<p>For the development of many cell types, terminal differentiation and continued cell cycle progression are incompatible processes. Rb, p107 and p130 comprise a gene family encoding transcriptional regulators that act within a complex network to control exit from, and progression through the cell cycle. The distinct requirements for p130 and p107 were assessed in vivo using homologous recombination in embryonic stem (ES) cells. p130 expression is ubiquitous, although the level of expression varies between tissues. As well, its induction during neuronal cell differentiation is consistent with p130 function accompanying terminal differentiation. p130 deficiency was incompatible with embryo survival in the hybrid 129Sv;Balb/c genetic background. The mutant embryos died between E11-13 and exhibited striking delays in growth and development at 10.5 dpc with specific deficits in neurogenesis, somitogenesis and cardiogenesis. p130 appears to be required for the maintenance and survival of specific cell types, most notably neuronal cells. The data indicate that the p130 gene is essential for normal development, but in a strain-specific manner. On a hybrid 129Sv;C57B1/6 background, the p130 mutants exhibited no phenotype. The p107 mutants were viable and fertile, indicating that p107 function was adequately compensated by other proteins during development, potentially Rb and p130. The p107-/- mice did however exhibit a postnatal growth deficit and a diathetic myeloid proliferative disorder. The accelerated proliferation and cell-cycle kinetics of p107-/- EF indicated that p107 functions, in part, to regulate cyclin expression and cell cycle progression. p107-/- myoblasts also exhibited accelerated prliferation and aberrant in vitro differentiation. Lastly, the p107-/- phenotype was also dependent on the genetic strain, indicating the presence of modifying genes. The mice produced in these studies can be assessed for genes that modify the phenotypes in these different strains, potentially revealing epistatic relations to p107 and p130. Although both striking and subtle cell-specific phenotypes were exhibited, these experiments strongly reconfirm that functional overlap and compensation exist within the Rb family. The overlapping expression patterns and apparent functional relations indicate that p130, p107 and Rb regulate transitions in a concerted manner during cell proliferation, and cell cycle exit and entrance.</p> / Doctor of Philosophy (PhD)

<p>The spontaneously hypertensive rat (SHR) was used as a model of human essential hypertension. The overall hypothesis was that hypertrophy of the smooth muscle layer of small muscular arteries in essential hypertension results in greater contractility of these vessels that then results in elevated total peripheral resistance and higher blood pressures. Elevated total peripheral resistance and small artery hypertrophy are well documented in the SHR, however, it remains unknown if these changes are the cause of result of elevated blood pressure. For this reason, we have focused our studies on young SHR during the initiation of hypertension to attempt to separate cause from effect. Studies were done to determine when SHR's blood pressure begins to differ from its normotensive control the Wistar-Kyoto rat (WKY). We found that blood pressure began to diverge between SHR and WKY at four weeks of age. Structural and functional differences between small muscular arteries from the mesenteric vascular bed of 4-week old SHR and age matched WKY controls were studied using a new morphometric protcol involving confocal microscopy and a pressurized artery myograph. Arteries from SHR had a larger medial volume, increased number of smooth muscle cell layers, but similar lumen size when compared with WKY in the maximally relaxed condition. Functional studies showed that SHR arteries contracted more in response to stimulation by KC1 and norepinephrine, resulting in significantly smaller lumen size in these vessels as compared to WKY. We concluded that structural and functional differences in SHR arteries were primary changes which may contribute to the development of hypertension. Further studies were conducted to determine if a differential incidence of apoptosis during the development of SHR and WKY arteries contributes to the structural differences. One to two week old animals were used for these studies since at this time the structure was similar between the strains. To measure the incidence of apoptosis, we used both DNA laddering and end labeling. It was found that SHR had a significantly decreased incidence of apoptosis over WKY. The cellular nature of the medial layer hypertrophy in SHR at 4-weeks was also assessed. Numerical density of smooth muscle cell nuclei in the medial layer was measured with a three dimensional disector method under confocal microscopy. We found that the numerical density of medial smooth muscle cells was significantly less in SHR than WKY, and the number of smooth muscle cells was significantly less in SHR than WKY, and the number of smooth muscle cells was similar between the strains. The smooth muscle cell length from SHR was significantly longer than WKY. We concluded that increased smooth muscle cell length in prehypertensive SHR is responsible for their increased medial volume. These studies have shown that medial layer hypertrophy due to smooth muscle cell lengthening in the small muscular arteries of SHR which increases their contractile ability, occurs at the initiation of hypertension. This evidence demonstrates that structural and functional changes in these SHR arteries can not be the result of increased blood pressure but may be a factor causing hypertension by increasing the total peripheral resistance in these animals.</p> / Doctor of Philosophy (PhD)

<p>Food allergies are a significant clinical problem, with symptoms including diarrhea, vomiting, or systemic anaphylaxis. To elicit allergic reactions, antigens must first cross the intestinal epithelium. The purpose of my studies was to examine macromolecular transport across intestinal epithelium, and the effect of sensitization and immune activation on transepithelial antigen transport. Rats were sensitized to a model protein antigen, horseradish peroxidase (HRP) by injection with adjuvants. Intestinal segments were removed and mounted in Ussing chambers for the study for transepithelial movement of HRP. Electon microscopy analysis of HRP transport showed that specifically sensitized rats transported HRP across the epithelium in greater amounts, and more rapidly, than naive controls or rats sensitized to an irrelevant antigen. After the hypersensitivity response, there was a significant increase in HR flux across the intestinal epithelium in HRP sensitized, but not control rats. This was accompanied by an opening of the epithelial tight junctions to allow paracellular flow of antigen. Sensitized mast cell deficient rats also had an enhanced initial uptake of antigen, but did not develop a non-specific decrease in epithelial barrier function. The role of interleukin-4 (IL-4) in the regulation of transepithelial antigen transport was examined. Treatment of human epithelial monolayers with IL-4, or with serum from atopic patients, caused a significant increase in transepithelial transport of HRP. Antibodies against IL-4 abolished the effect of atopic serum on transepithelial HRP transport. Electron microscopy analysis showed an increase in both transcellular and paracellular HRP transport. These studies show that transepithelial transport of antigen is profoundly altered by sensitization, and that mast cells and interleukin-4 enhance the delivery of antigen across the intestinal epithelium.</p> / Doctor of Philosophy (PhD)

<p>Cancer of the cervix is the second most common form of cancer in women worldwide. The natural history of cervical cancer is thought to involve sequential changes from varying grades of precursor intraepithelial lesions called CIN. However, little is known of the risk factors that can predict the oncogenic potential of a particular CIN lesion. Based on their preferential occurrence in cervical cancers and their potential oncogenic properties, the human papillomaviruses (HPV) particularly the genotypes 16, 18, 31, 33, 35 and 42 have been implicated in the etiology of invasive cervical cancer. However, these viruses could occur as either secondary pathogen of cancer or as predictor of those CIN lesions that are likely to progress to invasive disease. The hypothesis that was verified in this study was that HPV 16,33 and 18 are likely to be predictive of CIN lesions that progress to invasive cancer.</p> <p>First of all, in order to characterize the prevalent type of HPV in the target study population of B.C., a cross-sectional study was conducted and the presence of specific HPV types ascertained by the tissue in situ hybridization. The frequency of HPV types 16, or 33, was found to vary with the severity of the CIN grades, in contrast to the frequency of HPV types 6/11 and 18 that segregated independently of the CIN grades.</p> <p>Next, a case-control study was undertaken to verify the main hypothesis of HPV being predictive of CIN lesion progression to invasion. It was reasoned that the particular HPV would occur at higher frequency in CIN biopsies of cervical cancer cases than in CIN biopsies of noncases (controls). A total of 47 cases and 94 controls were enrolled from patients registered by the Cancer Control Agency of the Province of British Columbia. A case was defined as a post-pubertal woman with invasive disease and who had a CIN diagnosis at least two years prior to the invasive disease. For each case an attempt was made to enrol two control matched on grade of CIN and year of diagnosis. On each subject attempt was made to gather demographic informations that are known to be associated with cervical cancer. The HPV probes that were used included HPV 16/33 and HPV 6/11. The relative frequency of occurrence of specific HPV in the preinvasive biopsies of cases and controls were as folow: HPV 16/33 occurred in 10.6% of controls and 12.8% of cases; HPV 18 was found in 3.2% of controls and 8.7% of cases and HPV 6/11 in 2.2% of controls and 8.7% of cases. Conditional Chi-square analysis showed that the difference in the proportions of HPV positivity between cases and controls was compatible with sampling variation. Hence, with a statistical power approximately 60%, it was concluded that particular HPV could not be predictive of CIN lesions progression in the sample of population that was studied.</p> <p>However an excess risk for incurring cervical cancer, by being exposed to particular HPV at the CIN stage, was noted. The relative risk for HPV 16/33 was 2.34, [95% CI 0.70 to 7.66]; for HPV 18 was 2.45, [95% CI 0.22 to 27.80]; for HPV 6/11 2.19, [95% CI 0.39 to 12.42] or for all HPV combined was 1.87, [95% CI 0.55 to 6.28].</p> <p>Interestingly, a comparison of the frequency of HPV occurrences in the case-control study with that in the cross-sectional study revealed a lower rate of HPV positivity in the case-control component. This could possibly be due to a cohort effect.</p> / Doctor of Philosophy (PhD)

<p>Rat transferrin (RTf) was used to study the interaction of transferrin' (Tf) with the liver, with special reference to the çlycan of Tf.</p> <p>Iron uptake from Tf by cells is believed to occur by Tf receptor (TfR)-mediated endocytosis. Results from the present studies revealed that over a large range of competing diferric RTf concentrations, a constant percentage of iron is uptaken from RTf by the liver in vivo, and by hepatocytes in vitro. Hepatocytes were found to contain ~129,000 TfR/cell with ~40% expressed on the surface as estimated by both a polyclonal anti-TfR antiserum, produced as part of this thesis work, and a monoclonal anti-TfR antibody. On the basis of this estimate it was calculated that TfRs are not present in sufficient quantities to account for the observed uptake of iron. Studies of uptake and release of polyvinyl-pyrrolidone (PVP), RTf, and iron (as diferric Tf) were carried out in suspended hepatocytes. These studies demonstrated that the most likely mechanism to account for the results and to explain the iron uptake is "mixed-type" pinocytosis. The uptake of iron was found to be modulated by the type of glycan on RTf.</p> <p>On the basis of glycan microheterogeneity, at least six subforms of RTf are found to exist in rat plasma. These subforms are RTf-1, RTf-2·and RTf-3 as resolved by concanavalin A; ~20% of each is fucosylated and ~80% non-fucosylated. (The presence of fucose was found to have no measurable effect on catabolic rate, plasma iron disappearance or iron donation to liver in vivo or hepatocytes in vitro.) The slalylated subforms have different half-lives (RTf-1>RTf-2>RTf-3) with RTf-1 being significantly longer than RTf-3. Comparison of plasma iron disappearance and rates of iron donation to liver in suggested a trend (RTf-l>RTf-3>RTf-2) which was reproduced and found to be significant in studies with hepatocytes: iron uptake by hepatocytes from RTf-1 and RTf-3 could be competitively inhibited by an excess of the homologous subform. Desialylation of the subforms (RAsTfs) significantly reduced the half-lives and altered the order (RAsTf-3>RAsTf-1>RAsTf-2) with RAsTf-3 being significantly longer than RAsTf-2. The desialylated subforms were superior donors of iron to the liver in vivo. Studies to explain the enhanced rate of iron delivery by RAsTf, discounted the possibility of differing rates of iron release, but allowed postulation of a synergistic dual receptor mechanism. Results from experiments with hepatocytes in vitro supported the proposed mechanism. It is concluded that subtle differences in glycan structure can result in functional differences between Tf subforms.</p> / Doctor of Philosophy (PhD)

<p>The studies composing this thesis were designed to address the question of changes in muscle excitability during fatigue and recovery: the indirectly-evoked muscle compound action potential (M-wave) was used as an index of excitability. Earlier studies from this laboratory indicated that the rate of fatigue in human skeletal muscle depends on the frequency of excitation. The present studies have extended these findings by using a wide range of stimulating frequencies (0-30 Hz) and by comparing the change in muscle excitability in fast-versus slow-twitch muscles; the effect of ischaemia was also studied as was recovery from fatigue. Ten subjects (out of a total of fifteen) each successfully completed five experiments, spaced at least one week apart, in which intermittent tetanic trains at different frequencies were used to fatigue the ishaemic ankle dorsiflexors. The effects of ischemia were studied by repeating one experiment under non-ischaemic conditions. Five out of the ten subjects also volunteered for the experiments comparing changes in muscle excitability in soleus, versus those in tibialis anterior, in response to intermittent fatiguing stimulation.</p> <p>It was found that maintenance of excitability was possible for one minute regardless of stiumulus frequency; thereafter stimulation at the highest frequencies induced the greatest change in the amplitude of the (M-wave). The amplitude decline was also dependent on the position of the M-wave within the train of potentials: thus, at 30 Hz stimulation, the first, fourth and seventh responses within the trained decreased by 50%, 80% and 95% respectively (p<.01). The decline in M-wave amplitude was always greater than the decline in the area of the compound action potential, indicating an increase in duration due to dispersion of single fiber action potentials. At 30 Hz stimulation the areas of the first, fourth and seventh responses decreased by 33%, 56% and 82% respectively.</p> <p>On the basis of animal studies, it was hypothesized that muscle excitability would be preferentially retained in the soleus muscle; however, no significant differences emerged in M-wave changes between soleus and tibialis anterior although the onset of decline was delayed in soleus. It is proposed that this delay was due to the early potentiating mechanisms observed in soleus but not in tibialis anterior. The presence of ischaemia significantly (p<.01) accelerated the decline in both amplitude and area of the tibialis anterior M-wave. Recovery of the M-wave was limited when the tetanic stimulation ceased but progressed rapidly after the circulation was restored. M-wave failure occurred at firing rates not normally associated with neuromuscular blockade, implying propagation failure along the sarcolemmal membrane.</p> / Doctor of Philosophy (PhD)

<p>The studies that make up this thesis have been designed to describe and analyse the changes in muscle action potentials associated with muscular activity. The initial experiments were carried out in human muscle to examine changes in the muscle compound action potential (M-wave) during both stimulated and voluntary activity. It was found that the M-wave increased in amplitude either during or following either type of activity, and that this increase in M-wave amplitude could not be explained by either a movement artefact or a greater synchronization of action potential firing. It was hypothesized that the potentiation of the M-wave was due to a hyperpolarization of the muscle fibre membrane which was increasing the amplitude of the individual fibre action potentials. Experiments were then conducted on rat soleus muscle (in vivo) in order to measure changes in resting membrane and action potentials associated with contractile activity. After 5 min of intermittent tetanic stimulation at 20 Hz, the mean resting membrane potential increased from a baseline value of 79.5 ± 4.8 mV to a mean maximum of -92.6 ± 4.2 mV 3-6 min post-stimulation (p<.01). There was a corresponding increase in action potential amplitude; it rose from a resting value of 82.2 ± 10.8 mV to 98.1 ± 7.8 mV in the recovery period (p<.01). It was hypothesized that increased activity of the electrogenic Na⁺-K⁺ pump was causing the hyperpolarization. A series of experiments utilizing inhibitors of the Na⁺-K⁺ pump were then conducted; it was found that the administration of ouabain (1.25 x 10⁻⁴M), cooling the bathing medium (from 37°C to 19°C) or removal of extracellular K⁺ prevented the hyperpolarization following repetitive stimulation (p<.05). The magnitude of the electrogenic contribution of the Na⁺-K⁺ pump was then estimated by exposing stimulated muscle fibres to a high K⁺ (20 mM) medium. While in the control (unstimulated) condition this caused an immediate depolarization of the muscle fibre membrane to approximately -58 mV, stimulated fibres maintained membrane potentials of -79.5 mV (± 8.6 mV) for at least 3 min, which was approximately -30 mV greater than that predicted by the Goldman-Hodgkin-Katz (GHK) equation. It is concluded that the Na⁺-K⁺ pump plays an important role in maintaining muscle fibre excitability during muscular activity, which is additional to its role in the restoration and maintenance of ionic gradients for Na⁺ and K⁺. The temporary hyperpolarization of the muscle fibre membrane during increased Na⁺-K⁺ activity offers an explanation for the potentiation of muscle compound action potentials observed during voluntary and stimulated contractions.</p> / Doctor of Philosophy (PhD)

<p>Dexamethasone (DEX) treatment in very preterm infants has proven to facilitate earlier weaning from mechanical ventilation and supplemental oxygen, thereby lessening the severity of lung disease incurred by long-term oxygen dependency. However, DEX therapy is not without negative side-effects; studies in preterm infants and piglets have reported DEX-induced impairments in growth and bone mineral metabolism. DEX may act by altering the concentration or activity of specific components of the growth hormone (GH)/insulin-like growth factor (IGF-I) axis which are essential for regulating growth and bone mass. The first study, in preterm infants, characterized how DEX alters the circulating components of the GH-IGF-I axis and suggested potential mechanisms by which DEX delays growth and bone development as both plasma IGF-I and biochemical markers of bone metabolism were reduced during DEX. The objectives of the piglet studies were to delineate the effectiveness of adjunctive GH or GH+IGF-I to counter the detrimental effects of DEX on growth, protein turnover and bone mass. In the first studies, we administered GH, GH+IGF-I or placebo to piglets while they received a two week course of DEX. GH and GH+IGF-I partially attenuated the reductions in growth and bone mas to a similar extent. Only with respect to protein metabolism was an additional benefit observed with combined treatment (GH+IGF-I). A dose-response study revealed the minimal effective GH dose, and demonstrated that bone cell activity and weight and length gain returned to control levels during a period of rehabilitation in which no DEX or GH were administered. Currently, it is uncertain if DEX-treated infants experience similar metabolic improvements in weight and length growth or bone mineral mass post-DEX treatment or whether the metabolic insults of DEX are sustained. Longer term follow-up of DEX-treated preterm infants is required to fully comprehend to long-term consequences of DEX on growth into childhood. If there are long-term effects on growth and bone development, future studies should focus on whether GH is more effective post-DEX compared to during DEX treatment or whether adjunctive administration of other anabolic agents will counter the negative effects of DEX during development.</p> / Doctor of Philosophy (PhD)

<p>The gastrointestinal tract is particularly sensitive to stress. Stress-induced gastric ulceration and stress-induced alterations in motility have been examined. The purpose of my studies was to define stress-induced changes in the intestinal epithelium and the mechanisms involved in the epithelial responses. Wistar Kyoto rats were stressed by restraint. Jejunal and colonic tissues from stressed or control rats were removed and parametres of epithelial physiology were studied in Ussing chambers. Acute stress caused a significant increase in intestinal chloride ion secretion. In addition, tissues from stressed rats demonstrated impaired responses to neural activation. Compared to controls tissues from stressed rats were also found to have increased permeability to ions, and small inert probes, and increased permeability to a macromolecular protein. In spite of these functional abnormalities, the mucosa showed no signs of damange. Our next series of experiments showed that peripheral cholinergic nerves and corticotrophin releasing factor were responsible for mediating these epithelial responses to acute stress. Pretreatment with atropine (jejunum) or a corticotropin-releasing factor antagonist (colon) prevented the stress-induced pathophysiology, while administering corticotropin-releasing factor mimicked the colonic responses. These studies also showed that the Wistar Kyoto strain of rats developed more extreme intestinal abnormalities to stress than the parental Wistar strain, most likely due to the fact that Wistar Kyoto rats have less cholinesterase activity. In summary, my studies showed that stress impaired epithelial function along the intestinal tract. We speculate that in susceptible individuals, acute stress can cause the epithelial barrier to become leakly allowing greater uptake of small proinflammatory molecules (bacterial products) as well as larger macromolecules (antigens) from the lumen. Subsequent stimulation of immunocytes may initiate or exacerbate inflammation.</p> / Doctor of Philosophy (PhD)

<p>Roberts syndrome (RS) is a rare, recessive condition characterized by growth retardation, developmental delay and tetraphocomelia. Some RS patients (RS+), but not others (RS-), exhibit a "puffing" pf the constitutive heterochromatic regions of their chromosones (the "RS effect"). Cells from RS+ patients also show cellular hypersensitivity to DNA crosslinking agents such as mitomycin C (MMC). In the present study, correction of both the RS effect and MMC hypersensitivityin somatic cell hybrids between RS+ and normal lymphoblastoid cells (LCLs) supported the hypothesis of an association between the RS effect and mutagen sensitivity in RS+ cells. Somatic cell hybrids between two RS+ LCLs derived from patients with diverse ethnic backgrounds exhibited both the RS effect and MMC hypersensitivity indicating that these patients represent a single complementation group. Somatic cell hybrids between one of the RS+ and two different RS- LCLs demonstrated complete complementation of both the RS effect and MMC hypersensitivity indicating that these patiens represent a single complementation group. Somatic cell hybrids between one of the RS+ and two different RS- LCLs demonstrated complete complementation of both the RS effect and MMC hypersensitivity. These findings suggest that RS+ and RS- patients belong to different complementation groups.</p> <p>Fanconi anaemia (FA) is another rare, recessive disorder characterized by growth retardation, developmental delay, limb abnormalities and progressive pancytopenia. Cells from FA pateints exhibit both chromosomal and celluar hypersensitivity to DNA crosslinking agents. A study of the sensitivity and mutanility of various LCLs by ethyl methanesulphonate (EMS) indicated an increased cellular sensitivity and decreased mutability of RS+ LCLs relative to control LCLs. RS-LCLs did not exhibit these phenomena. One FA LCL from complementation group A showed a slightly increased cellular sensitivity but normal mutability. These results suggest hypomutability by EMS may be associated with the RS effect and MMC hypersensitivity.</p> <p>Somatic cell hybrids were made between one of the RS+ LCLs and an LCL from each of the four known FA complementation groups. Hybrids were examined for correction of MMC hypersensitivity, the RS effect and diepoxybutane-induced chromosome aberrations.</p> <p>Complementation was observed in hybrids with FA LCLs from complementation groups A,B and D but incomplete correction of chromosomal and cellular sensitivities to crosslinking agents in RS+ x FA C hybrids suggested a genetic association.</p> / Doctor of Philosophy (PhD)