p> <p>The biological and clinical significance of these findings in relation to the reports of other investigators on human melanoma-associated antigens are discussed.</p> / Doctor of Philosophy (PhD)