Thesis (Ph. D.)--University of Texas at Austin, 1999. / Vita. Includes bibliographical references (leaves 97-104). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--University of Texas at Austin, 1999. / Vita. Includes bibliographical references (leaves 102-106). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--University of Texas at Austin, 2005. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--Physics, Georgia Institute of Technology, 2007. / Landman, Uzi, Committee Member ; Yannouleas, Constantine, Committee Member ; Bunimovich, Leonid, Committee Member ; Chou, Mei-Yin, Committee Member ; Pustilnik, Michael, Committee Member.

Orientador: Fernanda Ramos Gadelha / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-15T15:48:02Z (GMT). No. of bitstreams: 1 Silva_ThiagoMirandada_M.pdf: 571506 bytes, checksum: c09f01f1f6bb2c2777c4387f5674f71c (MD5) Previous issue date: 2010 / Resumo: O Trypanosoma cruzi é o agente etiológico da doença de Chagas (DC), cujo tratamento é feito através do uso do nifurtimox e benzonidazol. Esses medicamentos não são efetivos tornando a busca para novos alvos para o desenvolvimento de uma terapia mais específica uma prioridade. O alto grau de heterogeneidade existente entre as cepas representa um desafio para o desenvolvimento desta terapia, tornando a compreensão da biologia do parasita essencial nessa busca. O objetivo deste trabalho foi avaliar a funcionalidade do complexo I da cadeia respiratória de epimastigotas de T. cruzi ao longo da curva de proliferação (fases log, estacionária e estacionária tardia). Deste modo foi avaliado em duas cepas (Tulahuen 2 e Y) o consumo de oxigênio, potencial de membrana mitocondrial (??) e a atividade da enzima succinato desidrogenase (SDH), utilizando-se diferentes substratos respiratórios (malato/piruvato (M/P) malato/piruvato + malonato (MPM) ou succinato (SUC)). De um modo geral, em ambas as cepas o consumo de oxigênio foi maior na fase estacionária tardia em relação à log. A utilização de diferentes substratos não resultou em diferenças significativas nas taxas de respiração em ambas as cepas. Tulahuen 2 exibiu maiores taxas de consumo de oxigênio em relação à Y. Não foram observadas diferenças significativas nos valores de controle respiratório (-1,7) nas duas cepas, nas diferentes fases de proliferação. Na presença de um desacoplador da fosforilação oxidativa, as taxas não variaram na cepa Y, enquanto na Tulahuen 2 ocorreu um aumento em direção à fase estacionária tardia. A administração de malonato, inibidor competitivo da SDH, rendeu padrões diferenciados de inibição com a respiração sustentada por diferentes substratos que não variaram quando as células foram submetidas a um "jejum" (incubadas em PBS / 1 raM MgCb) por 3 horas. A atividade da SDH diminuiu em ambas as cepas na fase estacionária em relação à log, justificando a queda das taxas de inibição pelo malonato. Não foram registradas diferenças significativas com o aumento da concentração deste inibidor. A adição de cianeto de potássio não inibiu completamente a respiração, não importando o substrato utilizado ou a fase de proliferação, indicando que outras fontes além da cadeia respiratória estão consumindo oxigênio. Interessantemente, o ?? não variou entre as cepas em nenhuma fase de proliferação. Estes resultados fornecem novos dados sobre a cadeia respiratória do parasita, além de indicarem que não foi possível estabelecer a funcionalidade do complexo I, uma vez que o malonato não é um inibidor eficiente do complexo II. / Abstract: Trypanosoma cruzi is the etiological agent of Chagas' disease, where nifurtimox and benznidazole are used in treatment. These drugs are not efficient turning the search for new targets to be used in the development of a more effective therapy a priority. The high degree of heterogeneity among strains represents a challenge for the development of this therapy, and the comprehension of the parasite biology becomes essential in this search. The aim of this work was to evaluate the functionality of the respiratory chain complex I along the growth curve in T. cruzi epimastigotes. In this way it was analyzed in two strains (Tulahuen 2 and Y) the oxygen consumption, mitochondrial membrane potential (??) and succinate dehydrogenase (SDH) activity, using different respiratory chain substrates (Malate/Pyruvate, Malate/Piruvate + Malonate or Succinate). Generally, in both strains oxygen consumption was higher in the late stationary phase in relation to the log phase. The use of different substrates for the respiratory chain did not lead to significant variations in the respiratory rates in both strains. Tulahuen 2 showed higher oxygen consumption rates than the Y strain. No significant differences were observed in the respiratory control rates (~1,7) in both strains along the growth curve. In the presence of an uncoupler, the respiration rates did not vary in the Y strain while in Tulahuen 2 an increase towards the late stationary phase was observed. Addition of malonate, a SDH competitive inhibitor, resulted in distinct inhibition patterns when respiration was sustained by different substrates and did not change when cells were "starved" (incubated in PBS / 1 mM MgCb) for 3 hours. SDH activity decreased in both strains in the stationary phase in relation to log phase that could explain the decrease in the inhibition rates induced by malonate. No significant differences were observed with higher inhibitor concentration. Addition of potassium cyanide did not completely inhibit respiration in both strains regardless the substrate or growth phase, suggesting that other sources beyond the respiratory chain consume oxygen. Interestingly, ?? were similar between strains in all growth phases. These results provide new data about the parasite's respiratory chain indicating that complex I functionality was not possible to determine, once malonate is not a good inhibitor of complex II. / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

No description available.

Of the considerable challenges researchers face in the control and elimination of malaria, the development of antimalarial drug resistance in parasite populations remains a significant hurdle to progress worldwide. Atovaquone is used in combination with proguanil (Malarone) as an antimalarial treatment in uncomplicated malaria, but is rendered ineffective by the rapid development of atovaquone resistance during treatment. Previous studies have established that de novo mutant parasites confer resistance to atovaquone with a substitution in amino acid 268 in the cytochrome b gene encoded by the parasite mitochondrial genome, yet much is still unknown about how this resistance develops, and whether parasites are inherently predisposed to resistance development. Here we report phenotypic characterization of isolates from patients that failed treatment in the original atovaquone Phase II studies in Thailand by using a diverse series of chemotypes that target mitochondrial functions. We defined their structure-activity relationships and observed broad resistance (5-30,000 fold in atovaquone), suggesting that cytochrome b mutations alone are not sufficient to explain this spectrum of resistance. We also report the first known in vitro selection that recapitulates the clinical Y268S mutation using the TM90-C2A genetic background, the pre-treatment parent for TM90-C2B. Selection of the Y268S mutation in TM90-C2A and others indicates that the parasite genetic background is critical in the selection of clinical atovaquone resistance, since selection attempts in multiple other genetic backgrounds results in mutations at positions other than amino acid 268. We implicate mitochondrial heteroplasmy in the development of sporadic, rapid resistance to atovaquone, where pre-existing low-level mutations in the multi-copy mitochondrial DNA can be quickly selected for in parasite populations. High-coverage mitochondrial deep-sequencing data showed that low-level Y268S mutants were present in admission parasites from the atovaquone Phase II clinical trials in Thailand, and recrudescent parasites either maintained high level Y268S mutation frequencies or gradually returned to cryptic Y268S levels. The phenomenon of gradual heteroplasmic conversion back to wild-type was noted in some ex vivo patient isolated parasites as well as some in vitro selected lines, which suggests that other factors are at play that influence heteroplasmy stability. In addition to mitochondrial heteroplasmy, the total mtDNA copy number is likely influencing phenotypes in a gene dose-dependent fashion. Further, pressure on the DHODH enzyme that results in DHODH copy number amplifications/mutations has been shown to influence mitochondrial heteroplasmy directly. Last, mitochondrial diversity was shown to be vastly underestimated without heteroplasmic loci being taken into account, as seen in the re-analysis of the Pf3K MalariaGEN genome dataset we performed. The complex interactions between these drug resistance mechanisms reveal the phenotypic and genotypic plasticity that the Plasmodium falciparum parasite utilizes are a clear fitness advantage in the face of mitochondrial stress, and further studies are required to determine the impact of this wide-ranging phenotype on the development of new mitochondria-targeted drugs.

>Magister Scientiae - MSc / TiO2 nanoparticle films are important electron transport layers (ETLs) in photovoltaics such as dye-sensitised, perovskite and polymer hetero-junction solar cells. These films, however, have significant electron trap-sites as a result of the large density of oxygen vacancies present in nanosized TiO2. These trap-sites cause electron-hole recombination and ultimately lower photon-tocurrent conversion efficiency of the underlying cell during operation. Doping the TiO2 lattice with low atomic number elements such as nitrogen is a proven method to overcoming the charge transport inefficiency of TiO2 ETLs; another is the use of one-dimensional (1D) nanowires (NWs), instead of nanoparticles. Modification of TiO2 with non-metals leads to optical bandgap narrowing, improvement in electron conductivity and increased electron lifetime in the ETL layer. However, a lot of scope exists in understanding and fully quantifying the relationship between optical property, for example light transmission and bandgap modification, versus the doping concentration and type. Most doping approaches are in-situ and involve the addition of a dopant precursor (usually a salt) during the synthesis of TiO2 nanostructures – this invariably leads to uncontrolled doping levels, anion contamination and poor-quality materials – a need thus exists to develop simple, controllable doping approaches. One such approach, which forms the basis of this study, is ex-situ doping by means of plasma generated species in a controlled environment. This field of study is fairly novel and not widely studied, requiring more research to understand the doping mechanisms and influence on the optical and electronic properties of the underlying nanomaterials. In particular, controlled doping of TiO2 with nitrogen using radio-frequency generated (RF) plasma requires vigorous experimentation and characterisation. Inaccuracy of the deposition parameters during exposure remains a common drawback for this approach in addition to a lack of understanding of the surface interaction between the N2 species and specimen during irradiation.

In neurons, mitochondrial quantity and basal cellular respiration are maintained with age, but alterations in other key functions and quantities make these cells susceptible to the pathology of age-related neurodegenerative disease. We observed age-related decreases in cytochrome C, cardiolipin, cytochrome C oxidase (CCO) function, and glutamate response that render cells less capable of responding to stress. Rescue experiments showed that estrogen is a promising treatment in restoring neuron function with age. After finding key differences in CCO, we examined the electron transport chain more closely and found age-related deficits in quantity or function for each individual complex. Our experiments support a lack of endogenous substrates or a failure of upstream complexes to transport electrons to complex IV with age, ultimately leading to age-related neurodegeneration. Reactive oxygen species production may add to the problem by degrading macromolecules such as nucleic acid, cardiolipin, and proteins. Increased ROS may also lead to a redox imbalance in the neuron, reducing the potential for energy production. Also, epigenetic controls such as DNA methylation, histone acetylation ubiquitination and phosphorylation that persist in culture independent of aging hormone levels, vasculature, and immune system may be partly responsible for the observed age-related deficiencies as has been previously observed in aging human muscle (Ronn et al., 2008). This compelling cumulative evidence suggests an age-related deficiency in electron transport via quinones from complexes I to III, and age-related deficiencies in substrates, cofactors, and quantity or function for complex IV. These studies add to the growing body of evidence that dysfunction in the enzyme complexes of the electron transport chain lead to neurodegeneration in senescence-related diseases. In an attempt to integrate our age-related findings with Alzheimer's Disease (AD) pathology, we sequentially isolated the electron transport chain complexes using selective mitochondrial inhibitors in cortical neurons removed from the 3xTg-AD mouse model, which harbors mutations in the PS1, APPSwe and tauP301L genes and follows the proposed temporal development of human AD pathology (Oddo et al., 2003a; 2003b). Overall, we did not detect 3xTg-AD cortical neuron deficits at the four electron transport complexes of mitochondria or in NAD(P)H oxidase (NOX), an extramitochondrial oxygen consumer and regulator of NAD(P)+/NAD(P)H homeostasis (Morre et al., 2000).

>Magister Scientiae - MSc / TiO2 nanoparticle films are important electron transport layers (ETLs) in photovoltaics such as dye-sensitised, perovskite and polymer hetero-junction solar cells. These films, however, have significant electron trap-sites as a result of the large density of oxygen vacancies present in nano-sized TiO2. These trap-sites cause electron-hole recombination and ultimately lower photon-to-current conversion efficiency of the underlying cell during operation. Doping the TiO2 lattice with low atomic number elements such as nitrogen is a proven method to overcoming the charge transport inefficiency of TiO2 ETLs; another is the use of one-dimensional (1D) nanowires (NWs), instead of nanoparticles.