Os melanomas correspondem a 7% de todas as neoplasias malignas em cães e são principalmente encontrados em cavidade oral e lábios, correspondendo a 33% dos tumores de boca, possuem um prognóstico ruim devido ao fato de serem diagnosticados tardiamente, por sua grande capacidade de invasão local e formação de metástases, além de altas taxas de recidiva após o tratamento cirúrgico. A Galectina-3 (Gal-3) é uma proteína responsável por diversas funções fisiológicas como adesão, apoptose, angiogênese, proliferação e diferenciação. Em medicina veterinária existem poucos estudos relacionando à expressão da Gal-3 com prognóstico e a progressão da neoplasia. Realizamos imuno-histoquímica para Gal-3 em 27 melanomas orais caninos que foram avaliados de maneira semiquantitativa e quantitativa, e comparamos os resultados obtidos com a sobrevida, outros marcadores prognósticos (Ki67, índice mitótico e atipia nuclear), expressão de proteínas relacionadas à apoptose (BCL2 e CASP3) e parâmetros histopatológicos (grau de pigmentação e tipo histológico). Detectamos alta expressão de Gal-3 em melanomas com maior sobrevida pós-cirúrgica e uma alta expressão nuclear de Gal-3 em melanomas com menor sobrevida pós-cirúrgica. Além disso, houve correlação entre as expressões de Gal-3 e BCL2, assim como entre atipia nuclear e sobrevida pós-cirúrgica. É sabido que a Gal-3 é capaz de formar heterodímeros com a BCL2 no citoplasma para atuar na evasão da morte por apoptose, impedindo a liberação da citocromo C. Já no núcleo, a Gal-3 induz à parada do ciclo celular, reduzindo a taxa da proliferação. Apesar do reduzido número amostral devido à dificuldade nos acompanhamentos clínicos nossos dados permitem sugerir que a Gal-3 é possui potencial para ser um marcador prognóstico de sobrevida em casos de melanomas orais caninos. Novos estudos devem ser realizados afim de confirmar nossas observações e elucidar o papel da Gal-3 nesta neoplasia. / Melanomas are almost 7% of all malignant neoplasms in dogs. They are mainly found in the oral cavity and lips, corresponding to 33% of tumors of the oral cavity. They carry a poor prognosis because of late diagnoses, local invasiveness, high metastatic and recurrence rates after surgical treatment. Galectin-3 (Gal-3) is a protein with a variety of biological roles such as in adhesion, apoptosis, angiogenesis, proliferation and differentiation. In veterinary medicine, there are few studies comparing the expression of Gal-3 with prognosis and tumor progression. We performed immunohistochemistry for Gal-3 in 27 canine oral melanomas and evaluated the immunolabelling both semi-quantitatively and quantitatively. The results were compared with survival, other prognostic markers (Ki67, mitotic index and nuclear atypia), expression of proteins related to apoptosis (BCL2 and CASP3) and histopathological parameters (degree of pigmentation and histological type). We detected higher expression of Gal-3 in cases of melanoma that presented longer post-surgical survival and a higher nuclear expression of Gal-3 in dogs with melanoma that had shorter post-surgical survival. In addition, there was correlation between the Gal-3 and BCL2 expressions, as well as between nuclear atypia and post-surgical survival. It is known that Gal-3 is able to form heterodimers with BCL2 in the cytoplasm leading to evasion of apoptosis, through preventing mitochondrial cytochrome C release. Nuclear Gal-3 induces the cell cycle arrest, reducing the proliferation rate. Despite the small sample size due to the difficulty in clinical follow-up, our data suggest that Gal-3 has the potential to be a prognostic marker for survival in cases of canine oral melanomas. Further studies should be performed to confirm our observations and elucidate the role of Gal-3 in this neoplasm.

Radioactive beams of 8Li and 6He were produced using the double superconducting solenoid system of RIBRAS (Radioactive Ion Beams in Brasil) with a primary beam of 7Li of Elab = 30 MeV, at the São Paulo Pelletron Accelerator. The production reactions were 9Be(7Li, 8Li)8Be and 9Be(7Li, 6He)10B. The angular distributions of the elastic scattering of the 8Li radioactive beam of 26.0 MeV and 6He radioactive beam of 23.0 MeV were measured on 51V target of 1.9 mg/cm2.The elastic scattering angular distributions were analyzed using Optical Model, where the real and imaginary parts are described through a Non-Local Interaction Model called São Paulo Potential . The results were compared with the data present in the literature. The largest cross section, particularly for the halo 6He, shows evidence of the importance of the break up for these medium mass systems. [1]R. Lichtenthaler et al, Eur. Phys. J. A 25,s01,733 (2005); [2]L.C. Chamon et al, Phys. Rev. C66 (2002) 014610. / Radioactive beams of 8Li and 6He were produced using the double superconducting solenoid system of RIBRAS (Radioactive Ion Beams in Brasil) with a primary beam of 7Li of Elab = 30 MeV, at the São Paulo Pelletron Accelerator. The production reactions were 9Be(7Li, 8Li)8Be and 9Be(7Li, 6He)10B. The angular distributions of the elastic scattering of the 8Li radioactive beam of 26.0 MeV and 6He radioactive beam of 23.0 MeV were measured on 51V target of 1.9 mg/cm2.The elastic scattering angular distributions were analyzed using Optical Model, where the real and imaginary parts are described through a Non-Local Interaction Model called São Paulo Potential . The results were compared with the data present in the literature. The largest cross section, particularly for the halo 6He, shows evidence of the importance of the break up for these medium mass systems. [1]R. Lichtenthaler et al, Eur. Phys. J. A 25,s01,733 (2005); [2]L.C. Chamon et al, Phys. Rev. C66 (2002) 014610.

Cientes de que o histórico, a intensidade e a expansão espacial das atividades antrópicas afetam a resiliência e as taxas de recuperação de um ecossistema florestal, existe atualmente o desafio de determinar qual ou quais métodos de restauração ecológica serão os mais eficientes em termos de recursos em metas para cada local. Isso porque existem diversos tipos de métodos e estratégias de restauração ecológica, os quais variam desde apenas assistir um ecossistema se reestabelecer à alta intervenção as quais mudarão os rumos da sucessão ecológica. Neste contexto, o presente trabalho busca analisar se a nucleação aplicada, um método de restauração florestal de baixo custo e intervenção na área, restaurou e ocupou um antigo pasto abandonado numa área de preservação permanente dentro do campus da Escola Superior de Agricultura \"Luiz de Queiroz\". Para isso, foi implantado um experimento de nucleação aplicada, vizinho a um fragmento de floresta estacional semidecidual em maio de 2008 onde foram plantados 40 núcleos. Foram testadas 4 espécies (Inga laurina, Cordia trichotoma, Albizia niopoides e Jacaratia spinosa) com dez repetições cada e com apenas uma espécie por núcleo. Os núcleos foram então mensurados anualmente até 2013 e posteriormente no ano de 2016. Mensuramos o diâmetro na altura do peito (DAP), altura, área basal, biomassa, índice de área de vegetação (via fotografias hemisféricas) e área de copa (calculado como uma elipse). Depois, em 2017 alocamos 24 parcelas de 2 metros de raio no entorno dos núcleos para avaliar a densidade, riqueza e diversidade dos indivíduos regenerantes. Na análise dos dados comparamos os diferentes tratamentos através de análises de variância e dos testes de comparações de média de tukey para o último período de medição nos diferentes parâmetros dendrométricos. Procurando entender se alguma espécie plantada em núcleos monoespecíficos conseguiu atrair maior quantidade de regenerantes, espécies e diversidade comparamos entre os diferentes tratamentos esses parâmetros. Posteriormente, realizamos um modelo linear generalizado a fim de encontrar possíveis relações entre densidade e a riqueza dos indivíduos regenerantes com as variáveis dos núcleos, como altura máxima, área basal, índices de área de vegetação e área de copa. Os resultados encontrados nos dizem que existem diferenças no desenvolvimento dos diversos parâmetros analisados incluindo a ocupação da área pelas diferentes espécies plantadas na nucleação aplicada. Encontramos também para o presente projeto que a densidade de regenerantes no entorno dos núcleos é significantemente relacionada com a área basal e altura das árvores dos núcleos e a riqueza de espécies dos indivíduos em regeneração é significantemente relacionada com apenas a área área basal das árvores presentes nos núcleos. Sugerimos o uso de nucleação aplicada para restaurar áreas com grau médio de degradação usando uma espécie pioneira com altas taxas de crescimento. Os núcleos monoespecíficos devem ser utilizados apenas em paisagens com abundância de propágulos para que não haja baixa diversidade de espécies a médio prazo. / Despite the history that intensity and spatial expansion of anthropogenic activities affect the resilience and recovery rates of a forest ecosystem, we have the current challenge in forest restoration to determine which ecological restoration methods will be most efficient for each specific area, this in terms of resources and goals. In this context, the ecological restoration of degraded ecosystems must be specific to each site. The present work aimed to analyze applied nucleation, a method of low cost and low intervention in the area of forest restoration, occupied an abandoned pasture in a permanent preservation area inside the campus \"Luiz de Queiroz\" College of Agriculture in Piracicaba, São Paulo. For this, an applied nucleation experiment was implanted next to a semideciduous seasonal forest fragment in May 2008, where 40 nuclei were planted. Four species (Inga laurina, Cordia trichotoma, Albizia niopoides and Jacaratia spinosa) were tested with ten replicates each, with only one specie per nuclei. The nuclei were then measured annually until 2013 and later in 2016. In the individual\'s data, we measured the diameter at the chest height (DBH), height, basal area, biomass, vegetation area index (via hemispheric photographs) and Crown area (calculated as an ellipse). After that, in 2017, 24 subplots were allocated around the nuclei to evaluate the density, specie richness and diversity of regenerating individuals. In order to compare the different treatments used, we performed analysis of variance and the Tukey mean comparison test for the different dendrometrical parameters between species. Later, in order to understand the facilitation of density and specie richness by the different treatments of nuclei, we related the height, basal area, vegetation area index and area with these parameters through a generalized linear model. The results show that there are differences in the development of the various parameters analyzed including the occupation of the area by the different species planted in the applied nucleation. We also found for the present project that the density of regenerants found near the nuclei are significantly related to the sum of basal area and height of the nuclei and the species richness of regenerants are significantly related to the sum of the basal area of the individuals\' nuclei. We suggest the use of applied nucleation to restore areas with a medium degree of degradation using a pioneer species with high grown rates. The mono specific nuclei should be use just only in landscapes with plenty of propagules. So that there is no low diversity of species in the medium term.

O núcleo é uma estrutura organizada e possui verdadeiras organelas nucleares. Entre elas, estão os nucléolos e os corpúsculos de Cajal (CBs). Estes compartimentos nucleares são estruturas dinâmicas, mantidos pela associação de macromoléculas envolvidas na expressão gênica que interagem entre si delimitando-as. A principal proteína encontrada nos CBs é a p-80-coilin e, portanto, o principal epítopo capaz de marcar essas estruturas. Suas funções específicas ainda tem sido alvo de estudo. Existe uma proteína em comum a ambas as estruturas, a fibrilarina que participa no processamento de rRNA. Estes corpúsculos já foram descritos na periferia dos nucléolos ou mesmo fisicamente ligados a ele. Acredita-se que os corpúsculos de Cajal participem da síntese de rRNA, maturação, transporte e associação das subunidades ribossômicas.Diante esta relação, este trabalho visa estudar a inter-relação entre estas estruturas em células normais e as respectivas linhagens de células tumorais em cultura antes e após tratamentos com actinomicina D. Esta droga se usada em baixas concentrações, bloqueia a transcrição dos genes que foram decodificados pela RNA polimerase I e II, e -amanitin, por sua vez bloqueia a transcrição de genes decodificado pela RNA polimerase II . Além disso, também visa investigar uma relação entre a proliferação das linhagens estudadas e freqüência dos corpúsculos de Cajal nas células controle e tratadas. O microscópio confocal de varredura a laser permitiu o estudo dessas estruturas em preparações imunofluorescência fornecendo uma análise tridimensional destas estruturas quando utilizados anticorpos específicos. Linhagens de células que apresentaram um crescimento mais lento foram aquelas que tinham uma maior freqüência de corpúsculos por núcleo. Por outro lado, aquelas que apresentaram um crescimento mais intenso, foram aquelas que apresentaram maior variação no número de corpúsculos por núcleo. Após o tratamento com inibidores de síntese de RNA, tanto os corpúsculos de Cajal quanto os nucléolos, apresentaram alterações morfológicas, às vezes apresentando um grande acúmulo na região dos corpúsculos ou desorganizando os nucléolos. Mudanças no tamanho e forma também puderam ser destacadas. / The nucleus is a structure that has sub-compartments which can be called nuclear organelles. Among them, may be cited the nucleoli and the Cajal bodies (CBs). These nuclear compartments are dynamic structures, maintained by association and stock of macromolecules involved in gene expression. The main protein found in the CB is a p-80-coilin and therefore the main epitope able to label these structures. Their functions are still to be clarified. There is a protein in common to the nucleolus and Cajal bodies, the fibrillarin that takes part in the processing of rRNA. The CBs can be found at the periphery of the nucleoli or even physically connected to them. It is believed that the CBs may have role in the synthesis of rRNA and maturation, transport and association of ribosome subunits. In view of this relationship between Cajal bodies and nucleoli, this work aims to study the interrelationship between these structures in normal cells and their respective tumor cell line in culture before and after treatments with actinomycin D, which in low concentrations, blocks the transcription of genes that were decoded by the RNA polymerase I and II and -amanitin, which is responsible for blocking the transcription of genes decoded by the RNA polymerase II and find out a relationship between cell proliferation and Cajal bodies frequencies in control and treated cells. The confocal microscope of laser scanning enabled the study of these structures in preparations immunofluorescent providing a three-dimensional analysis of these structures when used specific antibodies before and after treatment. Cell lines that shown low cell grow, appears to have lass CB/ nucleus in the other hand, cell lines that have fastest grow shown nuclei with more Cajal bodies frequencies and more variation in the number of Cajal/nucleus After treatment with inhibitors, both Cajal bodies as nucleoli, made quite clear morphological changes, sometimes giving large accumulation of proteins in organelles and sometimes appeared disorganized in the nucleoplasm. Changes in the size and shape were also highlighted. The tumor cell lines also showed changes compared to their normal cell type.

O óxido nítrico (NO) é uma molécula gasosa descrita recentemente, com implicações sobre uma vasta quantidade de processos fisiológicos, incluindo transmissão de sinais no sistema nervoso central (SNC). A sinalização nervosa mediada pelo NO ocorre por meios extrassinápticos, na chamada neurotransmissão por volume. Há evidências de que o NO seja um importante fator de modulação no controle da motricidade. A presença de neurônios que produzem NO já foi descrita em várias espécies, e estruturas ligadas ao controle do movimento como os núcleos da base (NNBB) contêm células nitrérgicas em quantidades variadas. Não se conhece os efeitos do processo de envelhecimento sobre a estrutura e função destes neurônios produtores de NO. O objetivo geral deste estudo foi investigar se o envelhecimento provoca alterações nos neurônios nitrérgicos presentes nos NNBB do encéfalo humano. Além disso, busca agregar mais conhecimento a aspectos morfológicos e de distribuição das células que compõem o sistema nitrérgico nos NNBB em humanos. As amostras de estriado (caudado e putâmen), globos pálidos (GP), núcleo subtalâmico (NST), substância negra (SN) e núcleo pedunculopontino (NPP) de 20 indivíduos sem doenças neurológicas e psiquiátricas foram submetidas à avaliação histológica em secções, coradas por técnicas que localizam neurônios que expressam NO, como a histoquímica para NADPH-diaforase (NADPHd) e à imunohistoquímica para sintase do NO neuronal (nNOS), e parâmetros de densidade neuronal e morfometria foram comparados entre indivíduos adultos jovens e idosos. Análises de densidade neuronal e morfometria entre subdivisões topográficas e funcionais também foram realizadas. Foi visto que o envelhecimento não provoca modificações na densidade neuronal e morfometria nitrérgica nos NNBB em humanos. Adicionalmente, o trabalho mostrou que: (I) as regiões mais posteriores do estriado se destacaram por apresentarem uma elevada densidade neuronal, associada a neurônios menores, em comparação com as regiões mais anteriores; (II) as porções do estriado ligadas ao córtex límbico apresentam maiores densidades neuronais; (III) o NST é uma região em que cerca de 90% de seus neurônios expressam NO, e suas características morfológicas sugerem que estas células coexpressem glutamato; (IV) o NPP é extensamente povoado por neurônios nitrérgicos, principalmente no nível do colículo inferior; (V) a presença de células NO-positivas é preponderante nas lâminas medulares de ambos GP, porém notamos maior concentração de células nitrérgicas no GPi; (VI) não foi detectada presença de neurônios quem contém NO na SN. Nossos resultados mostram que há uma presença maciça de neurônios que expressam NO em núcleos-chaves envolvidos com processamento motor corticobasal, como o NST, o estriado e o NPP, sugerindo que a neurotransmissão nitrérgica seja peça fundamental da fisiologia dos NNBB, portanto, com considerável potencial terapêutico nas doenças que afetam estas estruturas. / The nitric oxide (NO) is a gaseous molecule recently described, with a role on several physiologic processes, including signal transmission in central nervous system (CNS). The NO-mediated brain signaling occurs by extrasynaptic mode, called volume transmission. There are evidences supporting the NO as a major neurotransmitter involved on motor control modulation. The presence of NO neurons was described in many species, and movement-related structures, as the basal nuclei (BN), also contains variable densities of nitrergic cells. It is unknown the effect of aging over the structure and function of these NO neurons. The objective of the study is to investigate if the aging causes abnormalities on human BN nitrergic neurons. Furthermore, we aimed to explore distribution and morphologic features of these cells in BN. The samples of striatum (caudate and putamen), globus pallidum (GP), subthalamic nucleus (STN), substantia nigra (SN) and pedunculopontine nucleus (PPN) of 20 human brains from subjects without neurologic or psychiatric disases were processed for histologic analysis, stained by 2 techniques which localizes NO neurons: histochemistry for NADPH-diaphorase (NADPHd) and immunohistochemistry for neuronal NO synthase (nNOS); the neuronal density and morphometric parameters were compared between young adults and aged subjects. The neuronal density and morphometric analysis between striatal and subthalamic topographic / functional subdivisions were also performed. Our data showed that aging does not change the neuronal density or morphometric parameters of nitrergic neurons in human BN. Additionally, other results were found: (I) the most posterior regions of striatum have a higher neuronal density and smaller neurons than the most anterior regions of this nucleus; (II) the limbic cortex-associated areas of striatum have higher neuronal density than others functional subdivisions; (III) the STN is a region in which about 90% of its neurons expresses NO, and its morphologic features suggest these neurons coexpress glutamate; (IV) the PPN has a massive nitrergic neuronal density, mostly in the inferior colliculus level; (V) in GP, there is a marked presence of NO neurons in laminae medullaris, and the internal GP has more NO-positive cells than the external GP; (VI) nitrergic neurons were not detected in SN. Our results showed a remarkable presence of neurons expressing NO in nuclei essential for motor corticobasal processing (striatum, STN, PPN), suggesting that the nitrergic neurotransmission has a fundamental role in BN physiology, therefore, with great therapeutic potential in diseases involving these structures.

The hippocampal-medial prefrontal circuit has been shown to serve a critical role in decision making and goal directed actions. While the hippocampus (HF) exerts a direct influence on the medial prefrontal cortex (mPFC), there are no direct return projections from the mPFC to the HF. The nucleus reuniens (RE) of the midline thalamus is strongly reciprocally connected with the HF and mPFC and represents the major link between these structures. We investigated the role of RE in functions associated with the hippocampus and the mPFC -- or their interactions. Using two different inactivation techniques (pharmacological and chemogenetic), we sought to further define the role of RE in spatial working memory (SWM) and behavioral flexibility using a modified delayed non-match to sample (DNMS) working memory task. We found that the reversible inactivation of RE with muscimol critically impaired SWM performance, abolished well-established spatial strategies and produced a profound inability to correct non-rewarded, incorrect choices on the T-maze (perseverative responding). We observed similar impairments in SWM following the chemogenetic (DREADDs) inactivation of RE or selective RE projections to the ventral HF. In addition, we showed that the inhibition of RE terminals to the dorsal or ventral HF altered task related behaviors by increasing or decreasing the time to initiate the task or reach the reward, respectively. Finally, we examined discharge properties of RE cells across sleep-wake states in behaving rats. We found that the majority of RE cells discharge at high rates of activity in waking and REM and at significantly reduced rates in SWS, with a subpopulation firing rhythmically in bursts during SWS. We identified five distinct subtypes of RE cells that discharged differently across vigilant states; those firing at highest rates in waking (W1, W2), in REM sleep (R1, R2) and SWS (S1). Given the differential patterns of activity of these cells, we proposed they may serve distinct functions in waking – and possibly in SWS/REM sleep. In sum, our findings indicate that RE is critically involved in mnemonic and executive functions and the heterogeneous activity of these cells support a role for RE in arousal/attention, spatial working memory and cognition. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

No presente trabalho medimos e analisamos distribuições angulares para o processo de espalhamento elástico do núcleo radioativo de 12B em alvo de 58Ni. As medidas foram realizadas nas energias de Elab=30.0 e 33.0 MeV no laboratório do acelerador Pelletron. Essas energias são próximas a barreira Coulombiana (VB=28.0 MeV) para esse sistema. Para a produção do feixe radioativo de 12B utilizamos o sistema RIBRAS instalado nesse laboratório. As distribuições angulares foram analisadas com o modelo ótico, utilizando potenciais de Woods-Saxon e Potencial de São Paulo. Para uma interpretação física mais consistente e um estudo da influência de outros canais de reação no espalhamento elástico analisamos também considerando o método de canais acoplados. Considerando o acoplamento dos canais de espalhamento inelásticos, reorientação e spin-órbita pudemos descrever a distribuição angular na energia de 30.0 MeV. No entanto esses canais não foram suficientes para descrever a distribuição angular na energia de 33.0 MeV, indicando que outros canais como de transferência e/ou break-up possam ser importes. A partir da análise das distribuições angulares com modelo ótico obtivemos também a seção de choque total de reação. Essas seções de choque foram comparadas com a de outros sistemas utilizando métodos de redução, indicando que o projétil 12B segue uma sistemática intermediária entre núcleos fortemente ligados e fracamente ligados. Finalmente, discutimos a sistemática dos resultados de canais acoplados para o espalhamento elásticos dos isótopos de Boro 8,10,11,12B no alvo 58Ni em termos da configuração de clusters dos projéteis. / In the present work we measure and analyzed angular distributions for the process of elastic scattering of the radioactive nucleus of 12B in a target of 58Ni. The measurements were performed in the energies of Elab = 30.0 and 33.0 MeV in the Pelletron accelerator laboratory. These energies are close to the Colombian barrier (VB = 28.0 MeV) for this system. For the production of the radioactive beam of 12B we used the RIBRAS system installed in this laboratory. The angular distributions were analyzed with the optical model, using potentials of Woods-Saxon and Potential of São Paulo. For a more consistent physical interpretation and a study of the influence of other reaction channels in the elastic scattering we also analyze the coupled channel method. Considering the coupling of the inelastic scattering channels, reorientation and spin-orbit we could describe the angular distribution in the energy of 30.0 MeV. However, these channels were not enough to describe the angular distribution in the energy of 33.0 MeV, indicating that other channels as transfer and / or break-up can be amounts. From the analysis of the angular distributions with optical model we also obtained the section of total reaction shock. These cross sections were compared with those of other systems using reduction methods, indicating that projectile 12B follows a systematic intermediate between tightly bound and weakly bonded cores. Finally, we discuss the systematics of the results of elastic scattering channels of the Boron isotopes 8,10,11,12B in the 58Ni target in terms of the cluster configuration of the projectiles.

Neste trabalho são apresentados os resultados obtidos do estudo da estrutura do núcleo \'ANTPOT. 58 CO\' utilizando a técnica de espectroscopia de raios gama em linha. Este núcleo foi produzido a partir da reação de fusão-evaporação \'ANTPOT. 51 V\'(ANTPOT. 10 B\', p2n), com energia de feixe de 33 MeV incidindo em três alvos de 200\'mü\'g/\'cm POT. 2\', realizada no acelerador Pelletron da Universidade de São Paulo. Foram feitas medidas em coincidência \'gama\'\'gama\'-partícula com o espectrômetro de raios \'gama\' Saci-Pererê, composto de 4 detectores de GeHP com blindagem Compton, e um sistema auxiliar para detectar partículas carregadas, consistindo de 11 telescópios cintiladores \'delta\'E-E tipo phoswich. Foram encontradas quarenta e sete transições novas que depopulam trinta e sete novos estados. O esquema de níveis proposto foi estendido até uma energia de excitação de 8 MeV e momento angular de \'J POT. pi\'=\'11 POT. +\'. A atribuição dos valores de spins foi baseada na razão DCO (Correlação Direcional de Estados Orientados). Os resultados experimentais foram comparados com os calculados pelo Modelo de Camadas em Larga Escala (LSSM) utilizando os códigos MSHELL e Antoine, assim como a interação residual GXPF1, desenvolvida para ser usada na camada pf Foram interpretados dezenove estados excitados a partir do LSSM. Foram medidas também as vidas médias para treze estados excitados do \'ANTPOT. 58 CO\' utilizando o Método de Deslocamento Doppler Atenuado (DSAM). Para este estudo, o núcleo \'ANTPOT. 58 CO\' foi produzido a partir da reação \'ANTPOT. 51 V\'(ANTPOT. 10 B\', p2n) com energia de feixe de 36 MeV, usando um alvo de 770\'mü\'g/\'cm POT. 2\' prensado em um suporte de Pb. Os valores experimentais também foram comparados aos cálculos do LSSM. Probabilidades de transição reduzidas B(Ml), e portanto as vidas médias, foram bem reproduzidas pelo modelo teórico para cinco níveis identificados. Os níveis de energia observados apresentaram funções de onda com grande mistura de configurações sendo a principal dada por \'pi\'\'f POT -1 IND. 7/2\' (PRODUTO VETORIAL) v \'p POT 2 IND. 3/2\' \'f POT 1 IND. 5/2\',. Os resultados indicam que a maior parte dos estados excitados do núcleo \'ANTPOT. 58 CO\' tem um comportamento esférico e são bem reproduzidos considerando excitações de partícula única / The excited states in the doubly odd nucleus \'ANTPOT. 58 CO\' have been studied using inbeam gama-ray spectroscopy. The \'ANTPOT. 58 CO\' nucleus has been produced with the fusionevaporation reaction \'ANTPOT. 51 V\'(ANTPOT. 10 B\', p2n) at 33 MeV bombarding energy, using the SMV Pelletron accelerator of the University of São Paulo. Gamrna-gamma-charged particle coincidences were measured with the Saci-Pererê gama-ray spectrometer composed of 4 Compton-suppressed GeHP and an ancillary charged-particle detector system composed of 11 plastic phoswich scintillator ~E-E telescopes. We have found 47 new gama-transitions de-populating 37 new excited states. A level scheme extending up to an excitation energy of about 8.0 MeV and spin \'J POT. pi\'=\'11 POT. +\' has been proposed. The spin assignments were based on the DCO (Directional Correlation from Oriented States) ratios. The experimental results were compared with Large Scale Shell Model (LSSM) calculations performed with the MSHELL and Antoine codes using the GXPFl effective interaction, developed for use in the pf shell. We have interpreted 19 excited states in the frame of the LSSM. We have measured also the lifetimes for 13 excited states of the \'ANTPOT. 58 CO\' nucleus. The lifetimes were measured with the Doppler Shift Attenuation Method (DSAM). For this study the \'ANTPOT. 58 CO\' nuclei were populated with the reaction \'ANTPOT. 51 V\'(ANTPOT. 10 B\', p2n) at 36 MeV bombarding energy, using a target consisting of a 770\'mü\'g/\'cm POT. 2\' foil with Pb backing. The experimental values were also compared with the LSSM calculations. Experimental B(M1) reduced transition probabilities, and thus the lifetimes, are well reproduced by the theoretical model for fi v e o f the identified levels. The observed levels presented wave functions with large configuration mixing with the main configuration being \'pi\'\'f POT -1 IND. 1/2\' (PRODUTO VETORIAL) v \'p POT 2 IND. 3/2\' \'f POT 1 IND. 5/2\',. The results indicate that most of the excited states in the \'ANTPOT. 58 CO\' nucleus has a spherical behavior and is well reproduced considering single-particle excitations.

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

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