Cellular ATP is synthesized by the mitochondrial oxidative phosphorylation (OXPHOS) system, composed of five enzyme complexes (Complexes I--V), which consist collectively of over 80 subunits. The majority of these subunits are encoded by nuclear genes, and 13 of them, by mitochondrial DNA (mtDNA). OXPHOS deficiencies resulting in mitochondrial disorders can be caused by either nuclear or mitochondrial mutations; however, most pathogenic mutations reported in adults occur in mtDNA. Such mutations often impair mitochondrial translation and are associated with a characteristic muscle pathology consisting of a mosaic pattern of normal fibers interspersed with fibers displaying mitochondrial proliferation and decreased OXPHOS activity. In this thesis, the molecular basis for a severe mitochondrial myopathy in two adult patients was investigated. All patient muscle fibers showed mitochondrial proliferation and barely detectable Complex IV activity (a measure of OXPHOS activity), a pattern never before reported. Biochemical studies demonstrated decreased activities of Complexes I and IV (5% of control) and Complex II+III (41% of control) in patient muscle. Immunoblot analysis of nuclear and mitochondrial subunits of Complexes 1, III and IV showed a greater than 90% decrease in the steady-state level of these subunits in mature muscle, but no change in nuclear-encoded subunits of Complexes II and V. A generalized mitochondrial translation defect was identified by pulse-label experiments in myotubes, but not in myoblasts cultured from both patients. This defect moved with the nucleus in patient cybrid cells. Myoblasts from one patient transplanted into the muscle bed of SCID mice differentiated into mature muscle fibers that displayed a defect similar to that seen in the patient muscle. Mapping of the defective gene in this patient was attempted using a functional complementation approach. Microcell-mediated transfer of mouse chromosomes in patient

Genetic predisposition to insulin-dependent diabetes mellitus (IDDM) involves one or more loci within the class II region of the major histocompatibility complex (MHC). The strongest MHC-linked determinants of disease susceptibility have been associated with polymorphisms in both the $ alpha$ and $ beta$ chain subunits of HLA-DQ (human) and I-A (mouse) class II molecules. Specifically, protection against IDDM development is associated with the presence of aspartic acid at position 57 of the $ beta$ chain while non-aspartic acid residues are found in diabetogenic $ beta$ chains. In humans, the greatest risk of disease development is observed in individuals with Arg 52$ rm sp+ alpha/Asp 57 sp- beta$ heterodimers. The BB rat is a useful system in which one can identify and characterize the genes necessary for the onset of IDDM. Although sequence data have been reported for various alleles of these class II genes, the effect of polymorphisms in the rat MHC is not nearly as well characterized as it is in humans or the NOD mouse. The rat MHC (RT1) contains two expressed class II loci, RT1.B and RT1.D, each encoding two class II molecules: RT1.B $ alpha$ and $ beta,$ and RT1.D $ alpha$ and $ beta.$ This study determined the nucleotide sequences of relevant regions from the RT1.B and RT1.D $ alpha/ beta$ genes in five rat strains (ACI, BB, Buffalo, Lewis, and Wistar-Furth) of varying susceptibility to IDDM to examine the role of specific polymorphisms in predisposition to disease. The data show that BB and Wistar-Furth rats (RT1$ sp{ rm u}$ haplotype) have identical class II sequences at all of the regions examined. Although no unique $ alpha$ chain sequences were found to associate with IDDM, we did confirm the association of aspartic acid at position 57 of RT1.B$ beta$ with susceptibility to diabetes. (Abstract shortened by UMI.)

Cyclical neutropenia is a disease in humans and grey collie dogs in which the circulating neutrophil numbers regularly oscillate below normal values. CN can be treated using the cytokine G-CSF which decreases the period, increases the mean value, and elevates the amplitude of the oscillations. It has been theorized that this disease is a result of a periodic output of the hematopoietic stem cells in the bone marrow or a destabilization in a peripheral feedback mechanism controlled by circulating neutrophil number. We use a simple, but physiologically realistic, mathematical model, which incorporates the distribution of maturation times of neutrophils in the bone marrow, to study the latter possibility. We find that a change in the distribution of maturation times from normal to CN is insufficient to destabilize the system, and that even after a destabilization occurs, the model results are dissimilar from the observed CN dynamics. Also, G-CSF has a destabilizing effect on the system. Thus, it seems likely that the oscillations of CN are generated within the pluripotential stem cell population.

Hippi, HIP-1 Protein Interactor, is a novel protein that mediates the cellular toxicity of the Huntingtin Interacting Protein-1 (HIP-1). In order to establish a role for this protein in the pathogenesis of Huntington's Disease, targeted deletion of the Hippi locus in mice will be accomplished. First the murine counterpart to the human Hippi gene was cloned from various cDNA library sources. Secondly, the genomic structure of the Hippi gene was characterized in multiple mouse strains. Thirdly, using these Hippi genomic sequences, the gene targeting strategy was developed. Finally, the required knock-out vectors for Hippi deletion were constructed.

Remodelling of extracellular matrix (ECM) is a prominent feature of atherosclerotic lesions and contributes to lipoprotein retention as well as smooth muscle cell (SMC) activation. To gain further knowledge about ECM, certain ECM components and their degrading enzymes were studied in injury-induced arterial neointima, which shares features with early atherosclerotic lesions. / It has been shown that synthesis of collagen and syndecan-1, a hybrid heparan/chondroitin sulfate proteoglycan, is enhanced. In situ hybridization indicates that syndecan positive cells are restricted to the arterial neointima. These data confirm the importance of arterial SMC in ECM metabolism and indicate that increased synthesis contributes to ECM accumulation in neointima. / Remodelling of ECM in atherogenesis refers not only to increased ECM deposition, but also involves enhanced ECM catabolism. A family of zinc-containing proteinases, termed matrix metalloproteinases (MMPs) has recently been implicated in atherosclerosis. Subsequently, we examined expression of two common MMPs, MMP-2 and MMP-9 in our model. The mRNAs for both MMPs are up-regulated, but their tissue distribution is different: MMP-2 positive cells are visible in neointima and in aortic media; whereas cells positive for MMP-9 are located only in neointima. MMPs are active at neutral pH and in tissue, their activity is regulated by tissue inhibitors of metalloproteinases (TIMPs) including TIMP-1. The enhanced MMP expression in neointima makes it relevant to examine the simultaneous expression of TIMP-1. To do this, we cloned rabbit TIMP-1 from neointima using a PCR-cloning technique. Transformation of the cloned gene resulted in synthesis of a TIMP-1 protein in E. Coli. The concentration of TIMP-1 in neointima was examined and a significant increase of both mRNA and protein levels was observed. It is suggested that the proteolytic activity of MMPs contributes to ECM breakdown. However, this digestion is limited, as continuous augmentation of TIMP-1 expression is observed after aortic de-endothelialization.

We identified two large French-Canadian (FC) pedigrees with idiopathic partial epilepsy. Family studies of over 500 members from these two families revealed over 63 individuals reported to have seizures or seizure-like histories. Pedigree analysis confirmed an autosomal dominant inheritance with reduced penetrance. Most of the affected individuals shared a number of clinical characteristics compatible with the definition of a novel genetic form of epilepsy syndrome. We termed it as familial partial epilepsy with variable foci (FPEVF), which was recently described by Scheffer et al. (1998) in one Australian family with eight possibly affected individuals. The most prominent clinical feature of FPEVF is the presence of variable seizure foci in different affected individuals within the same family. Most affected individuals in the two FC families present infrequent, brief, non-clustering, nocturnal, partial seizures. Age of onset is between 5 and 25 years. Affected individuals are neurologically intact without any detectable pathological lesion(s). / These two families were then subjected to a full-scale molecular genetic study. No linkage to any known loci for idiopathic partial epilepsy was detected, including the suggested chromosome (Chr) 2q locus for FPEVF in the Australian FPEVF family, the Chr 20q13.2 and 15q24 loci for autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) and the Chr 10q locus for autosomal dominant temporal lobe epilepsy with auditory symptoms (ADTLEAS). Significant linkage (LOD score = 8.60) was detected with markers on Chr 22 q12 during a genome scan. Collaborating with a group from Australia, we confirmed another two FC families and two non-FC families with compatible linkage to the same locus and a cumulative LOD score of 14.79 with marker D22S689. The minimum candidate region of FPEVF has been finally defined to an interval of 3.8 cM between markers D22S 1163 and D22S 1686 on Chr 22q12. We proceeded to search for the gene mutation by directly sequencing the coding regions of all candidate genes within the FPEVF region, an interval of 5.04 Mb harboring approximately 100 known or predicted genes. So far, 77 candidate genes have been sequenced, and no mutation has yet been found. Since there is no known ion channel gene mapped to this region, FPEVF is probably the first non-ion channel gene causing human idiopathic epilepsy*. / *After submission of this thesis for examination, Kalachikov et al. (2002) identified LGI1 (leucine-rich gene, glioma inactivated), a possible tumor suppressor, as the gene for ADTLEAS. This is the first evidence for a non-ion channel gene causing idiopathic epilepsy in humans.

Scoliosis is a deformity of the spine that predominately affects adolescent females. Mild scoliotic curves are most vulnerable to progression during the adolescent growth spurt, however, only an unpredictable 15-25% progress to large incapacitating deformities. The present objective was to identify mechanical factors associated with the adolescent growth spurt which are instrumental to curve progression in adolescent idiopathic scoliosis (AIS). An initially curved and twisted, spatial beam-column model of a spine with a mild scoliosis was developed. The spine was embedded in a three-dimensional elastic medium to represent the ribcage. A finite element model of a ribcage was developed to establish its three-dimensional stiffness through a series of numerical experiments. Parametric analyses of both the ribcage and spine models were conducted to elucidate a better understanding of this mechanical system. The geometry, material properties and applied loads of the spine were then systematically changed to simulate both normal and aberrant growth patterns during the adolescent years. The three-dimensional stiffnesses of the ribcage were found to vary significantly with rib level and orientation, and were most sensitive to changes in the gross ribcage geometry and the material properties of the costotransverse joints. The parametric analysis of the whole spine model indicated that the progression of a mild scoliosis was most sensitive to the initial Cobb angle, the spine length, the body weight and the lateral translational stiffness of the ribcage. The progression of a mild scoliotic curve (Cobb angle $<$ 20$ sp circ$) was found to be small due to mechanical changes associated with the normal adolescent growth spurt in both males and females. For an initial Cobb angle of 30$ sp circ$, significant progression was predicted for a female during normal growth. The mechanical changes associated with reported aberrant growth patterns could be key factors in the progression

Secondary, or AA, amyloidosis is a systemic disease characterized by the extracellular tissue deposition of insoluble, fibrillar amyloid A protein. Aberrant metabolism of serum amyloid A protein by reticuloendothelial cells in the presence of amyloid enhancing factor (AEF) is thought to result in the accumulation of fibrils within the tissue. Treatment of mice with AEF, in conjunction with an inflammatory stimulus, induces amyloid deposition within 48-72 hours. In vivo examination of the effect of AEF on spleen macrophage activation-associated phenotype and functions reveal that AEF has no apparent effect on the ability of spleen and liver macrophages to phagocytose or kill Listeria monocytogenes. It did appear to block enhanced respiratory burst function as well as result in a decrease in the number of cells expressing the MHC Class II surface antigen. Polyacrylamide gel electrophoretic purification of AEF from crude amyloidotic tissue extracts attributed AEF activity to a region apparently consisting of 3 protein species with molecular weights ranging from 63.2 KDa to 47.9 KDa. Ex vivo studies of macrophage function with protein eluted from this "AEF-active" region did not indicate an inhibition of respiratory burst suggesting that the action of AEF may be dependent on the presence of additional, possibly inflammation-associated, component(s).

<p> Clinical laboratories are facing increasing challenges to provide accurate and timely test results. In order to improve the quality and turnaround time of laboratory results, improvement opportunities should be sought in all phases of laboratory testing. This thesis demonstrates the applicability of quality tools within the Deming Plan, Do, Study, and Act cycle to the implementation of autoverification. </p><p> Autoverification is a post-analytical laboratory process improvement tool, which uses computer algorithms to allow qualifying test results to pass directly from automated instruments to the patient's medical record without intervention by a laboratory technologist. This can result in significant time-savings, improved turnaround time, and improved consistency in result handling. This thesis investigates the current status of autoverification in Southern California hospitals and the perceived barriers to the use of this process. The experience of a 350-bed community hospital is presented as an example to assist other laboratories in overcoming these barriers.</p>

<p> Sterol 12a-hydroxylase (CYP8B1) is involved in cholic acid synthesis and plays a role in intestinal cholesterol absorption and pathogenesis of cholesterol gallstone disease and dyslipidemia. In this study, we investigated the underlying mechanism of a fasting-induced and cholesterol activated nuclear receptor and core clock gene RORa in regulation of circadian rhythm and fasting induction of CYP8B1 expression. In free fed mice, CYP8B1 expression was reduced to the lowest level at the onset of the dark cycle when RORa expression was the lowest. However, fasting stimulated, while re-feeding reduced expression of CYP8B1 mRNA and protein expression. Interestingly, fasting and feeding had little effect on the diurnal rhythm of RORa mRNA expression, but fasting increased, whereas feeding decreased RORa protein levels in mouse liver. Adenovirus-mediated transduction of RORa to mice strongly induced CYP8B1 gene expression, increased 12a-hydroxylated bile acids in bile acid pool and serum and liver cholesterol. Reporter assay and mutagenesis analysis of the CYP8B1 promoter identified a functional RORa response element. Mammalian two-hybrid assay showed strong interaction of RORa with cAMP response element binding protein-binding protein (CBP). Chromatin immune-precipitation assay showed that RORa recruited CBP to the CYP8B1 promoter to stimulate histone acetylation. CAMP-activated protein kinase A phosphorylates RORa and increases its half-life. In conclusion, RORa is a key regulator of circadian expression and fasting induction of CYP8B1 to increase 12a-hydroxylated bile acids in the bile acid pool, and serum and liver cholesterol. This study contributes to our understanding of the molecular mechanism by which bile acid synthesis and composition regulates hepatic metabolic homeostasis. Overall this study sheds light on the mechanism of development of hypercholesterolemia in diabetic patients. Therefore, antagonizing RORa activity may be a therapeutic strategy for treating inflammatory diseases such as non-alcoholic fatty liver disease.</p>