Thesis (Ph. D.)--Case Western Reserve University, 2005. / [School of Medicine] Department of Genetics. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Thesis (M.S.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 57 pages. Includes Vita. Includes bibliographical references.

Sepsis is an exaggerated inflammatory syndrome involving cell, organ and system dysregulation. This dysregulation is detrimental to the patient, mainly as it causes imbalances in the immune system. Monocytes and neutrophils are key cell players in the pathogenesis of sepsis; Two populations that follow divergent apoptotic fates; most importantly delayed neutrophil apoptosis has been demonstrated in sepsis and may contribute to organ damage. These two subpopulations have recently been referred to as specific targets of leukocyte filtration, a noveltherapeutic approach in septic patients. As the molecular mechanisms driving the survival/death switches of these cells in sepsis is still controversial, we decided to investigate the role of a new family of proteins predicted to be involved in inflammation and cell death mechanisms; the ApoLs. We studied ApoL expression in whole blood leukocytes and purified populations of neutrophils and monocytes from healthy volunteers and patients with or without sepsis. We then correlated ApoL expression to the degree of inflammation (CRP levels) and cellular apoptosis. We showed a down regulation in ApoL expression in septic and non-septic patients as compared to healthy volunteers, whereas purified monocytes showed an up regulation of ApoL1 and ApoL2 expression in septic patients as compared to non-septic patients. Given that these cells behave differently in the septic frame, we aim to investigate and invalidate the role of ApoLs in conditions related to cell activation and death. Given the high degree of variability in human samples, we resorted to an in-vitro cell line, the PLB 985 myelomonoblastic cell line with granulocytic differentiation potential. Culturing these cells in RPMI 1640 in the presence of specific differentiation agents resulted in mature terminally differentiated cells with phenotypic and functional similarities to human neutrophils. This was determined by monitoring specific leukemic and granulocytic markers. This cell model will help us to better understand the role ofApoLs in regulating neutrophil half-life particularly in inflammatory conditions. It can also be employed to characterize “The cytokine(s)” that is inducing a change in ApoL expression and ultimately uncover the signaling pathway dictating both ApoL expression and apoptosis/survival. / Le sepsis est defini comme une reponse inflammatoire exageree, associee a une dysfonction d'organes faisant suite a un desequilibre du systeme immunitaire. En effet, les monocytes et les neutrophiles representent les acteurs cle dans la physiopathogenese du sepsis. Etant donne que les mecanismes moleculaires regulant la balance de survie / mort cellulaire de ces deux populations de cellules restent jusqu'a present controverses, nous nous sommes interesses a etudier l'implication d'une nouvelle famille de proteines: les apols, susceptibles d'intervenir dans le processus inflammatoire et de la mort cellulaire. Nous avons explore l'expression des apols dans les leucocytes totaux et dans les populations de neutrophiles et de monocytes purifiees a partir de donneurs sains et de patients septiques et non septiques. Par la suite, nous avons etablit le lien de correlation entre l'expression des apols, le degre d'inflammation systemique (dosage de la crp) et l'apoptose. Nos resultats ont demontre une baisse de l'expression des apols dans les leucocytes totaux el les neutrophiles purifies, chez les patients septiques et non septiques, en comparaison avec les donneurs sains. En revanche, nous remarquons une surexpression de l'apol1 et l'apol2 chez les patients septiques par rapport aux patients non septiques. Etant donne la grande variabilite dans les echantillons preleves, nous avons procede a la differenciation d'une lignee myelomonoblastique ayant un pouvoir de differenciation granulocytaire. La mise en culture de cette lignee dans les conditions de differenciation adequates, nous a permis d'obtenir des cellules ayant des similarites fonctionnelles et phenotypiques aux neutrophiles humains. Ce modele cellulaire va nous aider d'une part, a mieux comprendre l'implication des apols dans le controle de la viabilite cellulaire dans les conditions inflammatoires; et d'autre part, a caracteriser la / (les) cytokine(s) susceptibles de moduler l'expression des apols; et finalement a decortiquer la voie de signalisation impliquant les apols dans le controle du processus de survie ou de mort cellulaire. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

LDL-receptor mutations and familial defective apolipoprotein B-100 (codon 3500) (FOB), the known causes of monogenic hypercholesterolemia (MH), have similar clinical features. The nature of the mutations responsible for MH in South Africans of Indian origin was previously unknown. Similarly, the mutations in the LDL-receptor gene of a South African Black FH homozygote had also not been characterised. The aim of this thesis was to identify and analyse the LDL-receptor mutations in the Indian homozygotes NS, D, AV and AA and in the Black homozygote JL. In addition, the possible importance of FOB as a cause of MH in South Africans was also assessed. The patient NS was characterized as having two "Null" LDL-receptor alleles. His skin fibroblasts expressed no detectable LDL-receptor protein and very low levels of LDL-receptor mRNA of approximately normal size. Since NS' s LDLreceptor promoter sequence was normal, his alleles are likely to harbour exonic point mutations or minor rearrangements that cause premature stop codons. The patient D was found to be a heteroallelic homozygote. Two new point mutations in the LDL receptor, Asp₆₉ -Tyr and Glu₁₁₉-Lys, were identified. D's fibroblasts expressed about 30% of the normal surf ace complement of receptors that bound LDL poorly. This low number could at least be partially explained by their decreased stability. These mutations were not identified in any other Indian FH or hypercholesterolemic patients. Patients AV and AA were both shown to be homoallelic homozygotes for the Pro₆₆₄ -Leu mutation. This mutation was identified in 4 unrelated Muslim families of Gujerati origin suggesting that the mutation arose from this area in India. Contrary to previous reports (Knight et al. 1990, Soutar et al. 1989), neither LOL nor β-VLDL binding were shown to be affected by this mutation. These mutant receptors were rapidly degraded. Thus the disease FH in these subjects is presumably due to the low steady-state level of mature receptors that are functionally normal but exhibit accelerated turnover. The Pedi FH homozygote, JL, expressed very few LOL receptors due to decreased receptor synthesis associated with low mRNA levels and not due to enhanced degradation. One of JL's LOL receptor alleles has a 3 b.p. deletion in repeat 1 of the promoter (G. Zuilani, H. Hobbs and L.F. de Waal, personal communication). The nature of the defect in his other allele is unknown. The importance of FOB as a cause of monogenic hypercholesterolemia in the South African Indian, "Coloured" and Afrikaner populations was determined by screening hypercholesterolemic subjects with or without xanthomata. The absence of FOB in such patients, in whom the relevant common or founder South African mutations were excluded, suggested that this disorder was rarer in these groups than in North America and Europe. FOB was identified in two different families of mixed British and Afrikaner ancestry. One family contained individuals who were heterozygous for the FOB mutation, as well as the FH Afrikaner-1 and the FH Afrikaner-2 LOL-receptor mutations. In addition, 4 compound heterozygotes, who had both FOB and the FH Afrikaner-1 mutation and one individual whu inherited all 3 defects, were identified. This family allowed us to characterise the compound heterozygotes with one mutant LOLreceptor allele and FOB as having a condition that was probably intermediate in severity between the FH heterozygote and homozygote states.

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published_or_final_version / Chemistry / Master / Master of Philosophy

Genetic polymorphism of apolipoprotein (apo) E has been shown to account for a significant amount of variance in plasma lipid and lipoprotein levels, thereby contributing to the incidence of cardiovascular disease across populations. In this cross-sectional study apo E genotypes were determined in a sample of 173 healthy, middle-aged Indian women using a restriction isotyping method, in which DNA was amplified by PCR and the Cfol restricted DNA fragments were separated on a polyacrylamide gel, allowing unambiguous typing of the common apo E genotypes. Considering the three common alleles, e2, e3 and e4, a reduced frequency of the e2 allele was observed in the study population in comparison to other populations around the world. This finding underlines the heterogeneity of apo E allele frequencies in different populations. This study also investigated possible effects of apo E genotype on lipoprotein changes in this sample of women spanning the menopause. Apo E polymorphism was associated with significant differences in plasma lipid levels. Notably, total and low density lipoprotein cholesterol and more especially plasma triglyceride concentrations were increased in carriers of the e3/4 genotype. Two-way analysis of variance of the effect of apo E genotype and menopausal status on the lipid profile showed no significant interaction effect, indicating that the effects of apo E genotype on the lipid profile do not differ significantly between premenopausal and postmenopausal women. Age and to a lesser extent the waist hip ratio also correlated with lipid concentrations, but menopausal status had no apparent effect in this sample. This study confirms the potentially deleterious effect of the e4 allele, in a vulnerable population. The reduced occurrence of the E2 isoform, which is considered to offer a measure of protection against cardiovascular disease, may contribute to the relatively high incidence of coronary heart disease in the South African Indian population. However, the relatively low incidence of the e2 allele may protect this population against the occurrence of type III hyperlipoproteinaemia precipitated by diabetes and obesity in e2/2 homozygotes. / Thesis (M.Med.)-University of Natal, Durban, 1993.

by Ma Suk Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 106-129). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / Publications --- p.vi / Abbreviations --- p.vii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Epidemiology of AD --- p.2 / Chapter 1.2 --- Clinical and pathological features of AD --- p.3 / Chapter 1.2.1 --- Clinical features of AD --- p.3 / Chapter 1.2.2. --- Pathological features of AD --- p.3 / Chapter 1.3 --- Diagnosis of AD --- p.4 / Chapter 1.4 --- Classification of AD --- p.5 / Chapter 1.5 --- Causes of AD --- p.5 / Chapter 1.6 --- Risk factors --- p.5 / Chapter 1.6.1 --- Age --- p.5 / Chapter 1.6.2 --- Family history --- p.6 / Chapter 1.6.3 --- Genetics --- p.6 / Chapter 1.6.3.1 --- Autosomal dominant mutations --- p.6 / Chapter 1.6.3.2 --- Genotypes of Apolipoprotein E --- p.6 / Chapter 1.6.4 --- Environmental factors --- p.7 / Chapter Chapter 2 --- Pathology in Alzheimer's disease --- p.8 / Chapter 2.1 --- Overview of Alzheimer's disease pathology --- p.8 / Chapter 2.2 --- Amyloid plaques --- p.8 / Chapter 2.2.1 --- Amyloid precursor protein --- p.8 / Chapter 2.2.2 --- Processing ofAPP --- p.9 / Chapter 2.2.3 --- Amyloid β (Aβ) --- p.12 / Chapter 2.2.4 --- APP mutations and AD --- p.12 / Chapter 2.3 --- Neurofibrillary tangles (NFT) --- p.15 / Chapter 2.3.1 --- Tau --- p.15 / Chapter 2.3.2 --- Tau mutation and neurodegeneration --- p.17 / Chapter 2.4 --- Hypotheses for AD pathology --- p.18 / Chapter 2.4.1 --- Amyloid cascade hypothesis --- p.18 / Chapter 2.4.2 --- Inflammatory hypothesis --- p.20 / Chapter 2.4.2.1 --- Microglia and astrocytes --- p.21 / Chapter 2.4.2.2 --- Inflammatory cytokines --- p.23 / Chapter 2.4.2.3 --- Inflammation and AD --- p.25 / Chapter 2.4.3 --- ApoE hypothesis --- p.27 / Chapter 2.4.3.1 --- Apolipoprotein E --- p.27 / Chapter 2.4.3.2 --- ApoE and AD --- p.28 / Chapter 2.5 --- Theory towards the pathology of AD --- p.30 / Chapter Chapter 3 --- ApoE genotyping --- p.32 / Chapter 3.1 --- Introduction --- p.32 / Chapter 3.2 --- Materials and methods --- p.32 / Chapter 3.2.1 --- Patients and control subjects --- p.32 / Chapter 3.2.2 --- Blood sampling --- p.33 / Chapter 3.2.3 --- DNA genotyping --- p.34 / Chapter 3.2.4 --- Statistical analysis --- p.35 / Chapter 3.3 --- Results --- p.35 / Chapter 3.. --- Discussion --- p.38 / Chapter Chapter 4 --- IL-1β polymorphism in relation to the risk of ADin Chinese --- p.39 / Chapter 4.1 --- Introduction --- p.39 / Chapter 4.2 --- Materials and methods --- p.44 / Chapter 4.2.1 --- Patients and control subjects --- p.44 / Chapter 4.2.2 --- Blood sampling --- p.44 / Chapter 4.2.3 --- DNA genotyping --- p.44 / Chapter 4.2.4 --- Statistical analysis --- p.48 / Chapter 4.3 --- Results --- p.48 / Chapter 4.4 --- Discussion --- p.53 / Chapter Chapter 5 --- TNFα polymorphism in relation to the risk of ADin Chinese --- p.63 / Chapter 5.1 --- Introduction --- p.63 / Chapter 5.2 --- Materials and methods --- p.66 / Chapter 5.2.1 --- Patients and control subjects --- p.66 / Chapter 5.2.2 --- Blood sampling --- p.66 / Chapter 5.2.3 --- DNA genotyping --- p.66 / Chapter 5.2.4 --- Haplotype determination --- p.70 / Chapter 5.2.5 --- Statistical analysis --- p.70 / Chapter 5.3 --- Results --- p.70 / Chapter 5.4 --- Discussion --- p.75 / Chapter Chapter 6 --- LRP8 polymorphism in relation to the risk of ADin Chinese --- p.81 / Chapter 6.1 --- Introduction --- p.81 / Chapter 6.2 --- Materials and methods --- p.87 / Chapter 6.2.1 --- Patients and control subjects --- p.87 / Chapter 6.2.2 --- Blood sampling --- p.87 / Chapter 6.2.3 --- DNA genotyping --- p.87 / Chapter 6.2.4 --- Statistical analysis --- p.89 / Chapter 6.3 --- Results --- p.91 / Chapter 6.4 --- Discussion --- p.98 / Chapter Chapter 7 --- Conclusions and prospects for future work --- p.102 / Chapter 7.1 --- Conclusion --- p.102 / Chapter 7.2 --- Prospects for future work --- p.105 / Reference --- p.106

Apolipoprotein E (apoE) is a lipid transport protein which plays a key role in lipid metabolism. In addition to the well known polymorphic coding alleles epsilon2, epsilon3 and epsilon4, APOE promoter single nucleotide polymorphisms (SNPs) have also been reported to modify disease susceptibilities in humans. / In a case-control study involving 710 Chinese type 2 diabetes and 198 non-diabetic subjects, genotyping of three SNPs (-491A/T, -219G/T and +113G/C) within the APOE proximal promoter identified that -491A was associated with increased risk for type 2 diabetes in women (OR=2.44, 95%CI=1.15-5.19, p=0.017). However, the three tested SNPs were not associated with the risk of diabetic nephropathy (DN). Yeast one-hybrid screening of the human brain cDNA library using the polymorphic DNA sequences spanning the APOE promoter -491 site as the 'baits' identified one of the interacting transcription factors being the activating transcription factor 4 (ATF4). Electrophoretic-mobility-shift assay confirmed the physical interaction of the purified recombinant ATF4 protein and APOE promoter -491 A/T spanning region (-521 to -461). The binding of ATF4 to the -491T-containing sequence was stronger than that of the -491A-containing sequence. Chromatin immunoprecipitation (ChIP) assay further confirmed the interaction between ATF4 and APOE promoter -491-spanning region in vivo. The functional significance of APOE -491A/T polymorphism was supported by the dual-luciferase reporter assay showing that -491 A to T single nucleotide substitution significantly decreased the activity of the cloned APOE promoter (-1019 to +407) in human kidney (293), liver (WRL-68) and astrocyte (U-87) cell lines. Further analysis showed that ATF4 over-expression significantly down-regulated the activities of the cloned APOE promoter. The suppression of ATF4 on APOE promoter with -491A allelic form was significantly stronger than that with -491T allelic form in 293 cells (p<0.05). Interestingly, overexpression of recombinant ATF4 stimulated endogenous APOE transcription by about 10% in WRL-68 cells. / In conclusion, APOE promoter -491A/T polymorphism modifies the risk of type 2 diabetes in Hong Kong Chinese women. The -491A/T polymorphism controls APOE promoter activity and is interactive with transcription factor ATF4. / My thesis project aimed at testing two hypotheses: (1) APOE promoter SNPs associate with the risks of type 2 diabetes and diabetic nephropathy, (2) APOE promoter SNPs modify transcriptional control of the gene. / Geng, Hua. / "September 2007." / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4559. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 140-151). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.