The fastidious bacterium Campylobacter jejuni has recently been identified as a leading cause of human bacillary enteritis. Delays in recognition of this important pathogen reflect inadequate isolation techniques, which cannot recover environmentally stressed viable but non-culturable forms. Therefore, a non-culture based detection systems for C. jejuni would be invaluable. Studies were undertaken to develop a PCR based detection assay for C. jejuni. Fingerprints enriched for repetitive C. jejuni chromosomal elements were generated using arbitrarily primed PCR. Dot blot screening of fingerprint products for specificity to C. jejuni identified a 496 bp product which hybridized with all C. jejuni isolates examined. No binding to other Campylobacter species or enteric genera screened was observed. The product was cloned, sequenced, and primers synthesized to three overlapping regions of the probe. A primer pair was identified which directs amplification of a 265 bp product from C. jejuni alone. Sensitivity studies demonstrated that the C. jejuni specific PCR generated product from as few as 100 lysed bacteria. The ability of C. jejuni to penetrate normally non-phagocytic host cells is believed to be a key virulence determinant. Kinetics of C. jejuni of intracellular survival have been described and indicate that the bacterium can persist and multiply within epithelial cells and macrophages in vitro. Studies by Pesci et al. demonstrate that super-oxide dismutase contributes to intra-epithelial cell survival, suggesting that bacterial factors which combat reactive oxygen species enable the organism to persist inside host cells. Experiments were conducted to determine the contribution of catalase to C. jejuni intracellular survival. The gene encoding catalase (katA) was cloned via functional complementation, sequenced, and isogenic katA mutant strains constructed. Kinetic studies of bacterial viability indicate that catalase provides resistance to hydrogen peroxide in vitro but does not have a role in intra-epithelial cell survival as growth curves for katA mutant and wild type strains generated from long term culture within HEp-2 cells are roughly identical. Catalase does however contribute to intra-macrophage survival as katA mutants were recovered from cultured peritoneal macrophages at significantly reduced numbers (p = 0.00246) relative to the wild type strain after 72 hr incubation with these cells.

This thesis represents an effort to examine certain aspects of the differentiation potential of normal and neoplastic (spontaneously- and chemically-transformed) cultured rat pancreatic ductal cells under the influence of two microenvironments. / The technique of in vivo implantation of cells to subcutaneous and intraperitoneal sites is used as it not only reveals the intrinsic potential of the implanted cells but also reflects the effects of the microenvironment on phenotypic expression. / The results of these studies indicate that when implanted in vivo normal propagable cultured cells derived from the duct epithelium of adult rat pancreas develop phenotypic features of a hepatocyte, and that the extent of this phenotypic expression is influenced by the microenvironment in which these cells are implanted. When localized subcutaneously, the cells displayed partial differentiation toward hepatocytes but retained some of their ductal phenotype. In contrast, when the same cells were implanted intraperitoneally, they expressed the full phenotypic properties of mature hepatocytes. Both spontaneously- and chemically-transformed pancreatic ductal cell lines did not display phenotypic differentiation along the hepatocytic lineage after in vivo implantation. It is concluded that (1) pancreatic ductal cells can be the progenitor cell for pancreatic hepatocytes; (2) Neoplastic transformation of these cell lines results in partial or total loss of hepatoctyic differentiation.

The excessive deposition of redox-active iron has been amply documented in the basal ganglia of subjects with Parkinson's disease (PD). Yet, much remains to be learned regarding the cellular and subcellular distribution of this metal and the precise role(s) it plays in the pathogenesis of PD. Cysteamine (CSH) induces the appearance of peroxidase-positive cytoplasmic granules in cultured neonatal rat astroglia which are identical to glial inclusions that progressively accumulate in the aging subcortical brain. These inclusions are derived from degenerate mitochondria which sequester iron and other transition metals before undergoing fusion with lysosomes in an autophagic process. Dr. Schipper has previously demonstrated that iron-mediated peroxidase activity in these cells is capable of oxidizing dopamine and other catechols to potentially neurotoxic semiquinone radicals (Schipper et al., 1991). In the present study, we co-cultured PC12 cells, a catecholamine-secreting cell line, atop confluent monolayers of either CSH pre-treated (iron-enriched) or control neonatal rat astroglia. We observed that the PC12 cells grown on the surface of iron enriched (senescent-like) astroglia were far more susceptible to dopamine/H2O 2-related killing than PC12 cells cultured atop control glial substrata. Augmented killing of PC12 cells in the former paradigm was inhibited by the antioxidants, ascorbate, melatonin or resveratrol implicating a free radical mechanism of action. The aging-associated accumulation of iron in mitochondria of subcortical astroglia may facilitate the oxidation of dopamine to neurotoxic free radical intermediates and thereby predispose the senescent nervous system to PD and other neurodegenerative afflictions.

The most common cause of human hereditary neuropathies is a duplication in the peripheral myelin protein-22 (PMP22) gene. PMP22 is an integral membrane glycoprotein expressed primarily in the compact myelin of the mammalian peripheral nervous system. The naturally-occurring Trembler, and Trembler-J mouse models of hereditary neuropathies carry non-conservative point mutations in the PMP22 gene and have been essential in developing the understanding of the cell biology of PMP22 mutants. However, the greatest limitation to the study of PMP22 mutant trafficking has been the lack of a good model of myelination. To address this, we have developed replication-defective recombinant adenoviruses to study the intracellular trafficking of epitope-tagged wild-type, Trembler, and Trembler-J PMP22 in vivo. We have determined that in myelinating Schwann cells in vivo, newly synthesized epitope-tagged wild-type PMP22 is incorporated into myelin whereas the PMP22 mutants, Trembler and Trembler-J, are intracellularly retained in the ER.

This thesis describes the changes in cerebral morphology studied with magnetic resonance imaging (MRI) in two common forms of partial epilepsy, temporal lobe epilepsy (TLE) and frontal lobe epilepsy (FLE). In addition, the contribution of structural abnormalities to the observed distribution of fluorodeoxyglucose (FDG) in positron emission tomography (PET) was examined in patients with TLE. / In the first study, a quantitative morphological examination of the lateral temporal lobe in patients with mesial TLE is described. A significant reduction of white matter volume ipsilateral to the seizure focus and bilateral gray matter volume reduction were observed. In addition, abnormalities of the structure of the gray matter/CSF interface were found by surface curvature analysis. / The results of the first study raised the possibility that the apparently reduced uptake of FDG observed in interictal PET studies in TLE may in part be due to partial volume effects (PVE). The second study examines the contribution of volume loss and PVE to the asymmetry in the uptake of FDG seen in interictal PET. The results indicated that PVE contributes significantly to the observed asymmetry but does not explain it entirely. This suggests that additional metabolic or physiological factors are responsible for the observed abnormalities. / On the basis of the changes in white matter volume ipsilateral to the seizure focus, a loss of temporal lobe projection fibers resulting in local changes in the morphology of the corpus callosum at a site corresponding to the location of temporal crossover fibers was predicted. A method of detecting localized changes in the midsagittal corpus callosum was devised. Focal thinning in the corpus callosum was observed in patients with TLE compared to normal controls, supporting the biological significance of temporal lobe white matter changes in patients with TLE. The location of temporal lobe crossover fibers in the anterior to midbody of the corpus callosum was demonstrated. Progressive regional thinning of the anterior corpus callosum was demonstrated after temporal lobe resection in the same location as the thinning seen in TLE. / In the final study, the quantitative techniques developed in the previous morphometric studies of the cortex in TLE were applied to patients with FLE with normal MR images. White matter volume asymmetries corresponding to the clinical and electrographic lateralization of the site of seizure onset were observed in patients with normal MRI scans on visual inspection. These findings indicate that in patients with FLE, subtle structural lesions may be present despite a normal appearance on visual inspection of MR images. / In addition to their vital clinical role, neuroimaging techniques such as MRI and PET, are valuable research tools in the study of epilepsy. They yield a diverse range of biological information; in the studies describe, they have provided insight into aspects of the morphology of the normal brain and the structural changes of partial epilepsy.

The object of this thesis is to describe tissue classification software that was developed specifically for the identification of cerebral white matter lesions found in patients with multiple sclerosis (MS). Multiple sclerosis is a debilitating disease of the central nervous system (CNS) which results in a pathology observable macroscopically by magnetic resonance imaging (MRI). Lesion volumes are used as a surrogate of disease progression in clinical trials for MS treatment. The availability of accurate, reliable and reproducible measurements is invaluable in the advancement of patient care and research into this disease. This thesis documents a comprehensive approach to achieve such results which to date has proven to be challenging. A novel fully automated Bayesian classifier was developed which utilizes a variety of techniques to more accurately model brain tissue, and performs lesion identification at a level comparable to human experts. A new validation model that affords a better estimation of ground truth is introduced, providing a better means to measure classification accuracy. It is hoped that this document will be practical in nature, so that people who continue this work will have a step upon which to start.

Degenerating spinal motor neurons in amyotrophic lateral sclerosis (ALS) are characterized by a reduced expression of neurofilament light (NF-L) mRNA and by the presence of inclusions composed of peripherin and neurofilament (NF) proteins. This thesis examines questions related to the formation and toxicity of inclusions containing peripherin. Transfection studies demonstrated that the organization of the penpherin network is disrupted by the larger NF proteins NF-M and NF-H in context of NF-L deficiency. To further investigate the in vivo assembly of peripherin, transgenic mice overexpressing the mouse peripherin gene were generated and then bred with NF-L null mice. Mice overexpressing peripherin developed a late onset motor neuron disease characterized by the formation of peripherin inclusions and by the degeneration of motor axons in two years old mice. Remarkably, a deficiency of the NF-L protein enhanced the formation of inclusions and accelerated the onset of the peripherin-mediated disease by about 15 months. Transgenic mice overexpressing the human NF-H protein in the absence of NF-L (hH;L-/- mice) were then generated to further examine the impact of a NF-L deficiency upon the formation and toxicity of protein inclusions. These mice were characterized by the formation of large perikaryal inclusions containing peripherin and NF-H. Unlike the axonal and perikaryal inclusions formed in peripherin transgenic mice, the inclusions of hH;L-/- mice were not associated with loss spinal motor neuron, thus suggesting that the toxicity of inclusions in peripherin transgenic mice may be related to their axonal localization. Finally, a last series of experiments demonstrated that stab injuries and ischemia can also trigger the formation of peripherin inclusions in brain neurons that are normally silent for peripherin expression. The combined results suggest that peripherin inclusions may participate to the pathology of ALS and perhaps to neurodegeneration in other dis

The roles of some nutrients and environmental factors on CEA synthesis and release has been determined for the LS180 colon cancer cell line. When these cells were grown on various carbohydrate sources, cells grown on mannose exhibited the highest release rates at 6.5 ng CEA/mg protein/hour. When 25mM glucose with 10% FBS was used, the release rates were 3.8 ng CEA/mg protein/hour, the lowest of any of the cultures. The effects of nutrient depletion revealed that replacing the media at 145 hours after inoculation inhibited both the rate of synthesis and release of CEA. Feeding the cells at 100 hours eliminated antigen synthesis and release. Elevating incubation temperatures from 37$\sp\circ$C for 30 minutes at 100 hours increased the rates of both CEA synthesis and release from near zero to 150 ng CEA/mg protein/hour for cells heated to 45$\sp\circ$C and 42$\sp\circ$C. (Abstract shortened with permission of author.)

Pulsations in arterial blood flow expose the endothelium to diverse mechanical forces. I hypothesize that the temporal variations in pulsatile, arterial shear stress differentially regulate endothelial gene expression compared to steady, arterial shear stress. A computer-controlled system was developed to mimic the common carotid artery flow waveform and shear stress levels or to provide steady flow of the same mean shear stress in a parallel plate flow chamber. The pseudo-steady state shear stress was determined from real-time pressure gradient measurements and compared to the Navier-Stokes equation solution. Spectral analysis of the pseudo-steady state shear stress compared well with the power spectrum of published in vivo measurements of the common carotid artery flow rate. Total RNA from human umbilical vein endothelial cells (HUVEC) subjected to 24 hrs of pulsatile, arterial shear stress (average = 13 dyne/cm2, range = 7 to 25 dyne/cm 2; 1 Hz); steady, arterial shear stress (13 dyne/cm2), or static condition was collected for microarray analysis. Normalized intensities were filtered for 1.5 fold (shear vs. static) and subjected to ANOVA with Benjamini and Hochberg multiple testing correction at a false discovery rate of 5%. Compared to static condition, pulsatile arterial shear stress differentially regulated over 1,000 genes while steady arterial shear stress differentially regulated over 1,100 genes. More than 200 genes were differentially regulated by pulsatile arterial shear stress compared to steady arterial shear stress, but hierarchal cluster analysis indicates HUVEC respond similarly to both types of shear stress (Pearson correlation coefficient = 0.785). Quantitative real time polymerase chain reaction verified the trend in the gene expression results from the microarray analysis. Data mining of the differentially expressed genes with Ingenuity Pathways Analysis and with Expression Analysis Systematic Explorer identified several prominent biological themes differentially regulated by mechanical forces including glycosylphosphatidylinisotol anchor bioprocesses, nitric oxide signaling, and metal ion processes. Collectively, these results demonstrate that the common carotid flow waveform elicits subtle changes in HUVEC responses to arterial levels of shear stress. These differences reflect a shift away from quiescence and may be mediated by nitric oxide bioavailability.

Two distinct patterns of neurological disease have been identified among Cree Indian children in Chisasibi, Nouveau Quebec and among Native Indian infants in Northern Manitoba. Both affect the cerebral white matter. The fifteen patients in the first group may suffer from an autosomal recessive defect causing defective development of cerebral myelin. The eleven patients in the second group have systemic immune dysfunction and meningoencephalitis. Together, these diseases are responsible for over half the deaths of infants and young children in Chisasibi. Inbreeding, and the familial occurrence of cases, suggest a genetic cause in both groups, in whom no gestational or perinatal causes can be identified. The first disease pattern may be due to hypomyelination of the central nervous system. The etiology of the second group may be a genetic susceptibility to viral infection.