Polyglutamine disease is a class of neurodegenerative diseases, which is manifested by the atrophy of nervous system that results in dementia and/or motor dysfunction. The major pathological characteristics include progressive loss of neuronal cells as well as the appearance of insoluble nuclear inclusions in degenerating neuronal cells. Polyglutamine disease is caused by CAG triplet expansion in the genome. When translated, such expansion leads to the formation of expanded polyglutamine domain within the respective disease proteins and promotes abnormal protein conformational changes. Owing to their misfolded nature, the expanded polyglutamine proteins form insoluble nuclear inclusions. These insoluble nuclear inclusions are heterogeneous in nature, in which polyglutamine protein and molecular chaperones are the recruited components. All eukaryotic cells express molecular chaperones which function to mediate the proper folding of proteins. The recruitment of molecular chaperones into nuclear inclusions that contain misfolded triplet-expanded proteins strongly suggests the involvement of molecular chaperones in polyglutamine disease progression. It has been shown that over-expression of molecular chaperones in polyglutamine disease models can lead to a suppression of polyglutamine toxicity and a concomitant increase in the solubility of disease proteins, i.e. the solubility of polyglutamine disease protein is related to its toxicity. Intrigued by these observations, I aimed at elucidating the mechanism of polyglutamine disease pathogenesis by first studying the cellular regulation of endogenous chaperone expression in neurodegeneration in a transgenic Drosophila model of polyglutamine disease. A biphasic regulation of Hsp70 expression was observed, which the regulation was at the transcription level. Moreover, over-expression of Hsp70 could alter the endogenous Hsp70 protein and mRNA level of polyglutamine disease fly model. The study may help the understanding of how the chaperone expression is regulated under the effects of polyglutamine expression and thus to find out the mechanism of pathogenesis. In addition, cellular proteins that change in solubility other than disease protein will also be identified. Small heat shock proteins, glutathione S transferase and alpha 4 proteasome subunit, etc., showed change in solubility or expression by 2D gel electrophoresis analysis. Identifying the proteins that change in solubility or expression may help the finding of the interplay of proteins and thus the pathways involve in the mechanism of polyglutamine disease pathogenesis. Understanding pathogenic pathways can give ideas on how polyglutamine lead to the disease, up- or down-regulation of those protein interplays may provide direction and therapeutic candidates to suppress polyglutamine disease. / Huen Ngar Yee. / "September 2006." / Advisers: Ho Yin Chan; Siu Kai Kong. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1465. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 134-146). / 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.

by Wong Ho Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 107-129). / Abstract --- p.i / List of Abbreviations --- p.ii / Contents --- p.iii / Chapter Chapter 1. --- Introduction / Chapter 1.1 --- Introduction of aging in central nervous system --- p.1 / Chapter 1.2 --- Introduction of hippocampus / Structure of the hippocampus --- p.4 / Function of hippocampus --- p.6 / Chapter 1.3 --- A literature review of aging in hippocampus / Cell loss in aging --- p.8 / Ultrastructural changes in aging --- p.9 / Changes in neurotransmitter system --- p.10 / Neuroglial change --- p.11 / Change in potentiation --- p.13 / Chapter 1.4 --- A literature survey of Nitric Oxide Synthase (NOS) / General introduction of Nitric Oxide Synthase --- p.15 / Introduction of nNOS --- p.15 / Introduction of iNOS --- p.16 / Introduction of eNOS --- p.17 / Similarities and differences among isoforms --- p.18 / Role of NO/NOS in neurotransmission --- p.19 / Role of NO in neurotoxicity --- p.23 / Chapter 1.5 --- Aim of study --- p.25 / Chapter Chapter 2. --- Change of nNOS in aging / Chapter 2.1 --- Purpose and approach --- p.27 / Chapter 2.2 --- Basic principle of the techniques / Basic principle of immunohistochemistry --- p.28 / Basic principle of RT-PCR --- p.28 / Chapter 2.3 --- Experimental procedure / nNOS immunohistochemistry --- p.32 / RT-PCR of nNOS --- p.34 / Chapter 2.4 --- Result / nNOS immunohistochemistry --- p.38 / RT-PCR of nNOS --- p.44 / Chapter Chapter 3. --- Expression of iNOS in aging / Chapter 3.1 --- Purpose and approach --- p.50 / Chapter 3.2 --- Experimental procedure / iNOS immunohistochemistry --- p.50 / RT-PCR analysis of iNOS --- p.51 / Chapter 3.3 --- Result / iNOS immunohistochemistry --- p.52 / RT-PCR analysis of iNOS --- p.56 / Chapter Chapter 4. --- Verification of the RT-PCR product of iNOS / Chapter 4.1 --- Purpose and approach --- p.58 / Chapter 4.2 --- Basic principle --- p.58 / Chapter 4.3 --- Experimental procedure / Elution of PCR product from PAGE gel --- p.60 / Restriction digestion of the eluted PCR product --- p.61 / Chapter 4.4 --- Result --- p.62 / Chapter Chapter 5. --- Identification of the iNOS-positive cells / Chapter 5.1 --- Purpose and approach --- p.64 / Chapter 5.2 --- Experimental procedure --- p.64 / Chapter 5.3 --- Result --- p.65 / Chapter Chapter 6. --- Quantitation of astrocyte in aging hippocampus / Chapter 6.1 --- Purpose and approach --- p.67 / Chapter 6.2 --- Experimental procedure --- p.68 / Chapter 6.3 --- Result --- p.69 / Chapter Chapter 7. --- Detection of apoptosis in aging / Chapter 7.1 --- Introduction of apoptosis --- p.74 / Chapter 7.2 --- Purpose and approach --- p.75 / Chapter 7.3 --- Basic principle --- p.76 / Chapter 7.4 --- Experimental procedure / TUNEL method --- p.77 / DNA gel electrophoresis --- p.78 / Chapter 7.5 --- Result / TUNEL method --- p.80 / DNA gel electrophoresis --- p.82 / Chapter Chapter 8. --- Discussion / Chapter 8.1 --- Pattern of neuronal NOS in aging / Localization of nNOS --- p.84 / Decrease in staining of nNOS in the hippocampus during aging --- p.87 / No change in nNOS mRNA level --- p.88 / nNOS in aging - past and present works --- p.89 / Implication of the result --- p.91 / Chapter 8.2 --- Increased iNOS expression in aging / Neurotoxicity of iNOS --- p.93 / Circumstances of iNOS expression --- p.95 / Discussion of the present study --- p.96 / Chapter 8.3 --- Detection of apoptosis in aging --- p.103 / Chapter Chapter 9. --- Conclusion --- p.106 / Biblography --- p.107 / Appendix --- p.130 / Acknowledgements --- p.134

Ion channels are membrane proteins that facilitate electrical signaling in important physiological processes, such as the rhythmic contraction of the heart. KCNQ1 is the pore-forming subunit of a voltage-gated potassium channel that assembles with the β-subunit KCNE1 in the heart to generate the IKs current, which is critical to cardiac action potential repolarization and electrical conduction in the heart. Mutations in IKs subunits can cause potentially lethal arrhythmia, including long QT syndrome, short QT syndrome, and atrial fibrillation. Each channel consists of four voltage-sensing domains and a central pore through which ions permeate. Voltage-dependent gating occurs when movement of voltage sensors cause pore opening/closing through coupling mechanisms. Although KCNQ1 by itself is able to form a voltage-dependent potassium channel, its assembly with KCNE1 is essential to generating the physiologically critical cardiac IKs current, characterized by a delay in the onset of activation, an increase in current amplitude, and a depolarizing shift in the current-voltage relationship. KCNE1 is thought to have multiple points of contact with KCNQ1 that reside within both the voltage-sensing domain and the pore domain, allowing for extensive modulation of channel function. Atrial fibrillation is the most common cardiac arrhythmia and affects more than 3 million adults in the United States. Much rarer, genetic forms of atrial fibrillation have been associated with gain-of-function mutations in KCNQ1, such as two adjacent mutations, S140G and V141M. Both mutations drastically slow channel deactivation, which underlies their pathophysiology. Deactivation slowing causes accumulation of open channels in the context of repeated stimulation, which abnormally increases the repolarizing K+ current, excessively shortens the action potential duration, and predisposes to re-entry arrhythmia such as atrial fibrillation. Although both mutations are located in the voltage-sensing domain, their mechanisms of action remain unknown. Understanding the gating mechanisms underlying deactivation slowing may provide key insights for the development of mechanism-based pharmacologic therapies for arrhythmias associated with KCNQ1 mutations. In addition to gain-of-function mutations, molecular activators of KCNQ1 can slow deactivation and increase channel activity. An existing problem in the pharmacologic treatment of arrhythmia is that many antiarrhythmic drugs do not have specific targets and cause undesired side effects such as additional arrhythmia. Thus, developing mechanism-based therapies may optimize clinical treatment for patients with specific forms of channel dysfunction. Two KCNQ1 activators, ML277 and R-L3, have been previously shown to slow current deactivation, but the underlying gating mechanisms remain known. Although these modulators are unlikely to serve directly as antiarrhythmic therapy, investigating their mechanisms will likely provide fundamental insights on channel modulation and guide future efforts to develop personalized therapies for arrhythmia, such as congenital long QT syndrome. Given the central importance of deactivation slowing in both pathophysiology and pharmacology, we focused on investigating gating mechanisms that underlie deactivation slowing. To this end, we utilized voltage clamp fluorometry, a technique that simultaneously assays for voltage sensor movement and ionic current through the channel pore. In Chapter 1, we begin our study by examining the gating mechanisms of KCNQ1 atrial fibrillation mutations in the absence of KCNE1. We show that S140G slows voltage sensor deactivation, which indirectly slows current deactivation. On the other hand, V141M neither slows voltage sensor nor current deactivation. This is followed by Chapter 2, where we examine the gating mechanisms underlying deactivation slowing by atrial fibrillation mutations in the presence of KCNE1. We show that both S140G and V141M slow IKs deactivation by slowing pore closing and altering voltage sensor-pore coupling. Based on these findings, we proposed a molecular mechanism in which both mutations disrupt the orientation of KCNE1 relative to KCNQ1 and thus impede pore closing, implying that future efforts to modulate KCNQ1 function can benefit from targeting the β-subunit. Finally, in Chapter 3, we explore the gating mechanisms underlying deactivation slowing for two small-molecule activators of KCNQ1. We show that ML277 predominantly slows pore transitions, whereas R-L3 slows voltage sensor deactivation, which indirectly slows current deactivation. Taken together, these studies guide future efforts to develop mechanism-based therapies for arrhythmia.

Pan, Tak Yin. / Thesis submitted in: November 2007. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references. / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract (English) --- p.ii / Abstract (Chinese) --- p.iv / Content --- p.v / Abbreviations --- p.x / List of Figures --- p.xi / List of Tables --- p.xii / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Alzheimer's Disease --- p.1 / Chapter 1.1.1 --- Cholinergic hypothesis --- p.2 / Chapter 1.1.2 --- p-amyloid hypothesis --- p.2 / Chapter 1.1.3 --- Taupathy hypothesis --- p.3 / Chapter 1.1.4 --- Current therapies --- p.4 / Chapter 1.2 --- Proteins Involved in Alzhemer's Disease --- p.5 / Chapter 1.2.1 --- Acetylcholinesterase (AChE) --- p.5 / Chapter 1.2.2 --- p-amyloid --- p.6 / Chapter 1.2.3 --- Paired helical filaments (PHF) --- p.7 / Chapter 1.2.4 --- Protein kinases --- p.8 / Chapter 1.2.4.1 --- Glycogen synthase kinase-3 (GSK-3) --- p.9 / Chapter 1.2.4.2 --- Cyclin-dependent kinase-5 (CDK-5) --- p.9 / Chapter 1.2.5 --- Protein phosphatase (PP) --- p.10 / Chapter 1.2.5.1 --- Protein phosphatase 1 (PP-1) --- p.11 / Chapter 1.2.5.2 --- Protein phosphatise 2A (PP-2A) --- p.12 / Chapter 1.2.5.3 --- Protein phosphatise 2B (PP-2B) --- p.13 / Chapter 1.2.6 --- Apoptotic and Anti-apoptotic proteins --- p.14 / Chapter 1.2.6.1 --- Caspase-3 --- p.15 / Chapter 1.2.6.2 --- Bcl-2 --- p.15 / Chapter 1.3 --- Flavonoids --- p.16 / Chapter 1.3.1 --- Biosynthesis of flavonoids --- p.17 / Chapter 1.3.2 --- Biological functions of flavonoids in plants --- p.18 / Chapter 1.3.3 --- Beneficial effects of flavonoids on human health --- p.19 / Chapter Chapter 2: --- Materials and Methods --- p.20 / Chapter 2.1 --- Differentiation of SHSY-5Y cells --- p.20 / Chapter 2.1.1 --- SHSY-5Y cell culture --- p.20 / Chapter 2.1.2 --- Counting cells --- p.20 / Chapter 2.1.3 --- Retinoic acid differentiation --- p.21 / Chapter 2.2 --- Western blot analysis --- p.21 / Chapter 2.2.1 --- Extraction of proteins from mammalian cells --- p.21 / Chapter 2.2.2 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.22 / Chapter 2.2.3 --- Semi-dry protein transfer to nitrocellulose membrane --- p.23 / Chapter 2.2.4. --- Membrane blocking and immunostaining --- p.24 / Chapter 2.3 --- MTT assay --- p.25 / Chapter 2.4 --- Hoechst 33342 Nuclei staining --- p.25 / Chapter 2.5 --- Cell cycle analysis --- p.25 / Chapter 2.5.1 --- Ethanol fixation --- p.25 / Chapter 2.5.2 --- Propidium iodide staining --- p.26 / Chapter 2.6 --- Annexin V-FITC & Propidium iodide staining --- p.26 / Chapter 2.7 --- DNA fragmentation analysis --- p.26 / Chapter 2.7.1 --- Phenol/Chloroform extraction of DNA --- p.26 / Chapter 2.7.2 --- Ethanol precipitation of DNA --- p.27 / Chapter 2.7.3 --- Agarose gel electrophoresis of DNA --- p.27 / Chapter 2.8 --- Proteomic analysis --- p.28 / Chapter 2.8.1 --- First dimension: isoelectric focusing --- p.28 / Chapter 2.8.2 --- Second dimension: SDS PAGE --- p.29 / Chapter 2.8.3 --- Gel staining --- p.30 / Chapter 2.8.3.1 --- Silver staining --- p.30 / Chapter 2.8.3.2 --- SYBRO Ruby staining --- p.31 / Chapter 2.8.4 --- Gel scanning and image analysis --- p.31 / Chapter 2.8.5 --- ln-gel digestion --- p.32 / Chapter 2.8.6 --- Zip Tip for desalting the digested sample --- p.33 / Chapter 2.8.7 --- Protein identification with mass spectrometry and database search --- p.33 / Chapter Chapter 3: --- Characterization of Okadaic acid-induced tail hyperphosphorylation in SHSY-5Y cells --- p.35 / Chapter 3.1 --- Introduction --- p.35 / Chapter 3.2 --- Objectives --- p.37 / Chapter 3.3 --- Results --- p.38 / Chapter 3.3.1 --- Differentiation of SH-SY5Y cell --- p.38 / Chapter 3.3.2 --- Changes of protein expression after okadaic acid treatment --- p.40 / Chapter 3.3.3 --- Neurite Retraction Induced by okadaic acid --- p.42 / Chapter 3.3.4 --- Okadaic acid-induced Cell Death measured by MTT assay --- p.44 / Chapter 3.3.5 --- Hoechst 33342 Nuclei Staining --- p.44 / Chapter 3.3.6 --- Cell cycle analysis by propidium iodide staining --- p.47 / Chapter 3.3.7 --- Early Apoptotic cells detection by Annexin V/PI staini --- p.49 / Chapter 3.3.8 --- DNA fragmentation --- p.51 / Chapter 3.4 --- Discussion --- p.53 / Chapter Chapter 4: --- Flavonoids screening for protecting neuronal death by preventing tau hyperphosphorylation --- p.57 / Chapter 4.1 --- Introduction --- p.57 / Chapter 4.2 --- Objectives --- p.58 / Chapter 4.3 --- Tested flavonoids --- p.59 / Chapter 4.4 --- Results --- p.60 / Chapter 4.4.1 --- Toxicity of flavonoids --- p.60 / Chapter 4.4.2 --- Effects of flavonoid pre-treatment on OA-induced neu retractions and cell death --- p.62 / Chapter 4.4.3 --- Western blot analysis --- p.65 / Chapter 4.4.4 --- The effect of different concentrations of hesperidin or OA treatment --- p.70 / Chapter 4.4.5 --- Proteomic analysis --- p.74 / Chapter 4.5 --- Discussion --- p.78 / Chapter Chapter 5: --- General Discussion --- p.82 / References

Tsang, Chi Keung. / "November 2011." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 103-116). / Abstracts in English and Chinese. / Abstract --- p.I / 摘要 --- p.III / Acknowledgement --- p.IV / Table of content --- p.V / List of Figures --- p.VIII / List of Tables --- p.XI / Abbreviations --- p.XII / Chapter Chapter 1: --- INTRODUCTION --- p.1 / Chapter 1.1. --- Hepatocellular carcinoma --- p.2 / Chapter 1.1.1. --- Etiological factors --- p.11 / Chapter 1.1.1.1. --- Chronic Hepatitis and Liver Cirrhosis --- p.13 / Chapter 1.1.1.2. --- HBV --- p.13 / Chapter 1.1.1.3. --- HCV --- p.17 / Chapter 1.1.1.4. --- Male gender --- p.20 / Chapter 1.1.1.5. --- Aflatoxin B1 exposure --- p.21 / Chapter 1.2. --- Genomic abnormalities in HCC --- p.23 / Chapter 1.3. --- GEF-H1 --- p.24 / Chapter 1.4. --- RhoA --- p.26 / Chapter 1.5. --- Epithelial-Mesenchymal Transition (EMT) --- p.29 / Chapter 1.6. --- Aims of Thesis --- p.31 / Chapter Chapter 2: --- MATERIALS AND METHODS --- p.32 / Chapter 2.1. --- Materials --- p.33 / Chapter 2.1.1. --- Chemicals and Reagents --- p.33 / Chapter 2.1.2. --- Buffers --- p.35 / Chapter 2.1.3. --- Cell Culture --- p.37 / Chapter 2.1.4. --- Nucleic Acids --- p.38 / Chapter 2.1.5. --- Enzymes --- p.39 / Chapter 2.1.6. --- Equipments --- p.40 / Chapter 2.1.7. --- Kits --- p.41 / Chapter 2.1.8. --- Antibodies --- p.42 / Chapter 2.1.9. --- Software and Web Resources --- p.43 / Chapter 2.2. --- Fluorescence In Situ Hybridization (FISH) --- p.44 / Chapter 2.2.1. --- Probe Preparation --- p.44 / Chapter 2.2.1.1. --- Human Bacterial Artificial Chromosome (BAC) probe preparation --- p.44 / Chapter 2.2.1.2. --- Nick translation --- p.44 / Chapter 2.2.2. --- Hybridization --- p.45 / Chapter 2.3. --- Genomic DNA extraction --- p.47 / Chapter 2.4. --- Copy number analysis --- p.48 / Chapter 2.5. --- Exon Sequencing analysis --- p.49 / Chapter 2.5.1. --- PCR amplification of GEF-H1 exons --- p.49 / Chapter 2.5.2. --- Cycle sequencing --- p.49 / Chapter 2.6. --- Ectopic expression of GEF-H1 in immortalized hepatocyte cell line --- p.52 / Chapter 2.6.1. --- Construction of GEF-H1 expressing vector --- p.52 / Chapter 2.6.2. --- Sub-cloning --- p.52 / Chapter 2.6.3. --- Transfection and clonal selection --- p.53 / Chapter 2.7. --- Gene Expression Analysis by Quantitative RT-PCR --- p.55 / Chapter 2.7.1. --- Total RNA extraction --- p.55 / Chapter 2.7.2. --- qRT-PCR analysis for gene expression --- p.55 / Chapter 2.8. --- Western blot --- p.58 / Chapter 2.9. --- Functional Analysis --- p.60 / Chapter 2.9.1. --- Cell viability (MTT) assay --- p.60 / Chapter 2.9.2. --- Cell proliferation assays (BrdU-incorporation) --- p.60 / Chapter 2.9.3. --- Mitomycin C treatment --- p.61 / Chapter 2.9.4. --- Migration and Invasion assays --- p.63 / Chapter 2.9.5. --- Wound healing assay --- p.65 / Chapter 2.9.6. --- Transient knock-down of RhoA --- p.65 / Chapter 2. --- 10. Immuno-fluorescent imaging --- p.66 / Chapter 2. --- 11. In vivo tumorigenic study of GEF-H1 by subcutaneous injection --- p.68 / Chapter 2. --- 12. Statistical analysis --- p.69 / Chapter Chapter 3: --- RESULTS --- p.70 / Chapter 3.1. --- Verifying copy number gain of GEF-H1 in high GEF-H1 expressing HCC --- p.71 / Chapter 3.2. --- Verifying if there is any GEF-H1 exon point mutation in HCC --- p.75 / Chapter 3.3. --- Functional roles of GEF-H1 in HCC --- p.77 / Chapter 3.4. --- GEF-Hl-induced functions were RhoA independent --- p.83 / Chapter 3.5. --- GEF-H1 Induction of Epithelial-mesenchymal transition in HCC --- p.88 / Chapter 3.6. --- GEF-H1 induced tumorigenicity of MIHA cells --- p.95 / Chapter Chapter 4: --- DISCUSSIONS --- p.96 / Chapter 4.1. --- GEF-H1 in HCC and other cancers --- p.97 / Chapter 4.2. --- GEF-H1 promotes cell motility --- p.98 / Chapter 4.3. --- GEF-H1 induced tumorigenicity --- p.100 / Chapter Chapter 5: --- CONCLUSIONS AND PROPOSED FUTURE INVESTIGATIONS --- p.101 / Chapter Chapter 6: --- REFERENCES --- p.103

Introdução- A hiperglicemia (HG), durante o infarto agudo do miocárdio (IAM), está associada com aumento de mortalidade hospitalar em pacientes diabéticos e não diabéticos. Entretanto, não é conhecido o mecanismo responsável por esta associação. Assim estudou-se, simultaneamente, a correlação entre a glicemia e marcadores bioquímicos relacionados ao sistema neuro-humoral de estresse, metabolismo glicídico e lipídico, sistema de coagulação e inflamatório. Métodos- 80 pacientes foram incluídos consecutiva e prospectivamente. Foram realizadas duas coletas de sangue, a primeira com 24h a 48h do início dos sintomas do IAM (fase aguda) e a segunda após 3 meses do IAM (fase crônica), sempre com 12h de jejum. Foram analisados os seguintes parâmetros: glicose, cortisol, noradrenalina, hemoglobina glicada (HbA1c), insulina, LDL minimamente modificada eletronegativa, ácidos graxos livres (AGL), adiponectina, factor VII da coagulação, fibrinogênio, inibidor do ativação do plasminogênio tipo 1, proteína C reativa ultra-sensível (PCRus), colesterol total (c) e frações e triglicérides. Nas correlações univariadas entre glicemia e as variáveis contínuas empregou-se o teste de correlação de Pearson. As análises multivariadas foram feitas através de regressão logística (variáveis qualitativas) e modelo linear generalizado (quando as variáveis independentes incluídas foram quantitativas e nominais). Resultados- Na fase aguda, a glicemia correlacionou-se significativamente com HbA1c (r=0,75, p<0,001), insulina (r=0,25, p<0,001), AGL (r=0,3, p=0,01), adiponectina (r=-0,22, p=0,05), LDL-c (r=-0,25, p=0,03), VLDL-c (r=0,24, p=0,03) e triglicérides (r=0,27, p=0,01). No modelo multivariado, as variáveis correlacionadas de forma independente com a glicemia, na fase aguda, foram: HbA1c (p<0,001), insulina (p<0,001), e AGL (p=0,013). Para analisar uma variável de confusão, a história de diabetes mellitus (DM), incluiu-se esta variável num modelo, juntamente com as variáveis acima e todas mostraram associação significativas com glicose: HbA1c (p<0,001), insulina (p=0,001), AGL (p=0,013) e história de DM (p=0,027). Na fase crônica, glicose correlacionou-se com: cortisol (r=0,31, p=0,01), noradrenalina (r=0,54, p<0,001), HbA1c (r=0,78, p<0,001) e PCRus (r=0,46, p<0,001). Na análise multivariada, somente HbA1c (p<0,001) e noradrenalina (p<0,001) mantiveram correlação independente. Conclusão- A HbA1c foi a única variável que correlacionou-se de forma significativa e independente com a glicemia, tanto na fase aguda, quanto na crônica, mostrando que a hiperglicemia, durante o IAM, pode representar uma alteração crônica, sub-diagnosticada, do metabolismo glicídico. / Introduction- Hyperglycemia (HG) is an important prognostic factor in acute myocardial infarction (AMI). However, the pathophysiology is poorly understood. So we proposed a simultaneous correlation between glycemia and biochemical markers of stress, glucose and lipid metabolism, coagulation and inflammation system. Methods- Eighty AMI patients were included prospectively. Blood were collected between 24h and 48h from the pain (acute phase), and 3 months post AMI (chronic phase), with 12-h fasting. These parameters were analyzed: glucose, cortisol, norepinephrine, hemoglobin glycated (HbA1c), insulin, minimally modified electronegative LDL, free fatty acids (FFA), adiponectin, factor VII coagulant, fibrinogen, plasminogen activator inhibitor-1, high sensitive C reaction protein (hsCRP), total cholesterol (c) and fractions and triglyceride. The relationships between glucose and continuous variables were assessed by Pearsons correlation coefficient (r) and multivariate analysis with linear regression. Results- At acute phase, glucose correlated significantly with HbA1c (r=0.75, p<0.001), insulin (r=0.25, p<0.001), FFA (r=0.3, p=0.01), adiponectin (r=-0.22, p=0.05), LDL-c (r=-0.25, p=0.03), VLDL-c (r=0.24, p=0.03) and triglyceride (r=0.27, p=0.01). In a multivariate model, variables correlated were: HbA1c (p<0.001), insulin (p<0.001), and FFA (p=0.013). At the chronic phase, glucose correlated significantly with cortisol (r=0.31, p=0.01), norepinephrine (r=0.54, p<0.001), HbA1c (r=0.78, p<0.001) and hsCRP (r=0.46, p<0,001). By multivariable analysis, only HbA1c (p<0.001) and norepinephrine (p<0.001) remained correlated. Conclusion- HbA1c was the main variable that correlated significantly and independently with glycemia at acute and chronic phases, suggesting that HG during AMI can represent an exacerbation of abnormal glucose metabolism previously not diagnosed.

Alzheimer’s disease is the most common form of dementia among older adults, and directly contributes to the third leading cause of death in the United States. Although amyloid plaques and tau-loaded neurofibrillary tangles have been identified as the main pathological features of Alzheimer’s disease for more than one hundred years, the molecular mechanism is still poorly understood and treatments are limited to palliative care. Oligomeric Amyloid beta plays a crucial synaptotoxic role in Alzheimer’s disease, and hyperphosphorylated tau facilitates Amyloid beta toxicity, but the link between the two remains controversial. Since tau is a microtubule associated protein and microtubules are critical for neuronal functions, regulation of dynamic microtubules may serve as the link between Amyloid beta and tau. Here I propose a model in which Amyloid beta can induce changes in MT dynamics in dendrites and axons that are primary to tau hyperphosphorylation, while these MT changes are sufficient to cause tau hyperphosphorylation and necessary for Amyloid beta synaptotoxicity through tau. My thesis work further characterizes mammalian excitatory presynaptic boutons as hotspots for activity-dependent dynamic microtubule nucleation that is required for synaptic transmission during neuronal activation or Amyloid beta-induced neuronal injury through tau.

Little is known about the physiology or biochemistry of hypoxia (reduced levels of oxygen) tolerance during development in vertebrate embryos. In most species, relatively brief bouts of severe hypoxia are lethal or teratogenic. An exception to such hypoxia intolerance is the annual killifish Austrofundulus limnaeus, in which populations persist in hypoxic environments. This species inhabits seasonal ponds in Venezuela, surviving through the dry season in the form of diapausing embryos. Embedded in the pond sediment, embryos of A. limnaeus are routinely exposed to hypoxia and anoxia (lack of oxygen) as part of their normal development. Here, we exposed embryos to various levels of PO2 (21.2, 15.6, 10.8, 8.4, 6.1, 3.6, and 2.2 kPa) at two different temperatures (25°C and 30°C) to study the effects on developmental rate and heart rate. We also measured enzyme activity and quantified DNA content of individual embryos to compare differences among the varying levels of hypoxia and temperature. Hypoxia caused a significant decline in developmental rate and caused a stage-specific decline in heart rate. Higher temperature caused an increase in the developmental rate for those embryos incubated at PO2 of 6.1 kPa and greater. Temperature had a negative effect by hindering development below a PO2 of 6.1 kPa. Total embryonic DNA content was reduced at low partial pressures (15.6, 10.8, 8.4, 6.1, 3.6, and 2.2 kPa) of oxygen. Citrate synthase, lactate dehydrogenase, and phosphoenolpyruvate carboxykinase were all down-regulated indicating a complete lack of enzymatic metabolic compensation to combat reduced oxygen levels.

Embryos of the annual killifish Austrofundulus limnaeus display a remarkable tolerance to anoxia during development, most notably during embryonic diapause. Little is known about the metabolic or enzymatic changes that accompany this state of anoxia tolerance. This study examined the metabolic changes associated with exposure to anoxia by measuring the activity of the enzyme phosphoenolpyruvate carboxykinase (PEPCK), and by profiling the concentration of 31 metabolites ranging from amino acids to citric cycle intermediates at 4 different developmental stages, diapause 2 (DII), 4 days post diapause (dpd), 12 and 22 dpd. Embryos of A. limnaeus showed stage specific changes in concentrations of several metabolites. The most notable changes in metabolite concentration in response to anoxia were the increases of lactate, alanine, GABA and succinate as well as a pronounced decrease in aspartate concentrations. However, a complete understanding of the mechanisms by which anoxia tolerance is achieved remains elusive. Further studies into the tissue specific responses of anoxia would enable greater resolution when attempting to explain changes in concentrations of metabolites both during development and in response to anoxic insult.

Approximately 10% of cystic fibrosis (CF) cases remain undiagnosed until age 18 or later and medical professionals are still unclear about the needs of these individuals. Adults with CF iii must either adapt to life with a chronic disease or transition into adulthood with CF. The purpose of this study was to address the impact of age-at-diagnosis on quality of life in a CF adult population. The psychological functioning of 21 adult CF patients (seven adult-diagnosed (AD) and 14 pediatric-diagnosed (PD)) was assessed using the Beck Depression Index (BDI)-II. A descriptive review of demographic and clinical data, which included an assessment of the financial impact of active outpatient medications, was also conducted among patients in both groups. The incidence of depression among participants was relatively low and appeared independent of age-at-diagnosis. Differences between AD and PD patients with CF in terms of demographic and clinical parameters were also minimal. (96 pages).