Polyketides are a group of bioactive natural products synthesized by bacteria, fungi and plants with various acyl-CoA precursors, such as malonyl-CoA, methylmalonyl-CoA and ethylmalonyl-CoA. A sufficient supply of these precursors is a prerequisite for the high level production of polyketide products. A thorough understanding of relative roles of various metabolic pathways involved in precursor supply makes increased production by genetical manipulation, and thus rational strain improvement, a reality. Monensin A is a polyketide antibiotic assembled from one ethylmalonyl-CoA, seven methylmalonyl-CoA and five malonyl-CoA molecules by Streptomyces cinnamonensis. In the present work, the origin of these biosynthetic precursors was investigated using an industrially mutagenized monensin producer and industrial fermentation conditions. A hitherto disregarded metabolic pathway was discovered to play a significant role in providing methylmalonyl-CoA for monensin biosynthesis by gene disruption, isotope-labeling of monensin and analysis of in vivo acyl-CoA pools. This pathway starts from biosynthesis of butyryl-CoA from two molecules of acetyl-CoA, and goes through the intermediate of isobutyryl-CoA, and finally produces methylmalonyl-CoA by direct oxidation of the pro-S methyl group of isobutyryl-CoA.Industrial fermentation of the industrially mutagenized monensin producer yields significantly more monensin than the routine laboratory fermentation. This suggested the presence of abundant in vivo malonyl-CoA and methylmalonyl-CoA in this process and presented an opportunity to utilize it as a biological system for the high-titer production of heterologous polyketides derived from malonyl-CoA and/or methylmalonyl-CoA. The tetracenomycin C polyketide synthase (PKS) synthesizes tetracenomycin C, a polyketide with ten molecules of malonyl-CoA. In this work, the tetracenomycin C PKS gene cluster was introduced into two industrially mutagenized strains of Streptomyces cinnamonensis. Unprecedented multi-gram/liter of tetracenomycin production was observed in the resulting two strains, indicating the high potential of industrially mutagenized monensin production strains as efficient hosts for the production of malonyl-CoA-derived polyketides. For additional improvement in tetracenomycin yield, we attempted to increase malonyl-CoA supply to tetracenomycin C PKS by genetically manipulating metabolic pathways affecting production of malonyl-CoA and eliminating competition from monensin PKS for malonyl-CoA. However, only decreased tetracenomycin production was observed, demonstrating that the regulation of malonyl-CoA-related metabolic pathways is a complex process.

Hippocampal neurons are vulnerable to injury, as indicated by the prevalence of learning and memory deficits following traumatic brain injury. Research indicates that proliferation of neural precursor cells increases following brain injury, which implies that there is an endogenous response in the hippocampus to replenish neurons and restore cognitive function. Studies show that mitogenic growth factors may drive this proliferative response; one of which is epidermal growth factor. Because adults and the elderly manifest the most enduring deficits following TBI, it is critical to investigate how EGF expression following injury may relate to injury-induced cell proliferation and the degree of cognitive recovery observed with aging. In the current study, we assessed the temporal and spatial expression of EGF in the injured hippocampus with age. Our results suggest that EGF expression increases following TBI, and this increase is more significant in the younger brain. Additionally, we investigated the phenotype and localization of cells that express EGF following injury.

Resumo: Dentre os compostos mais pesquisados atualmente encontram-se os eletrólitos sólidos com elevada condutividade iônica, pois estes apresentam potenciais aplicações em baterias de estado sólido, entretanto, na literatura, há poucos trabalhos que descrevam o processo de preparação e caracterização de pós com estrutura tipo perovskita para aplicações como catodos em baterias de lítio. Assim, este projeto teve como objetivo o desenvolvimento do processo de preparação do pó de La0.50Li0.50TiO3 pelo método de precursores poliméricos. O comportamento térmico do pó obtido a 350ºC foi avaliado através das técnicas de Análise Termogravimétrica (TGA) e Análise Térmica Diferencial (DTA); já a evolução térmica do pó obtido a 350 e 700ºC foi analisada por meio das técnicas espectroscópicas de Infravermelho (IR) e Raman. O processo de cristalização do pó foi realizado por Difração de Raios X (DRX), em conjunto com o Método de Rietveld que identificou uma fase pervskita altamente cristalina durante o processo de cristalização, no entanto, uma fase secundária, LiTi2O4, também foi detectada. A morfologia do pó foi observada por Microscopia Eletrônica de Varredura (MEV-FEG), que revelou uma microestrutura de grãos esféricos e homogêneos. Quanto às medidas eletroquímicas, foram utilizadas as técnicas de Titulação Galvanostática Intermitente (GITT), Cronopotenciometria e Voltametria Cíclica que evidenciaram a presença de dois processos cinéticos diferentes / Abstract: Among the most researched compounds currently are the solid electrolytes with high ionic conductivity, because of their potential applications in solid state batteries, however, in the literature there are few studies that describe the preparation and characterization of powders with perovskite structure for applications such as cathodes in lithium batteries. Thus this project aimed to develop the process of preparing the powder La 0.50Li0.50TiO3 by the polymeric precursor method. The thermal behavior of the power obtained at 350ºC was evaluated using the techniques of Thermogravimetric analysis (TGA) and Differential Thermal Analysis (DTA), whereas the thermal evolution of the power obtained at 350 and 700ºC was analyzed by spectroscopic techiniques IR and Raman. The crystallization process was analyzed by X-ray powder diffraction together with the Rietveld Method that identified a highly crystalline perovskite phase during the crystallization process; however, a secondary phase LiTi2O4 was also detected. The morphology of the powder was observed by field emission gun scanning electron microscopy (FEG-SEM), which revealed a microstructure of spherical and homogeneous grains. As for the eletrochemical measurements, we have used the Galvanostatic Intermittent Intermittent Titration Technique (GITT), Chronopotenciometry and Cyclic Voltammetry demonstrating the presence of two different kinetic processes / Orientador: Carlos Frederico de Oliveira Graeff / Coorientador: Alejandra Hortencia Miranda González / Banca: Fritz Cavalcante Huguenin / Banca: Fenelon Martinho Lima Pontes / Banca: Roberto Manuel Torresi / Banca: Maria Aparecida Z. Bertochi / O programa de Pós graduação em Ciência e Tecnologia de Materiais, POSMAT, tem carater institucional e integra as atividades de pesquisa em diversos materiais de diversos campi da Unesp / Doutor

La réduction de l’impact environnemental est aujourd’hui l’un des défis cruciaux de l’industrie aéronautique. La poursuite d’une moindre consommation des aéronefs a conduit à concevoir des systèmes propulsifs en géneral, et des étages de compression en particulier, toujours plus compacts et chargés. Cette tendance dans la conception des moteurs est directement responsable de l’accentuation du caractère instationnaire des écoulements internes ainsi que de la favorisation dans l’émergence de phénomènes entrainant la perte de stabilité. L’étude expérimentale, conduite pendant ce projet de thèse, porte sur la caractérisation des écoulements dans un étage de compresseur axial en phase émergente et stabilisée du décrochage tournant, grâce à des mesures instationnaires de pression pariétale et de vitesse. L’étude doit son originalité à l’utilisation et au développement de techniques de post-traitement non-standard. La transformée par ondelettes se révèle être un outil particulièrement intéressant à la détection de structures cohérentes de brève durée, telles que le précurseur de type « spike » ainsi que les caractéristiques instantanées d’une cellule de décrochage tournant. A côté de cette approche d’analyse d’un signal localisé, différentes procédures de calcul de champs en moyenne de phase ont été mises au point, chacune adaptée aux spécificités du phénomène étudié et de la procédure expérimentale suivie. Il a été ainsi possible de suivre l’évolution des caractéristiques du champ de pression du régime nominal jusqu’à l’installation du décrochage tournant. L’alignement de la trajectoire du tourbillon de jeu avec la section d’entrée du rotor est associé au déclenchement du décrochage par précurseur de type « spike ». La comparaison entre les champs en phase transitoire et en décrochage établi, amène à affirmer que le précurseur n’est que le stade embryonnaire d’évolution du phénomène du décrochage. L’approche a, en outre, permis d’apprécier la complexité de la structure « interne » de la cellule qui apparait comme la succession d’une phase de propagation de décollement, une zone fortement débitante à charge presque nulle et une phase de ré-attachement de l’écoulement. / The reduction of the environmental impact is nowadays one of the crucial challenges of the aeronautic industry. The quest to lower the consumption of aircrafts has led to more compact and higher loaded engines in general, and especially compressor stages. This leads an increase of the internal flow unsteadiness and to the occurrence of unstable phenomena. The experimental study, performed during this work, concerns the characterization of flows in an axial compressor stage during both the emergence of rotating stall and its stabilized phase, by means of unsteady pressure and velocity measurements. The originality of the work proposed resides in the use and the development of non-standard data processing methods. The wavelets transform reveals to be an interesting tool for the detection of short coherent structures, like the spike-type precursor as well as the instantaneous features of a rotating stall cell. Beside this local approach, different procedures for phase-locked field measurements were developed, according to the specification of each studied phenomenon and the experimental proceedings. Thanks to these methods, it was possible to highlight the pressure field evolution until the development of the rotating stall regime. The alignment of the tip leakage vortex with the rotor inlet section forecasts a spike type stall onset. The comparison between transitional phase and fully developed stall fields conducts to assert that the precursor represent only the embryonic stage of the rotating stall evolution. This approach led to appreciate the complexity of the internal structure of the cell that appears to be the succession of stall propagation phase, zero-loaded high flow rate region and reattachment phase

Amostras nanoestruturadas do sistema SrTi1-xMnxO3 (STM) com x = 0%, 0.5%, 1%, 2.5%, 5% e 10% na forma de pó foram sintetizadas através do método dos precursores poliméricos. As propriedades térmicas, estruturais e ópticas das amostras STM calcinadas em diferentes temperaturas foram analisadas a temperatura ambiente através de técnicas de análise térmica, difração de raios-x, espectroscopia de absorção de raios-x e fotoluminescência. Os resultados obtidos através das técnicas de analise térmica mostraram que o íon manganês causa um efeito retardante, aumentando a temperatura onde ocorre o início da cristalização. Os resultados de DRX mostraram que no limite de solubilidade estudado, o íon manganês foi incorporado em solução sólida no sítio B na rede do SrTiO3 (ST), assumindo estados de oxidação entre +3,4 e +3,7, indicando que ocorre a criação de defeitos para compensação de cargas, mantendo a neutralidade elétrica do material. A análise dos espectros de fotoluminescência das amostras STM amorfas mostrou que o aumento da quantidade de manganês até 1% leva a um aumento da intensidade fotoluminescência em relação a amostra SrTiO3. Entretanto, a adição de manganês acima desta quantidade leva a uma diminuição pronunciada da intensidade fotoluminescente. Nas amostras amorfas não foi observada uma variação significativa da ordem local ao redor dos átomos de titânio bem como no estado de oxidação dos átomos de manganês à medida que a quantidade de manganês aumenta. Desta forma, a variação da intensidade fotoluminescente não pôde ser atribuída a estes fatores. O aumento da temperatura de calcinação leva a uma diminuição significativa da intensidade fotoluminescente. Uma análise dos espectros XANES medidos na borda K do titânio destas amostras mostrou que ocorre uma maior ordenação dos átomos de Ti nos octaedros de oxigênio [TiO6] a medida que a temperatura de calcinação aumenta, ou seja, à medida que aumenta o grau de cristalinidade das amostras. / Nanostructured SrTi1-xMnxO3 (STM) powder samples with x = 0%, 0.5%, 1%, 2.5%, 5% and 10% were synthesized by the polymeric precursors method. The thermal, structural and optical properties of STM samples heat-treated at different temperatures were analyzed by thermal analysis, X-ray diffraction, X-ray absorption spectroscopy and photoluminescence techniques. The thermal analysis results have shown that manganese ion causes a retarding effect, increasing the onset of the crystallization temperature. The XRD results showed that manganese ion is incorporated in a solid solution in the B site of the SrTiO3 (ST) network for the range of solubility studied, with oxidation states between +3.4 and +3.7, indicating the creation of charge compensation defects, keeping the materials electrical neutrality. The analysis of the photoluminescence (PL) spectra of the STM amorphous samples showed that the PL intensity increases when the amount of manganese is up to 1%. However, higher manganese concentrations leads to a pronounced decrease of the photoluminescence intensity. The amorphous samples showed no significant change of the local order around the titanium atoms, observed as well as in the oxidation state of the manganese atoms as the amount of manganese increases. Thus, the variation of the photoluminescence intensity cannot be explained by these factors. Increasing the calcination temperature leads to a significant decrease of the photoluminescence intensity. An analysis of the XANES spectra measured at the titanium K edge of these samples showed that as the calcination temperature increases, i. e., as it increases the degree of crystallinity, the ordering of the Ti atoms in the oxygen octahedra TiO6 of the samples increases.

Diversos anodos para célula a combustível de óxido sólido (SOFC) têm sido estudados devido aos problemas de deterioração dos anodos tradicionalmente usados, os compósitos à base de zircônia estabilizada/Ni (YSZ/Ni). Estudos prévios evidenciaram que a perovskita La0,75Sr0,25Cr0,50Mn0,50O3 (LSCM) possui desempenho similar em SOFCs usando hidrogênio e metano como combustível, tornando essa cerâmica um possível substituto dos compósitos à base de níquel. No presente estudo, foram sintetizados compostos La0,75Sr0,25Cr0,50-xMn0,50- yRux,yO3 (LSCM-Ru) pelo método dos precursores poliméricos. Análises termogravimétrica e térmica diferencial (TG/ATD) simultâneas e difração de raios X (DRX) foram utilizadas para monitorar a evolução térmica das resinas precursoras e a formação de fase dos compostos. As propriedades elétricas de amostras sinterizadas foram estudadas pela técnica de 4 pontas de prova dc na faixa de temperatura entre 25 °C e 800 °C. Os resultados experimentais indicaram a formação de fase única dos compostos LSCM-Ru calcinados a ~1200 °C. Os parâmetros de rede, calculados a partir dos dados de DRX, revelaram que a substituição parcial dos íons Cr ou Mn pelo Ru não altera significativamente a estrutura cristalina do LSCM até x,y ~ 0,10; uma característica consistente com os raios iônicos similares dos cátions Cr, Mn e Ru com número de coordenação seis. Medidas de resistividade elétrica ao ar mostraram que o mecanismo de transporte não é alterado e que o efeito da substituição de Ru nas propriedades elétricas do composto depende do íon substituído (Cr ou Mn) de maneira consistente com suas substituições parciais. Os testes de SOFCs unitárias revelaram que células com os anodos constituídos por uma camada coletora de corrente do anodo cerâmico LSCM-Ru e uma camada funcional de YSZ/Ni têm desempenho superior a células contendo apenas o anodo cerâmico. As células contendo os anodos cerâmicos LSCM-Ru foram testadas em hidrogênio e etanol, entre 800 °C e 950 °C, e mostraram desempenho em etanol superior ao em hidrogênio; uma característica que foi associada às propriedades de transporte eletrônico dos compostos LSCM-Ru em atmosfera redutora. Os resultados sugerem que os compostos LSCM com substituição parcial de Ru são anodos promissores para SOFC operando com etanol. / Several anodes for solid oxide fuel cell (SOFC) have been studied because of serious degradation exhibited by the traditionally used yttria-stabilized zirconia/Ni cermets (YSZ/Ni). Previous studies showed that the perovskite La0.75Sr0.25Cr0.50Mn0.50O3 (LSCM) has similar performance in SOFCs running on hydrogen and methane fuels, making such a ceramic a potential alternative to YSZ/Ni cermets. In the present study, compounds La0.75Sr0.25Cr0.50- xMn0.50-yRux,yO3 (LSCM-Ru) were synthesized by the polymeric precursor method. Simultaneous thermogravimetric and differential thermal analysis (TG/DTA) and X-ray diffraction (XRD) were used to monitor the thermal evolution of the precursor resins and the formation of crystalline phases. The electrical properties of sintered samples were studied by the 4-probe dc technique in the temperature range between 25 °C and 800 °C with controlled atmosphere. The experimental results showed the formation of single phase LSCM-Ru compounds after heat treatment at ~ 1200 °C. Lattice parameters, calculated from the XRD data, revealed that the partial substitution of Cr or Mn by Ru has no significant effect on the crystal structure of LSCM up to Ru x,y ~ 0.10; in agreement with the similar ionic radius of Cr, Mn and Ru with coordination number six. Electrical resistivity measurements showed that the transport mechanism is unchanged and that the effect of Ru addition on the electrical properties of the compound depends on the substituted ion (Cr or Mn). Electrochemical tests of SOFCs demonstrated that single cells comprised of a current collector layer of LSCM-Ru ceramic anode and a functional layer for YSZ/Ni have superior performance when compared to single cells with only one layer of the ceramic anode. Single cells with the LSCM-Ru ceramic anode layer were tested under both hydrogen and ethanol fuels, in the 800 °C - 950 °C temperature range. The main results showed that the single fuel cells exhibited higher performance under ethanol than under hydrogen; a feature that was related to the enhanced electronic transport properties of LSCM-Ru in reducing atmosphere. The experimental results suggest that the LSCM-Ru compounds are promising anodes for ethanol fueled SOFCs.

Schizophrenia is a major psychiatric disorder that affects 1% of the world population and is among the 10 leading worldwide causes of disability. Disrupted-In- Schizophrenia (DISC1) is one of the most studied risk genes for mental illness and is disrupted by a balanced translocation between chromosomes 1 and 11 that co-segregates with major mental illness in a single large Scottish family. DISC1 is a scaffold protein with numerous interactors and has been shown to hold key roles in neuronal progenitor proliferation, migration, cells signalling and synapse formation and maintenance. The studies herein provide the platform in order to investigate the molecular and cellular consequences of the t(1;11) translocation using induced pluripotent stem cells (iPSCs)-derived neural precursor cells and neurons from within-family carriers and controls. Towards this end, several iPSC lines have been converted into neural progenitor cells (NPCs) and differentiated into physiologically active forebrain neurons following well-characterised protocols. These cells were characterised in terms of basic marker expression at each developmental stage. Inter-line variation was observed in all subsequent experiments but overall t(1;11) lines did not generate less neuronal or less proliferating cells compared to control lines. Furthermore, the expression pattern of genes disrupted by the t(1;11) translocation was investigated by RT-qPCR. DISC1 was reduced by ~50% in the translocation lines, both neural precursors and neurons. This observation corresponds to previous findings in lymphoblastoid cell lines (LBCs) derived from members of the same family. Moreover, DISC1 expression was found to increase as neural precursors differentiation to neurons. Two other genes are disrupted by the t(1;11) translocation;DISC2 and DISC1FP1. Their expression was detectable, but below the threshold of quantification. Similarly, DISC1/DISC1FP1 chimeric transcripts corresponding to such transcripts previously identifies in LBCs from the family were detectable, but not quantifiable. A fourth gene, TSNAX, was also investigated because it is located in close proximity to, and undergoes intergenic splicing with, DISC1. Interestingly, TSNAX was found to be altered in some but not all time points studied, in the translocation carriers compared to control lines. In addition to breakpoint gene expression profiling, iPSC-derived material was used to investigate neuronal differentiation. There seemed to be attenuation in BIII-TUBULIN expression at two weeks post-differentiation, while NESTIN, MAP2 and GFAP expression was similar between translocation carrier and control lines at all time points studied. I also had access to targeted mice designed to mimic the derived chromosome 1 of the t(1;11) balanced translocation. Using RT-qPCR Disc1 expression was found to be 50% lower in heterozygous mice compared to wild types, and I detected a similar profile of chimeric transcript expression as detected in translocation carrier-derived LBCs. These observations support my gene expression studies of the human cells and indicate that the iPSC-derived neural precursors and neurons can be studied in parallel with the genome edited mice to obtain meaningful insights into the mechanism by which the t(1;11) translocation confers substantially elevated risk of major mental illness. In conclusion, the studies described in this thesis provide an experimental platform for investigation of the effects of the t(1;11) translocation upon function and gene and protein expression in material derived from translocation carriers and in brain tissue from a corresponding mouse model.

Chung, Po Yin. / Thesis submitted in: December 2006. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 128-155). / Abstracts in English and Chinese. / Thesis Abstract --- p.i / 論文摘要 --- p.iv / Acknowledgements --- p.vi / Abbreviations --- p.vii / Thesis Content --- p.xi / List of Figures --- p.xv / List of Tables --- p.xvii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1. --- Normal Hematopoiesis --- p.1 / Chapter 1.2. --- Hematological Malignancy and the Aberrant Development of Blood Cells --- p.2 / Chapter 1.3. --- Leukemia and Its Classification --- p.3 / Chapter 1.4. --- Childhood Acute Lymphoblastic Leukemia (ALL) --- p.5 / Chapter 1.4.1. --- Epidem iology --- p.5 / Chapter 1.4.2. --- Causes and Risk Factors --- p.6 / Chapter 1.4.3. --- Molecular Pathophysiology --- p.7 / Chapter 1.4.4. --- Clinical Presentation --- p.9 / Chapter 1.4.5. --- Classification --- p.10 / Chapter 1.4.5.1. --- Immunophenotyping --- p.10 / Chapter 1.4.5.2. --- French-American-British (FAB) Classification --- p.12 / Chapter 1.4.6. --- Diagnosis and Prognosis --- p.14 / Chapter 1.4.6.1. --- Morphological and Cytochemical Analysis --- p.15 / Chapter 1.4.6.2. --- Cytogenetic and Molecular Genetic Characterizations --- p.16 / Chapter 1.4.7. --- Treatment --- p.19 / Chapter 1.5. --- Overview of Epigenetics --- p.21 / Chapter 1.6. --- Concepts ofDNA Methylation --- p.23 / Chapter 1.6.1. --- CpG Islands --- p.23 / Chapter 1.6.2 --- Mechanisms of DNA Methylation --- p.24 / Chapter 1.6.3 --- Physiological Roles of DNA Methylation --- p.28 / Chapter 1.6.4 --- Initiation of Aberrant DNA Methylation --- p.30 / Chapter 1.7. --- DNA Methylation in Tumorigenesis --- p.31 / Chapter 1.7.1. --- Regional Hypermethylation --- p.33 / Chapter 1.7.2 --- Global and Regional Hypomethylation --- p.34 / Chapter 1.7.3 --- Microatellite Instability and Oncogeneic Mutation --- p.35 / Chapter Chapter 2 --- Literature Review --- p.37 / Chapter 2.1. --- Aberrant DNA Methylation in Childhood ALL --- p.37 / Chapter 2.1.1. --- Cell Cycle --- p.39 / Chapter 2.1.2. --- Apoptosis --- p.41 / Chapter 2.1.3. --- Tissue Invasion and Metastasis --- p.42 / Chapter 2.1.4. --- Transcription Factors and Metabolic Enzymes --- p.44 / Chapter 2.1.5. --- Putative Tumor Suppressor Genes --- p.44 / Chapter 2.1.6. --- Chromosome Instability --- p.46 / Chapter 2.2. --- Methodologies in DNA Methylation Analysis --- p.50 / Chapter 2.2.1. --- Principle of Methylation-sensitive Arbitrarily Primed PCR (MS-AP PCR) --- p.50 / Chapter 2.2.2. --- Combined Bisulfite Restriction Analysis (COBRA) --- p.53 / Chapter 2.2.3. --- Cloned Bisulfite Sequencing --- p.55 / Chapter 2.2.4. --- Experimental Use of Demethylating Agents --- p.55 / Chapter Chapter 3 --- Background of Research --- p.58 / Chapter 3.1. --- Current Methylation Studies in Childhood ALL --- p.58 / Chapter 3.2. --- Objectives of Research --- p.60 / Chapter 3.3. --- Study Approach and Experimental Design --- p.61 / Chapter Chapter 4 --- Materials and Methods --- p.63 / Chapter 4.1. --- Clinical Samples and ALL Cell Lines --- p.63 / Chapter 4.1.1. --- Clinical Samples from Pediatric Patients with ALL and Normal Healthy Donors --- p.63 / Chapter 4.1.2. --- ALL Cell Lines --- p.63 / Chapter 4.2. --- Genomic DNA Isolation from Clinical Samples and Cell Lines --- p.64 / Chapter 4.2.1. --- Ficoll Gradient Centrifugation --- p.64 / Chapter 4.2.2. --- DNA Extraction --- p.64 / Chapter 4.3. --- MS-AP PCR --- p.65 / Chapter 4.3.1. --- Methylation-sensitive Restriction Enzyme Digestion of Genomic DNA --- p.65 / Chapter 4.3.2. --- Arbitrarily Primed Polymerase Chain Reaction --- p.66 / Chapter 4.3.3. --- Isolation of Differentially Methylated DNA Fragments --- p.69 / Chapter 4.4. --- Cloning of Differentially Methylated DNA Fragments --- p.70 / Chapter 4.4.1. --- TA Cloning --- p.70 / Chapter 4.4.2. --- Screening of Positive Clones --- p.71 / Chapter 4.4.3. --- Preparation of Plasmid DNA by Alkaline Lysis Method --- p.72 / Chapter 4.5. --- DNA Sequence Analysis of Differentially Methylated DNA Fragments --- p.72 / Chapter 4.5.1. --- Dye-terminator Cycle Sequencing --- p.72 / Chapter 4.5.2. --- CpG islands Analysis of Differentially Methylated Sequences --- p.73 / Chapter 4.6. --- DNA Methylation Analysis --- p.74 / Chapter 4.6.1. --- Sodium Bisulfite Modification --- p.74 / Chapter 4.6.2. --- Combined Bisulfite Restriction Analysis --- p.75 / Chapter 4.6.3. --- Cloned Bisulfite Genomic Sequencing --- p.76 / Chapter 4.7 --- Gene Expression Study --- p.76 / Chapter 4.7.1. --- RNA Extraction from Clinical Samples and ALL Cell Lines --- p.76 / Chapter 4.1.2. --- Reverse Transcription PCR --- p.77 / Chapter 4.7.3. --- Semi-quantitative RT-PCR --- p.78 / Chapter 4.7.4. --- 5-aza-2 '-deoxycytidine Demethylation Treatment --- p.79 / Chapter Chapter 5 --- Results --- p.80 / Chapter 5.1. --- Generation of DNA Methylation Pattern by MS-AP PCR --- p.80 / Chapter 5.1.1. --- Differential Methylation Patterns of Childhood ALL --- p.84 / Chapter 5.1.2. --- Methylation Patterns of B and T lineages Childhood ALL --- p.86 / Chapter 5.2. --- UCSC BLAT Analysis of Differential Methylated DNA Sequences / Chapter 5.3. --- Identification of Candidate Gene --- p.89 / Chapter 5.4. --- Fibrillin 2 --- p.90 / Chapter 5.4.1. --- FBN2 CpG Islands: UCSC BLAT Search Analysis --- p.90 / Chapter 5.4.2. --- Verification ofFBN2 by ALL Cell Lines --- p.91 / Chapter 5.4.2.1. --- Semi-quantitative RT-PCR --- p.91 / Chapter 5.4.2.2. --- COBRA --- p.92 / Chapter 5.4.2.3. --- Cloned Bisulfite Sequencing --- p.94 / Chapter 5.4.2.4. --- Demethylation Treatment Resorted FBN2 mRNA Expression in ALL Cell Lines --- p.98 / Chapter 5.4.3. --- Studies ofFBN2 in Childhood ALL --- p.99 / Chapter 5.4.3.1. --- Methylation Analysis --- p.99 / Chapter 5.4.3.2. --- Semi-quantitative RT-PCR --- p.105 / Chapter Chapter 6 --- Discussion --- p.107 / Chapter 6.1. --- Genome-wide Screening Approach: MS-AP PCR --- p.107 / Chapter 6.2. --- Sample Selection in this Study --- p.109 / Chapter 6.2.1. --- MS-AP PCR --- p.109 / Chapter 6.2.2. --- Methylation Studies --- p.109 / Chapter 6.2.3. --- Studies in ALL Cell Lines --- p.110 / Chapter 6.3. --- Methylation Patterns in Childhood ALL --- p.111 / Chapter 6.4. --- Candidate Genes Selection Strategies in MS-AP PCR --- p.112 / Chapter 6.5. --- Fibrillin 2: mRNA Expression and Methylation Studies --- p.113 / Chapter 6.5.1 --- ALL Cell Lines --- p.113 / Chapter 6.5.2 --- Childhood ALL --- p.113 / Chapter 6.5.2.1 --- mRNA Expression and Methylation Studies --- p.113 / Chapter 6.5.2.2 --- Statistical Analysis --- p.115 / Chapter 6.5.3. --- Possible Roles of FBN2 in Leukemogenesis --- p.116 / Chapter 6.6. --- Clinical Application of FBN2 Aberrant Methylation --- p.119 / Chapter 6.6.1. --- Tumor Markers --- p.119 / Chapter 6.6.2. --- Use of Demethylating Drugs in Chemotherapy --- p.121 / Chapter 6.7. --- Limitations of Methylation Studies --- p.122 / Chapter 6.7.1. --- MS-AP PCR --- p.122 / Chapter 6.7.2. --- Techniques Used in Methylation Study --- p.122 / Chapter 6.7.3. --- Problems in Methylation Study --- p.123 / Chapter 6.8. --- Future Studies --- p.125 / Chapter Chapter 7 --- Conclusion --- p.127 / References --- p.128 / Appendix --- p.155

In certain neurodegenerative diseases such Dementia with Lewy Bodies (DLB), it is hypothesised that misfolded α-synuclein (α-syn) and β-amyloid both contribute to pathology. α-Syn and β-amyloid have been suggested to synergistically promote one another’s accumulation and aggregation, but the mechanisms are unknown. β-Amyloid is generated from β-/γ-secretase-mediated processing of the amyloid precursor protein (APP). This study investigated how α-syn overexpression in cells affects β-amyloid production from APP, using multiplex assays, luciferase reporter assays, and western blotting. Wildtype α-syn expression induces β-amyloid generation from APP in SH-SY5Y human neuroblastoma cells, and similar changes to APP processing occur in another neuronal cell model. Dominant-negative overexpression of α-syn mutants revealed that disrupting the N-terminal domain can increase APP amyloidogenic processing. Secretase enzymes that perform APP processing were next investigated. γ-Secretase activity, measured by a luciferase reporter, was not increased by α-syn overexpression. A higher ratio of β- to α-secretase processing was hypothesised, which led to expression and activity studies of the major β- and α-secretases, BACE1 and ADAM10 respectively. It was shown that the BACE1 protein expression is post-transcriptionally upregulated in α-syn cells, with increased APP cleavage in cells. ADAM10 protein expression is transcriptionally suppressed in wild-type α-syn cells, reducing total levels of catalytically active enzyme. However the change in ADAM10-mediated APP processing may be negligible since, critically, plasma membrane expression of ADAM10 appears to be maintained. To aid understanding of the mechanism that connects α-syn to APP processing, BACE1 expression was used in pharmacological studies of cell stress signalling. This approach revealed that in α-syn cells BACE1 lysosomal and/or proteasomal degradation may be disturbed. Additionally, BACE1 expression is induced by translational de-repression mediated by eIF2α ser-51 phosphorylation, which was increased in α-syn cells. Although preliminary, the data suggests a role for oxidative stress mediating the increased BACE1 expression in wild-type α-syn cells.

FE65 es una proteína adaptadora que actualmente se ha relacionado con la formación del péptido β-amiloide y la hiperfosforilación de tau, procesos que se ven incrementados en pacientes con Alzheimer, probablemente como consecuencia de un aumento en la actividad transcripcional de FE65. Una de las principales interacciones que se asocian con la activación de ésta proteína es la unión de su dominio PTB2 con el dominio citoplasmático de la proteína precursora del amiloide (AICD), interacción que se ve estabilizada por la proteína adaptadora 14-3-3γ. En esta tesis se llevó a cabo la construcción de un modelo por homología de PTB2 y un segmento de AICD, correspondiente a la secuencia de interacción con FE65. A partir de éstos, mediante técnicas de docking se determinó el modo conformacional de interacción más probable para el complejo, cuya interfaz de interacción fue estudiada mediante un análisis de geometrías de interacción, principalmente de formación de puentes de hidrógeno y también mediante el estudio del cambio en el área de superficie accesible a solvente entre los monómeros y el complejo. Una vez determinados los aminoácidos que interaccionan en la interfaz se realizó un estudio de la contribución de éstos en la estabilidad del complejo mediante una técnica conocida como Alanine Scanning. Se logró determinar la importancia de cada uno de estos aminoácidos en la formación del complejo, presentando así las bases necesarias para el futuro diseño de ligandos inhibidores de la interacción como estrategia farmacoterapéutica para combatir la patología del Alzheimer. A su vez se presenta un estudio preliminar de otro complejo involucrado en la interacción de FE65 y AICD, aquel formado entre 14-3-3γ con AICD, pudiendo determinar dos posibles sitios de unión. Por otra parte se realizó un breve estudio de un potencial sitio de unión de PTB2 a fosfoinositidos de membrana, interacción que se ha señalado como probable, pero cuyo posible papel en los procesos moleculares de FE65 no ha sido determinado aun.