Ion channels mediate the electrical properties of neurons and other excitable cells. Mutations in ion channel genes have been linked to several neurological disorders. For example, a rare familial form of epilepsy, generalized epilepsy with febrile seizures plus (GEFS+), is associated with mutations in voltage gated sodium channels. We examined how two such mutations (C121W, D188V) alter the functional properties of the channel through voltage-clamp studies in Xenopus oocytes and HEK cells respectively. D188V is located in the alpha subunit and C121W in the auxiliary beta1 subunit of the sodium channel. / The C121W mutation causes a 100 fold reduction in efficacy of current modulation as well as a reduction of current amplitude. This may cause increased sodium currents via a negative shift of the steady-state inactivation curve. alpha-D188V channels recover faster from the inactivated state which causes a resistance to frequency-dependent cumulative inactivation of current amplitude. This may contribute to cellular hyperexcitability resulting in ictal events in the epileptic patient.

Parkinson's disease (PD) is a neurodegenerative disease characterized by progressive loss of dopaminergic neurons of the substantia nigra pars compacta. Symptoms usually include akinesia, bradykinesia, muscle rigidity, postural imbalance and tremor. Despite numerous studies on the physiology and pathophysiology of tremor, its influence on voluntary motor behavior remains unclear. Accordingly, the main objectives of the present thesis were to (a) determine if a clinical relationship existed between tremor and performance of voluntary movements, and (b) characterize the impact of ventrolateral (VL) thalamotomy on tremor and voluntary motor behavior. Results indicate that age-related change of the supraspinal component of normal physiological tremor (NPT) has no influence on the performance of healthy elderly subjects making rapid alternating movements (RAM). Our results also show that subsets of patients presenting different tremor amplitudes and/or tremor power characteristics had similar RAM performance, hence negating any direct relationship between the supraspinal oscillator(s) generating tremor and RAM performance. Our results demonstrate that tremor can be detected during manual-tracking movements performed by patients with early PD, but this tremor has little consequence on the accuracy of these patients. / As for the impact of VL thalamotomy on tremor, our results show that the thalamic lesion eliminates selectively PD tremor oscillations, in addition to preventing a resurgence of the supraspinal component of physiological tremor. The surgical procedure did not however improve or worsen RAM performance, suggesting that tremor probably plays little role in bradykinesia. Accuracy during the manual-tracking task is nonetheless greatly improved post-surgery, hence confirming the anticipated clinical benefits of the surgical procedure. In conclusion, despite previous evidence that tremor and RAM may share common neural networks and that tremor may be pathophysiologically linked with bradykinesia, the aforementioned results suggest that there is little clinical relationship between tremor and bradykinesia observed in patients with PD making RAM.

The appropriateness of three animal models in mimicking pathophysiology and symptoms of Alzheimer's disease was assessed. Behavioural tasks provided measures of cognitive function, affective behaviour and locomotor ability. Adult and aged transgenic mice, expressing the C104 fragment of the human $ beta$-Amyloid Precursor Protein, displayed increased anxiety by seven months of age in the Thatcher-Britton Novelty Conflict paradigm, memory impairments in the Forced Alternation T-Maze, the Porsolt Forced Swim test and the Recognition test, and decreased hippocampal acetylcholinesterase staining. BIBN99, a muscarinic M2-antagonist that increases release of acetylcholine, failed to ameliorate these changes. Apolipoprotein E deficient mice showed impairments of spatial memory in the Morris swim maze, but were unaffected by the administration of tacrine, an acetylcholinesterase inhibitor. Segmental trisomy 16 mice, a model of Down Syndrome and Alzheimer's disease, showed increases in hippocampal choline acetyl-transferase activity and mild memory impairments in the swim maze. BIBN99 had no effect on this deficit. These findings suggest that each of these models is beneficial in its own right for studying the functional deficits associated with Alzheimer's disease.

To further our understanding of the complex pathophysiology of human sickle cell disease (SCD) and to develop better therapies, three transgenic mouse lines have been produced that survive on mostly human hemoglobin (Hb). These lines have been generated by crossing HbSAD (SAD) transgenic mice with other mice carrying a null deletion of the endogenous globin genes (alpha or beta), SAD/homozygous alpha-globin null (SADalpha -/-) mice display a SCD-like phenotype and have a dramatically reduced mean lifespan; therefore, this line provides a novel model for assessment of anti-sickling protocols or identifying epistatic modifiers genes. Intriguingly, SAD/heterozygous alpha-globin null (SADalpha+/-) mice have a mild phenotype compared to SAD mice. In stark contrast, the highest cellular HbSAD fraction and degree of in vivo sickling was obtained in SAD/compound heterozygous alpha- and beta-globin null (SADalpha +/-beta+/-) mice. These SADalpha +/-beta+/- mice display chronic anemia and delayed growth indicative of a severe phenotype, similar to the human HbSS phenotype.

Retinopathy of prematurity (ROP) is a potentially blinding retinal disorder, which results from the exposure of the premature infant to hyperoxia. The diagnosis and the assessment of severity of ROP are based on the degree of retinal vascular abnormalities (such as avascular peripheral retina and neovascularisation), which are observed upon fundus examination. However, even if the vascular consequences of ROP are resolved, functional sequels such as high myopia, anisometropia and strabismus, may persist through adulthood. In severe cases, even more dramatic consequences, namely retinal detachment and blindness, may also occur. In oxygen-induced retinopathy (OIR), the animal model of ROP, hyperoxia-induced vasoobliteration and neovascularisation have been studied extensively in mice, rats, cats and dogs. However, the functional and ultrastructural changes that might result from the exposure to hyperoxia have not yet been examined in details. In this study, we report functional, as assessed with the electroretinogram (ERG), and structural, determined with histological sections of the retina, consequences of postnatal hyperoxia which took place during a period of intense retinal maturation, that is, the first 14 days of life of Sprague Dawley rats. / Our results indicate that there is a sigmoidal dose-response correlation between the increasingly longer duration of oxygen exposure, and the decrease in the postreceptoral retinal activity, as revealed by the gradual reduction in the amplitudes of rod and cone dominated b-waves and oscillatory potentials. Furthermore, the cone function appeared to be slightly more affected than that of the rod. Also, of all the ERG components the oscillatory potentials, especially the short latency ones, appeared to be the most sensitive to the hyperoxia. The gradual thinning of the outer plexiform layer (OPL) is also correlated with the duration of oxygen exposure, while the reduction in the number of horizontal cells is not. / Secondly, our experimental approach has allowed us to evidence a period of higher oxygen susceptibility (a window), which takes place during the second week of life of the newborn rats, more specifically around postnatal day 10. Several oxygen exposures of short duration, which included this period, were shown to be more detrimental to the rod and cone function than longer exposures taking place prior to or after this window. This would suggest that during the normal maturation process, the immature retina is not equally sensitive to the hyperoxic insult, presumably because specific retinal structures (synapses in OPL) are targeted by oxygen. / Finally, Trolox C, a potent water-soluble antioxidant, partially prevents the functional and structural consequences of OIR. The prophylactic effect appears to benefit more the rod function than that of the cone. / To conclude, we believe that this new knowledge will help us understand the pathophysiological processes at the origin of oxygen toxicity in the immature retina, thus, significantly increasing our insights on the human form of this disease, namely ROP, which as a result, will be instrumental in devising new therapeutic avenues to help fight this potentially debilitating retinal disorder.

Nitric oxide (NO) is an atypical signaling molecule in both the peripheral and central nervous system, which is produced by three isoforms of nitric oxide synthase (NOS). In the brain, NO has been shown to possess various neurophysiological roles, including its involvement in cognitive functioning. NO can also be neurotoxic and has been implicated in neurodegenerative processes. / The present thesis examined the effects of aging on the NO producing systems in the brain and the putative associations between NO, cognitive impairments, aging, and Alzheimer's disease (AD). In situ hybridization experiments demonstrated that neuronal NOS (nNOS) mRNA expression was significantly increased in aged rats, and that cognitively impaired aged rats showed higher nNOS mRNA expression than aged animals without cognitive deficits. Moreover, mRNA expression of heme oxygenase 2 (HO-2) also showed age-related changes. Western blotting experiments and NOS assays showed that cortical and hippocampal nNOS expressions and activities were decreased in aged animals. In a primary rat cortical mixed neuronal and glial culture model, amyloid (Abeta) peptide increased NO production by nNOS and inducible NOS (iNOS) significantly. Furthermore, NO was neurotoxic in this model, and specific iNOS inhibitors, a NO scavenger, as well as an antioxidant were able to prevent and reverse NO and peroxynitrite toxicity. Western blotting and NOS assay experiments on the brains of patients with AD revealed significant decline in hippocampal nNOS expression along with increased hippocampal and frontal cortical iNOS expressions, as well as the predominance of iNOS activity. / Taken together, the studies presented here have indicated the existence of complex relationships between aging, cognitive impairments, and NOS. Moreover, iNOS could be a significant "intermediate" element in AD pathogenesis.

The development of animal models for pancreatic cancer has magnified the controversy regarding the cell of origin of the tumor in animals and man. In rats, most pancreatic tumors have an acinar histology. By contrast, tumors in hamsters have a ductular morphology, resembling the predominant type of human exocrine pancreatic cancer. While some workers advocate a ductular origin for hamster tumors, others insist that the progenitor is an acinar cell. / With the use of normal adult rat pancreatic ductular cell lines, this work shows that four representative pancreatic carcinogens all display dose-related genotoxicity to rat pancreatic ductular cells in vitro. Further, repeated exposure to sublethal doses induced anchorage-independent growth. Transformed cells, injected subcutaneously into isogeneic newborn rats, produced malignant tumors with ductular morphology. A novel achievement of this work was the production of ductular adenocarcinomas using streptozotocin, previously reported as only inducing islet cell tumors.

In order to study the role of cellular adhesion in the process of lymphatic metastasis, in vitro models were developed from isolated components of the lymphatic system. Cultures of rat lymphatic endothelial cells and of rat lymph node stromal cells were obtained. The endothelial cells expressed von Willebrand factor (vWF) and angiotensin converting enzyme (ACE), and took up acetylated-low density lipoprotein (Ac-LDL). The stromal cells were composed mainly of Ac-LDL$(-),$ vWF$(-)$ cells which produced in vitro a reticular mesh of fibers, and expressed fibronectin, laminin and type IV collagen. The adhesion of tumor cells derived from two rat breast carcinoma lines of divergent metastatic potentials to the lymphatic cells and to extracellular matrix (ECM) proteins has been tested. / The capacity for lymphatic metastasis was found to correlate with tumor cell adhesion to fibronectin, a major component of the lymph node ECM. Inhibition experiments showed that tumor cell adhesion to fibronectin was mediated by a $ beta sb1$ integrin(s). In contrast no correlation was found between tumor cell potential for lymphatic metastasis and tumor cell adhesion to the lymphatic cells. / It is postulated that, in the present system, adhesion to fibronectin is one of the factors regulating tumor potential for lymph node metastasis.

The expression of the c-fos proto-oncogene during normal and pathological bone development was studied. The regulation of c-fos expression in bone cells by calcitriol and its contribution to the etiology of fibrous dysplasia was addressed. We have shown that treatment of osteoblasts with calcitriol, the active form of vitamin D, transiently stimulated the expression of c-fos, fos-B, c-jun and jun-B in a specific and dose-dependent manner. The expression of the Fos protein correlated with the expression of the c-fos gene. Finally, calcitriol appeared to modulate c-fos transcription in osteoblasts, whereas it stimulated c-jun and jun-B expression by a post-transcriptional mechanism distinct from mRNA stabilization. The transcriptional stimulation of c-fos was explored further and we identified a novel vitamin D response element (VDRE) in the c-fos promoter. We report that a 36 bp sequence centered around position-161 upstream of the c-fos transcription start site is responsive to 1,25-(OH)$ rm sb2D sb3$ in osteoblastic cells. This sequence binds the VDR, and mutations that abrogate binding to this element also abolish transcriptional activation by 1,25-(OH)$ sb2D sb3,$ demonstrating that we have identified a functional VDRE. Structure-function analysis revealed that the c-fos VDRE has an unusual structure that does not correspond to the classical direct repeat (DR)-type elements. Supporting this observation, our results show that the VDR requires a novel dimerizing partner to bind the c-fos VDRE. Finally, we report increased expression of c-fos in bone lesions from patients with fibrous dysplasia. The elevated c-fos mRNA levels were detected only in fibrous dysplasia lesions and not in samples from patients with other bone diseases where high bone turnover and fibrous marrow tissue are present. Our results support the implication of c-fos in the etiology of fibrous dysplasia.

Pyrithiamine-induced thiamine deficiency (PTD) in the rat results in a metabolic disorder associated with selective cerebral vulnerability. The basis of the histological lesions that develop in areas that include the thalamus, medial geniculate, and inferior colliculus is undetermined but an underlying glutamate-mediated form of excitotoxicity may be responsible. This work examines the possibility that PTD leads to changes in extracellular glutamate concentration, membrane polarization status, and gene expression consistent with a decreased ability to maintain homeostatic integrity. / At the acute symptomatic stage, extracellular glutamate by concentration as determined microdialysis was found to be elevated in the thalamus but within normal limits in the non-vulnerable parietal cortex. This result suggests an involvement of glutamate in the pathogenesis of thiamine deficiency lesions. An in vivo examination of L-type voltage-sensitive calcium channel activity using quantitative autoradiography and ($ sp3$H) -nimodipine at the same stage of PTD revealed that increased activity was present in and localized to the thalamus, but was absent in areas resistant to histological damage. The finding indicates a likelihood of regional depolarization. Finally, induction of the proto-oncogene c-fos was detected in the thalamus and inferior colliculus with quantitative in situ hybridization analysis at the acute symptomatic stage, thereby confirming the association of PTD with depolarization-related events occurring in advance of the appearance of frank infarction. / Together, these results suggest that vulnerability in the thalamus may be determined by an inability to maintain spatial buffering of extracellular glutamate under energy-compromised conditions. Such an environment may be sufficient to set into motion event cascades in these cells that lead to the demise of the structure as a whole.