Thesis (Ph. D.)--University of Oregon, 1999. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 101-110). Also available for download via the World Wide Web; free to University of Oregon users. Address: http://wwwlib.umi.com/cr/uoregon/fullcit?p9957566.

Thesis (Ph.D)--Mechanical Engineering, Georgia Institute of Technology, 2010. / Committee Chair: Harvey Lipkin; Committee Member: Joel S. Sokol; Committee Member: Michael J. Leamy; Committee Member: Nader Sadegh; Committee Member: Stephen C. Harvey. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Thesis (Ph.D.)--Drexel University, 2010. / Includes abstract and vita. Includes bibliographical references (leaves 192-215).

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 128-142).

Thesis (Ph.D.)--University of Delaware, 2006. / Principal faculty advisor: Anne S. Robinson, Dept. of Chemical Engineering. Includes bibliographical references.

Thesis (Ph. D.)--University of Delaware, 2007. / Principal faculty advisor: Colin Thorpe, Dept. of Chemistry & Biochemistry. Includes bibliographical references.

Undergraduate honors paper--Mount Holyoke College, 2007. Program in Biochemistry. / Includes bibliographical references (leaves 64-67).

The kinetochore is a highly complex proteinaceous structure located at the primary constriction of mitotic chromosomes. Here, it performs an essential role in accurate chromosome segregation. Recently, much interest has been directed towards the Constitutive Centromere Associated Network (CCAN) components, as they participate in the formation of a scaffold involved in kinetochore assembly. It is therefore important to fully understand their role, and their distribution, at the kinetochore. Although many kinetochore proteins have already been identified, it is still unclear how centromeric chromatin folds to form the structure of the inner kinetochore. This is an interesting yet still open field of study, where the literature reports are still quite divided. In this study we take advantage of the high homologous recombination efficiency in DT40 B-lymphoma chicken cell lines, allowing the generation of conditional knockouts and deletion cell lines of several centromere proteins, subsequently engineered to stably express GFP:CENP-A. In the parental cell line the unfolding properties of the centromeric region were investigated by using TEEN buffer. Using fluorescence microscopy we were able to measure the length of many unfolded centromeric chromatin fibres, from both interphase and mitotic samples, based on the signal of GFP:CENP-A. A multi-peak analysis revealed the presence of discrete populations of fibres, recognised as peaks, in both interphase and mitotic samples. Compared with interphase, mitotic centromeres showed a greater level of compaction. Next, mutants for CCAN components, blocked in mitosis, were subjected to centromere chromatin unfolding. Results revealed that mitotic kinetochores depleted of CENP-C and CENP-S behaved similarly to the parental interphase samples, suggesting a role of those proteins in maintaining kinetochore structure. In contrast, CENP-O, CENP-H and CENP-I depletion did not seem to weaken the structure of the kinetochore. Additionally, we tested a hypothesis revealed by the multi-peak analyses, that chromatin layers exist in the inner kinetochore. Our data, when combined with published electron microscopy and crystallography measurements of centromere/kinetochore components, allowed us to assemble a robust and mathematically viable model that supports a boustrophedon organisation of the kinetochore chromatin. Finally, characterization studies of the novel kinetochore protein CENP-Z were performed. An involvement of CENP-Z in controlling the levels of di-methylation on lysine 4 of histone H3 was shown. This work represents an advance in our understanding of kinetochore structure in vertebrates.

Orientador: Makoto Yoshida / Banca: Valter Luiz Líbero / Banca: João Ruggiero Neto / Resumo: Estudamos o enovelamento de proteínas através da termodinâmica de uma cadeia heteropolimérica. Para isso, adaptamos um método de Monte Carlo introduzido por Wang e Landau para o estudo de transições de fase em sistemas magnéticos para estudarmos a mecânica estatística dessa cadeia. Trata-se de um método que se vale do passeio randômico no espaço de energias para determinação da densidade de estados acessíveis à cadeia e com ela determinar grandezas termodinâmicas do sistema em estudo. Para obtermos essa densidade de estados, adotamos modelos de rede para gerarmos todas as conformações geométricas possíveis de uma cadeia de 27 monômeros em uma rede cúbica. Estudamos várias seqüências de monômeros adotando os modelos de rede mais relevantes utilizados para a investigação do enovelamento de proteínas tais como o modelo HP e modelos mais elaborados que utilizam as interações de Miyazawa-Jernigan entre monômeros. Calculamos diversas grandezas termodinâmicas essenciais para a compreensão do enovelamento de proteínas e com isso mostramos a eficiência do método Wang-Landau. / Abstract: We studied the protein folding through investigating the thermodynamics of a hetero-polymeric chain. For this purpose, a Monte Carlo method introduced by Wang and Landau to study the phase transitions in magnetic systems, was adapted to investigate the statistical mechanics of this chain. The method is based on the random walk in the energy space to determine the density of states of the chain and important thermodynamics quantities. For this purpose, we adopted the lattice model and generate all geometric conformations of a 27-monomer chain in a cubic lattice. We studied several sequences of monomers adopting the most relevant lattice models, such as the HP model and more elaborated models considering the Miyazawa-Jernigan interactions. We computed several thermodynamics quantities to help us to understand the protein folding and show the efficiency of the Wang-Landau method. / Mestre

We present a new method for predicting the secondary structure of RNA sequences. Using our method, each RNA nucleotide of an RNA Sequence is represented as a point on a 3D triangular lattice. Using the Simulated Annealing technique, we manipulate the location of the points on the lattice. We explore various scoring functions for judging the relative quality of the structures created by these manipulations. After near optimal configurations on the lattice have been found, we describe how the lattice locations of the nucleotides can be used to predict a secondary structure for the sequence. This prediction can be further improved by using a greedy, 2-interval post-processing step to find the maximum independent set of the helices predicted by the lattice. The complete method, DeltaIS, is then compared with HotKnot, a popular secondary structure prediction program. We evaluate the relative effectiveness of DeltaIS and HotKnot by predicting 252 sequences from the Pseudobase Database. The predictions of each method are then scored against the true structures. We show DeltaIS to be superior to HotKnot for shorter RNA sequences, and in the number of perfectly predicted structures.