Thesis (Ph. D.)--University of Missouri--Columbia, 1996. / Typescript. Vita. Includes bibliographical references (leaves: 108-124). Also available on the Internet.

Thesis (Ph. D.)--Case Western Reserve University, 2005. / [School of Medicine] Department of Pharmacology. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Thesis (Ph. D.)--University of Oregon, 1999. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 90-101). Also available for download via the World Wide Web; free to University of Oregon users. Address: http://wwwlib.umi.com/cr/uoregon/fullcit?p9947978.

Thesis (Ph.D.)--University of Alberta, 2009. / Title from pdf file main screen (viewed on Sept. 18, 2009). "A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Virology, Medical Microbiology and Immunology, University of Alberta." Includes bibliographical references.

Thesis (Ph.D. in Molecular Biology) -- University of Colorado, 2005. / Typescript. Includes bibliographical references (leaves 100-113). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Theses (Ph. D.)--Marshall University, 2005. / Title from document title page. Includes abstract. Includes vitae. Document formatted into pages: contains xi, 149 p. Bibliography: p. 122-135.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 159-176).

Orientadores: Christine Hackel, Juergen K. V. Reichardt, Maricilda P. Mello / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T00:27:44Z (GMT). No. of bitstreams: 1 Ferraz_LucioFabioCaldas_D.pdf: 2604031 bytes, checksum: c313be68a5b9599e035866a8ee12ef8c (MD5) Previous issue date: 2006 / Resumo: O hormônio androgênico di-hidrotestosterona (DHT) possui fundamental importância na diferenciação sexual masculina e no desenvolvimento e manutenção da próstata. Duas enzimas atuam diretamente na concentração deste andrógeno nas células: 1) com uma função anabólica, a enzima 5α-redutase tipo 2 (gene SRD5A2) é responsável pela síntese de DHT ao converter testosterona (T) em 5α-di-hidrotestosterona e 2) com uma função catabólica, a enzima 3β- hidroxi desydrogenase/Δ5-Δ4-isomerase de esteróides tipo 2 (gene HSD3B2) é responsável pela degradação do DHT, além de contribuir para síntese indireta de testosterona por uma via anabólica. Isto exposto, cenários distintos se apresentam considerando as atividades deficientes dessas enzimas: i) a deficiência congênita da enzima 5a-redutase tipo 2 conduz a uma forma específica de pseudohermafroditismo masculino (PHM) no qual a conversão de T em DHT está nula ou defeituosa, inviabilizando a virilização normal da genitália externa em indivíduos com cariótipo 46,XY e ii) em razão das propriedades bifuncionais da enzima 3β-HSD2, tanto na via de síntese quanto de degradação de andrógenos, sua deficiência congênita pode conduzir a quadros clínicos distintos de ambigüidade genital. No adulto, mutações somáticas que afetem sua atividade enzimática podem contribuir para a manifestação do câncer de próstata, pelo acúmulo do DHT. O presente trabalho aborda as duas enzimas esteroidogênicas envolvidas com o metabolismo da DHT, buscando caracterizar mutações germinativas e/ou somáticas que conduzem a deficiências enzimáticas relacionadas a diferentes condições clínicas. Com relação à deficiência em 5a-redutase tipo 2, investigou-se a presença de mutações germinativas no gene SRD5A2 em amostras de DNA 20 pacientes de sexo genético masculino com suspeita de deficiência em 5α-redutase tipo 2, pertencentes a 18 famílias brasileiras, por meio de sequenciamento direto dos produtos de PCR dos cinco exons do gene e de suas regiões flanqueadoras. Foram identificadas alterações moleculares em 18 desses pacientes, compreendendo tanto mutações não anteriormente referidas na literatura (G158R, del642T, 217_218insC e IVS3+1G>A), como mutações recorrentes já descritas em outros grupos étnicos ou em indivíduos de outras regiões geográficas. Os resultados detalhados, bem como a discussão, acham-se apresentados no Capítulo III.1, sob a forma de artigo publicado. (...continua) / Abstract: The androgenic hormone dihydrotestosterone (DHT) has fundamental relevance in normal male sexual differentiation and in prostate development and maintenance. Two enzymes act directly on the regulation of DHT concentration at cellular level: 1) with an anabolic function the steroid 5α-reductase type 2 enzyme (SRD5A2 gene) leads to DHT synthesis by converting testosterone (T) in 5α-dihydrotestosterone and 2) with a catabolic pathway the 3β-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase type 2 enzyme (HSD3B2 gene) is responsible for DHT degradation, besides contributing to indirect synthesis of testosterone in an anabolic pathway. Thus, different scenarios can be considered regarding the deficiencies in the activities of these enzymes: i) congenital steroid 5α-reductase type 2 enzyme deficiency leads to a specific form of male pseudohermaphroditism (MPH), where the conversion of T into DHT is defective or inexistent, preventing normal virilization of the external genitalia in individuals with a 46,XY karyotype; and ii) due to the bi-functional properties of the 3β-HSD2 enzyme, either at synthetic or degradation androgen pathways, its congenital deficiency can lead to distinct manifestations of genital ambiguity. Furthermore, in the adult, somatic mutations that affect 3β-HSD2 enzymatic activities could contribute to prostate cancer manifestation, due to DHT accumulation. The present work approaches these two steroidogenic enzymes involved with the DHT metabolism, aiming to characterize germinal and/or somatic mutations leading to enzymatic deficiencies related to different clinical conditions. Concerning the steroid 5α- reductase type 2 deficiency, we screened for germinal mutations on SRD5A2 gene in DNA samples of 20 patients from 18 Brazilian families with suspected SRD5A2 deficiency, by directly sequencing of the PCR products from the five exons and flanking regions of the gene. Molecular alterations were detected in 18 of these patients, comprising either mutations not previously reported in the literature (G158R, del642T, 217_218insC e IVS3+1G>A) as well as recurring mutations already described in other ethnical groups or in individuals from other geographical regions. The detailed results and corresponding discussion are presented at Chapter III.1, as a published paper. (¿to be continued) / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular

Normal DNA replication is blocked by DNA damage in the template strand. Translesion synthesis is a major pathway for overcoming these replication blocks. In this process, multiple non-classical DNA polymerases form a complex at the stalled replication fork called the mutasome. This complex is structurally organized by the replication accessory factor PCNA and the non-classical DNA polymerase Rev1. One of the non-classical DNA polymerases within the mutasome then catalyzes replication through the damage. Each non-classical DNA polymerase has one or more cognate lesions, which the enzyme bypasses with high accuracy and efficiency. Thus, the accuracy and efficiency of translesion synthesis depends on which non-classical DNA polymerase within the mutasome is chosen to bypass the damage. In this thesis, I discuss how the most appropriate polymerase is chosen. In so doing, I examine the components of the mutasome; the structural motifs that mediate the protein interactions in the mutasome; the methods used to study translesion synthesis; the definition of a cognate lesion; the intrinsically disordered regions that tether the polymerases to PCNA and to one another; the multiple architectures that the mutasome can adopt, such as PCNA tool belts and Rev1 bridges; and the kinetic selection model in which the most appropriate polymerase is chosen via a competition among the multiple polymerases within the mutasome. Taken together, this thesis provides and inclusive review of the current state of what is known about translesion synthesis with conclusions at its end suggesting what major questions remain and ideas of how to answer them.

Trypanosoma brucei is the causative agent of Human African Trypanosomiasis (HAT), also known as African sleeping sickness. T. brucei is unique in several ways that distinguish this organism from other eukaryotes. One of the unique features of T. brucei is the organism’s mitochondrial DNA, which is organized in a complex structure called kinetoplast DNA (kDNA). Since kDNA is unique to the kinetoplastids, kDNA may serve as a good drug target against T. brucei. Previews studies have shown that kDNA has 4 different family A mitochondrial DNA polymerases. Three of these mitochondrial DNA polymerases (POLIB, POLIC, and POLID) are essential components of kDNA synthesis and replication. POLID and POLIC dynamically localize throughout the cell cycle. POLID is found dispersed in the matrix before the kDNA has undergone replication and is re-localized at the antipodal sites when the kDNA is dividing. POLIC is found in the kinetoflagellar zone (KFZ) at low concentrations when the kDNA is not replicating and relocalizes to the antipodal sites when dividing. Based on the dynamic localization of these two DNA polymerases, we hypothesize that POLIB undergoes dynamic localization at some point during the cell cycle stage. Here, a POLIB/PTP single expressor cell line was analyzed by immunofluorescence microscopy in an unsynchronized population. We characterized the localization pattern of POLIB-PTP at different cell cycle stages and found different localization patterns throughout cell cycle. Cells at 1N1K (the majority of cell in an unsynchronized population) have single foci, but at 1N1Kdiv two different patterns are mainly observed, diffuse and segregated. When the kDNAs are separated POLIB-PTP is again seen as a distinct foci in each kDNA. By doing TdT labeling and a quantitative analysis, we found that at early stages of minicircles replication POLIB-PTP start relocalizing to the kDNA disk with a diffuse pattern being the main. By the time the minicircles are being reattached in the disk (late TdT), POLIB is seen in the disk as a bilobe shape.