Tissue inhibitors of metalloproteinases (TIMPs) comprise a family of four proteins in humans that modulate the turnover of the extracellular matrix by regulating the activities of endopeptidases that catalyze its degradation, especially the matrix metalloproteinases (MMP). In general, the four TIMPs are broad-spectrum tight binding inhibitors of MMPs with individual differences in specificity. In this study, we attempted to understand the basis of such variation by using membrane type-1 MMP (MT1-MMP) as a model, since it is inefficiently inhibited by TIMP-1 in contrast with the other TIMPs. We designed and engineered mutations in the N-domain of TIMP-1, based on current knowledge of TIMP interactions. By measuring inhibition levels of each mutant against several MMPs, including MT1-MMP, we were able to obtain a triple mutant with an vii improved affinity for MT1-MMP. / Our results, along with previous data, confirm that multiple residues in the critical interface segments between TIMPs and MMPs, namely at positions 2, 4, 5, 6, and 98, are key in determining the basic interaction between the two molecules. The second part of this work focused on naturally occurring mutations in TIMP-3 which cause an early form of macular degeneration called Sorsby's Fundus Dystrophy (SFD). The TIMP-3 mutants identified so far share certain features but the mechanism by which they result in macular disease is not yet understood. As an initial step, we expressed recombinant TIMP-3 carrying a truncation mutation, glutamic acid 139 to a stop codon (E139X), and assessed its activity towards representative MMPs and tumor necrosis factor-(Sa (Bconverting enzyme, another metalloproteinase normally inhibited by TIMP-3. Our results indicate that this mutation does not impair the inhibitory activity of TIMP-3. / Expression of this mutant in mammalian retinal cells revealed a difference in localization between wild-type and E139X mutant TIMP-3. Therefore, we concluded that the SFD mutations may actually influence the processing and/or binding properties of TIMP-3 in the retina. / by Asmaa Bilal Hamze. / Vita. / Thesis (Ph.D.)--Florida Atlantic University, 2008. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2008. Mode of access: World Wide Web.

Articular cartilage damage and degeneration is a siginficant clinical problem which no technique has been able to adequately and reliably repair or regenerate. Recent research has investigated the use of cell-based therapies to treat focal cartilage lesions. In clinical practice proliferated autologous chondrocytes are used and clinical trials are investigating the use of mesenchymal stem cells. The aim of this thesis was to assess aspects of current cell-based therapy and to investigate the potential of perivascular stem cells for articular cartilage repair. The phenotype of expanded matrix-applied autologous chondrocytes utilised in current cell therapies was confirmed using immunocytochemistry and polymerase chain reaction (PCR) expression of hyaluronan and proteoglycan link protein 1 (HAPLN1), transcription factor sox-9 (SOX9) and aggrecan (ACAN). Quantitative real-time PCR demonstrated that they were down-regulated for expression of COL2A1, SOX9 and ACAN but up-regulated for COL1A1 compared to unproliferated chondrocytes. Confocal laser-scanning microscopy (CLSM) demonstrated a significant decrease in cell viability and density when the membranes were subjected to levels of trauma similar to those that could be experienced during surgery. Hyperosmolar solutions did not confer a chondroprotective effect. Pericytes and adventitial cells, collectively termed perivascular stem cells (PSCs), from the infra-patellar fat pad were identified using immunohistochemistry and isolated using enzymatic digestion and fluorescence-activated cell sorting (FACS). Cell identity was ascertained using PCR, FACS and mesenchymal differentiation (osteogenesis, adipogenesis and chondrogenesis). Quantitative real-time PCR analysis of micromass cultures indicated that PSCs displayed increased chondrogenic potential compared to mesenchymal stem cells. An ovine model of perivascular stem cells was developed and a pilot study using three sheep was undertaken to confirm the viability of the model. Autologous ovine PSCs were isolated and re-implanted into articular cartilage defects. Green fluorescent protein transfected cells were identified in the cartilage defect four weeks following re-implantation using CLSM. This thesis has examined aspects of matrix-applied autologous chondrocyte implantation for cell based cartilage repair and has identified a new source of prospectively identified and purified stem cells that have demonstrated increased chondrogenic potential compared to mesenchymal stem cells, which are commonly used in clinical research. The methods to identify and purify ovine perivascular stem cells were developed to investigate the use of autologous PSCs and to track the cells following implantation.

Os receptores para mineralocorticoides (MR) e glicocorticoides (GR) representam importantes sítios de ligação para os glicocorticoides. Enquanto a ativação crônica e excessiva de GR leva à atrofia do hipocampo, a ativação do MR é neuroprotetora. Considerando as alterações no giro denteado (GD), decorrentes do envelhecimento, e a possível participação do MR e GR neste processo, o objetivo deste trabalho foi correlacionar o desbalanço de tais receptores com a degeneração neuronal nesta região do hipocampo de cães idosos. Para isso, utilizaram-se cortes histológicos do hipocampo de 6 cadáveres caninos jovens/adultos (até 8 anos de idade) e 12 idosos (idade igual ou superior a 10 anos), de ambos os sexos e de qualquer raça, os quais foram submetidos à: coloração de Nissl, para a identificação de células nervosas; imuno-histoquímica, para o estudo da expressão do MR e GR; e marcação fluorescente (fluoro-jade B), para detecção de neurônios em degeneração. A camada polimórfica do GD de cães idosos apresentou redução (em 16%) na contagem de neurônios e maior número de neurônios em degeneração. Em conjunto com estas alterações celulares, a menor marcação/expressão do MR e a menor relação MR:GR foram correlacionadas com a degeneração neuronal na camada polimórfica do GD de cães idosos. Além disso, cadelas idosas apresentaram menor densidade celular na camada granular, quando comparadas aos machos idosos, sugerindo uma ação diferencial dos esteroides sexuais nas alterações do GD. Os resultados indicam que o desbalanço na relação MR:GR pode interferir na sobrevivência neuronal no GD de cães idosos. / The mineralocorticoid (MR) and glucocorticoid (GR) receptors bind the glucocorticoid hormones. The chronic and excessive GR activation leads to hippocampus atrophy. By contrast, MR activation is neuroprotective. Considering the aging changes in the dentate gyrus (DG) and the possible participation of MR and GR in this process, the objective of this study was to correlate the unbalance of these receptors with the neuronal degeneration in this hippocampal region of aged dogs. For that purpose, cadaveric histologic sections of hippocampus of 6 young/adult dogs (until 8 years old) and 12 aged dogs (more than 10 years old), of both sex and any breed, were included. The Nissl staining and imunochemistry were performed to identify nerve cells and to study the MR and GR expression, respectively. Moreover, fluorescent labeling (fluoro- Jade B) was used to detect degenerating neurons. The polimorfic layer of the DG of aged dogs showed reduction (of 16%) on the neurons counting and more degenerating neurons. Together with this cells changes, the less MR expression and MR:GR relation were correlated with the neuronal degeneration in the polimorfic layer of DG of aged dogs. Besides that, aged females presented lower cell density in the granular layer, when compared to aged males, suggesting a differential sex steroid action on changes in the GD. Our results indicate that unbalance on the MR:GR relation may interfere with neuronal survival in the DG of aged dogs.

A Doença de Wilson (DW) é uma moléstia hereditária, caracterizada pela deficiência de excreção do cobre pelo fígado devido à mutação do gene A TP7B. O distúrbio do olfato ocorre com frequência em doenças neurodegenerativas como na doença de Parkinson (DP) e na doença de Alzheimer (DA). A análise do olfato tem sido utilizada como um instrumento útil no diagnóstico diferencial das diversas formas de parkinsonismo degenerativo, e, especialmente, na diferenciação entre DP e tremor essencial. O diagnóstico precoce na DW é a chave para o sucesso do tratamento. Na hipótese de haver comprometimento do olfato em fases iniciais da doença, esse poderia ser um dado a mais para auxiliar no diagnóstico. Até o presente, há apenas um estudo relacionando a DW com a disfunção do olfato. O objetivo deste estudo foi avaliar o olfato em um grupo de pacientes com DW e confrontar com grupo- controle. No presente estudo, foram analisados 37 portadores de DW com manifestação neurológica, 24 portadores de DW sem manifestação neurológica e 59 controles. Todos os indivíduos foram analisados com relação à idade, ao gênero, ao grau de escolaridade, ao uso de tabaco e ao miniexame do estado mental (MEEM), e os portadores de DW foram avaliados quanto ao tempo de doença, tratamento medicamentoso e escore neurológico. O olfato foi avaliado por meio do teste de identificação de odor 8niffin\' 8ticks (88-16 canetas numeradas e quatro opções de resposta para cada uma). Vinte e quatro indivíduos eram pacientes da DW sem manifestação neurológica (45,83% do gênero feminino) e 37 pacientes apresentavam manifestações neurológicas (56,76% do gênero masculino). O qrupo-controle foi composto por 59 indivíduos, 35 (59,33%) do gênero masculino. As médias de- idade foram de 33,38 ± 9,79 anos no grupo de portadores de DW com manifestação neurológica; 29 ± 9,61 anos no grupo de portadores de DW sem manifestação neurológica e 33,81 ± 10,67 anos no grupo-controle. Todos os pacientes com DW estavam em tratamento: 47(77%) com penicilamina, 7 (11,5%) com trientine e 7 (11,5%) com sais de zinco. As médias de respostas corretas no teste de identificação do odor 88-16 foram: 12,03 ± 2, 21 no grupo de portadores de DW com manifestação neurológica, 12, 15 ± 2,07 no grupo de portadores de DW com manifestação hepática e 12,70 ± 2,03 para o grupo- controle. Na avaliação objetiva do olfato com o teste de identificação do odor SS-16, não foi evidenciada diferença significativa entre os três grupos analisados, mas observou-se que o MEEM e o grau de escolaridade influenciaram significativamente no escore do 88-16 na comparação do grupo de pacientes com DW com manifestação neurológica com os outros dois grupos (grupo-controle e o grupo de portadores de DW com manifestação hepática). No presente estudo, não foi evidenciada disfunção olfatória nos pacientes com DW, mas foi observada diminuição da percepção do olfato em alguns pacientes com DW (com e sem manifestação neurológica). Em relação à disfunção olfatória evidenciada em alguns pacientes com DW na presente análise, algumas considerações são pertinentes e poderiam ter influenciado na identificação do olfato neste grupo de pacientes com DW. O acúmulo de cobre e a produção de radicais livres no sistema nervoso central (SNC) podem desencadear processos de neurodegeneração em estruturas envolvidas no olfato, alterações metabólicas, acúmulo de substâncias neurotóxicas (amônia e manganês) e alterações de neurotransmissores, e contribuir para o surgimento da disfunção olfatória / Wilson\'s disease (WO) is a hereditary disease due to a mutation in ATP7B gene, characterized by deficiency of copper excretion by the liver. Smell disorders are frequently encountered in neurodegenerative diseases, such as Parkinson\'s disease (PO) and Alzheimer\'s disease (AO). Smell analysis has been a useful tool for the differential diagnosis of several forms of degenerative parkinsonism, and especially for the differentiation between PO and essential tremor. Early diagnosis in WO is the key for a successful treatment. If there were smell impairment in the early stages of the illness, it could be used as another clue to help on its diagnosis. To the present date, there is only one study connecting WO with smell problems, the aim of this study was to evaluate smell function in a group of WO patients and compare them with a control group. We analyzed 37 WO patients with and 24 WO patients without neurologic symptoms, and 59 controls. Ali subjects were evaluated regarding age, gender, schooling, tobacco use, Mini Mental State Examination (MMSE), and the WO patients were also evaluated regarding duration of the illness, medication and neurologic scoring. Smell was analyzed by means of Sniffin\' Sticks smell identification test (SS-16 numbered pens and four options of answer for each pen). Twenty-four subjects with WO had no neurologic symptoms (45.83% female), and 37 patients had neurologic impairment (56,76% male). The control group was composed by 59 individuais, 35 (59,33%) male. Their age average were 33,38 ± 9,79 years for WO neurologic symptoms; 29 ± 9,61 years for WO without neurologic symptoms; and 33,81 ± 10,67 years for the control group. Ali WO patients were on treatment: 47(77%) with penicillamine, 7(11,5%) with trientine, and 7(11,5%) with zinc salt formulations. The average of correct answers in the SS-16 were: 12,03 ± 2,21 for the WO with neurologic symptoms group; 12,15 ± 2,07 for the WO without neurologic symptoms; and 12,70 ± 2,03 for the control group. In the smell testing with SS-16, there was no significant difference among the three groups, but the MMSE scoring and schooling had a significant influence over SS-16 score when comparing WO with neurologic symptoms patients with the other groups (WO patients without neurologic symptoms and control group). There was no smell dysfunction in WO patients in this study, but diminished smell perception was observed in some WO patients (either with or without neurologic impairment). Regarding smell impairment observed in some WO patients in the current analysis, some considerations must be made that could have influenced smell identification in these individuais. Copper accumulation and free radicais production in the central neNOUS system can trigger neurodegeneration processes in structures involved in srnell perception, metabolic impairment, building up of neurotoxic substances (such as ammonia and manganese), and neurotransmitter disorders, contributing to the emergence of srnell dysfunction

A doença de Wilson, moléstia hereditária, caracteriza-se pela deficiência de excreção de cobre pelo fígado, originária da mutação do gene ATP7B. As manifestações neurológicas na doença de Wilson são pleomórficas, observando-se distúrbios do movimento com início insidioso e em idade variável - geralmente na segunda ou terceira décadas de vida. Este estudo, dividido em duas partes, descreve as manifestações neurológicas iniciais em 119 pacientes com doença de Wilson (93 casos-índice e 26 familiares acometidos), avaliados entre 1963 e 2004 dos quais 109 foram através de análise retrospectiva dos prontuários médicos, enquanto aos 10 pacientes restantes se dispensou avaliação clínica prospectiva, a partir de 2002. O início dos sintomas ocorreu na média etária dos 19,4 anos (7-37), e o tempo médio do surgimento dos sintomas ao diagnóstico de 1,1 +/- 1,2 anos (0-5 anos). Entre as manifestações neurológicas mais freqüentes, observaram-se: disartria (91%), distúrbios da marcha (75%), risus sardonicus (72%), distonia (69%), rigidez (66%), tremor (60%) e disfagia (50%). A incidência das manifestações coréia e atetose, 16% e 14%, respectivamente, foi baixa. Manifestações atípicas incluíram convulsões (4,2%) e sinais piramidais (3%). A segunda parte do estudo trata da investigação do genótipo ATP7B em 41 pacientes e suas possíveis correlações com o fenótipo neurológico. Encontraram-se 23 mutações distintas, a mais comum das quais (p.A1135fs) com freqüência alélica de 31,7%. Expressiva associação (p<0,05) se deu entre essa mutação e a manifestação disfagia, ainda que limitada por amostra restrita de pacientes. Também sugestiva foi a associação entre a mutação p.A1135fs e quadros neurológicos precoces e graves. Este é o primeiro estudo a comparar o genótipo ATP7B com as manifestações neurológicas na doença de Wilson / Wilson disease, a rare inborn metabolic error, is characterized by deficient hepatic copper excretion, due to mutations in ATP7B gene. Neurological manifestations may vary, although there is commonly a movement disorder starting in the second or third decade of life. This study is divided in two parts, and it describes the neurological manifestations in 119 patients with Wilson disease (93 index cases and 26 affected family members), which were seen between 1963 and 2004 a retrospective analysis in 109 medical records and prospective clinical evaluation in 10 patients since 2002. The average age of symptoms onset was 19.4 years (ranging from 7 - 37 years), and the mean time between the first symptom and diagnosis was 1, 1 +/- 1, 2 years. The most frequent neurological manifestations observed were: dysarthria (91%), gait disturbance (75%), risus sardonicus (72%), dystonia (69%), rigidity (66%), tremor (60%), and dysphagia (50%). Less frequent manifestations were chorea (16%), and athetosis (14%). Rare neurological presentations were seizures (4,2%), and pyramidal signs (3%). In the second part of this study, we ascertain ATP7B genotype correlations with distinct neurological phenotypes in 41 Wilson disease patients. A total of 23 distinct mutations were detected, and the p.A1135fs frameshift had the highest allelic frequency (31.7%). An association between a p.A1135fs mutation and dysphagia was detected (p<0, 05), but the limited number of patients restricts valuable conclusions. This analysis also suggests an association between this mutation and early and severe neurological presentation. This present study is the first one to evaluate an ATP7B genotype correlation with specific neurological profile in Wilson disease

The term 'Retinitis pigmentosa' (RP) represents a group of inherited, late-onset diseases characterised by progressive retinal degeneration due to photoreceptor death. Mutations in the RP2 gene are found in 7-18% of patients with X-linked RP, one of the most severe forms. The RP2 gene product is a membrane-associated protein which encompasses two distinct domains. The N-terminal domain is well characterised as possessing GTPase-activating protein (GAP) activity towards the small GTPase ARL3 and thus regulate the transport of lipid-modified proteins within the photoreceptor cell. However, it is not known if the loss of this particular function of RP2 is the sole reason that causes the disease, while the role of the protein's C-terminus remains unknown. This thesis focuses on the characterisation of two novel protein-protein interactions of RP2 with the aim to investigate novel roles of the protein. Firstly, evidence is provided that a highly-conserved cluster of RP2 residues that span both the N- and C-terminus participate in direct interaction with Osteoclast-stimulating factor 1 (OSTF1). Two hypotheses are explored about the potential role of the complex in SRC-mediated RP2 phosphorylation and the regulation of cell motility. Secondly, the catalytic subunit of DNA-dependent protein kinase (DNA PK) is identified as a novel interaction partner of RP2 in cultured cells. The two proteins are shown to co-localise in the nuclear and membrane compartments of a retinal-derived cell line and might engage in a kinase-substrate relationship. So far, no evidence was found that RP2 participates in the canonical function of DNA PK which is the regulation of DNA double-stranded breaks. Finally, the CRISPR/Cas9 genome editing method was applied on zebrafish embryos to generate a novel vertebrate animal model for the loss of RP2 function. One out of three different zebrafish lines with rp2 mutations was shown by histology to have mild late-onset thinning of the photoreceptor outer segments. The present thesis reports previously unexplored aspects of RP2's function and will, therefore, contribute to understanding the molecular mechanisms that underlie RP. Moreover, this thesis will contribute to the discussion about the usefulness of zebrafish as an RP model.

多聚谷氨酰胺疾病 (Polyglutamine diseases) 是一類在各自的致病基因編碼區的CAG重複編碼擴張造成的顯性遺傳神經退退化疾病。已擴大的CAG訊息核醣核酸 (Expanded CAG transcripts) 在多聚谷氨酰胺蛋白疾病作出細胞毒性作用。從基因減弱篩查中，我發現U2AF50能修飾已擴大的CAG訊息核醣核酸的毒性。並發現U2AF50能與已擴大的CAG訊息核醣核酸作實體互動，能參與已擴大的CAG訊息核醣核酸的核出口 (Nuclear export)。U2AF50的基因減弱增強已擴大CAG訊息核醣核酸在細胞核的累積和毒性。這突出核醣核酸的核出口在多聚谷氨酰胺疾病的重要性，並暗示細胞核是已擴大的CAG訊息核醣核酸毒性的起源地。此外，我鑑定已擴大的CAG訊息核醣核酸在亞細胞的分佈，並發現它們特別累積在核仁 (Nucleolus) 內。核仁是核糖體核醣核酸（rRNA）的轉錄場所。我發現已擴大的CAG訊息核醣核酸減弱rRNA基因啟動子 (rRNA promoter) 的活性並且抑制核糖體核醣核酸的轉錄。 核糖體核醣核酸基因轉錄的抑制，促進核糖體蛋白RpL23和E3連接酶MDM2蛋白作實體互動，從而增強p53的穩定性導。穩定的p53能夠轉移至線粒體 (Mitochondria)。我還發現，線粒體內的p53能打亂Bcl-xL與 Bak的實體互動，導致細胞色素C釋放到細胞質，這導致凋亡蛋白酶 (Caspases) 的活化和細胞凋亡。我的研究，首次證明核仁參與在多聚谷氨酰胺疾病的發病機制中，揭示了在多聚谷氨酰胺疾病中的新致病機制。 / Polyglutamine (polyQ) diseases are a class of dominantly inherited neurodegenerative disorders caused by the expansion of CAG-repeat encoding glutamine within the coding region of the respective disease genes. Expanded CAG transcripts have been reported to contribute to cytotoxicity in polyQ diseases. From a candidate gene knockdown screen, I identified U2AF50 as a modifier of RNA toxicity. U2AF50 has been reported to be involved in RNA nuclear export, and I showed that it interacted specifically with expanded CAG transcripts. Knockdown of U2AF50 expression enhanced nuclear accumulation of expanded CAG transcripts and neurotoxicity. This part of my work highlights the role of RNA nuclear export in polyQ degeneration and implies that the nucleus is a major site for RNA toxicity. In addition, I determined the subcellular distribution of expanded CAG transcripts and found that they particularly localized in the nucleolus. The nucleolus is a critical sub-nuclear compartment for ribosomal RNA (rRNA) transcription. I discovered that expanded CAG transcripts in nucleolus inhibited rRNA transcription by inactivating the rRNA gene promoter activity. Inhibition of rRNA transcription promoted the interaction between ribosomal protein L23 and the ubiquitin E3 ligase MDM2, which led to the stabilization of p53 and its accumulation in mitochondria. I also found that mitochondrial p53 disrupted the interaction between the anti-apoptotic protein, Bcl-xL, and pro-apoptotic protein, Bak, subsequently causing Cytochrome c release, caspase activation, and apoptosis. In summary, my study first describes the involvement of nucleolar function in polyQ pathogenesis and uncovers a new pathogenic mechanism in polyQ diseases. / Detailed summary in vernacular field only. / Tsoi, Ho. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 220-228). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Thesis Committee --- p.ii / Declaration --- p.iii / Acknowledgement --- p.iv / Abstract --- p.v / Abstract in Chinese --- p.vii / List of Abbreviations --- p.viii / List of Figures --- p.x / List of Tables --- p.xvi / Table of Contents --- p.xvi / Chapter 1 --- Introduction / Chapter 1.1 --- Introduction to Polyglutamine Diseases --- p.1 / Chapter 1.1.1 --- Etiology of Polyglutamine Diseases --- p.1 / Chapter 1.1.2 --- Common Features of Different Types of Polyglutamine Disease --- p.1 / Chapter 1.2 --- Pathogenic Mechanisms of Expanded Polyglutamine Proteins --- p.4 / Chapter 1.2.1 --- Pathogenesis of Polyglutamine Diseases --- p.4 / Chapter 1.2.1.1 --- Loss-of-function toxicity --- p.4 / Chapter 1.2.1.2 --- Gain-of-function toxicity --- p.4 / Chapter 1.3 --- Expanded CAG Transcript-mediated Pathogenic Mechanism --- p.6 / Chapter 1.3.1 --- Identification of the Toxic Role of Expanded CAG Transcripts --- p.6 / Chapter 1.3.2 --- Nuclear Foci Formation of Expanded CAG Transcripts and Polyglutamine Pathogenesis --- p.8 / Chapter 1.4 --- Receptor-mediated RNA nuclear export Transport --- p.9 / Chapter 1.4.1 --- Introduction to RNA Nuclear Export --- p.9 / Chapter 1.4.2 --- Regulation of RNA Nucleocytoplasmic Transport and Human Diseases --- p.11 / Chapter 1.5 --- Function of Nucleolus --- p.12 / Chapter 1.5.1 --- Ribosomal RNA Transcription --- p.12 / Chapter 1.5.2 --- Nucleolar Stress and Apoptosis --- p.15 / Chapter 1.6 --- Research Plan --- p.17 / Chapter 1.6.1 --- Project Objective --- p.17 / Chapter 1.6.2 --- Experimental Model --- p.17 / Chapter 1.6.2.1 --- In vivo Drosophila Model --- p.17 / Chapter 1.6.2.2 --- In vitro Cell Culture Model --- p.19 / Chapter 1.6.2.3 --- Transgenic Mouse Model --- p.20 / Chapter 1.6.3 --- Significance of the Present Study --- p.21 / Chapter 2 --- Materials and Methods / Chapter 2.1 --- Molecular Cloning --- p.22 / Chapter 2.1.1 --- Polymerase Chain Reaction (PCR) --- p.22 / Chapter 2.1.2 --- Primers Used for PCR --- p.29 / Chapter 2.1.3 --- Restriction Digestion --- p.31 / Chapter 2.1.4 --- Agarose Gel Electrophoresis --- p.32 / Chapter 2.1.5 --- Preparation of genomic DNA from A Single Adult Fly --- p.34 / Chapter 2.1.6 --- Removal of 5' Phosphate Groups on Linearized Plasmids --- p.35 / Chapter 2.1.7 --- Addition of 5' Phosphate Group to Linker Oligonucleotides --- p.35 / Chapter 2.1.8 --- Ligation Reaction --- p.37 / Chapter 2.1.9 --- Bacterial Transformation --- p.37 / Chapter 2.2 --- Mammalian Cell Culture --- p.40 / Chapter 2.3 --- Drosophila Culture --- p.44 / Chapter 2.4 --- Semi-quantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.48 / Chapter 2.5 --- Microscopy --- p.51 / Chapter 2.6 --- Protein Sample Preparation and Concentration Measurement --- p.53 / Chapter 2.7 --- Co-immunoprecipitation --- p.57 / Chapter 2.8 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Immunoblotting --- p.62 / Chapter 2.9 --- Bacterial Protein Purification --- p.65 / Chapter 2.1 --- DNA Methylation Assay --- p.68 / Chapter 2.11 --- Mitochondrial Fraction Isolation --- p.79 / Chapter 3 --- U2 Small Nuclear Riboprotein Auxiliary Factor 50 Modulates Polyglutamine Diseases Toxicity by Altering the Subcellular Localization of Expanded CAG Transcripts in vivo / Chapter 3.1 --- The Nuclear Accumulation of Expanded CAG Transcripts Correlates with the Neurodegeneration in vivo --- p.72 / Chapter 3.1.1 --- Expanded CAG Transcripts Predominantly Localize in the Nucleus in Drosophila Model of Machado-Joseph Disease --- p.72 / Chapter 3.1.2 --- Nuclear Accumulation of Expanded CAG Transcripts Correlates with the Neurodegeneration in an Inducible Model of Machado-Joseph Disease --- p.73 / Chapter 3.1.3 --- Nuclear Accumulation of Expanded CAG Transcripts Correlates with the Neurodegeneration in Inducible DsRed[subscript CAG100] Model. --- p.76 / Chapter 3.1.3.1 --- Expanded CAG Transcripts Induce the Expression of Pro-apoptotic Genes --- p.77 / Chapter 3.1.3.2 --- Co-expression of p35 Suppresses the Toxicity Induced by the Expanded CAG Transcripts --- p.80 / Chapter 3.2 --- A Candidate-gene RNA Interference Approach was Employed to Identify Genetic Factors Involved in Nuclear Export of Expanded CAG Transcripts --- p.80 / Chapter 3.3 --- Confirmation of the Modulatory Effect of U2 Small Nuclear Riboprotein Auxiliary Factor 50 on Machado-Joseph Disease in vivo --- p.84 / Chapter 3.4 --- The Modulatory Effect of U2 Small Nuclear Riboprotein Auxiliary Factor 50 on Different Drosophila Models of Polygultamine Diseases --- p.84 / Chapter 3.5 --- U2 Small Nuclear Riboprotein Auxiliary Factor 50 Specifically Modulates Expanded CAG Transcript-induced Toxicity in vivo --- p.87 / Chapter 3.5.1 --- Knockdown of U2 Small Nuclear Riboprotein Auxiliary Factor 50 Enhances Expanded CAG Transcript-induced Toxicity --- p.87 / Chapter 3.5.2 --- Knockdown of U2 Small Nuclear Riboprotein Auxiliary Factor 50 Does Not Modulate Expanded PolyQ Protein-induced Toxicity --- p.89 / Chapter 3.5.3 --- Knockdown of U2 Small Nuclear Riboprotein Auxiliary Factor 50 Does Not Alter the Expression Level of Expanded CAG Transcripts in vivo --- p.89 / Chapter 3.5.4 --- Knockdown of U2 Small Nuclear Riboprotein Auxiliary Factor 50 Does Not Modulate the Toxicity in Fragile X syndrome in vivo --- p.91 / Chapter 3.6 --- Over-expression of Human U2 Small Nuclear Riboprotein Auxiliary Factor 65 Does Not Modulate Expanded CAG Transcript-induced Toxicity in Drosophila --- p.91 / Chapter 3.7 --- Expanded CAG Transcripts Does Not Compromise Endogenous Function of U2 Small Nuclear Riboprotein Auxiliary Factor 50 --- p.94 / Chapter 3.8 --- A Correlation between Nucleocytoplasmic Localization of Expanded CAG Transcripts and Its Induced Toxicity --- p.97 / Chapter 3.8.1 --- Knockdown of U2 Small Nuclear Riboprotein Auxiliary Factor 50 Enriched DsRedCAG100 Transcripts in the Nucleus in vivo --- p.99 / Chapter 3.8.2 --- Knockdown of U2 Small Nuclear Riboprotein Auxiliary Factor 50 Enriched MJDCAG78 Transcripts in the Nucleus in vivo --- p.99 / Chapter 3.9 --- Expanded CAG-repeat on the Transcripts Interact with U2 Small Nuclear Riboprotein Auxiliary Factor 50/65 in vivo and in vitro --- p.102 / Chapter 3.9.1 --- Expanded CAG Transcripts Interact with U2 Small Nuclear Riboprotein Auxiliary Factor 50 in vivo --- p.102 / Chapter 3.9.2 --- Expanded CAG Transcripts Interact with U2 Small Nuclear Riboprotein Auxiliary Factor 65 in vitro --- p.103 / Chapter 3.9.3 --- Expanded CAG Transcripts Directly Interact with U2 Small Nuclear Riboprotein Auxiliary Factor 65 in vitro --- p.103 / Chapter 3.10 --- Identification of Expanded CAG Transcripts Interacting Domain on U2 Small Nuclear Riboprotein Auxiliary Factor 65 --- p.107 / Chapter 3.10.1 --- Generation of Different Myc-tagged U2 Small Nuclear Riboprotein Auxiliary Factor 65 Expression Constructs --- p.107 / Chapter 3.10.2 --- RNA Recognition Motif 3 on U2 Small Nuclear Riboprotein Auxiliary Factor 65 Is Essential for the Interaction with Expanded CAG Transcripts --- p.109 / Chapter 3.11 --- Nuclear RNA Export Factor 1 is Involved in U2 Small Nuclear Riboprotein Auxiliary Factor 65-mediated Nuclear Export of Expanded CAG Transcripts --- p.113 / Chapter 3.11.1 --- The Effect of Full Length U2 Small Nuclear Riboprotein Auxiliary Factor 65 and its Corresponding Deletion Mutants on Nuclear Export of Expanded CAG Transcripts --- p.113 / Chapter 3.11.2 --- Formation of Complexes Composed of Nuclear RNA Export Factor 1/U2 Small Nuclear Riboprotein Auxiliary Factor 65/Expanded CAG Transcripts in HEK293 Cells --- p.115 / Chapter 3.12 --- The Nuclear Export of Expanded CAG Transcripts is Mediated by U2 Small Nuclear Riboprotein Auxiliary Factor 65 and Nuclear RNA Export Factor 1 --- p.120 / Chapter 3.13 --- Aging Compromises the Nuclear Export of Expanded CAG Transcripts in Polyglutamine Disease Mouse Model --- p.123 / Chapter 3.13.1 --- Expanded CAG Transcripts Accumulate in the Nucleus of Polyglutamine Disease Mouse Model --- p.123 / Chapter 3.13.2 --- Expression Level of U2 Small Nuclear Riboprotein Auxiliary Factor 65 Declines with Age in Mice --- p.124 / Chapter 3.14 --- Discussion --- p.127 / Chapter 3.14.1 --- Expanded CAG Transcripts Induce Nuclear Toxicity through a Mechanism Independent on Pathogenic Mechanism Mediated by Other Trinucleotide Repeats Expansion --- p.127 / Chapter 3.14.2 --- Nuclear Accumulation of Expanded CAG Transcripts Leads to Neurodegeneration --- p.128 / Chapter 3.14.3 --- U2 Small Nuclear Riboprotein Auxiliary Factor 50 Modulates Expanded CAG Transcript-induced Toxicity by Mediating the Subcellular Localization of Expanded CAG Transcripts --- p.129 / Chapter 3.14.4 --- U2 Small Nuclear Riboprotein Auxiliary Factor 65 and Nuclear RNA Export Factor 1 Regulate the Nuclear Export of Expanded CAG Transcripts --- p.130 / Chapter 3.14.4.1 --- U2 Small Nuclear Riboprotein Auxiliary Factor 50/65 Interacts with Expanded CAG Transcripts and Mediates the Subcellular localization of Expanded CAG Transcripts --- p.130 / Chapter 3.14.4.2 --- U2 Small Nuclear Riboprotein Auxiliary Factor 65 Requires Nuclear RNA Export Factor 1 to Mediate the Nuclear Export of Expanded CAG Transcripts --- p.131 / Chapter 3.14.4.3 --- Developmental Decline of U2 Small Nuclear Riboprotein Auxiliary Factor 65 Protein Level is a Factor That Leads to Progressive Neurodegeneration in Polyglutamine Diseases --- p.134 / Chapter 4 --- Expanded CAG Transcripts Induce Nucleolar Stress / Chapter 4.1 --- Expanded CAG-repeat Sequence Mediates the Nucleolar Localization of RNA Transcripts in vitro --- p.135 / Chapter 4.1.1 --- Machado-Joseph Disease Cell Model --- p.135 / Chapter 4.1.2 --- EGFPCAG Cell Model --- p.137 / Chapter 4.2 --- Expanded CAG Transcripts Suppress Nucleolar Function in vitro and in vivo --- p.140 / Chapter 4.2.1 --- Expanded CAG Transcripts Suppress Ribosomal RNA Transcription in vivo --- p.140 / Chapter 4.2.1.1 --- Drosophila Model of Machado-Joseph Disease --- p.140 / Chapter 4.2.1.2 --- Drosophila Model of DsRedCAG --- p.142 / Chapter 4.2.1.3 --- Transgenic Mouse Model of PolyQ Disease --- p.142 / Chapter 4.2.2 --- Expanded CAG Transcripts Suppress rRNA Transcription in vitro --- p.145 / Chapter 4.2.2.1 --- Machado-Joseph Disease Patient-derived Fibroblast Cell Lines --- p.145 / Chapter 4.2.2.2 --- Expanded CAG Transcript-expressing HEK293 Cells --- p.145 / Chapter 4.3 --- Expanded CAG Transcripts Disrupt the Interaction between RNA Polymerase I and rRNA Promoter in vitro --- p.148 / Chapter 4.4 --- Expanded CAG Transcripts Disrupt the Interaction between Upstream Binding Factor and Upstream Control Element in vitro and in vivo --- p.149 / Chapter 4.4.1 --- Expanded CAG Transcripts Compromise the Interaction between Upstream Binding Factor and Upstream Control Element in vitro --- p.149 / Chapter 4.4.2 --- Expanded CAG Transcripts Compromise the Interaction between Upstream Binding Factor and Upstream Control Element in vivo --- p.151 / Chapter 4.5 --- Expanded CAG Transcripts Induce DNA Hyper-methylation on Upstream Control Element in vitro and in vivo --- p.151 / Chapter 4.5.1 --- The HpaII-PCR Assay for DNA Methylation --- p.154 / Chapter 4.5.2 --- Expanded CAG Transcripts Lead to DNA Hyper-methylation of Upstream Control Element in vitro --- p.154 / Chapter 4.5.2.1 --- Expanded CAG Transcript-expressing HEK293 Cells --- p.154 / Chapter 4.5.2.2 --- Machado-Joseph Disease Patient-derived Fibroblast Cell Lines --- p.156 / Chapter 4.5.3 --- Expanded CAG Transcripts Lead to DNA Hyper-methylation of Upstream Control Element in vivo --- p.156 / Chapter 4.5.4 --- Expanded CAG Transcripts Disrupt the Regulatory Mechanism of Upstream Control Element Methylation in vitro --- p.159 / Chapter 4.6 --- Expanded CAG Transcripts Induce Nucleolar Stress and Apoptosis --- p.161 / Chapter 4.6.1 --- Expanded CAG Transcripts Induce Nucleolar Stress in vitro and in vivo --- p.162 / Chapter 4.6.1.1 --- Expanded CAG Transcript-expressing HEK293 Cells --- p.162 / Chapter 4.6.1.2 --- Transgenic Mouse Model of PolyQ Disease --- p.162 / Chapter 4.6.2 --- Expanded CAG Transcripts Lead to Stabilization of p53 in vitro and in vivo --- p.165 / Chapter 4.6.2.1 --- Expanded CAG Transcripts Lead to Stabilization of p53 in vitro --- p.165 / Chapter 4.6.2.2 --- Expanded CAG Transcripts Lead to Stabilization of p53 in vivo --- p.167 / Chapter 4.6.3 --- Expanded CAG Transcripts Enrich p53 in Mitochondria in vitro --- p.167 / Chapter 4.6.4 --- Expanded CAG Transcripts Lead to Disruption of interaction between Bcl-xL and Bak by p53 in mitochondria in vitro --- p.169 / Chapter 4.6.5 --- Expanded CAG Transcripts Lead to Release of Cytochrome c in vitro --- p.171 / Chapter 4.6.6 --- Expanded CAG Transcripts Lead to Activation of Caspase 3 in vitro --- p.173 / Chapter 4.7 --- Discussion --- p.176 / Chapter 4.7.1 --- Expanded CAG Transcripts Compromise Nucleolar Function --- p.176 / Chapter 4.7.2 --- Expanded CAG Transcripts Induce Apoptosis via Nucleolar Stress --- p.176 / Chapter 4.7.3 --- The Origin of Nucleolar Stress Induced by Expanded CAG Transcripts --- p.178 / Chapter 5 --- Expanded CAG Transcripts Interact with Nucleolin and Deplete It from Upstream Control Element to Suppress Ribosomal RNA Transcription / Chapter 5.1 --- Nucleolin is an Interacting Partner of Expanded CAG Transcripts --- p.180 / Chapter 5.1.1 --- Nucleolin is Pulled down by S1-tagged Expanded CAG Transcripts in vitro --- p.180 / Chapter 5.1.2 --- Expanded CAG Transcripts Interact with Endogenous Nucleolin in vitro --- p.181 / Chapter 5.1.3 --- Expanded CAG Transcripts Directly Interact with Nucleolin in vitro --- p.184 / Chapter 5.2 --- RNA Recognition Motifs 2 and 3 on Nucleolin Interact with Expanded CAG Transcripts --- p.184 / Chapter 5.2.1 --- Generation of Expression Constructs Carrying Full Length Nucleolin and its Deletion Mutants --- p.184 / Chapter 5.2.2 --- Mapping of Domains on Nucleolin Required for Interacting with Expanded CAG Transcripts --- p.187 / Chapter 5.3 --- Nucleolin Regulates Ribosomal RNA Transcription by Mediating the DNA Methylation of Upstream Control Element in HEK293 Cells --- p.187 / Chapter 5.3.1 --- Nucleolin is involved in Regulating the Interaction between Upstream Binding Factor and Upstream Control Element in vitro --- p.191 / Chapter 5.3.2 --- Nucleolin is Involved in Regulating DNA Methylation Level of Upstream Control Element in vitro --- p.191 / Chapter 5.3.3 --- Nucleolin Associates with Upstream Control Element in vitro --- p.194 / Chapter 5.4 --- Expanded CAG Transcripts Deplete Nucleolin from Upstream Control Element in vitro and in vivo --- p.194 / Chapter 5.4.1 --- Expanded CAG Transcripts Compete Nucleolin with Upstream Control Element in vitro --- p.197 / Chapter 5.4.2 --- Expanded CAG Transcripts Compete Nucleolin with Upstream Control Element in vivo --- p.197 / Chapter 5.4.3 --- Expanded Polyglutamine Proteins does not Interact with Nucleolin in vitro --- p.200 / Chapter 5.5 --- Over-expression of Nucleolin Counteracts the Effect of Expanded CAG Transcripts on Ribosomal RNA Transcription in vitro --- p.200 / Chapter 5.5.1 --- Over-expression of Nucleolin Restores the Methylation Level of Upstream Control Element in Dose-dependent Manner in vitro --- p.200 / Chapter 5.5.1.1 --- The Dosage Effect of Nucleolin on DNA Hyper-methylation of Upstream Control Element Induced by Expanded CAG Transcripts in vitro --- p.202 / Chapter 5.5.1.2 --- Does-dependent Expression of Nucleolin in vitro --- p.202 / Chapter 5.5.1.3 --- The Effect of Nucleolin Over-expression on DNA Hyper-methylation of Upstream Control Element Induced by Expanded CAG Transcripts is Dose-dependent in HEK293 cells --- p.205 / Chapter 5.5.2 --- Over-expression of Nucleolin Does Not Alter the Expression Level of Expanded CAG Transcripts in vitro --- p.205 / Chapter 5.5.3 --- Over-expression of Nucleolin Relieves the Nucleolar Stress induced by Expanded CAG Transcripts in vitro --- p.208 / Chapter 5.6 --- Discussion --- p.212 / Chapter 5.6.1 --- The Physical Interaction between Expanded CAG Transcripts and Nucleolin Leads to Suppression of Ribosomal RNA Transcription --- p.212 / Chapter 5.6.2 --- Expanded CAG Transcripts Deprive Upstream Control Element of Nucleolin to Induce Toxicity --- p.212 / Chapter 5.6.3 --- Nucleolin Suppresses Expanded CAG Transcript-induced Cell Death --- p.213 / Chapter 5.6.4 --- Expanded CAG Transcripts Employ both p53-dependent and p53-independent pathways to Induce Cell Death --- p.214 / Chapter 6 --- Concluding Remarks --- p.216 / References --- p.220

Retinal hypoxia and inadequate oxygen delivery have been implicated as causal for the development of several eye diseases, including diabetic retinopathy, glaucoma, and retinopathy of prematurity. The imaging of oxygen tension in the retina, generated from a measure of the phosphorescence lifetimes of bolus-injected palladium-porphyrin probes, has been used successfully to study retinal oxygen dynamics in numerous animal models. However, the specific parameters for applying this technique in the mouse have not been thoroughly investigated. The goals of this project were to calibrate a newly-constructed phosphorescence lifetime imaging instrument and data analysis software against known oxygen concentrations, to determine specific parameters for probe excitation and image collection and analysis in the mouse eye, and to assess any damage caused to the eye by the technique using histological analysis. An in vitro system was developed for calibration of the probe and for estimation of power of excitation light and camera settings necessary to produce acceptable oxygen maps. In vivo experiments were then performed, and plots indicating camera settings necessary for producing varying qualities of oxygen maps were constructed. Trypsin digestion of retinal tissue was used in an attempt to assess any damage to experimental subjects, but this histological technique was deemed inadequate for analyzing the capillary structures of the mouse eye. Alternatively, damage was assessed using the instrument itself to calculate changes in oxygen tension during the experimental process. The results of this work will allow the phosphorescence lifetime imaging system to be used in the mouse to study how changes in retinal oxygen tension correlate with the progression of eye diseases where oxygen is implicated, including diabetic retinopathy.

INTRODUCTION: Retinal Pigment Epithelia (RPE) degeneration is a key event in the development of age-related macular degeneration (AMD). RPE dysfunction in AMD is thought to occur through the accumulation of reactive oxygen species (ROS) and oxidative damage. The transcriptional co-activators, PGC-1α and PGC-1β, are important regulators of mitochondrial biogenesis and anti-oxidant capacity. Our group has previously shown that the PGC-1α protein promotes RPE oxidative metabolism and that overexpression of the PGC-1α gene protects cells from AMD-associated pro-oxidants. On the other hand, PGC-1β gene expression has been found to be upregulated in patients with neovascular AMD, and in-vitro overexpression of PGC-1β damages cells and induces pro-oxidant conditions. OBJECTIVE: Given the divergence of PGC-1α and PGC-1β functions in RPE and their clinical relevance in AMD pathogenesis, this project will seek to investigate the impact of the upregulation of PGC-1α and PGC-1β in RPE metabolism. PGC-1α will be upregulated through treatment with compound ZLN005. A new methodology for PGC-1β expression will be developed to closely modulate in-vitro PGC-1β induction. METHODS: In-vitro experiments were performed on the ARPE-19 cell line. Cells were treated with 10µM of ZLN005 for 24 hours. Oxidative stress was induced by exposure to H2O2 and NaIO3 under serum-free conditions. Lactate dehydrogenase (LDH) levels were used to quantify cell death. Quantitative PCR (qPCR) and Western Blot were performed to measure changes in gene and protein expression respectively. Superoxide production by the mitochondria was measured to evaluate ROS levels within the cell. Intravitreal injections of 20µM ZLN005 were performed on eight-week old male C57BL/6J mice. After 24 and 72 hours of treatment, the mice were euthanized and the enucleated eyes were dissected to obtain the RPE and neural retina layers. Total RNA was extracted from these layers and qPCR was performed to measure gene expression. A tetracycline-inducible PGC-1β plasmid was designed and transfected into ARPE-19 cells. The cells were exposed to 0.01-100µg/ml doxycycline for 48-hours and qPCR was used to measure gene expression. Transfected cells were treated with ZLN005 and cell death upon exposure to oxidative stress was quantified. RESULTS: Gene expression analysis on ARPE-19 cells treated with ZLN005 showed robust upregulation of PGC-1α, PGC-1β and their associated transcription factors and enzymes. Induction of PGC-1α at the protein level was also confirmed. ZLN005 efficiently protected ARPE-19 cells from H2O2 and NaIO3 cytotoxicity and its protection was negated in PGC-1α-silenced cells. Treatment with ZLN005 also decreased mitochondrial superoxide production. ZLN005 intravitreal injections were safely administered to the animals and did not cause cataracts or other damage to the ocular tissues. While statistical significance in gene expression changes was limited due to the small sample size, anti-oxidants GPX1 and TXN2, and electron transport chain gene, ATP50, were found to be potentially induced in the neuro-retina, while FOXO3 was found to be downregulated. Evaluation of our novel tetracycline-inducible PGC-1β adenoviral vector showed that upregulation of PGC-1β was efficiently controlled by the addition of doxycycline to transfected cells. Upon exposure to H2O2, transfected cells treated with doxycycline experienced greater cell death than transfected cells not exposed to doxycycline. ZLN005 treatment was able to decrease cell death in both conditions. CONCLUSION: The present study shows that ZLN005 efficiently protects RPE cells from oxidative damage through selective induction of PGC-1α. While still preliminary, the in-vivo study indicates that ZLN005 is safe to be injected into the eye and may be able to increase the expression of mito-protective and anti-oxidant genes in the neuronal retina. In addition, our design of the tetracycline inducible PGC-1β plasmid allows for tight control over PGC-1β expression through doxycycline addition. Upregulation of PGC-1β at levels similar to those observed in clinical conditions caused increased pro-oxidant induced cell death and treatment with ZLN005 was able to protect against cell death. / 2021-06-30T00:00:00Z

Genome-wide association studies (GWAS) have delivered many novel insights about the etiology of many common heritable diseases. However, in most disorders studied by GWAS, the known single nucleotide polymorphisms (SNPs) associated with the disease do not account for a large portion of the genetic factors underlying the condition. This suggests that many of the undiscovered variants contributing to the risk of common diseases have weak effects or are relatively rare. This thesis introduces novel adaptations of techniques for improving detection power for both of these types of risk variants, and reports the results of analyses applying these methods to real datasets for common diseases. Chapter 2 describes a novel approach to improve the detection of weak-effect risk variants that is based on an adaptive sampling technique known as Distilled Sensing (DS). This procedure entails utilization of a portion of the total sample to exclude from consideration regions of the genome where there is no evidence of genetic association, and then testing for association with a greatly reduced number of variants in the remaining sample. Application of the method to simulated data sets and GWAS data from studies of age-related macular degeneration (AMD) demonstrated that, in many situations, DS can have superior power over traditional meta-analysis techniques to detect weak-effect loci. Chapter 3 describes an innovative pipeline to screen for rare variants in next generation sequencing (NGS) data. Since rare variants, by definition, are likely to be present in only a few individuals even in large samples, efficient methods to screen for rare causal variants are critical for advancing the utility of NGS technology. Application of our approach, which uses family-based data to identify candidate rare variants that could explain aggregation of disease in some pedigrees, resulted in the discovery of novel protein-coding variants linked to increased risk for Alzheimer's disease (AD) in African Americans. The techniques presented in this thesis address different aspects of the "missing heritability" problem and offer efficient approaches to discover novel risk variants, and thereby facilitate development of a more complete picture of genetic risk for common diseases.