<p>In our studies, we have used adenoviruses containing the genes for Angiostatin (Ad-Angiostatin) and IL-12 (Ad-IL12) for tumor therapy. The biological activity of the Ad-Angiostatin has been characterized using an artificial matrix which promotes the growth of blood vessels (Matrigel). We observed suppression of vessel growth in the Matrigel following treatment with Ad-Angiostatin demonstrating the inibitory activity of this virus. Treatment of growing tumor in mice with either Ad-Angiostatin or Ad-IL12 resulted in modest delays in tumor growth compared to tumors treated with viruses which did not contain any genes. Ad-IL-12 treatment also causes tumors to regress in a small fraction of the treated animals (13%). However, when used in combination, treatment with Ad-Angiostatin and Ad-IL12 resulted in tumor regression in 54% of the cases, a strong anti-tumor response by killer white blood cells, and the cured animals were resistant to further outgrowth of tumor. These results are the first to demonstrate the usefulness of combining angiogenesis inhibitors with cytokines using gene therapy. The rationale for this therapy is to limit the tumor size by attacking the vasculature with angiostatin, allowing the Interleukin-12 to activate killer white blood cells to respond against foreign products present in the tumor tissue. In this manner, the potential for the white blood cells to reject the tumor is increased as there is less tumor mass present. (Abstract shortened by UMI.)</p> / Doctor of Philosophy (PhD)

<p>Lipopolysaccharide (LPS), a component of the outer membrane of gramnegative bacteria, is a potent inflammatory stimulus responsible for a number of clinically critical airway conditions including gram-negative pneumonia, gram-negative septic lung injury and septic adult respiratory distress syndrome (ARDS). These conditions are all characterized by activation of alveolar monocytes/macrophages and massive infiltration of polymorphonuclear leukocytes (PMNs). While it is generally agreed that alveolar macrophages have a central role in the initiation of neutrophil accumulation and that the contents released from PMNs play a major part in causing tissue injury, the molecular mechanisms underlying these processes remain incompletely understood. Recently, some cytokines (polypeptide hormones) such as interleukin-1 (IL-1), tumor necrosis factor α (TNFα), interleukin-8 (IL-8) and interleukin-6 (IL-6), have been implicated in these processes, yet the expression of these cytokines by airway cells in response to LPS still remains to be fully elucidated. Thus, both in vitro and in vivo studies were undertaken to investigate the effect of LPS on cytokine gene expression and protein production by airway cells and tissues.</p> <p>To investigate the regulation of cytokine expression in alveolar macrophages by LPS and extracellular matrix (ECM) components, LPS stimulated rat alveolar macrophages were maintained on different substrates: plastic, collagen and airways fibroblast-derived ECM (fECM). It was found that adherence to plastic induced IL-1β expression and that LPS further enhanced this expression in a time-dependent manner. In contrast, significant expression of IL-6 mRNA was observed only in LPS-stimulated alveolar macrophages. Adherence to collagen or fECM evoked a stronger IL-1β mRNA expression as compared to adherence to plastic. Only cells cultured on fEeM, however, retained maximal responsiveness to LPS stimulation over a period of five days in culture.</p> <p>To determine whether LPS can directly act on airway-derived fibroblasts, nasal, bronchial and lung fibroblasts were exposed to LPS and expression of several cytokines including granulocyte/macrophage colony-stimulating factor (GM-CSF), IL-8 and IL-6 was assessed. While fibroblasts of all three anatomic sites produced significantly increased amounts of cytokines in response to IL-1, only upper airway-derived nasal fibroblasts could synthesize significant quantities of cytokines upon LPS stimulation.</p> <p>Furthermore, the interaction of airways structural epithelial cells and fibroblasts with monocytes/macrophages was examined. Human peripheral blood monocytes were cultured with either nasal epithelial cell- or fibroblast-derived conditioned medium, and survival, proliferation and differentiation of monocytes then analyzed. Both macrophage colony-stimulating factor (M-CSF) and particularly, GM-CSF released by airway structural cells were found to dramatically promote survival and differentiation, but not proliferation, of monocytes.</p> <p>To investigate the temporal sequence of cytokine induction upon LPS exposure in vivo, a rat model of acute lung inflammation was established. Kinetics and cellular origin of cytokines were examined. LPS markedly evoked an early expression of TNFα and macrophage inflammatory protein-2 (MIP-2; the rodent functional equivalent of human IL-8), followed by IL-1β and IL-6 but not RANTES (a T cell/monocyte chemotactic cytokine) or transforming growth factor β₁ (TGFB₁) expression. Alveolar macrophages represented the most significant source of cytokines shortly after LPS challenge. At later times, the infiltrating PMNs were the most significant source of cytokines in the lung.</p> <p>In conclusion, these data suggest that (a) the alveolar macrophage can respond directly to LPS stimulation by elaborating and releasing cytokines; (b) this cytokine response can be modulated by the extracellular environment in which alveolar macrophages reside; (c) only the fibroblast population in the upper airway, in close proximity to the external environment, is capable of responding to LPS exposure by cytokine release. This particular fibroblast population may be thus directly involved in the initiation of acute airways inflammation; (d) monocytes/macrophages and airway structural cells communicate with each other. This interaction is accomplished in part through cytokines released from the structural cells; and (e) LPS evokes a sequential and specific cytokine response in the airways in vivo. TNFa and MIP-2 appear to play a major early role in eliciting the neutrophilic response. Consistent with in vitro findings, alveolar macrophages serve as a significant source of cytokines in vivo prior to recruitment of PMNs. Thereafter, PMNs serve as the other significant source of cytokines in vivo, suggesting that these cells likely contribute to the amplification of the response to LPS in an autocrine and paracrine fashion.</p> / Doctor of Philosophy (PhD)

<p>Herpes simplex virus type 1 and adenovirus type 5 are nuclear DNA viruses that encode a number of regulatory polypeptides that serve to facilitate expression of the viral genome at the expense of host gene expression. These viruses depend on cellular transcriptional apparatus, and viral regulatory proteins flmction primarily to ensure that host factors engage the viral template and that viral genes are expressed in the correct spatial and temporal sequence. The results presented in this thesis demonstrate that regulatory proteins encoded by herpes simplex virus and adenovirus modulate the expression of two classes of cellular genes: the globin genes and Alu repetitive sequences. Herpes simplex virus gene products were required to stimulate the expression of human α-globin, rabbit β-globin genes and Alu elements introduced into cells as part of the herpes genome. In addition, infection with herpes simplex virus or adenovirus stimulated expression of host α-globin and Alu sequences and de novo synthesized viral gene products were required for induction of these cellular genes. Viral induction of α-globin and Alu expression are both unusual instances of activated gene expression: viral infection allows the α-globin gene to escape its erythroid~restricted transcription pattern, and the viral activation of RNA polymerase III transcription of Alu sequences is the first instance of high level class III transcription of these repetitive DNA elements in vivo. The identification of the herpes implex virus and adenovirus gene products which mediate the transcriptional activation of these host sequences has contributed to the understanding of the mechanisms which regulate expression of α-globin genes and Alu repetitive elements and also of the mechanisms by which viral regulatory proteins modulate gene expression.</p> / Doctor of Philosophy (PhD)

<p>Intestinal disaccharidases are enzymes located in the apical membrane of enterocytes of the small intestine. These proteins are responsible for the breakdown of dietary carbohydrates. Despite the central importance of mouse genetics to modern biomedical research, little is known concerning the structure and regulation of the expression of murine intestinal disaccharidases. Moreover, limited information exists on the variations in molecular and genetic characteristics that the murine enzymes display between mouse strains.</p> <p>In this thesis a partial characterization of murine intestinal disaccharidases expressed by seven different mouse strains was performed. Data obtained in these studies indicated the existence of two independent congenital deficiencies of murine intestinal disaccharidases. The two described murine deficiencies constitute the only cases in which normal and altered phenotypes are available in genetically related strains of animals. Therefore, these two deficiencies may serve as excellent models for the study of disaccharidase deficiencies.</p> <p>One of the deficiencies consisting of relative low levels of sucrase activity, was found to be present in CBA/Ca mice and other two CBA/Ca-derived strains. These CBA/Ca mouse strains displayed aproximatedly 50% of intestinal sucrase activity compared to the other four mouse strains analysed. Experiments using papain-solubilized intestinal disaccharidases, molecular sieving HPLC, heat-inactivation and kinetic analysis of the sucrase activity, demonstrated that the deficiency was most likely caused by a structural alteration of the SucraseIsomaltase complex expressed by the CBA/Ca and derived mouse strains.</p> <p>The second observed deficiency consisted in a virtual absence of y-Glucoamylase maltase activity exclusively in CBA/Ca mice. When analysed by heat-inactivation, molecular sieving HPLC and 2D-electrophoresis, the absence of y-Glucoamylase maltase activity was demonstrated to be caused by a lack of synthesis of y-Glucoamylase molecules in CBA/Ca mice.</p> <p>The genetic background of the two disaccharidase deficiencies was analysed by cross-breeding CBA/Ca mice with CBA/J mice; the first strain displaying low sucrase and lack of y-GA expression and the second displaying normal phenotypes. The levels of activity of intestinal sucrase, maltase and palatinase expressed in the resulting F1 mice indicated that each of these deficiencies is most probably caused by a single defective gene, respectively. In addition, the defects were of a somatic nature and codominantly expressed with the respective normal phenotype.</p> <p>Finally, data obtained from a partial molecular characterization of murine intestinal y-Gluccamylase indicated that the enzyme may be originally synthezised as a single polypeptide with approximated M.W. of 410 kDa. This polypeptide constitutes a precursor of four additional polypeptides with M.W. of 275, 260, 140 and 130 kDa, respectively, generated by proteolytic cleavage at two alternative points along the precursor molecule. The enzyme contains one single type of active site and variable proportion of N-linked carbohydrates. These data provide additional information on the inter-species structural variation of this enzyme and probable posttranslational processing experienced by y-Glucoamylase.</p> / Doctor of Philosophy (PhD)

<p>The SH2 containing inositol polyphosphate 5-phosphatase (SHIP) has been implicated in the negative regulation of growth and differentiation of multiple hematopoietic cell types. In this thesis, I describe the cloning and biochemical characterization of the human SHIP cDNA. SHIP expression was found to be restricted to cell lines of hematopoietic origin at both the protein and RNA level. The K562 erythroleukemia cell line was identified which lacked detectable SHIP protein and message. The absence of endogenous SHIP in K562 cells provides a useful in vitro system to study the contribution of SHIP to the process of growth and differentiation in this cell line. Hemin stimulation of the K562 cell line results in the induction of an erythroid differentiation program. When stably overexpressed in K562 cells, SHIP was found to be constitutively tyrosine phosphorylated and associated with endogenous Shc, Grb-2 and Bcr/Abl. Overexpression of SHIP did not affect the overall growth rate of the cells but resulted in decreased synthesis of hemoglobin protein and [varepsilon]-globin mRNA in response to hemin stimulation. This effect was not due to increased cell death or cell cycle arrest in the SHIP-expressing lines following hemin stimulation, but was likely the result of an impaired differentiation program in these cells. Mutational studies indicated that SHIP must retain both an intact catalytic domain and Shc binding site to efficiently inhibit K562 erythroid differentiation.</p> / Doctor of Philosophy (PhD)

<p>In spontaneously hypertensive rats (SHR), treatment with the arterial vasodilator minoxidil does not prevent or attenuate the progression of cardiovascular hypertrophy despite blood pressure control, and the reason is generally unknown. The purpose of this study, was to examine temporal changes of cardiac and mesenteric arterial structure with respect to changes in volume load and cardiac and arterial sympathetic activity, during chronic treatment of normotensive and SHR with minoxidil alone, or in combination with the diuretic hydrochlorothiazide (HCTZ). The hypothesis to be tested is that an increase in the sympathetic activity and/or cardiac and intravascular volume is involved in causing these structural changes. In normotensive rats, minoxidil induced (i) right ventricular hypertrophy (RVH), (ii) eccentric left ventricular hypertrophy (LVH), (iii) medial hypertrophy of the superior mesenteric artery, (iv) intravascular volume expansion, (v) increases in cardiac filling pressures, (vi) increases in ventricular and arterial norepinephrine turnover rates. In SHR, minoxidil (i) decreased blood pressure, (ii) potentiated RVH, (iii) caused the development of eccentric LVH superimposed on the preexisting hypertrophy, (iv) increased the lumen of the superior mesenteric artery, (v) prevented further increases in medial hypertrophy of the large and small mesenteric arteries, (vi) induced intravascular volume expansion, (vii) increased ventricular but decreased arterial norepinephrine turnover rates, and (viii) increased elastin content and decreased elastase activity in the large conducting vessels (aorta, superior mesenteric artery). In SHR and normotensive rats, concomitant diuretic treatment prevented intravascular volume expansion, caused concentric LVH rather than eccentric LVH and no longer increased the medial and luminal areas of the superior mesenteric artery. These results suggest that there are regional differences in the response of the cardiovascular system to minoxidil in SHR and normotensive rats. Some of these differences may relate to differences in regional sympathetic activity, whereas volume load appears to play a modulatory role.</p> / Doctor of Philosophy (PhD)

<p>A prospective 2-year study was designed to test the hypothesis that 3 core factors influence the postmenopausal decline in bone mineral: adequate supply of calcium to the skeleton, endogenous estrogen production by lean and fat tissue mass, and mechanical stress imparted by physical activity and body mass. The rate of change in bone mass (ΔBMD) was established for 61 postmenopausal women from semi-annual measurements of bone mineral density (BMD) at the proximal femur and lumbar spine using dual energy X-ray absorptiometry (DXA). Whole body BMD and body composition were also assessed annually using DXA. Serum estradiol was determined at baseline by radioimmunoassay. Calcium intake was evaluated using a food frequency questionnaire. Calcium absorption was measured using a single isotope technique. Grip strength was measured using a Jamar hand dynamometer. Aerobic fitness was determined using a submaximal 1-mile walk test. Habitual daily activity was assessed using a portable accelerometer. At baseline, the strongest associations were between BMD and body mass values. These explained 22 to 25% of the variance in BMD. Estradiol was an independent predictor of BMD of the whole body. No physical activity variable was independently predictive of BMD. Of the dietary variables, only tea consumption was independently predictive of BMD at the femoral and whole body sites. With age, bone loss was attenuated. At the lumbar spine, ΔBMD was also positively influenced by lean mass, weight gain, protein intake and increased calcium intake. The contribution of estradiol was borderline (p = 0.06). Weight gain was similarly influential at the femur. There was no positive influence of any physical activity measure on ΔBMD at any site. Lean mass and weight gain had the greatest positive influence on BMD and ΔBMD. A hormonal rather than mechanical explanation was favoured. Trabecular bone may also be responsive to dietary perturbations.</p> / Doctor of Philosophy (PhD)

<p>Bone structure is compromised in individuals with osteoporosis. Indices of trabecular bone structure which reflect the connectivity (CI), the average dimensions of the marrow holes (HA ) and the area of the largest hole (HM ) at the distal radius can determined in vivo . This work evaluates whether these structural indices (1) are influenced by gender, aging and mechanical loading and (2) aid in the discrimination of individuals with low bone mass most at risk for fracture. Gender and age-related patterns of bone structure in the nondominant distal radius measured using peripheral quantitative computed tomography (pQCT) demonstrate the expected trends in a cross-sectional study of 145 healthy adults. Men have a better connected, less porous trabecular network. The age-related decrease in CI in men (-0.8%/yr, p < 0.05) is less pronounced than in women (-2.2%/yr, p < 0.001). Similarly, there are significant age-related increases in HA (+2.2%/yr, p < 0.01) and H M (+1.1%/yr, p < 0.01) in women but not in men. To investigate the impact of mechanical loading associated with hand dominance on indices of bone structure, bilateral images for 106 healthy adults were acquired. For all subjects, HM is significantly smaller in the dominant radius (p < 0.01). Right handed subjects (n = 96) have greater CI (p < 0.05) and smaller HM (p < 0.01) in the dominant limb. The effect of altered mechanical loading on bone structure was assessed by immobilizing the nondominant limb of 10 healthy volunteers in a plaster cast for 6 weeks followed by a remobilization period of 1 year. HM increases (p = 0.04) during immobilization and recovers within 3 months of remobilization. The ability of indices of trabecular structure to discriminate individuals with recent wrist fracture (n = 22) from controls matched for bone density (n = 22) was evaluated. The fracture group has a larger mean HA than the group without fractures (p < 0.05). The relative odds of wrist fracture is 6.9 (95% CI: 1.3 to 37.3) for individuals with a HA ≥ 6 mm2 . These data show that indices of trabecular bone structure, derived from pQCT images, reflect the expected patterns with respect to gender, age and mechanical loading. Preliminary results suggest that measuring HA at the distal radius may aid in the identification of individuals with low bone mass who will sustain a fracture.</p> / Doctor of Philosophy (PhD)

<p>The purpose of this research was to determine the physiological mechanism(s) underlying the reduction in voluntary electromyographic (EMG) activity with maximal contractions during fatigue. The specific hypothesis was that this reduction in EMG activity results from reflex inhibition of homonymous motoneurons by afferents from the fatigued muscles. The experiments were conducted on the human ankle dorsiflexor and plantarflexor muscles, with the use of eschemia to accelerate the fatigue process. In the first part of the study it was shown that, within 3-4 minutes, repetitive indirect stimulation of the dorsiflexor muscles via the peroneal nerve, at either 15 Hz or 30 Hz, could abolish dorsiflexion torque elicited by single shocks and trains of stimuli. During the 15 Hz fatiguing frequency the relative loss of dorsiflexion torque was greater than the decline in the amplitude of the evoked muscle compound action potentials (M-waves). Tus 15 Hz was the rate of fatiguing stimulation used in the remainder of the study. In the second part of the study, fatiguing indirect stimulation at 15 Hz caused significant reduction in both the dorsiflexion torque and EMG activity associated with subsequent maximal voluntary contractions (MVCs) of 66.1 ± 16.4% and 50.3 ± 27% respectively. The relative preservation of M-wave amplitudes in the fatigued muscle (reduction of 13.1 ± 22.9%) indicated that most of the loss of EMG activity was not due to inexcitability of the neuromuscular junctions or muscle fibre membranes. Nor could the reduction in voluntary EMG activity have been due primarily to failure of subjects to exert maximal voluntary effort since supraspinal motor pathways had not been involved in the fatigue process. Furthermore, during the MVC, no additional force was evident with a supramaximal interpolated stimulus. Hence, reflex inhibition appeared to be the most likely mechanism. The third part of the study explored the possibility that the reduction in voluntary EMG activity during fatigue was due to a lower level of alpha motoneuron excitability; for these experiments recordings were made of the electrically-induced homonymous response (H-reflex) of the soleus muscle. It was shown that the H-reflex excitability was depressed by 44.4 ± 25.1% during fatigue of the right soleus muscle using 15 Hz stimulation under ischemic conditions; the H-reflex excitability in the nonfatigued left soleus did not change significantly. The maximum M-wave amplitudes demonstrated a mean decline of only 8.8 ± 11.9% indicating good peripheral excitability of the muscle fibre membranes. Control experiments performed under ischemic conditions alone (without induced fatigue) or with electrical stimulation alone (without ischemia and hence without fatigue) failed to demonstrate any significant changes in reflex excitability. Thus, in the absence of fatigue, the depression could not be accounted for on the basis of either ischemia or electrical stimulation; instead the findings were consistent with the presence of an inhibitory reflex from the fatigued muscle onto the homonymous alpha motoneurons. In the final part of the study it was demonstrated, by means of pressure-induced impulse blockade of large myelinated afferents in the sciatic nerve prior to fatigue, that the mean plantarflexor torque produced during MVCs decreased by 38.0 ± 18.6% from the postblock value compared to a decrease of 5.2 ± 7.0% in the ischemia control; the mean EMG activity decreased from postblock values by 43.4 ± 15.6% following fatigue and by only 6.6 ± 5.5% following ischemia alone. These results were very similar to those demonstrated without any blockade of large diameter afferents. This suggested that the afferents involved in the putative reflex inhibition of the alpha motoneuron pool with fatigue were likely to have been those with small diameters (Group III and IV). This research provided evidence suggestive that the reduction in voluntary EMG during fatigue, in both tibialis anterior and soleus muscles, resulted from reflex inhibition of the motoneuron pool.</p> / Doctor of Philosophy (PhD)

<p>The gastrointestinal (GI) tract is a hollow tubular organ that runs through the length of the central body. To move, mix, and compartmentalize ingesta through this tract, different patterns of motility are needed. This thesis is concerned with the myogenic control of motility through the pacemaker network of interstitial cells of Cajal (ICC) and the smooth muscle cells (SMC). Using patch clamp techniques, the electrophysiological properties of single ICC and SMC were examined. Previous research suggested the possibility of a specialized cell type generating the pacemaker slow wave potentials: the network of ICC that resides in the Auerbach's plexus region of the small intestine. An isolation procedure was developed and optimized to harvest single ICC that can survive short term culture and allow examination by patch clamp. Single cell patch clamp recordings demonstrated the presence of slow wave-like voltage oscillations driven by active current oscillations that match all properties seen in whole intestinal tissue slow waves. With different recording modes, whole cell currents, voltage and current oscillations were recorded from the same cell, showing that ICC are electrophysiologically unique and that the active inward current driving the slow wave-like oscillations are not voltage dependent. The isolated single ICC were demonstrated to have a specific tyrosine receptor marker protein for ICC, Kit , by selective RT-PCR amplification. The slow wave-like oscillations had a reversal potential consistent with a non-specific cation conductance. Although previous research had been done on single smooth muscle cells, there is currently no consensus on the cellular ionic currents present. In this thesis, analysis of different recordings demonstrated that there are at least four main groups of SMCs with different whole cell current profiles. Different cellular ionic currents were found specifically in different groups, and can be confirmed by reconstructing single channel recordings. One cellular outward current was chosen for further investigation-a fast activating and inactivating transient outward current. This current was characterized by common protocols and with a novel ramp analysis. Characterization revealed two distinct transient outward currents with different kinetic properties. Finally, spontaneous transient outward currents (STOCs) have been recorded in 25% of smooth muscle cells, reflecting quantal Ca2+ release from the intracellular stores to the plasmalemma calcium dependent potassium channels. Therefore, the study of STOCs gives direct information not only on the activities of intracellular Ca2+ stores, but also on the kinetics of Ca2+ release and reuptake in the microenvironment where STOCs originate. From these results, a simple model for GI motility was developed to account for the cellular interactions between nerve, ICC, and smooth muscle.</p> / Doctor of Philosophy (PhD)