Mitochondria not only supply the energy for cell function, but also take part in cell signaling. This review describes the dysfunctions of mitochondria in aging and neurodegenerative diseases, and the signaling pathways leading to mitochondrial biogenesis (including PGC-1 family proteins, SIRT1, AMPK) and mitophagy (parkin-Pink1 pathway). Understanding the regulation of these mitochondrial pathways may be beneficial in finding pharmacological approaches or lifestyle changes (caloric restrict or exercise) to modulate mitochondrial biogenesis and/or to activate mitophagy for the removal of damaged mitochondria, thus reducing the onset and/or severity of neurodegenerative diseases.

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A thesis submitted to the Faculty of Medicine, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. / The subject of this thesis is mitochondrial DNA (mtDNA) variation in southern African populations. The purpose of this study was twofold. Firstly, mtDNA variations were used to investigate the genetic affinities of Negroid, Khoisan, Caucasoid and "Coloured" populations in an attempt to refine theories on southern African population affinities and prehistory. MtDNA variations were detected using two different methods. The first method makes use of restriction fragment length polymorphisms (RFLPs) detected with the restriction enzymes Hpal, BamUI, Haell, Mspl, Avail and Hindi in 795 unrelated individuals from twenty ethnic groups within the Khoisan, Negroid, Caucasoid and "Coloured" populations from South Africa and Namibia. The combinations of the various restriction enzyme patterns (morphs) for the enzymes Hpal, Bam HI, Hae II, Mspl, Avail and Hindi (in this order), were used to derive the mtDNA type for each individual studied. This resulted in the discovery of 52 distinct mtDNA types: 30 of which had been previously reported, 28 out of 32 resulted from new combinations of enzyme morphs and 4/32 were due to the discovery of new enzyme morphs (MspI-17 in the Ashkenazi Jewish population and AvaII-31, AvaII-32 and AvaII-33 in the South African "Coloured" population). The second method involves sequencing approximately 750 base pairs of mtDNA contained within the two hypervariable segments within the non-coding control region of the mtDNA molecule in 144 individuals, most of whom where investigated for mtDNA RFLP variations. Pairwise comparisons of mtDNA sequences revealed 119 variant sites which gave rise to 129 unique mtDNA types. / WHSLYP2017

Bobcats (Lynx rufus) are generalist carnivores with an expansive range from southern Canada to northern Mexico, in which 11 - 12 subspecies have been described. Since European settlement, bobcat habitat has become increasingly fragmented due to urbanization and development of agricultural land. Presently, there is little information on the genetic structure of bobcat populations at large spatial scales. Furthermore, it is unknown whether the current isolation of bobcat populations is a historic feature or whether recent landscape alterations have disrupted dispersal among previously connected populations. To address these questions, microsatellite and mitochondrial DNA data were used for relatedness measurements, spatial autocorrelation examinations, Bayesian assignment testing and sequence analysis of bobcats across their range. These analyses showed that, within southern Illinois, females were closely related to one another and males tended to disperse from their natal area. On a regional scale, microsatellite data revealed several distinct genetic groups within the midwest, notably eastern and western bobcat populations. In contrast, mitochondrial DNA analyses resolved little differentiation among bobcat populations, elucidating two-three phylogeographic groups. Taken together, these data reveal that bobcats have not historically, experienced large barriers to dispersal. Rather, recent habitat alterations may be disrupting dispersal over large scales. Genetically defined groups are potential conservation units and should be used for regional management of bobcats.

Changes in gene expression in response to mitochondrial dysfunction are mediated by components of the retrograde signaling pathway. The mitochondrial signal is recognized and transferred to the nucleus by dynamic interactions between regulatory proteins Rtg2p, Mks1p and Bmh1p. Retrograde signaling genes have been well characterized in the budding yeast Saccharomyces cerevisiae but very little is known about the retrograde response of other fungi. To identify retrograde signaling proteins in other fungi, the protein sequence encoded by the S. cerevisiae RTG2 gene was used to search for fungal homologs using NCBI BlastP and the T-Coffee Multiple Sequence Alignment program. We selected four species having uncharacterized ORFs with more than 66% amino acid identity to Rtg2p for further analysis: Ashbya gossypii, Candida glabrata, Vanderwaltozyma polyspora and Kluyveromyces lactis. In S. cerevisiae, cells deleted for RTG2 are glutamate auxotrophs, and have reduced expression of Aco1p and Cit2p proteins. To determine whether the putative RTG2 genes we identified encode bonefide regulators of the retrograde response pathway, we used standard yeast genetic approaches and molecular biology tools to investigate their ability to complement the defects associated with the rtg2Ä mutant using our S. cerevisiae RTG2 shuffle strain. We investigated functional roles of Rtg2p homologs by comparing Cit2p and Aco1p protein levels, glutamate auxotrophy, as well as analyzing the interaction between Rtg2p homologs and Mks1p. We also analyzed sensitivity of mutant strains under various stress conditions to address possible signaling cross talk between the retrograde signaling pathway and the TOR pathway. Our data show that the fungal Rtg2p homologs from C. glabrata, V. polyspora and K. lactis are functional in mediating the mitochondrial signal through known components of the retrograde signaling cascade. Our immunoprecipitation data suggest that TOR and retrograde signaling may exhibit cross pathway activation under rapamycin treatment. We show that Mks1p, the negative regulator of retrograde signaling pathway is required for Cit2p expression under rapamycin treatment. Given that all Rtg2p homologs showed low affinity for Mks1p which was in turn paralleled by a higher affinity of Mks1p for Bmh1p suggests that Rtg2p may have an additional functional role in influencing the association of Mks1p with Bmh1p.

The overall objective of this thesis was to examine mitochondrial bioenergetics in muscle biopsies from humans with type 1 diabetes (T1D) to gain a deeper understanding of the cellular mechanism(s) underlying changes to skeletal muscle health reported in T1D, a phenotype we have referred to as ‘diabetic myopathy’. It was hypothesized that humans with T1D, compared to their matched counterparts without diabetes (control), would demonstrate significant deficiencies in muscle mitochondrial function and ultrastructure/content as determined by the gold-standard in vitro methodology: high-resolution respirometry and transmission electron microscopy, respectively. It was further hypothesized that sex differences would not exist in mitochondrial function with T1D, and mitochondrial deficiencies would be more dramatic at an earlier age with T1D. Adults with uncomplicated T1D and strictly matched controls (age, sex, BMI, self-reported physical activity levels) were recruited from surrounding university-dwelling communities. Site-specific deficiencies in mitochondrial respiration, H2O2 emission, and calcium retention capacity were found in young, physically active adults with T1D despite normal mitochondrial content. Further experiments revealed that muscle mitochondrial respiration in women and men differentially adapt to the T1D environment where men with T1D have lower complex II but higher complex I respiration compared to women with T1D, while women (irrespective of T1D) have lower ADP sensitivity. Women with T1D also demonstrated lower H2O2 emission compared to men with T1D. In contrast, despite a lower mitochondrial content in middle- to older-aged adults with T1D, mitochondrial respiration (normalized to content) was either normal or increased in adults with T1D compared to control, with observable differences between sexes. Overall, this research has demonstrated that despite being recreationally to physically active, adults with uncomplicated T1D with moderately well-managed glycemia demonstrate alterations in skeletal muscle mitochondrial function and ultrastructure, including differences between sexes. / Dissertation / Doctor of Science (PhD) / Type 1 diabetes (T1D) is a complex disease that still has no known cure. Current treatment focuses on managing blood sugar levels with exogenous insulin injections and frequent blood sugar checks. However, over time, people with T1D still develop serious complications that inevitably impact their quality of life and lifespan. A potential adjuvant therapy to prevent complications in T1D is improving the health of skeletal muscle through exercise given its role in stabilizing blood sugar/lipid levels and whole-body insulin sensitivity. However, this area continues to be severely understudied in the T1D population. Thus, this thesis examined skeletal muscle metabolic ‘health’ from adults with T1D who do not have major diabetes complications and manage their blood glucose moderately-well. Through a series of novel experiments, we found that young and middle- to older-aged adults with T1D have alterations in the metabolic engines of their muscles, and depending on biological sex, the alterations manifest as either heightened or degraded cellular function. These findings are the first to provide a comprehensive cellular investigation of the impact of T1D on the metabolic health of skeletal muscle in people with T1D and provide the foundation for future research examining skeletal muscle as an essential and early adjuvant therapy in this population.

Mitochondrial DNA (deoxyribo nucleic acid) is typically used in forensic casework when small quantities of high molecular weight quality DNA is not expected to be present thus negating the chances of obtaining usable nuclear DNA. Typical samples that utilized mitochondrial DNA analysis are: hair, bones, teeth, ancient remains (samples or remains that are at least 100 years old) or very old samples (samples that are less than 100 but greater than 10 years old). The current method used to evaluate mitochondrial DNA is Sanger sequencing. Although robust, it is also time consuming and labor intensive, on the other hand pyrosequencing is a nonelectrophoretic, rapid, reliable, and sensitive sequencing method which can be easily automated. Therefore pyrosequencing could enable the widespread use of mitochondrial DNA in forensic casework and reduce the amount of time spent on each sample without compromising quality. The aim of this study is to evaluate the efficacy of pyrosequencing for forensic DNA applications, in particular mitochondrial DNA. Two dispensation orders, cyclic and directed, were examined to determine if there is any effect on the sequence generated. The accuracy of pyrosequencing was evaluated by sequencing samples of known sequence provided by the FBI. The sensitivity of pyrosequencing was evaluated by sequencing samples at different DNA concentrations and inputs. Experiments were conducted to determine the ability of pyrosequencing to detect mixtures and heteroplasmy. Additionally, the ability of pyrosequencing to sequence damaged/degraded DNA was evaluated using blood, semen, and saliva samples that were subjected to three different environmental conditions. A blind study will be conducted to confirm the accuracy of pyrosequencing. Finally, a comparison study will be conducted in which pyrosequencing will be compared to Sanger sequencing.

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