Thesis (Ph. D.)--Illinois State University, 1999. / Title from title page screen, viewed July 24, 2006. Dissertation Committee: David W. Borst (chair), Angelo P. Capparella, Sabine S. Loew, Edward L. Mockford, Carleton J. Phillips, Douglas W. Whitman. Includes bibliographical references (leaves 77-89) and abstract. Also available in print.

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

Thesis (Ph. D.)--University of Montana, 2007. / Title from title screen. Description based on contents viewed Aug. 20, 2007. Includes bibliographical references (p. 116-120).

Thesis (Ph. D.)--University of Oregon, 2006. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 63-68). Also available for download via the World Wide Web; free to University of Oregon users.

Orientador: Fabíola Freitas de Paula Lopes / Resumo: A função do espermatozoide pode ser comprometida por condições ambientais adversas. Estudos já demonstraram que a exposição in vivo e in vitro de espermatozoides bovinos a temperatura elevada induz a morte celular, reduz a motilidade espermática e o potencial fertilizante do espermatozoide. No entanto, as alterações celulares induzidas pelo choque térmico em espermatozoides bovinos ainda são controversas. Portanto, o objetivo deste estudo foi determinar o efeito do choque térmico em espermatozoides de touros holandeses na motilidade espermática, produção de espécies reativas de oxigênio (EROs), atividade mitocondrial, atividade de caspase, potencial de fertilização, cinética e desenvolvimento embrionário pré-implantacional. As amostras de sêmen foram descongeladas e submetidas ao gradiente de Percoll. Na amostra controle (sem incubação) os espermatozoides foram avaliados imediatamente após o gradiente de Percoll. Posteriormente, as amostras foram incubadas à 35 °C (Controle da temperatura testicular), 38,5 °C (temperatura corporal) e 41 °C (choque térmico) por 4 horas. O choque térmico de 41 °C reduziu a motilidade espermática após 2 h de incubação em comparação com 35 e 38,5 °C. A exposição de espermatozoides a diferentes temperaturas aumentou a produção de EROs, sendo este efeito mais acentuado no grupo 41°C em relação aos demais tratamentos. O aumento das EROs em espermatozoides submetidos ao choque térmico foi seguido da redução na atividade mitocondrial espermática e au... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

The present thesis focuses on the elucidation of human mitochondrial inner membrane presequence-translocation machinery with implications on cancer cell proliferation. Mitochondria are the endosymbiotic organelles in an eukaryotic cell performing a vast repertoire of functions and require approximately 1500 proteins. However, the mitochondria genome contains only 13 protein-coding genes primarily transcribing the complexes of the electron transport chain. Therefore, it is evident that most of the mitochondrial proteome is encoded by the nucleus and synthesized on cytosolic ribosomes. Chapter 1: Mechanism of mitochondrial inner membrane protein translocation and its oncogenic connection. Mitochondria consist of different routes of directing proteins to their intramitochondrial destinations. The presequence pathway, mediated by the inner membrane TIM23 complex, is responsible for the import of matrix and a number of single transmembrane helixes containing inner membrane proteins. This pathway accounts for approximately 60% of the total proteome imported into the organelle and hence, is the major focus of discussion in the present study. The components of the TIM23 complex can be subdivided into two groups, the protein conducting channel and the import motor. The initial translocation across the TIM23 channel utilizes the electrochemical membrane potential that exists across the inner membrane whereas the final step of the translocation process is driven by energy from ATP hydrolysis. MtHsp70 forms the central component of the import motor, and its function is regulated by the J-proteins. Pam18 stimulates the ATPase activity of mtHsp70. Pam16, on the other hand, forms a subcomplex with Pam18 and exerts an inhibitory effect its ATPase stimulatory activity, in turn regulating the activity of the import motor. The stoichiometric coupling with the substrate binding-release cycle of mtHsp70 drives the import process. Although the organization of presequence translocation machinery and its functional annotations have been described in detail in yeast system, little information is available on its organization in human. It is difficult to contemplate the existence of similar machinery in human mitochondria with complex and diversified functions. Human mitochondria apart from regulating the metabolic pathways are involved in progression of cancer, neurodegenerative disorders, responses to xenobiotic stress and induction of apoptosis. Numerous reports have shown that mutations and overexpression of human orthologs of translocase components are associated with various cancer subtypes. Such disease condition also involves targeting of specific cell signaling molecules that reprogram organellar functions and alter the cellular phenotype. Based on this evidence we defined our study into four broad objectives – 1) identify the components of human presequence translocase as Chapter two and three, 2) characterize the subunit organization of human presequence translocation machinery in Chapter four, 3) determine the functional connection between the translocase components and the cancer phenotype in Chapter four and five and 4) understand how the functions of J-proteins have evolved across the species as Chapter six. Chapter 2: Unraveling the role of Magmas in human mitochondrial protein transport. Pam16 plays a critical role in regulation of import process by governing the activity of the import motor. Proteins orthologous to Pam16 had been reported earlier to be overexpressed in various metabolically active tissues and cancer subtypes. We found that in humans a protein named as Mitochondria Associated Granulocyte Macrophage colony Stimulating factor signaling molecule (Magmas) showed significant sequence similarity with yeast Pam16 at its C-terminal region. Magmas was initially discovered as a protein that was overexpressed in neoplastic prostrate and when the cells were exposed to GM-CSF. Our experiments suggested that Magmas localized in human and yeast mitochondria and it was associated with the inner mitochondrial membrane. Magmas could complement the growth of yeast cells that were deleted for the essential gene PAM16 and could import precursor proteins into the mitochondria. Like Pam16, Magmas was able to form a stable heterodimeric subcomplex with yeast Pam18 and human Pam18 ortholog DnaJC19 (JC19). We found that J-domain forms the minimal region required for heterodimer formation between Magmas and Pam18/JC19. Mutations in Magmas J-like domain resulted in temperature sensitive growth phenotypes in yeast cells and associated import defect in translocating precursor proteins into the organelle due to inability to form a stable subcomplex with Pam18 and JC19, resulting in loss of import function. Loss of subcomplex formation leads to dissociation of Pam18 from the translocation machinery highlighting the importance of Magmas in tethering Pam18/JC19 to the presequence translocase. Magmas, showing characteristic of a J-like protein, was unable to stimulate the ATPase activity of mtHsp70. However, it exerted an inhibitory effect on the ATP stimulatory effect of the J-protein Pam18/JC19, indicating that Magmas has a regulatory effect on the overall activity of import motor. In contrast Magmas mutants those are incapable of forming a stable heterodimer with Pam18 were unable to regulate the activity of Pam18 resulting in import defects. In summary, our results highlight that Magmas is an ortholog of yeast Pam16 performing similar functions at the import channel. Chapter 3: Existence of two J-protein subcomplexes at the translocation channel with distinct physiological functions. JC19 has been regarded as the human ortholog of Pam18 whose loss of function was associated with dilated cardiomyopathy and ataxia syndrome. However, immunoprecipitation analysis using anti-Magmas antibody revealed the presence of a second J-protein identified as DnaJC15 (JC15) that shared a highly similar J-domain with JC19. JC15 was initially identified as a protein whose loss in expression resulted in development of a chemoresistant phenotype in ovarian carcinoma cells exposed to chemotherapeutic treatment. We found that JC15 localizes in mitochondria where it was associated with the inner membrane. Similar to Pam18 and JC19, JC15 heterodimerized with Magmas/Pam16 through its J-domain and associated with the presequence translocase of the inner membrane. A loss of function mutation at the J-domain of JC15 destabilizes its interaction with Magmas resulting in protein translocation defects and temperature-sensitive growth phenotype in yeast cells. The JC15 mutant showed inability to get associated with the translocation channel and had dysregulated stimulation of mtHsp70 activity leading to decreased mitochondria biogenesis and loss of mitochondrial membrane potential. In summary, our results showed that JC15 is the second human ortholog of Pam18 with similar functions. In contrast to yeast, in human mitochondria JC15 and JC19 were found to form two separate and distinct J-protein subcomplexes with Magmas at the mitochondrial import motor. The essentiality of the J-proteins for normal human mitochondria function was addressed through siRNA mediated downregulation of Magmas, JC19 and JC15. We found that Magmas and JC19 are essential for normal mitochondrial function and cell viability whereas JC15 is dispensable and might have a supportive role. Interestingly, both JC19 and JC15 interacted with Magmas with equal affinity and stimulated mtHsp70’s ATPase activity by equivalent levels. This shows that both JC19 and JC15 share similar properties in terms of their functions at the import channel, and the differences might be in a much broader perspective in terms of their association with the translocation channel. Chapter 4: Architecture of human mitochondrial inner membrane presequence -translocation machinery. In yeast, there exists a single J-protein subcomplex formed by Pam16 and Pam18, which is recruited to the sole translocase. However, humans present a completely different scenario where there exists a two distinct subcomplexes formed by Magmas with either of the J-proteins. So the question arises how the individual subcomplexes is recruited to the translocation machinery; whether they are associated to one or differentially recruited to two different translocases. We identified the existence of three distinct translocases in the human system constituted by the two J-proteins along with the Tim17 paralogs. JC15 along with Tim17a forms the translocase A of size similar to that of the yeast system, and it forms the ancestral translocase in the humans. Tim17b isoforms, on the other hand, associates with JC19 to form mammalian specific translocases B1 and B2. The association of the J-proteins at the translocation channel was found to be mediated by Magmas as a subcomplex. Downregulation of Magmas resulted in dissociation of both the J-proteins, and its overexpression resulted in redistribution of J-proteins at the translocases. We found that translocase B imported precursor proteins at a comparatively higher rate as compared to translocase A. Disruption of translocase B had deleterious effects on cell viability, respiratory chain complex's activities, Fe-S cluster biogenesis, mitochondria morphology, regulation of free radical levels and maintenance of mitochondrial genome. In contrast, depletion of translocase A did not significantly alter the survivability of cells, mitochondrial activity and maintenance of organellar morphology. This shows that translocase B is essential and performs the constitutive import function in the mammalian system whereas translocase A is dispensable and might have a supportive role in maintenance of mitochondrial function. However, translocase A play a specific role in human mitochondria in context to cancer cells. We observed that the elevated level of Tim17a found in cancer cells is responsible for maintenance of higher mitochondrial DNA copy number and higher proliferative potential of cancer cells. Additionally, translocase A also plays a specific role in translocation of cell signaling proteins that lack a mitochondrial targeting sequence into the mitochondria, highlighting the possible role of this translocase in neoplastic transformation. Chapter 5: Mechanistic insights into the role of JC15 as a part of translocase A in chemoresistant phenotype. JC15 had been initially identified to be associated with development of chemoresistance in cancer cells. However, the molecular mechanism followed by the protein has not been elucidated yet. Our studies have shown that overexpression of JC15 leads to increased sensitivity of cells to chemotherapeutic drug cisplatin and are coupled with complete loss of membrane potential, mitochondrial swelling and cytochrome c release. However, this chemosensitive phenotype was partially ameliorated upon preexposing the cell to cyclosporine A which is an inhibitor of cyclophilin D, a critical component of mitochondrial membrane transition pore (MPTP) complex. A similar reversal of phenotype was observed upon depleting cyclophilin D even under JC15 overexpressing background. This highlighted a possible functional connection between these two proteins. In order to check this hypothesis other way around, we overexpressed cyclophilin D in the cells which resulted in constitutive opening of the MPTP complex, enhanced mitochondrial swelling and reduced cell viability. In contrast, the gain of function anomalies of cyclophilin D overexpression was significantly reversed upon JC15 depletion. We observed through co-immunoprecipitation analysis that JC15 activates cyclophilin D by releasing it from the inhibitory effects of TRAP1 and couples it to the MPTP complex. Additionally, we have also shown that the J-domain of JC15 is critical for its interaction with cyclophilin D and loss of function mutation at the J-domain of JC15 disrupts its interaction with cyclophilin D. As a result the JC15 mutant is not able to mount a chemosensitive response to cisplatin drug. Chapter 6: Identification of regions determining the divergence of J-proteins functions at the mitochondrial import motor. The above studies show ample evidence to suggest that the two human J-proteins have undergone significant divergence in their function in human mitochondria in spite of having a highly similar J-domain. Therefore, we asked the question that how the human J-proteins have evolved and diversified from the primitive yeast protein Pam18 and what are the regional determinants in the protein sequence that dictate the function of the J-domain. We utilized a purely genetic approach to address the problem. We observed that JC19 was unable to rescue the growth of yeast cells deleted for the essential gene Pam18 and JC15 expression resulted in cold sensitive phenotype. We used JC15 as the model protein for our assays and applied three methodologies. First, generation and isolation of a series of mutations in JC15 that could rescue the cold sensitive phenotype, and the growth of the cells were similar to the wild type. Second, to identify the regulatory residues by isolation of second site suppressors that could be the suppressor the mutant phenotypes isolated earlier. Third, we utilized a purely evolutionary approach by swapping the individual domains between the three J-proteins- Pam18, JC19 and JC15. Our genetic data support the idea that the partial loss of function of human J-protein in the yeast system is due to altered subcomplex dynamics with Pam16. The altered dynamics of the subcomplex is mainly regulated by the residues in the arm, linker and helical regions of the J-domain, especially the helix II regions. Our analysis has also uncovered a critical role of the targeting (T) region of J-proteins which along with inter-membrane space (IMS) domain share significant sequence diversity among J-proteins in yeast and humans. The T-region in conjunction with the IMS domain plays a crucial role in regulating the J-domain’s function across the kingdoms and within the species. Although, our genetic data needs to be supplemented with biochemical evidence, this study provides significant insights into the diversity of J-protein function across the species and mode of their regulation through regions flanking the J-domain.

Abstract Analyses of mitochondrial control region sequences were used to infer phylogeny of Anser species, phylogeography of the lesser white-fronted goose, and genetic background of a captive stock. The genetic distances among the Anser species ranged from 0.9 to 5.5% in the complete control region sequences and supported the view of close relatedness of these species. Among the four most closely related species, the bean, pink-footed, white-fronted and lesser white-fronted goose, the branching order is uncertain. The short internal branches and low support for the branching order suggest that the species have diverged recently within short time-intervals. The mtDNA tree obtained is incongruent with the traditional view of the species relationships, but the reasons for this remain to be clarified. Two diverged mitochondrial lineages were found in the lesser white-fronted goose and a refugial origin was proposed. Basal haplotypes are geographically widespread and indicate a recent common ancestry for populations. The derived haplotypes are confined to singular breeding populations and suggest restrictions to the present female gene flow. A shift in the frequency of the mtDNA lineages approximately coincides with a migratory divide in the Taimyr Peninsula. Low mtDNA diversity and significant difference in the haplotype frequencies observed in Fennoscandian subpopulation suggested that it should be considered as a management unit. The fossil record was examined to gain additional information about the colonisation history of the species, but was found to be of limited use. The captive lesser white-fronted goose stock used for reintroduction/restocking was shown to be incompatible with the Fennoscandian wild population. Some captive individuals carried the mtDNA of the white-fronted goose suggesting a hybrid origin. Hybridisation has probably occurred during captive propagation, but to clarify further the extent of introgression, nuclear markers should be applied. The structure and evolution of the control region were studied by comparing complete avian sequences. Saturation was found to occur at pairwise divergences of 10% as shown for third codon positions of the mitochondrial genes previously. In pairwise comparisons of the control region and cytochrome b sequences, the rate of divergence varied among the lineages. Two conserved sequence blocks showed considerable sequence conservation when compared to mammalian sequences.

Abstract Genetic diversity in three taiga specialists – the Siberian tit (Parus cinctus), the Siberian jay (Perisoreus infaustus) and the Siberian flying squirrel (Pteromys volans) – was assessed by comparing DNA sequence variation across the mitochondrial control region and allele frequencies of microsatellites from samples collected from Fennoscandia and Siberia. Population sizes of these species have declined in association with fragmentation and loss of suitable forest habitat due to modern forestry practices in Fennoscandia. The red squirrel (Sciurus vulgaris) served as a reference for the flying squirrel. Genetic differentiation among species studied ranged from a panmictic population in the Siberian tit to that of the strong differentiation of populations (θST = 53%) in the flying squirrel in Finland. MtDNA and microsatellite data, together with assignment studies, showed the Siberian jay population to be significantly genetically structured and supported the existence of a metapopulation like structuring in Fennoscandia. Division of genetic variation among flying squirrel populations along the ancient shoreline of the Littorina Lymnea Sea stage of the Baltic Sea (7000 BP) and two geographically associated branches in the minimum spanning network supported a two-way colonisation history for the species. The Finnish inland appears to have been colonised from the east in association with the arrival of Norway spruce. At the same time, Coastal Finland was colonised from the south-east through the Karelian Isthmus. Gene flow of the species appeared female biased and restricted. Species exhibiting more restrictive dispersal characteristics and habitat requirements possessed stronger population genetic structure than those with opposite characteristics. Growth or contractions in population size leave characteristic signatures in mtDNA that can be studied by comparing different sequence diversity estimates among populations. I applied this method to the species studied. Significant differences in nucleotide diversities indicated restrictions in gene flow among populations in all species studied. Half of the Siberian jay populations gave a signal of population size bottleneck. All the species studied showed differences in their population genetic structures across their entire distribution ranges consistent with the multirefugia model, most likely to be attributable to differences in their ecological characteristics and Pleistocene histories.

Abstract Mitochondrial DNA sequences were used to study the phylogeny, population structure and colonisation history of Parus species. The phylogenetic relationships of seven European and three American species were examined by sequencing a part of the cytochrome b gene. Phylogenetically the closest species were the great tit (Parus major) and the blue tit (P. caeruleus). Subgenus Poecile was divided into two clades, one consisting of the Siberian tit (P. cinctus), the Carolina chickadee (P. carolinensis) and the Black-capped chickadee (P. atricapillus) and the other consisting of the marsh tit (P. palustris) and the willow tit (P. montanus). The coal tit (P. ater) and the crested tit (P. cristatus) did not group with any of the species studied. The population structure and the colonisation history of the willow tit, the great tit and the blue tit were examined by using control region sequences. The results suggest that the historical effective population size in the willow tit has been large and not contracted by the last ice age. Current gene flow must also be extensive as no population structuring was detected. No population structuring was evident either in the great tit and the populations showed distinctive signs of a recent population expansion. The patterns of genetic variation probably reflect a population bottleneck during the ice age, and a recolonisation of the European continent thereafter, presumably from a refugium situated in the Balkans. Two maternal lineages were found in the blue tit. The southern lineage was restricted to the Iberian peninsula whereas the northern lineage was detected from all the populations. The colonisation history has been similar to the one suggested for the great tit. The southern lineage, however, may have survived the ice age in a different refugium in the Iberian peninsula and was not as successful as the northern lineage in colonising available regions when the ice retreated. Both, the blue tit and the great tit have continued to expand their distribution northwards during this century and gene flow plays an important role in homogenising the populations.

Abstract This study describes clinical and biochemical consequences of the 3243A→G mutation in the tRNALeu(UUR) gene of the mitochondrial DNA. Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS syndrome) is usually caused by this mutation. Demyelinating polyneuropathy was observed as a novel feature in a patient with the mutation. Based on electrodiagnostic examination the polyneuropathy was defined as being of the demyelinating, mixed (motor more than sensory) type. In a 1 year follow-up an approximately 7% reduction in both the motor and sensory nerve conduction velocities were observed. The effect and mechanism of action of nicotinamide treatment in a MELAS patient with the 3243A→G was studied. The blood NAD concentration increased linearly, being 24-fold elevated at 6 weeks of treatment. Blood lactate and pyruvate concentration decreased by 50% within three days and 24 h urine lactate content within 2 weeks. A clinical improvement together with a decrease in the lesion volume in magnetic resonance imaging within the first month were observed. Alleviation of the lactate accumulation during the nicotinamide treatment suggested that an increase in the cellular NAD+NADH concentration led to enhancement of the oxidation of reducing equivalents, suggesting that complex I of respiratory chain operates at non-saturating substrate concentration. Myoblasts cultured from patients carrying the 3243A→G mutation and from controls were used to measure ATP, ADP, catalase and superoxide dismutase activity, population growth, apoptotic cell death and the morphology of cytoskeletal components. ATP and ADP concentrations were decreased, suggesting a decrease in the adenylate pool. The superoxide dismutase and catalase activities were higher than in control cells, suggesting an increased production of reactive oxygen species due to respiratory chain dysfunction. No increase in apoptotic cell death was observed in proliferating myoblasts, but randomization of vimentin filament direction and length was observed and decreased population growth was associated with the mutation. The results show that the 3243A→G mutation leads to numerous secondary pathophysiological events. Based on the literature and the results of this study, similarities were found between the pathophysiology of 3243A→G mutation and other neurodegenerative diseases and aging.