Würzburg, Univ., Diss., 2002.

Thesis (M.S.)--University of North Texas, May, 2007. / Title from title page display. Includes bibliographical references.

Thesis (M. Sc.)--University of Canterbury, 2006. / Typescript (photocopy). Includes bibliographical references (leaves 103-111). Also available via the World Wide Web.

Th. 3e cycle--Biochim., microbiol. Lille 1, 1979. N°: 778.

The emergence of antimicrobial resistance is a major global threat to modern medicine. The rapid dissemination of resistant pathogens and the associated loss of efficacy of many important drugs needs to be met with the development of new antibiotics and alternative treatment options. A better understanding of the evolution of resistance could help in developing strategies to slow down the spread of antimicrobial drug resistance. In this thesis we investigated the evolution of resistance to two important antibiotics, rifampicin and ciprofloxacin, paying special attention to the resistance patterns occurring with high frequency in clinical isolates. Rifampicin is a first-line drug in tuberculosis treatment and resistance to this valuable drug limits treatment options. Our work on rifampicin resistance helps to explain the extreme bias seen in the frequency of specific resistance mutations in resistant clinical isolates of M. tuberculosis. We identified an important interplay between the level of resistance, relative fitness and selection of fitness-compensatory mutations among the most common resistant isolates. Fluoroquinlones are widely used to treat infections with Gram-negatives and the frequency of resistance to these important drugs is increasing. Resistance to fluoroquinolones is the result of a multi-step evolutionary process. Our studies on the development of resistance to the fluoroquinolone drug ciprofloxacin provide insights into the evolutionary trajectories and reveal the order in which susceptible wild-type E. coli acquire multiple mutations leading to high level of resistance. We found that the evolution of ciprofloxacin resistance is strongly influenced by the mutation supply rate and by the relative fitness of competing strains at each successive step in the evolution. Our data show that different classes of resistance mutations arise in a particular, predictable order during drug selection. We also uncovered strong evidence for the existence of a novel class of mutations affecting transcription and translation, which contribute to the evolution of resistance to ciprofloxacin.

No description available.

abstract: Proteins are a fundamental unit in biology. Although proteins have been extensively studied, there is still much to investigate. The mechanism by which proteins fold into their native state, how evolution shapes structural dynamics, and the dynamic mechanisms of many diseases are not well understood. In this thesis, protein folding is explored using a multi-scale modeling method including (i) geometric constraint based simulations that efficiently search for native like topologies and (ii) reservoir replica exchange molecular dynamics, which identify the low free energy structures and refines these structures toward the native conformation. A test set of eight proteins and three ancestral steroid receptor proteins are folded to 2.7Å all-atom RMSD from their experimental crystal structures. Protein evolution and disease associated mutations (DAMs) are most commonly studied by in silico multiple sequence alignment methods. Here, however, the structural dynamics are incorporated to give insight into the evolution of three ancestral proteins and the mechanism of several diseases in human ferritin protein. The differences in conformational dynamics of these evolutionary related, functionally diverged ancestral steroid receptor proteins are investigated by obtaining the most collective motion through essential dynamics. Strikingly, this analysis shows that evolutionary diverged proteins of the same family do not share the same dynamic subspace. Rather, those sharing the same function are simultaneously clustered together and distant from those functionally diverged homologs. This dynamics analysis also identifies 77% of mutations (functional and permissive) necessary to evolve new function. In silico methods for prediction of DAMs rely on differences in evolution rate due to purifying selection and therefore the accuracy of DAM prediction decreases at fast and slow evolvable sites. Here, we investigate structural dynamics through computing the contribution of each residue to the biologically relevant fluctuations and from this define a metric: the dynamic stability index (DSI). Using DSI we study the mechanism for three diseases observed in the human ferritin protein. The T30I and R40G DAMs show a loss of dynamic stability at the C-terminus helix and nearby regulatory loop, agreeing with experimental results implicating the same regulatory loop as a cause in cataracts syndrome. / Dissertation/Thesis / Ph.D. Physics 2011

No description available.

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / Apoptosis, or programmed cell death, is a genetically controlled form of cell suicide used to rid an organism of superfluous or damaged cells and serves as one of the major mechanisms for patterning during the development of complex animal structures. The antenna and ovary of the fruitfly, Drosophila melanogaster, were chosen as model systems in which to examine the molecular mechanisms of developmental apoptosis. The caspases are a family of cysteine proteases required for the execution of cell death, while the inhibitor of apoptosis protein Diap-1 is responsible for repressing the function of caspases. Diap-1, also known as Thread, was discovered in 1922 with the isolation of thread^1, a homozygous viable mutant whose molecular nature is unknown. thread was given its name due to its branchless or thread-like arista, the featherlike structure at the tip of the antenna. thread^1 antennal imaginal discs show excessive cell death during a brief period of larval development, which corresponds to a significant decrease in levels of Thread protein. Caspase activity is found more broadly than apoptotic DNA fragmentation in thread1 imaginal discs, suggesting that the mutant fails to inhibit caspases in many cells, but only a fraction succumb to apoptosis. These findings point to a narrow window of development in which regulation of programmed cell death is essential to the formation of the arista. Additionally, a candidate mutation has been discovered in a transcriptional regulatory region of the thread gene. Proof that this mutation is responsible for the branchless aristal phenotype may have important implications for understanding tissue-specific gene regulation during organogenesis. In the ovary, apoptotic roles for the DP subunit of the E2F transcription factor and the actin binding protein Profilin were investigated. While mutants for DP affect the regulation of Cytochome c and caspase activity in nurse cells, Profilin mutants show milder effects. To further characterize the role of apoptosis in the ovary, an ethylmethane sulphonate (EMS) mutagenesis screen was conducted and has led to the identification of several new genes. Future study of the apoptotic pathway will assist in developing treatments for diseases associated with its misregulation. / 2031-01-02

Genome wide association studies have shown that common variants in the human fat mass and obesity-associated (FTO) gene predispose to obesity and increased fat mass. Mice globally lacking Fto are lean, while mice globally overexpressing Fto have increased body weight due to increased fat mass. FTO protein was shown to localise to the nucleus and demethylate ssDNA and ssRNA. However, the mechanisms by which FTO mediates its effects on body phenotype remain unknown. In this thesis, I found that native FTO can be detected in both the nucleus and the cytoplasm during interphase, and that nuclear FTO was exported through the nuclear membrane during early prophase of the mitotic cell division. I developed co-immunoprecipitation (Co-IP) protocol to pull-down native FTO and identified a large number of new candidate binding partners (CBPs). Computational analysis predicted a role for FTO, and many CBPs, in RNA post-transcriptional modification and processing. I confirmed that the E3 ubiquitin-protein ligase TRIM21 interacts with FTO in multiple mouse tissues and binds FTO through its SPRY domain. Importantly, TRIM21 ubiquitinated FTO which did not lead to its degradation. FTO partially co-localised with TRIM21 and the decapping enzyme DCP2 in mRNA processing bodies (p-bodies). Overexpression of TRIM21 led to the accumulation of FTO outside the nucleus, but was reversed when both proteins were overexpressed. Additionally, I created a muscle specific Fto knock-out mouse model and found that lack of FTO in muscle did not result in the body composition phenotype reported in global Fto knock-out mice. Taken together, FTO can function in both the cytoplasm and the nucleus, where it interacts with TRIM21 which ubiquitinates FTO and potentiates its cytoplasmic localisation. Moreover, function of FTO in muscle does not mediate the obesity phenotype in mice.