Yu Man Him. / Thesis (M.Sc.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 45-50). / Abstracts in English and Chinese. / Abstract --- p.ii / Acknowledgement --- p.ii / Chapter 1. --- Introduction / The mitochondrion --- p.1 / The mitochondrial genome --- p.3 / Mitochondrial DNA and diseases --- p.7 / Circulating plasma DNA --- p.8 / Project aims --- p.8 / Chapter 2. --- Materials and Methods / Choice of gene quantification system --- p.9 / Real time quantitative PGR --- p.11 / 7700Sequence Detection System --- p.14 / The LightCycler ´ؤ an alternative fast analyzer --- p.15 / Quatntitation of starting copy number using real time PCR --- p.17 / Primer and probe design rules --- p.18 / Hot start PCR technique --- p.19 / Other anti-contamination measures --- p.20 / Test subjects --- p.21 / Sample processing --- p.23 / DNA extraction --- p.24 / Chapter 3. --- System Development / Choice of primer and probe sequences --- p.28 / Optimization of PCR conditions --- p.30 / Imprecision of TaqMan assays --- p.33 / MTRNR2 vs. MTCYB probes --- p.33 / Construction of standard curve --- p.35 / Chapter 4. --- Research Application / The trauma model --- p.37 / Plasma DNA as a marker for trauma severity --- p.37 / Results --- p.38 / Disussion --- p.41 / Chapter 5. --- Conclusion --- p.44 / Chapter 6. --- References --- p.45

Ho, Ka Chai. / Thesis submitted in: November 2006. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 127-145). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract (Chinese) --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Tables --- p.ix / List of Figures --- p.x / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Molecular phylogeny of Metanephrops --- p.1 / Chapter 1.2 --- Identification of Nephropidae using DNA barcodes --- p.3 / Chapter Chapter 2 --- Literature Review --- p.5 / Chapter 2.1 --- Molecular phylogenetic studies of crustaceans --- p.5 / Chapter 2.1.1 --- Molecular phylogeny and reasons of using molecular markers in phylogenetic studies --- p.5 / Chapter 2.1.2 --- Characteristics of animal mitochondrial genome --- p.7 / Chapter 2.1.3 --- Examples of crustacean phylogenetic studies derived from mitochondrial DNA --- p.8 / Chapter 2.2 --- Identification of species based on DNA barcode --- p.17 / Chapter 2.2.1 --- Traditional taxonomy and its current practice --- p.17 / Chapter 2.2.2 --- Needs for DNA barcode --- p.18 / Chapter 2.2.3 --- Molecular identification based on DNA barcodes --- p.21 / Chapter 2.3 --- Taxonomy of Nephropidae --- p.28 / Chapter 2.3.1 --- Classification and phylogenetic relationship of Nephropidae --- p.28 / Chapter 2.3.2 --- Classification and distribution of Metanephrops --- p.31 / Chapter 2.3.3 --- Evolutionary history of Metanephrops --- p.36 / Chapter Chapter 3 --- Molecular Phylogeny of Metanephrops --- p.38 / Chapter 3.1 --- Introduction --- p.38 / Chapter 3.2.1 --- Species studied and sample collection --- p.41 / Chapter 3.2.2 --- DNA extraction --- p.43 / Chapter 3.2.3 --- Amplification of mitochondrial genes --- p.43 / Chapter 3.2.4 --- Nucleotide sequencing --- p.46 / Chapter 3.2.4.1 --- Asymmetric PCR --- p.46 / Chapter 3.2.4.2 --- Purification of asymmetric PCR products --- p.47 / Chapter 3.2.5 --- Sequence alignment --- p.47 / Chapter 3.2.6 --- Phylogenetic analyses --- p.48 / Chapter 3.3 --- Results --- p.50 / Chapter 3.3.1 --- PCR products of 16S rRNA and COI genes --- p.50 / Chapter 3.3.2 --- Nucleotide composition of 16S rRNA gene alignments --- p.52 / Chapter 3.3.3 --- Nucleotide composition of COI gene alignments --- p.54 / Chapter 3.3.4 --- Intraspecific and interspecific genetic variation --- p.56 / Chapter 3.3.5 --- Phylogenetic analysis based on 16S rRNA gene sequences --- p.61 / Chapter 3.3.6 --- Phylogenetic analysis based on COI gene sequences --- p.68 / Chapter 3.3.7 --- Phylogenetic analysis based on combined data set --- p.74 / Chapter 3.4 --- Discussion --- p.80 / Chapter 3.4.1 --- Interspecific genetic divergence --- p.80 / Chapter 3.4.2 --- Monophyly of the four species groups --- p.81 / Chapter 3.4.3 --- Phylogenetic relationship in Metanephrops --- p.84 / Chapter 3.4.4 --- Evolutionary history of Metanephrops --- p.90 / Chapter Chapter 4 --- Molecular Identification of Nephropidae --- p.92 / Chapter 4.1 --- Introduction --- p.92 / Chapter 4.2 --- Materials and methods --- p.93 / Chapter 4.2.1 --- Species studied and sample collection --- p.93 / Chapter 4.2.2 --- DNA extraction --- p.95 / Chapter 4.2.3 --- Amplification of genes --- p.95 / Chapter 4.2.4 --- PCR profiles for mitochondrial genes --- p.97 / Chapter 4.2.5 --- Nucleotide sequencing --- p.97 / Chapter 4.2.6 --- Purification of asymmetric PCR products --- p.97 / Chapter 4.2.7 --- Sequence alignment --- p.97 / Chapter 4.2.8 --- Cluster analysis --- p.97 / Chapter 4.2.9 --- Graphical summary of species similarity --- p.98 / Chapter 4.2.10 --- Testing of molecular identification system in Nephropidae --- p.98 / Chapter 4.3 --- Results --- p.100 / Chapter 4.3.1 --- PCR products and sequence alignments of 16S rRNA and COI genes --- p.100 / Chapter 4.3.2 --- Species identification for clawed lobsters --- p.100 / Chapter 4.3.2.1 --- 16S rRNA profile --- p.100 / Chapter 4.3.2.2 --- COI profile --- p.108 / Chapter 4.4 --- Discussion --- p.116 / General Conclusion --- p.124 / Literature Cited --- p.127 / Appendices --- p.146

Les mitochondries jouent un rôle majeur dans le métabolisme de l'ATP des cellules eucaryotes. Le maintien de l'ADN mitochondrial (ADNmt) est fondamental pour la production d'énergie chez les organismes aérobie stricte. De grandes délétions de ADNmt sont à l'origine d'anomalies mitochondriales entrainant des maladies chez l'homme. Plusieurs gènes nucléaires impliqués dans le métabolisme de l’ADNmt ont été identifiés et caractérisés chez l'homme. Cependant, l’ensemble des facteurs et leurs activités requis pour le maintien de l'ADNmt reste largement inconnu. L'identification de ces facteurs et la détermination de leurs activités dans des systèmes modèles simples peuvent contribuer à l’étude du maintien de l'ADNmt et à la compréhension des mécanismes induisant des délétions de l’ADNmt chez l'homme. Le champignon filamenteux Podospora anserina est un modèle d'étude du maintien de l’ADNmt. Chez P. anserina, l’accumulation de délétions région-spécifiques de l’ADNmt (Δmt) est corrélée à la présence de la mutation AS1-4 dans le gène nucléaire codant la protéine cytosolique ribosomale S15. L'altération de la protéine S15 pourrait modifier la traduction de transcrits codant des protéines impliquées dans le maintien de l'ADNmt et indirectement causer l'accumulation des Δmt. Par une approche globale (translatome), nous avons analysé l’ensemble des transcrits associés aux ribosomes AS1-4 en cours de traduction. A partir des données obtenues, deux gènes candidats, PaIML2 et PaYHM2 potentiellement impliqués dans le maintien de l'ADNmt, ont été identifiés et étudiés. L'analyse fonctionnelle a été principalement développée pour PaYHM2. La protéine PaYHM2 partage 68% d’identité avec la protéine mitochondriale bi-fonctionnelle Yhm2 de levure, impliquée dans le transport de métabolites dans la mitochondrie et possèdant un domaine de liaison à l'ADN. J'ai démontré que le gène PaYHM2 est essentiel pour P. anserina, un organisme aérobie stricte et que la protéine PaYHM2 est mitochondriale. Par mutagénèse, j'ai montré que c'est la fonction de transport qui est essentielle à la survie du champignon et non pas la putative capacité à se lier à l'ADN. Les résultats obtenus suggèrent également que PaYHM2 participe au métabolisme de l'acétyl-CoA chez P. anserina. / Mitochondria play main role as adenosine triphosphate (ATP)-energy factories of the eukaryotic cells. To ensure energy production, mitochondrial DNA (mtDNA) maintenance is essential for all obligate-aerobe eukaryotic organisms. Large-scale mtDNA deletions are major causes of mitochondrial dysfunction in human diseases. Several nuclear genes implicated in mtDNA metabolism were identified and characterized in human. Nuclear-encoded factors and their activities required for mtDNA maintenance are, however largely unknown. Identification of these factors and discovery of their activities in simple model systems can contribute to the comprehension of mtDNA maintenance and of the mechanisms leading to mtDNA deletions in human. The filamentous fungus Podospora anserina is a useful model system for studying mtDNA maintenance. An S15 cytosolic ribosomal protein mutant in P. anserina, named AS1-4 mutant, shows a positive correlation with the accumulation of specific large mtDNA deletion (Δmt) at the time of death. Alteration of S15 protein might modify translation of transcripts encoding proteins related to mtDNA maintenance and indirectly cause Δmt accumulation. Polysome profiling (called translatome), a global approach giving genome-wide informations about modified transcripts on translation, was performed on AS1-4 mutant. From the data of this translatome, two candidate genes potentially related to mitochondrial DNA maintenance, the PaIML2 gene and PaYHM2 gene has been identified and functionally analyzed. The function of the PaYHM2 gene has been especially characterized in this project. This gene encodes a protein sharing 68% of identity with yeast Yhm2, a bi-functional protein as a mitochondrial carrier and as a protein with DNA-binding activity. I demonstrated that the PaYHM2 gene is essential for P. anserina, an obligate-aerobe organism and that the PaYHM2 protein localizes to mitochondria. Through mutagenesis approach, I showed that the transport function decides the essentiality of mitochondrial carrier PaYHM2 while the putative DNA binding activity of PaYHM2 protein is important for P. anserina. Furthermore, I found that the function of PaYHM2 probably participates in the cytosolic acetyl-CoA metabolism.

The F-box and leucine-rich repeat factor (FBXL4) locus is altered in two distinct diseases, a pediatric mitochondrial encephalopathy associated with early death, and the highly aggressive hematological malignancy T-cell Acute Lymphoblastic Leukemia (T-ALL). As an F-box protein, FBXL4 is predicted to target specific protein substrates for proteasomal degradation. Notably, not much is known about the roles of FBXL4 in homeostasis or disease, and thus I generated conditional Fbxl4 knockout mice to characterize the contributions of Fbxl4 to mitochondrial encephalopathy and to T-ALL. Homozygous mutations in FBXL4 are associated with pediatric-onset mitochondrial encephalopathy, but the molecular and cellular mechanisms driving disease pathogenesis are unknown. Here, I show that constitutive loss of Fbxl4 recapitulates key features of human mitochondrial encephalopathy, including microcephaly, failure to thrive, and perinatal lethality. Moreover, Fbxl4 inactivation drives profound metabolic alterations in the perinatal period. On the cellular level, loss of Fbxl4 results in mitochondria DNA depletion and disrupts oxidative phosphorylation and mitochondria membrane potential. Isolation of the FBXL4 protein complex reveals that FBXL4 interacts with a diverse set of mitochondrial factors crucial for normal mitochondrial function. Overall, these findings underscore the importance of FBXL4 in development, metabolism, and mitochondrial dynamics, and may be used to develop novel therapies for patients with mitochondrial encephalopathy associated with FBXL4 mutations and for patients with 6q- T-ALL.

The present work addresses outstanding questions within the field of primary mitochondrial disease biology and treatment, by incorporating methods from structural biology, molecular biology, and animal studies. First, we utilize a mouse model of mitochondrial deoxyribose nucleic acid (mtDNA) disease to demonstrate the potential therapeutic benefit of low-dose chronic rapamycin treatment. Interestingly, rapamycin therapy significantly extends survival, but does so in the absence of correcting the underlying mitochondrial defect. Next, we focus on human cellular models of mtDNA-based diseases, and show that rapamycin treatment does not induce mitochondrial quality control-mediated clearance of pathogenic mtDNA mutation-harboring organelles. Finally, we investigate a mitochondrial disease phenotype at the level of the organelle, by utilizing in situ cryo-electron tomography to demonstrate the ultrastructural consequences of a pathogenic mutation affecting mitochondrial energy production. We conclude by highlighting the insights into disease biology and treatment that can be gained through a multi-level approach integrating techniques from multiple biomedical fields.

No description available.

It is well known that the mitochondrial genome has a much higher spontaneous mutation rate than the nuclear genome. mtDNA mutations have been identified in association with many diseases and aging. mtDNA replication continues throughout the cell cycle, even in post-mitotic cells. Therefore, a constant supply of nucleotides is required for replication and maintenance of the mitochondrial genome. However, it is not clear how dNTPs arise within mitochondria nor how mitochondrial dNTP pools are regulated. Recent evidence suggests that abnormal mitochondrial nucleoside and nucleotide metabolism is associated with several human diseases. Clearly, to uncover the pathogenesis of these diseases and the mechanisms of mitochondrial mutagenesis, information is needed regarding dNTP biosynthesis and maintenance within mitochondria, and biochemical consequences of disordered mitochondrial dNTP metabolism. The studies described in this thesis provide important insight into these questions. First, we found that a distinctive form of ribonucleotide reductase is associated with mammalian liver mitochondria, indicating the presence of de novo pathway for dNTP synthesis within mitochondria. Second, we found that long term thymidine treatment could induce mtDNA deletions and the mitochondrial dNTP pool changes resulting from thymidine treatment could account for the spectrum of mtDNA point mutations found in Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) patients. These results support the proposed pathogenesis of this disease. Third, we found that normal intramitochondrial dNTP pools in rat tissues are highly asymmetric, and in vitro fidelity studies show that these imbalanced pools can stimulate base substitution and frameshift mutations, with a substitution pattern that correlates with mitochondrial substitution mutations in vivo. These findings suggest that normal intramitochondrial dNTP pool asymmetries could contribute to mitochondrial mutagenesis and mitochondrial diseases. Last, Amish lethal microcephaly (MCPHA) has been proposed to be caused by insufficient transport of dNTPs into mitochondria resulting from a loss-of-function mutation in the gene encoding a mitochondrial deoxynucleotide carrier (DNC). We found that there are no significant changes of intramitochondrial dNTP levels in both a MCPHA patient's lymphoblasts with a missense point mutation in Dnc gene and the homozygous mutant cells extracted from Dnc gene knockout mouse embryos. These results do not support the proposed pathogenesis of this disease and indicate that the DNC protein does not play a crucial role in the maintenance of intramitochondrial dNTP pools. / Graduation date: 2005

Caenorhabditis elegans is arguably the best understood animal on the planet. Used for over 50 years to study development, we have a vast amount of knowledge of the inner workings of this worm. Our knowledge is incomplete, however, without placing this organism in its evolutionary and ecological context. In this body of work, I focused on examining the evolutionary forces shaping Caenorhabditis nematodes, with a particular emphasis on C. briggsae. In the first part, I examined the evolution of mitochondrial genomes throughout the genus. I tested for signatures of selection and examined the evolution of mitochondrial genome architecture. Through this, I have shown that the mitochondrial genomes of Caenorhabditis nematodes appear to be primarily influenced by purifying selection and that molecular evolutionary inference is greatly limited by mutational saturation. The evolutionary forces acting on mitochondrial genomes have been examined before, however, this study, extensively examining this within a single genus, provides a much better characterization than any of the studies to date. In the second part, I characterized the evolutionary dynamics of mitochondrial pseudogenes in C. briggsae and its closest relatives. I showed that these elements, while they might not evolve under strictly neutral terms, are still quite useful in uncovering cryptic diversity and population structure. I also observed that they appear/disappear in a manner that appears inconsistent with one commonly held model for mitochondrial pseudogene evolution. In the final part, I examined the evolution of C. briggsae in response to a biotic environment. I showed that fitness in a parasite-containing environment incurs a trade-off with fitness in the absence of parasites. Together, the chapters of this dissertation demonstrate the strength of Caenorhabditis, and in particular C. briggsae, for examining evolutionary questions and advances this system as a tool for evolutionary biology research. / Graduation date: 2013

Thesis (M.Sc.)--York University, 2004. Graduate Programme in Kinesiology and Health Science. / Typescript. Includes bibliographical references. Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL:http://gateway.proquest.com/openurl?url%5Fver=Z39.88-2004&res%5Fdat=xri:pqdiss&rft%5Fval%5Ffmt=info:ofi/fmt:kev:mtx:dissertation&rft%5Fdat=xri:pqdiss:MQ99349

Intraspecific relationships among the anchialine cave shrimp Typhlatya mitchelli were examined by sequencing a total of 1505 bp from portions of three mitochondrial DNA genes. Cytochrome b, cytochrome oxidase I, and 16S rRNA were partially sequenced and analyzed for specimens from six different cenotes (water-filled caves) across the Yucatan Peninsula, Mexico. The conspecific Typhlatya pearsei that is sympatric with T. mitchelli was also sequenced and used as the outgroup. Comparisons among specimens of T. mitchelli yielded low sequence divergence values (0-1.7%), with the majority being less than 0.4%. Phylogenetic tree topologies reconstructed with neighbor-joining, maximum likelihood, and maximum parsimony were in agreement in regards of the resolution of deep branches. Also, there was no obvious geographic differentiation among the majority of T. mitchelli samples, with the exception of specimens from Cenote San Antonio Chiich (Yokdzonot, Yucatan, Mexico) which all clustered into an extremely well supported monophyletic group. The level of differentiation of this group, together with the nearly total absence of differentiation among T. mitchelli from distant cave systems, suggests that this is an Evolutionary Significant Unit (ESU), which may correspond to a new species. This unidentified Typhlatya from Cenote San Antonio Chiich was helpful in establishing a period in which the epigean ancestor colonized the cenotes. Based on pairwise distance data and previously published shrimp molecular clocks (Baldwin et al., 1998), T. mitchelli and the putative new Typhlatya species last shared a common ancestor between 3-5 million years ago (mya), during the mid-Pliocene era, while T. mitchelli and T. pearsei was approximately 7-10 mya (middle to late Miocene). The ancestor to T. mitchelli and the unidentified Typhlatya species abandoned its shallow coastal water existence in the early Pliocene and eventually expanded its range across the peninsula. Approximately 4 mya, Cenote San Antonio Chiich became isolated from the remaining gene pool thereby halting gene flow. As the regional water table fluctuated in response to the rise and fall of Pleistocene sea levels, T. mitchelli actively colonized the peninsula. The discovery of a single, continuous subterranean freshwater system provides for a better understanding of anchialine biogeography within the Yucatan Peninsula.