Rhabdomyosarcoma is a class of malignant neoplasms composed of cells histologically resembling fetal striated muscle. It is the most common soft tissue tumor of children, adolescents, and young adults. In this Thesis, I demonstrate that the rhabdomyosarcoma tumor class is delimited in molecular genetic terms by the expression of the MyoD gene, and that loss of alleles on chromosome 11 distinguishes between the embryonal and alveolar subtypes. The elucidation of this genotypic distinction resolved the paradox between phenotypic variation and an apparent histogenetic relatedness between and among rhabodmyosarcomas. It also allowed for the construction of a two-tiered scheme for the unequivocal differential diagnosis of rhabdomyosarcoma and its subtypes. MyoD is syntenic to but distinct from Rd, a locus consistently involved in the etiology of the embryonal subtype. Abnormal segregation of alleles on chromosome 11p in both familial and sporadic embryonal rhabdomyosarcoma results in retention of only paternal alleles in tumors and supports a model of tumorigenesis involving unlinked modifier loci which inactivate Rd, perhaps by genome imprinting. Uniform loss of heterozygosity at loci on chromosome 11 in phenotypic variants of embryonal rhabdomyosarcoma that show simultaneous differentiation into another neoplastic tissue type implies that mutation at Rd is one of the earliest somatic events.

A mouse model of resistance and susceptibility to a pulmonary infection with Pseudomonas aeruginosa (PA), was used to study the inflammatory profile early following infection. This study allowed the identification of the mechanisms responsible for the resistant phenotype. We have first determined the pivotal role of the neutrophil recruitment in the alveolar space in the host defense against PA. This influx occurred earlier in the resistant BALB/c mice than in susceptible DBA/2 mice and occurred concommitantly with the beginning of the bacterial clearance. Furthermore, levels of TNFalpha were found to be higher in BALB/c mice than in DBA/2 mice. TNFalpha appears to mediate the production of chemokines responsible for neutrophil recruitment in the lungs. However, treatment of susceptible mice with TNFalpha led to an increase of neutrophil recruitment without any improvement in bacterial clearance. Lung homogenates from resistant mice also showed an increase in IL-10 and GM-CSF content. This was not seen in susceptible mice. This may lead to a more efficient neutrophil activation due to their effect on bacterial exotoxins and apoptotic gene regulation (via Fas pathway). TNFalpha, IL-10 and GM-CSF may represent valuable targets for treatment of lung infection with PA in patients unresponsive to antibiotherapy.

Duchenne muscular dystrophy (DMD) is common and fatal X-linked genetic disorder. The overall objective of this thesis was to carry out a prospective study for dystrophin gene therapy in dystrophic muscles, using the X-linked muscular dystrophy (mdx) mouse model. Recombinant adenovirus (AdV) is presently the vehicle of choice for gene therapy for a number of diseases including DMD. However AdV possess two major limitations when utilized as vectors in skeletal muscles: (i) the maturation-dependency of AdV-infectivity in skeletal muscles, (ii) the host immune response against adenoviral proteins as well as the transgene product. Thus, the work presented in this thesis addresses these two major limiting factors. By modifying either the host or the vectors, we have attempted to optimize AdV-mediated therapeutic gene transfer in dystrophic muscle. Our strategy has consisted firstly to evaluate the regenerative response of dystrophic muscle with advanced disease after experimentally-induced regeneration, in an attempt to recapitulate the myogenic program. We report that mdx mice with severe dystrophic pathology can still show a substantial level of muscle repair with attendant generation of immature myofibers. Secondly, by taking advantage of this level of regeneration and the concomitant generation of immature myofibers, we have upregulated expression of the coxsackie adenovirus receptor (CAR) present in mdx muscles. We have delivered AdV containing a dystrophin gene (AdV-Dys) at a period corresponding to this peak level of CAR expression and we have reported a significant increase of the number of dystrophin expressing myofibers with a net trend toward muscle function amelioration. / The work of this thesis also reports that the combined blockade of calcineurin and CD28 signaling, two key and distinct elements needed for an effective immune response, effectively blunted the immune-mediated destruction of dystrophin expressing myofibers expressed after AdV-Dys delivery. As an alternative to host modification (regeneration and immunosuppression) that can be associated with potential toxic effects, we have explored a strategy where by the recombinant AdV vector contains a less immunogenic transgene utrophin. We report that overexpression of utrophin and dystrophin by AdV-mediated gene transfer in adult immunocompetent mdx mice produces differential effects on muscle cell function in adult immunocompetent (mdx) mice. (Abstract shortened by UMI.)

Background. Susceptibility to a complex disease such as tuberculosis generally involves interactions among several genes and environmental factors. Several association studies have been conducted to examine the association between candidate genes and tuberculosis. However, the genetic risk factors are not fully understood. / Objective. To examine the effect of several candidate genes, including natural resistance associated macrophage protein 1 (NRAMP1), vitamin D receptor (VDR), surfactant proteins (SFTPA1), and mannose-binding lectin (MBL), and also to assess the effect of several risk factors on their association with tuberculosis. The other objectives were to test for mode of inheritance and also to estimate the relative risks of disease for different genotypes. / Methods. A prospective case-parental control study was conducted. Ninety-five nuclear families were selected from an existing database of families with tuberculosis in Ethiopia. Each family consisted of one affected child and two parents. The primary outcome was transmission/nontransmission of alleles from parents to affected offspring. / Results. The transmission disequilibrium test showed that marker SFTPA1-294 was significantly associated with the outcome (chi 2 = 4.297; p = 0.038). When other risk factors such as age, sex, ethnicity, certain symptoms or other genes were allowed to modify the transmission probabilities in a logistic regression model, several other markers were found to be significantly associated with the outcome. / Conclusions. Despite the limitations of this study, this thesis provided evidence for inheritance of susceptibility to tuberculosis in Hadiayan families in Ethiopia. To confirm the findings in this thesis, it would be useful to conduct similar research in populations with different ethnic origins, where genetic and environmental exposures can be examined and compared.

Increasing evidence indicates that cholesterol plays a central role in the pathophysiology of Alzheimer's disease (AD). Caveolin is a cholesterol-binding membrane protein involved in cellular cholesterol homeostasis and signalling. The global working hypothesis of the present project was to investigate on a possible role for caveolin in the pathophysiological events underlying AD. / More specifically, a first study was conducted to evaluate potential changes in the amount of brain caveolin in autopsy-confirmed AD and age-matched control subjects. Using rodents, the effect of age and apolipoprotein E (ApoE) deficiency on caveolin levels was also examined. In a second study, caveolin levels were investigated in an in vivo mouse paradigm of neuronal response to damage, the entorhinal cortex lesion. Moreover, the effect of caveolin on remodelling processes following neuronal lesion was examined in an in vitro model of reactive neuronal plasticity. Given the important role of caveolin in signalling, a third study was conducted to assess the potential functional interaction of caveolin with phospholipase A2 (PLA2), an enzyme implicated in synaptic plasticity and neurotransmission. / The results indicated an up-regulation of caveolin in the brain of AD patients, aged mice and apoE-deficient mice, that was potentially linked to alterations of cholesterol distribution in the plasma membrane of brain cells. Consistent with this idea, caveolin levels were increased during the neuronal membrane remodelling period following entorhinal cortex lesion in mice. In vitro, caveolin overexpression in neuron-like cells undergoing post-injury reactive plasticity caused significant biochemical and morphological alterations. Finally, a co-localisation and a functional interaction of caveolin with PLA2 was observed in hippocampal neurons. This interaction dramatically inhibited PLA2 enzymatic activity and consequently interfered with synaptic AMPA glutamate receptor binding properties. / These findings show that caveolin plays an important role in reactive neuronal remodelling process associated with age or damage. Its increased expression in brain cells, possibly ascribed to alterations in membrane cholesterol homeostasis, could negatively interfere with PLA2 and AMPA glutamate receptor functions during synaptic plasticity-related events. These findings are consistent with the hypothesis that part of the pathophysiology underlying AD may be related to cholesterol homeostasis, and indicate that caveolin may be an important and previously unrecognised player in this process.

Osteoarthritis (OA) is a degenerative, age related disease of diarthrodial joints. In OA, there is increased cleavage of type II collagen by collagenases in articular cartilage. This excessive cleavage can be caused by the altered feedback regulation of gene expression in chondrocytes by collagen type II or its degradation products which may serve as regulators of collagenase synthesis and activity through a cell surface receptor-mediated mechanism. The aim of the present study was to investigate the expression and production of collagen receptors, DDR-1 and DDR-2 in human adult normal and OA articular chondrocytes and to determine whether this correlates with the expression of disease related genes such as type X collagen, a specific marker for chondrocyte hypertrophy. (Abstract shortened by UMI.)

Arthritis is a chronic debilitating disease affecting millions of Canadians. Pain is a major symptom of arthritis and can be exacerbated during mechanical stimulation of the joint. In this project, we investigated whether changes occur in sensory neurotransmission in an animal model of monoarthritis and the neurochemicals that underlie such changes. Monoarthritis was induced by intra-articular injection of complete Freund's adjuvant (CFA) in the tibiotarsal joint of the rat. This model was characterised by a significant increase in ankle circumference and sensitivity to mechanical stimulus, as well as a decrease in ankle flexibility. Electrophysiological recordings from dorsal spinal cord neurones in intact animals demonstrated that the baseline activity of dorsal horn neurones was significantly higher in CFA-injected rats compared to vehicle-injected rats. Furthermore, the magnitude of the response of dorsal horn neurones to von Frey hair stimulation of the joint was significantly increased after CFA injection and was followed by a slowly decaying afterdischarge that was not present in control rats. Joint movement in arthritic rats caused an enhancement of the responses of wide dynamic range neurons to iontophoretic application of glutamate receptor agonists. This enhancement lasted 10--15 minutes and was blocked by pretreatment with the selective NK-1 receptor antagonist CP-96,345 (5mg/kg i.v.). These experiments suggest that joint movement in arthritic animals causes the release of substance P at central terminals within the dorsal spinal cord leading to a sensitisation of synaptic transmission. The enhancement of cellular excitability following an innocuous stimulus such as joint movement is at least partially mediated via activation of NK-1 receptors in the dorsal spinal cord and may underlie the exacerbation of the pain during arthritis.

Smooth muscle cell (SMC) proliferation in response to injury leads to the development of a neointima, which resembles, in many aspects, atherosclerotic lesions. Studies on experimental atherosclerosis have highlighted the role of SMC in atherogenesis. To gain further knowledge on the behaviour of SMC after arterial injury, SMC were cultured from neointimal explants with or without a covering of regenerated endothelium, 15 weeks after a single balloon catheter deendothelialization. Proliferation of these cells was studied by the examination of the cell growth curve, $ rm lbrack sp3H rbrack$-thymidine incorporation and determination of the cell cycle. The growth factors released from neointimal SMC and their effect on SMC proliferation were studied in neointimal SMC conditioned media. The production of proteoglycan and collagen by these SMC were also measured following the incorporation of radiolabelled precursors into these extracellular matrix components. The morphological features of the cells were observed using LM, TEM, SEM as well as immunofluorescence microscopy. Results revealed that neointimal SMC were characteristically different in their metabolic behaviour after injury. They have a higher proliferation rate, as indicated by higher $ rm lbrack sp3H rbrack$-thymidine incorporation and increased cell number. Neointimal SMC also exhibit alterations in their morphological features and synthesize significantly more proteoglycan and collagen. This study, demonstrated for the first time, that SMC have the ability for sustained proliferation, a long time (15 weeks) after injury. This persistent proliferation may be associated with some growth factors, such as PDGF-AB and TGF-$ beta sb1.$ These proliferating SMC and their cellular products contribute to the development of the neointima, which has many features in common with early atherosclerosis. Thus, endothelial injury is a strong and persistent stimulus for a sustained increase in thickness of the neointima,

Many hereditary peripheral neuropathies are characterized by abnormal myelination in peripheral nerves. Two structural myelin proteins, PMP22, a polytopic myelin protein, and P0, an Ig-like transmembrane protein, play a major role in myelin formation. A large number of mutations have been identified in P0 and PMP22 genes. Neuropathies associated with PMP22 and P0 mutations have varying severities suggesting their effects are pleiotropic. It is of great importance to explore molecular pathogenesis of mutated P0 and PMP22 proteins in order to understand the basic process of myelination and its pathology. We hypothesize that these mutations have a variety of effects on the cell biological processes, including activation of ER quality control mechanisms, abnormal intracellular trafficking, or disruption of normal function. In order to dissect these possible effects, we studied the intracellular distribution and trafficking of mutated forms of PMP22 and P0 proteins in mammalian cells. Our studies indicate that the various P0 and PMP22 mutants may exert their pathogenic effects in different compartments and by different mechanisms in the mammalian cell.

Heme oxygenase-1 is a 32 kDa protein that catalyzes the degradation of heme to biliverdin in brain and other tissues. Our laboratory had previously reported significantly decreased HO-1 protein levels in AD plasma and CSF and HO-1 mRNA in AD mononuclear cells (which may be chemically unstable relative to the levels seen in normal elderly control (NEC) subjects). We hypothesized that there exists a heme oxygenase-1 suppressor (HOS) factor in the plasma of AD patients. Using a novel, glial-based bioassay, we showed that plasma HOS activity was detected in 23/24 AD patients, 3/8 mild cognitive impairment (MCI) patients and in 0/21 NEC patients (HOS activity signifying HO-1 suppression of >50% compared to positive controls). Antioxidant exposure and hypercortisolemia did not account for the HOS activity in AD plasma. We investigated the specificity and selectivity of the HOS factor by analyzing plasma derived from patients with non-Alzheimer's dementia's and non-dementing neurological disorders. We conclude that a HOS factor exists in the plasma of sporadic AD and some MCI patients but is undetectable in NEC plasma. The presence of a circulating HOS factor may further signify systemic redox derangements in early sporadic AD and provide a possible biological marker for this condition.