<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)

<p>Alpha-l-protease inhibitor (αlPi) is a plasma glycoprotein which plays a major role in limiting proteolysis during inflammation due to its broad spectrum of inhibitory activity.</p> <p>The purification and partial characterization of αlPi have allowed for its investigation as an acute phase reactant during inflammation.</p> <p>Mouse αlPi is a glycoprotein of molecular weight 53,500 with a half-life of approximately 15.5 hours. On two dimensional immunoelectrophoresis, two variants of αlPi were seen which exhibited immunological non-identity. The two variants showed trypsin binding activity, both were synthesized by hepatocytes, and both behaved as acute phase reactants.</p> <p>The ubiquitous nature of alPi was evident from its presence in a wide variety of body fluids. Using an immunohistochemical stain, the inhibitor was shown to be normally present in the cytoplasm of hepatocytes, islet cells of pancreas and in some villous epithelial cells of the small intestine. The hepatocyte was shown to be the major source of serum αlPi. Alveolar macrophages, shown to contain αlPi histochemically in particular inflammatory states of the lung, synthesized minimal quantities.</p> <p>The acute phase response of αlPi was investigated in four models of inflammation consisting of either a subcutaneous injection of celite or infection with one of three parasites: Nippostrongylus brasiliensis; Trichinella spiralis; and Trypanosoma congolense. In addition, studies have been initiated with other mouse acute phase reactants, namely, complement component C3, serum amyloid P (SAP) and serum amyloid A (SAA).</p> <p>The induction of acute phase protein synthesis during inflammation was characterized by an increase in the number of hepatocytes staining for αlPi. During inflammation the level of hepatocyte staining was shown to correlate with the synthetic output of αlPi. The maximum staining activity for αlPi in hepatocytes preceded the peak increase in serum levels during inflammation. Moreover, there was a characteristic, progressive alteration in the distribution pattern of stained hepatocytes within the liver lobule. These results were consistent with an acute phase mediator perhaps originating from the site of inflammation, gaining access to the liver via the portal circulation causing an induction of acute phase protein synthesis.</p> <p>The synthesis of an acute phase mediator (APM), probably interleukin 1 (ILl), by alveolar macrophages and the in vitro induction by APM of αlPi synthesis by hepatocytes has been demonstrated.</p> <p>It was also shown that αlPi accumulated at the site of inflammation which may account for the apparent lack of increase in serum levels even though there was an increased hepatic output of alPi at that time.</p> <p>Collectively these results demonstrate that in the mouse the induction of synthesis of αlPi during an acute response to inflammation is mediated by an acute phase mediator originating from macrophages at the site of inflammation.</p> <p>The increased synthesis of αlPi has been shown in pivo by a measure of serum levels and immunohistochemical examination of liver tissue as well as in vitro by cultures of isolated hepatocytes.</p> <p>These studies constitute a basic framework upon which mechanisms for the induction and control of synthesis of acute phase proteins can be further explored.</p> / Doctor of Philosophy (PhD)

<p>The arenaviridae are a group of enveloped, negative-stranded RNA viruses. These viruses characteristically produce persistent infections in their natural host, which is how they are maintained in nature. Experimentally-induced arenavirus disease has been found to possess similar pathological features of human disease. In the inbred MHA strain of Syrian hamster, Pichinde virus causes a fatal infection when inoculated intraperitoneally. Investigation of the factors leading to a fatal Pichinde virus disease in Syrian hamsters revealed that susceptibility was genetically determined. Susceptibility of MHA hamsters to a lethal infection of the virus appeared to be related to the presence of a splenic target cell for Pichinde virus replication that was minimally expressed in resistant hamster strains. In addition, during studies on cell-mediated immune responses to Pichinde virus antigens, it was discovered that MHA hamsters survived infection when the virus was given in the footpad. However, unlike the resistant LSH and LVG strain hamsters, the MHA hamsters did not manifest a footpad swelling response.</p> <p>The present studies were initiated to determine some characteristics and possible mechanism(s) underlying the lack of footpad swelling response to a primary inoculation of Pichinde virus in the MHA strain hamster. MHA unresponsiveness was not due to a lack of immune recognition of Pichinde virus antigens, since this strain was shown to produce complement-fixing antibodies to Pichinde virus. Furthermore, footpad-inoculated MHA hamsters were protected against a subsequent intraperitoneal challenge with Pichinde virus.</p> <p>Experiments designed to elucidate the genetic basis for presence or absence of footpad swelling revealed that expression of footpad swelling to Pichinde virus was a dominant trait controlled by a single gene or closely linked genes. Furthermore, the lack of responsiveness in MHA hamsters appeared to be specific for Pichinde virus, since footpad swelling could be elicited in this strain by footpad injection of either vaccinia virus or vesicular stomatitis virus. Histological analysis of the virus-injected footpad showed that footpad swelling was associated with an influx of mononuclear cells at the site of injection. These observations suggested that MHA unresponsiveness could not have been caused by a general defect in effector mechanisms that mediate the footpad swelling response.</p> <p>A search for Pichinde virus-induced suppressor activity was then undertaken. Treatment of MHA hamsters with cyclophosphamide, a drug known to augment delayed hypersenstivity responses by inhibiting suppressor cell activity resulted in a significant footpad swelling response following Pichinde virus injection. Furthermore, transfer of lymphoid cells but not serum from 5 day footpad-inoculated MHA hamsters could specifically suppress footpad swelling to Pichinde virus in the LSH recipient.</p> <p>Aqother parameter of cell-mediated immunity studied was the generation of cytotoxic cells. Pichinde virus primed MHA and LSH hamsters were capable of generating cytotoxic cells, after restimulation in vitro with virus but the cytotoxicity lacked specificity. When spleen cells from 7 day footpad-inoculated MHA hamsters were added to spleen cells from LSH hamsters, the cytotoxic activity of the latter cells was significantly suppressed.</p> <p>These results taken together suggested that the absence of footpad swelling in the MHA strain hamster was caused by the generation of a cell-associated suppressor mechanism that appeared to be induced specifically by Pichinde virus.</p> / Doctor of Philosophy (PhD)

<p>Sexually-transmitted diseases (STDs) are among the most common causes of illness in the world. The annual incidence of STDs is rising, from 250 million in 1990 to 340 million in 2002. Viral infection is a frequent cause of STDs and the mucosal surfaces are the natural sites of transmission of viruses. Both genital and rectal tracts are involved in transmission of viral SIDs. To infect a host, a virus has to penetrate mucosal immunologic barriers, including lumenal immunoglobulin A (lgA) intervention, epithelial defenses, and lamina propria (LP) lymphocyte- ediated protection. The breach of the mucosal immune system can lead to virus spreading to the rest of the body, thereby causing life-threatening disease. Induction of effective mucosal immunity is essential for the host to control local viral infection and prevent SID development. Immune responses are initiated when pathogen-derived antigen (e.g., viral antigen) is encountered and taken by antigen-presenting cells (APes), especially dendritic cells (Des). After antigen processing, Des present immunogenic determinants to and activate naIve lymphocytes in mucosal lymphoid tissues. Activated lymphocytes leave the lymphoid tissue and, via the bloodstream, migrate to the LP. These effector cells exert a series of immune functions, such as cytokine production, cytotoxicity and antibody secretion. Accordingly, the mucosal immune system is divided into the inductive and effector site. Gut-associated lymphoid tissues (GALT) represent the inductive site of the gastrointestinal (GI) system. The GALT within the rectal mucosa mainly consists of iliac lymph nodes (lLN) , Peyer's patch-like aggregated lymphoid follicles (ALF) and isolated lymphoid follicles (ILF). The ILN have been identified as the principal inductive site, whereas the role for mucosally located lymphoid follicles remains poorly understood. To elicit specific mucosal immune responses against virus infection, a viral antigen must be introduced into the mucosal immune system, importantly the inductive site. Efficient antigen delivery ensures the success of inducing effective mucosal immunity. Both the mucus layer and the integral epithelial monolayers form barriers against the passage of proteins and particulate matter across the epithelium. Previous studies demonstrated that these barriers could be overcome by mucus removal with ethanol treatment followed by utilizing viral vectors such as replication-deficient adenoviral vectors (Adv) engineered to encode heterologous antigen genes. Adv can infect a broad range of mammalian cells, including human and ~ouse epithelial cells, and has proved to be efficient in transferring genes to the colonic mucosa. It has been discovered that mucosal immunity can be induced at multiple mucosal sites, but rarely via a systemic route. This phenomenon is largely due to the mechanism of the common mucosal immunologic system (CMIS), within which activated mucosal lymphocytes migrate from one mucosal site (e.g., the upper respiratory tract mucosa) to another (e.g. the genital tract). Thus, the concept of CMIS is used as a guiding principle for mucosal vaccine design. However, emerging results have suggested that distant mucosal immunization is less effective than local immunization and that CMIS might comprise several anatomical-based grouped networks having different lymphocyte homing mechanisms. In this context, the vaginal (local mucosal) immunization regimen might improve local protection against genital STDs. Following the similar logic, rectally-induced immune responses might have potential to combat rectal STD infections.As both genital and rectal mucosae are drained by the ILNs, the putative genito- ectal associated lymphoid tissue, rectal immunization might be an alternative solution to genital immunization, especially to deal with the problem of immunization in the male genitourinary tract. The major goal of this study was to evaluate the effects of Adv-based local mucosal immunization via the rectum of mice in the induction of mucosal immunity against virus infection at the rectal as well as genital mucosa. non-Invasive intrarectal (IR) delivery method (pipetting and Dermabond® following a ethanol enema) for Adv was developed in the present study to provide better gene transfer for induction of mucosal immune responses. The first approach was to reinvestigate gene transfer to the mouse colon after intrarectal (lR) administration. The transgene was found highly expressed at days 1-3 and mainly confined to the colon. Gene expression was not only identified within the epithelium but also immediately beneath the epithelium, probably due to the penetration of Adv through epithelial cells. Mucosal immune responses were examined in an antigen model using ovalbumin (OV A). After IR immunization with Adv encoding OVA (AdOV A), the frequency of LP interferon (lFN)y secreting cells was detected as early as day 4, and continually progressed upward. The appearance of ILN IFN-y-secreting cells was transient, and this was mirrored by the ILN cytolytic activities. CDS+ T cells were stimulated to produce IFN-y, and cytolytic activities depended largely on CDS. Also, IR immunization with Adv induced Thl T-cell responses and local production of specific IgA. When challenged with recombinant vaccinia virus expressing OV A, immunized mice completely controlled local viral infection and prevented virus dissemination from the rectal tissue. Thus, an infectious mouse model of herpes simplex virus type 2 (HSV -2) given via the rectum was developed in the study and used to further validate the IR immunization regimen. Mice immunized with Adv encoding glycoprotein B (AdgB) were protected from rectal challenge of HSV -2 at absolute lethal doses. Clinical pathology and virus replication were remarkably reduced and virus-shedding period was significantly lessened. CDS+ T cells, IFN-y and interleukin (lL)-12 appeared to play an essential role in such protective immunity. Protection of mice against HSV-2 challenge in the vaginal tract was also achieved, thus indicating that rectal immunization could also confer protective genital immunity. In comparison with the intranasal (distant mucosal) and subcutaneous (systemic non-mucosal) immunization, rectal immunization proved to be more effective in the induction of rectal immune responses including the frequency of IFN-y secreting cells and IgA production, and to provide better protection against rectal or vaginal HSV-2 challenge. All these results underscored the importance of applying local mucosal immunization to induce mucosal immunity at both rectal and genital tracts. In conclusion, IR administration with Adv by the new delivery method efficiently transferred antigen genes into the rectal mucosa and elicited protective local immunity to virus challenge. The present study provided evidence that rectal (local mucosal) immunization regimen was a better vaccination strategy than distant mucosal and systemic non-mucosal immunization to provide both rectal and genital protection against viral infection. Thus, the present approach supports the view that route is a critical determinant of vaccination and, furthermore, represents fertile ground for future studies of mucosal vaccination via the rectal mucosa.</p> / Doctor of Philosophy (PhD)

<p>Asthma and other allied allergic diseases represent a significant burden to health care and are recognized as endemic in the Western World. While a diverse array of effective pharmacopoeia provides reprieve from symptoms, no preventive or curative therapies are currently available. This is in part due to the paucity of understanding how allergic diseases develop. Recently, remarkable progress has been made in this regard, largely due to the discovery of regulatory mechanisms that control responsiveness to antigen in the airway. Indeed, to understand fully why people develop asthma requires an understanding of both allergic sensitization and tolerance. Work presented in this thesis contributes to our knowledge of inhalation tolerance. Using a classical mouse model of allergic airways inflammation, we show in Chapter 2 that inhalation tolerance is a persistent and active process as it prevents the generation of airway eosinophilia, antigenspecific IgE and airway hyperresponsiveness upon secondary immunogenic challenge, independently of IL-lO or IFN-y. Building on these observations, in Chapter 3 we show in a mucosal model of allergic sensitization that inhalation tolerance cannot be broken with the expression of GM-CSF, a potent growth factor and cytokine that has been associated with asthma and allergy in both human and animal subjects. However, concomitant expression of decorin, a natural inhibitor of TGF-f3, reverses inhalation tolerance, thus implicating TGF-f3 as putatively important in regulating responsiveness in the airway. In Chapter 4, we identify an alternative mode of tolerance. We show that chronic exposure to innocuous antigen in sensitized mice does not lead to chronic inflammation but to abrogated eosinophilia that can, nevertheless, be reversed with the expression of GM-CSF. Collectively, these findings enrich our understanding of tolerance and provide a framework for new discoveries that may, ultimately, lead to novel and powerful therapies for allergic disease.</p> / Doctor of Philosophy (PhD)

<p>Cancer cell metastasis to bone is a frequent occurrence with many common-human malignancies, including carcinomas of the prostate and breast. Bone metastasis may be promoted by the phenotypic responses of cancer cells to factors which are present within the bone microenvironment. This thesis describes in vivo and in vitro efforts to test the hypothesis that bone matrix proteins promote the growth and adhesiveness of metastatic cells. An animal model was developed to examine the growth properties of rat mammary cancer cells (Walker 256, W256) in metastatic target organs (Chapter 1). Rats were injected intramuscularly with W256 celIs, which formed spontaneous metastases in the skeleton, liver, lungs, and kidneys within 7-10 days. Skeletal metastasis was associated with a progressive loss of trabecular bone. Within the skeleton, W256 cells which were immediately adjacent to trabecular bone had a 30% greater growth rate than did W256 cells located >50 μm from bone surfaces (p<0.05). These data established the first animal model of spontaneous bone metastasis, and provided preliminary evidence that bone matrix is a possible mitogen for metastatic cancer cells in vivo.</p> <p>The mitogenic activity of bone matrix may be enhanced by osteoclastic bone resorption (1). To test this hypothesis, bone resorption was stimulated by injecting rats with Leydig tumor cells (Chapter 2). Leydig tumor burden was associated with decreased trabecular bone volume, fewer osteoblasts, and more osteoclasts. Leydig tumor burden was associated with a 56% increase in the growth rate of W256 cells in the skeleton (p<0.05), while W256 cell growth rate in the liver, lungs, and kidneys were not affected. The selective growth promotion of W256 cells in bone suggests the existence of a mitogen for W256 cells which is released from bone matrix during resorption.</p> <p>Previous in vitro data suggest that this putative bone-derived mitogen may be transforming growth factor β (TGF-β) (1), a factor which can stimulate oncogene expression. Media conditioned by resorbing fetal rat calvarial bones induced a rapid increase in W256 cell expression of c-myc oncogene mRNA and of nuclear c-myc protein (Chapter 3). Purified TGF-β also stimulated c-myc mRNA expression, suggesting that the mitogenic stimulation of W256 cells by bone-derived conditioned media may involve a signaling pathway which is also utilized during TGF-β-induced growth stimulation.</p> <p>Bone resorption may be a sufficient but unnecessary growth stimulus for metastatic cells in vivo. To test this hypothesis, bone resorption in rats was inhibited in vivo with the bisphosphonate APD prior to their inoculation with W256 cells (Chapter 4). APD blocked the pathologic bone resorption normally associated with W256 tumor burden. However, the skeletal tumor burden in APD-treated rats was 2.6-fold greater than in untreated rats. Tumor burden in other organs was unaffected by APD. W256 cells in the skeletons of APD-treated rats had a 55% greater growth rate than did W256 cells in the skeletons of controls. W256 cells located adjacent to trabecular bone had greater growth rates than did W256 cells>50 μm from bone, irrespective of APD treatment (p<0.05). These data suggest that bone matrix can exert a mitogenic influence on cancer cells without undergoing resorption.</p> <p>Bone-derived factors are also capable of promoting adhesion (2), and the adhesion of metastatic cells to bone matrix could promote their skeletal localization. An in vitro model of bone matrix was developed to identify potential ligands which could support the adhesion of metastatic cells (Chapters 5 and 6). The extracellular matrix deposited by human U2OS osteosarcoma cells supported the rapid adhesion of human PC-3 prostatic carcinoma cells. Antibodies directed against the integrin-type collagen receptor α2β1 inhibited PC-3 cell adhesion to U2OS matrix and to purified type I collagen, as did a collagen-derived peptide sequence (DGEA) which inhibits α2β1 function. TGF-β treatment caused a selective increase in α2β1 integrin expression, which was effected by increased rates of de novo synthesis of both α2 and β1 integrin subunits. The induction of α2β1 was associated with similar increases in the adhesion of PC-3 cells to U2OS matrix and to purified type I collagen. These data suggest that type I collagen may represent an important bone-derived adhesive ligand for metastatic cells. The abundant expression of type I collagen and of TGF-β in bone may promote bone metastasis by promoting the adhesion of metastatic cancer cells to bone matrix.</p> / Doctor of Philosophy (PhD)

<p>The general focus of my project was the production of recombinant adenovirus vectors for use in inmmunotherapy of cancer. The basic strategy involved the infection of tumour cells with Ad-vectors expressing cytokines, inducing a local anti-tumour response. The cytokine of interest for my project was tumour necrosis factor alpha (TNFα), which I used for treatment of a murine transgenic model of breast cancer. TNFα is a pluripotent cytokine with a wide variety of physiological functions including antitumour activity. TNFα was originally discovered through the anticancer activity of sera of mice treated with endotoxin (Carswell et al., 1975). There are two known cell surface receptors for TNFα termed p55 and p75. Both receptors signal a variety of functions and some redundancy exists between them. However the p55 TNF receptor is the major activator of cytotoxicity and cytokine secretion, while the p75 TNF receptor is primarily responsible for proinflammatory and lymphoproliferative signals. Perhaps the major limiting factor affecting the use of TNFα in tumour therapy is its systemic toxicity, through the induction of septic shock and cachexia (Tracey, 1995; Tracey et al., 1986). We have been investigating techniques which would reduce the systemic side effects of TNFα, while retaining its antitumour activity. We found that local expression of TNFα from within a tumour (transduced cells) alone is not enough to eliminate its lethal side effects, therefore two other approaches are being investigated in an attempt to deal with systemic toxicity induced by TNFα. The first was the construction of an Ad vector expressing a membrane bound mutant of murine TNFα (see chapter III). The second approach involved specific targeting of the two cell surface receptors of TNFα. To accomplish this, we used Ad vectors expressing human TNFα, and a novel p75 TNF receptor specific mutant of murine TNFα for use in tumour immunotherapy (See chapters IV and V). It was found that restricting TNFα to the membrane produced a marked reduction in lethality while retaining near normal antitumour activity. Targeting the p55 TNF receptor proved to be ineffective, while targeting the p75 TNF receptor drastically reduced the lethality of the cytokine while retaining some antitumour activity. However the overall efficacy of this therapeutic technique was poor, as only a low percentage of mice were cured with our Ad-TNF vectors.</p> / Doctor of Philosophy (PhD)

<p>The studies described in this thesis were undertaken to examine the mechanism of lactate production in exercise from a standpoint of enzymatic control and investigate the two rate-limiting enzymes which regulate glycogen breakdown and oxidation, glycogen phosphorylase and pyruvate dehydrogenase (PDH). The purpose was to establish the theory that it is the differential rate of flux through these enzymes which determines the extent of lactate accumulation in situations where significant lactate production occurs such as during 30-s bouts of maximal intermittent exercise and during exercise in hypoxia. In the first study, at the onset of maximal exercise, the transformation of PDH was delayed relative to phosphorylase accounting for a greater lactate accumulation. PDH was maximally transformed after 15s and remained elevated until 30s, whereas the transformation of phosphorylase remained elevated at 15s and reverted to resting levels after 30s. Before the third bout of exercise, PDH was nearly 50% transformed and was fully transformed after only 6s, whereas phosphorylase remained at resting levels. The regulation of these enzymes at the last 15s of the first bout and throughout the third bout is consistent with a reduction in glycogenolysis and increased pyruvate oxidation leading to less lactate accumulation. In the second study, at the onset of exercise in hypoxia, there was a delayed transformation of PDH relative to normoxia, accounting for the greater lactate accumulation typically observed in hypoxia. The results from these first two studies suggested that the metabolic inertia of PDH activation may be a factor in the mechanism of lactate production in both maximal exercise and in hypoxia. The third study sought to remove the metabolic inertia of PDH activation during exercise in hypoxia with the infusion of dichloroacetate (DCA). DCA fully transformed PDH at rest and at the onset of exercise in hypoxia. By removing the metabolic inertia of PDH activation with DCA or with prior exercise as in the third bout of maximal exercise in the first study, lactate production was significantly reduced. The findings from these studies demonstrate that the mechanism of lactate production is the result of a mismatch between the rates of pyruvate production and pyruvate oxidation due to the differential catalytic rates between glycogen phosphorylase and PDH under conditions where oxygen is presumed to be limiting such as maximal exercise and exercise in hypoxia. These results contradict the theory that an O2 limitation is the main cause for lactate production ("anaerobic metabolism") and suggest that the metabolic inertia of enzyme activation also plays a role. The data are indicative of a central role for PDH and phosphorylase in the mechanism of lactate production and further question whether O2 is limiting during exercise in healthy humans.</p> / Doctor of Philosophy (PhD)