Poly(ethylenimine) (PEI) is a polycation with proven success as a gene delivery vehicle both in vitro and in vivo. The work described herein addresses several aspects of PEI-mediated gene delivery. First, a model system was developed, and an assessment of what PEI molecular weights should be considered for optimal transfection was made. Next, PEI/DNA complexes were examined and their physical characteristics (such as surface charge concentration and buffering capacity) defined in an attempt to further improve PEI/DNA transfection efficiencies. After characterizations were completed, cellular trafficking studies were performed to yield a pictorial overview of the route taken by PEI/DNA complexes during transfection. With the general trafficking route in place, the stage was set for a more in-depth examination of the mechanism behind PEI-mediated transfection. The intracellular route of PEI/DNA complexes during transfection was then re-evaluated with respect to lysosomes, and a novel finding was made that disproves a popular hypothesis that has attempted to explain the success of PEI-mediated transfection. Additionally, the stability of PEI/DNA complexes was established, and applied toward the fact that the complexes are shuttled into cell nuclei during transfection. Finally, the effects of PEI's nuclear entry were addressed in terms of secreted cellular products and cell viability.

Mechanical forces such as shear stress and cyclic strain have been shown to regulate expression of many genes that can alter vascular functions such as cell proliferation, leading to the development of vascular diseases including atherosclerosis. Thrombin receptor gene, protease-activated receptor-1 (PAR-1), mediates many important vascular functions such as thrombin-stimulated thrombosis, inflammation, and proliferation of vascular cells; however, the regulation of PAR-1 by mechanical forces has not previously been studied. This thesis investigates effects of shear stress and cyclic strain on gene regulation of PAR-1 in human vascular cells such as endothelial (ECs) and smooth muscle cells (VSMCs) and the molecular mechanisms involved in this regulation. This work finds that shear stress and cyclic strain differentially regulated PAR-1 expression in vascular cells, leading to alterations of cell functions in response to thrombin, and that these processes were mediated through various signaling pathways. Cultured cells were exposed to different levels of shear stress or cyclic strain using the parallel flow plate chamber or uni-axial cyclic strain system. After exposure, PAR-1 mRNA and protein were quantified by Northern blot and flow cytometry, respectively. In addition, inhibitors of various signal pathways such as protein kinases were used to investigate the molecular mechanisms. Arterial shear stresses decreased PAR-1 mRNA and protein both time- and dose-dependently in both macro- and microvascular ECs, leading to attenuation of thrombin-stimulated nitric oxide and endothelin-1 releases. Furthermore, protein kinase C partly mediated shear-reduced PAR-1 expression in both cell types. As in ECs, shear-downregulated PAR-1 expression in VSMCs caused decreases in thrombin-stimulated calcium mobilization and cell proliferation. The transcription mechanism, but not mRNA stability, regulated shear-reduced PAR-1 expression in VSMCs. In contrast to shear stress, high levels of cyclic strain increased PAR-1 expression in VSMCs time-dependently, leading to induction of cell proliferation in response to thrombin, and this process was mediated by reactive oxygen species, possibly through the NADPH pathway. These findings indicate important roles of mechanical forces in regulating vascular functions and thus provide a better understanding of how mechanical factors act to promote vascular diseases.

A seizure warning system for intracerebral EEG is proposed. It is designed for clinical use with the intention of identifying sections of EEG containing seizure activity and alerting medical staff as a seizure occurs. The system is based on data filtering, spectral feature extraction, probability analysis using Bayes' theorem, spatial and temporal context analysis, and user tuneability. / The system was designed using 407 hours of EEG from 19 patients having 152 seizures. Once developed, the system was tested with a different set of EEGs from 19 patients having a total of 100 seizures during 389 hours. / Average results for the testing data were promising, with 86% sensitivity, a false detection rate of 0.47/hour, and a delay time of 16 seconds. Compared to current clinical systems, this shows a 9% enhancement in sensitivity, and a false detection improvement by a factor of 9.6. The aim of tuneability was also reached.

Objective. To develop predictive models for positive outcomes of upper and lower endoscopies in children. / Methods. Retrospective review of all endoscopies performed from January to December, 2000 at St. Justine Hospital. Predictive models for positive outcomes on endoscopy were constructed for upper and lower endoscopies separately using multiple logistic regression. / Results. Age greater than 13 years, hematemesis, epigastric tenderness and hypoalbuminemia were significant predictors of positive upper endoscopies. Male sex, age and rectal bleeding were significant predictors of positive lower endoscopies. Both models were significant at p < 0.0001 yet their receiver operating curves indicated that a model with sensitivity of 95% had a specificity of less than 40%. / Conclusion. Predictors of positive upper and lower endoscopy outcomes were found. The predictive models were statistically significant yet their performance did not reach clinical significance. A prospective, adequately powered study of predictors for endoscopy outcomes is needed to confirm these results.

Background. There are two specific scales designed to assess neck pain. They have weaknesses and limited assessment. A Functional Cervical Scale (FCS) based on neck disorders disability was developed after literature review and consultation with patients and medical specialists. Thirty-two items were tested. / Study design. Two hundred and ten patients with neck pain answered the FCS and other questionnaires on work, medication intake and quality of life. Another copy of the FCS was mailed to the subjects who had to return it in a pre-stamped envelope. / Methods. Item selection analyses were done. Test-retest and internal consistency were calculated to assess reliability. Univariate and multivariate analyses were conducted and correlations with other measures of neck pain were calculated to assess construct validity. / Results. Through item selection procedures, the number of items was reduced from 32 to 30. Intraclass correlation coefficient (ICC) for test-retest was 0.93 and Cronbach's alpha coefficient was 0.97. Correlations for validity were good. / Conclusion. FCS showed excellent reliability and good validity. The latter needs more assessment. The sensitivity to change should be estimated.

While parasympathetic activation is well-known to promote AF, the effects of sympathetic stimulation on AF have not been systematically studied. The present study was designed to study effects of sympathetic stimulation on AF, and the relative effects of sympathetic and vagal stimulation on AF duration for a comparable degree of wavelength and refractory period abbreviation. The results showed that, for a comparable degree of wavelength abbreviation, sympathetic stimulation is much less effective in promoting AF than vagal stimulation. Vagal stimulation markedly increased the heterogeneity of atrial refractoriness, while sympathetic stimulation did not. The heterogeneity in atrial refractory properties may be important in determining the ability to sustain AF and the effect of autonomic interventions on the arrhythmia.

The erectile mechanism is a sequential cascade of events, involving many chemical messengers, perhaps the most important one being nitric oxide (NO). NO is a putative neurotransmitter involved in the non-adrenergic non-cholinergic (NANC) system and is synthesized by the enzyme nitric oxide synthase (NOS). NOS has been localized in peripheral autonomic nerves innervating vascular and non-vascular smooth muscles in many organ systems. / The introduction of intracavernous injection (ICI) of vasoactive agents in 1981 was an effective means to restore erectile function for men with erectile dysfunction (ED). Recent evidence supports the theory that increased blood flow can induce the release of NO from vascular endothelium, suggesting that ICI may have positive benefits for the host. / Our study looked at (1) a new delivery system for intracavernous injection and (2) the effect of modulation of penile flow on the regulation of NOS content and activity. A canine model assessed the effectiveness of a subcutaneous drug delivery as an alternate means to ICI. Additionally, a paraplegic rat model was developed to assess the effects of chronic ICI of papaverine on the expression of NOS in the penile tissue. / Our first objective, testing a new subcutaneous drug delivery system, yielded no data due to technical difficulties. The experiment involving the rat model, our results demonstrated that ICI of papaverine significantly increased the number of NOS fibers within the penile shaft, indicating that an increase in the flow of blood within the penis can alter levels of NOS within penile tissue. This result may in part explain the observation seen in patients, whereby after 1 year of ICI, spontaneous erections return and ICI therapy may be discontinued.

The role of basic fibroblast growth factor (bFGF) in the malignant phenotype of human gliomas was examined in this dissertation. A bFGF ELISA, demonstrated to detect recombinant human bFGF protein, was used to quantitate bFGF protein in glioma tissue samples. Basic FGF protein was significantly elevated in low and high grade glioma tissues, suggesting increased bFGF expression could be an early event in the progression of gliomas. Because the tumor sections contained non-tumor elements, bFGF gene and protein expression were examined in established human glioma cell lines. Human glioma cells demonstrated extracellular release of bFGF and expression of higher molecular weight bFGF isoforms. Because secreted bFGF could be important in the malignant phenotype of gliomas, fibroblasts were stably transfected with an expression vector for a secreted bFGF. These cells were more invasive in vitro and in the brain. To confirm and extend these data, non-neoplastic human astrocytes were stably transfected with the bFGF secretion vector. These cells demonstrated features associated with malignant transformation, suggesting increased expression and extracellular release of bFGF could have transforming potential in human astrocytic cells. To determine if bFGF gene and protein expression were required for human glioma cell growth, bFGF-specific antisense oligodeoxynucleotides were tested. The antisense treated glioma cells demonstrated decreased bFGF gene and protein expression and significantly decreased proliferation. Taken together, the data suggests the potential importance of bFGF in glioma cell transformation and growth, and indicates anti-bFGF strategies could be useful in the management of glioma patients.

Neurodegenerative diseases are characterized by accelerated and/or inappropriate neuronal loss which is thought to be caused by genetic and environmental factors. At present predisposing factors remain unknown and effective therapies are not available for many of these diseases. However, recent work has described at least 4 pathogenic mechanisms. (1) Oxidative stress: mutations in the superoxide dismutase gene cause approximately 1% of ALS cases. (2) Neurofilament abnormalities: neurofilament overexpression causes an ALS-like phenotype in mice, while pathologically, neurofilament accumulation is observed in patients. (3) Deregulation of apoptosis: deletions in apoptosis-regulatory genes are present in spinal muscular atrophy. (4) Expansion of trinucleotide repeats---expansions are responsible for many neurodegenerative diseases including fragile X and Huntington's disease. / My work involved the investigation of these mechanisms in the pathogenesis of ALS and Parkinson's disease. Identification of molecular defects in genes regulating these pathways would provide direct evidence linking these mechanisms to neurodegeneration. Such evidence was not found. Mutations in the Mn SOD and catalase genes were absent in both disease populations. The Cu/Zn SOD gene was not mutated in Parkinson's patients. A novel allele at the NEFH KSP repeat locus was identified in Guam and Kii ALS patients but its presence in non-affected Guam and Japanese individuals eliminated it as a causative mutation. The same allele, absent in non-Asian Pacific populations, was identified in 7 affected individuals of an atypical Puerto Rican Parkinson-dystonia family. Further studies are necessary to determine whether the disease in this family is associated with this locus. / Results from the study of the SMA- and SBMA-causing mutations in ALS, revealed the presence of diagnostic errors within the ALS population. While not unexpected, the significant differences in prognosis between SMA or SBMA and ALS, make this differentiation important. Caucasian SBMA was found to be due to new mutations and not predisposing or founder chromosomes as reported in Japanese SBMA and other trinucleotide repeat diseases. / While the etiology of neurodegenerative disease is complex, my work has led to a significant increase in our understanding of this complex group of disorders. Much additional work needs to be done so that accurate diagnoses can be made and targeted effective treatments developed.

The purpose of this study was to show that nifedipine, a calcium channel blocker, can decrease gallbladder contractility in guinea pigs and in man. Gallbladder contraction was measured in response to repeated injections of cholecystokinin both before and after the injection of nifedipine in three groups of five animals each. The mean amplitude of gallbladder contraction in response to cholecystokinin was decreased by 45, 73 and 67% (p $<$ 0.01) in response to intravenous nifedipine doses of 100, 200 and 300 $ mu$g respectively. In nine healthy human volunteers, gallbladder emptying was measured by radionuclide cholescintigraphy in response to cholecystokinin infusion before and after a 10 mg oral dose of nifedipine. Gallbladder ejection fraction was significantly decreased by 29% (p $<$ 0.001). These data demonstrate that nifedipine is a potent inhibitor of gallbladder contractility.