The purpose of this study was to investigate the effects of hydroxytyrosol (HT) supplementation on markers of mitochondrial biogenesis, aerobic capacity, and endurance exercise performance in recreationally active men. Sixty-one (n = 61) subjects (21.46 ± 0.22 yrs, 179.46 ± 0.79 cm, 78.91 ± 1.19 kg) consumed either a high dose (HI) HT supplement (150 mg HT), a low dose (LO) HT supplement (50 mg HT), or a placebo (PLA) every day for 6 weeks. Muscle biopsies from the vastus lateralis were obtained at baseline and after 6 weeks of supplement consumption and analyzed for markers of mitochondrial biogenesis: succinate dehydrogenase (SDH), citrate synthase (CS), and peroxisome proliferator-activated receptor ɣ coactivator (PGC)-1α. Subjects completed exercise testing on a bicycle ergometer at baseline and after 3 and 6 weeks of supplement consumption to measure changes in maximal aerobic power (VO2MAX), lactate threshold, respiratory exchange ratio (RER), substrate utilization, and endurance exercise performance on a 20 km time trial course. The primary findings were that HT supplementation increased muscle oxidative enzyme activity suggesting increased oxidative capacity. HT also increased time trial performance at midpoint and endpoint and this corresponded with an improvement in lactate threshold and a lower RER for the LO HT treatment. Time trial performance was also improved at endpoint for PLA, however, unlike LO an HI HT, this was accompanied by a significant increase in rating of perceived exercise (RPE) and not associated with improvements in muscle oxidative capacity. Our results indicate that HT ranging from 50 to 150 mg/day for 6 weeks can improve muscle oxidative capacity and aerobic performance, and suggests that HT may be used chronically to improve mitochondrial function. HT may be used as an effective means to increase mitochondria to improve exercise performance, and limit diseases associated with mitochondrial dysfunction such as cardiovascular disease, type II diabetes, and some cancers. / text

The protein Liquid facets (Lqf) promotes endocytosis at the plasma membrane1. Lqf activity is required for proper Notch signaling, likely through facilitating the endocytosis of Notch ligand by indirectly linking ligand to clathrin. A genetic modifier screen to identify genes that interact with lqf was performed by a previous graduate student. Genes identified in the screen might provide new insights into how Lqf promotes endocytosis or how Notch signaling is regulated. In this work, I performed genetic mapping techniques to identify the genes mutated in each complementation group of the screen. I identified the gene mutated in complementation group 6 as mitochondrial alanyl tRNA synthetase (Aats-ala-m). tRNA synthetases link a tRNA to its cognate amino acid during translation. Mitochondrial tRNA synthetases function in the mitochondria in translation. Aats-ala-m genetically interacts with lqf, suggesting the two genes function in the same pathway. In this work, I also identified chromosomal regions where the genes mutated in complementation groups 1,2, and 9 are located. / text

Alzheimer’s disease (AD) is characterized pathologically by proteinaceous aggregates composed primarily of amyloid \(\beta (A \beta)\) and tau. Diseases characterized by abnormal deposition of tau are collectively termed “tauopathies.” \(A \beta\) acts upstream of tau in the AD pathogenesis pathway, but tau expression is required for the neurodegenerative effects of \(A \beta\). Mitochondrial abnormalities have been documented in Alzheimer’s disease and related tauopathies, but the causal relationship between mitochondrial changes and neurodegeneration, as well as specific mechanisms promoting mitochondrial dysfunction, are unclear. Mitochondrial morphology is regulated by fission and fusion events within and between individual mitochondria, and misregulation of this process has been observed in several neurodegenerative diseases. The contribution of mitochondrial dynamics to the pathogenesis of Alzheimer’s disease and tauopathy has not yet been determined. We have found that expression of tau promotes elongation of mitochondria in Drosophila and vertebrate neurons. Elongation is followed by mitochondrial dysfunction, aberrant cell cycle reactivation, and cell death, which can be rescued in vivo by genetically restoring the proper balance of mitochondrial fission and fusion. Tau induces mitochondrial elongation by inhibiting mitochondrial localization of DRP1, the primary effector of fission. We have previously demonstrated that direct tau-mediated stabilization of filamentous (F)-actin is critical for neurotoxicity. Here we show that actin stabilization is responsible for the mislocalization of DRP1 following tau expression. Additionally, we identify regulatory roles for F-actin and myosin II in DRP1 localization. Similarly to overexpression of human tau, loss of endogenous Drosophila tau (dtau) induces mitochondrial elongation, but through distinct mechanisms. Expression of human \(A \beta\)in Drosophila induces mitochondrial fragmentation and neuronal toxicity, which are reversed by depletion of dtau. Together, we demonstrate that human disease-associated tau induces neurotoxicity through disruption of mitochondrial dynamics, which can be mediated by enhanced actin stabilization. We also observe a novel role for dtau in the regulation of mitochondrial dynamics, a function critical to the ability of endogenous tau to mediate the effects of \(A \beta\). These findings offer new insights into the contribution of mitochondrial dysfunction to AD and tauopathy, and highlight the emerging role of mitochondrial dynamics in the pathogenesis of neurodegenerative disease.

published_or_final_version / abstract / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

No description available.

In an effort to better understand patterns of genetic variation in modern African populations, I surveyed nucleotide variability at four loci in five diverse sub-Saharan African populations. First, I analyzed the mitochondrial DNA (mtDNA) and the non-recombining portion of the Y chromosome (NRY), asking specifically if similar models of population size change could be fit to re-sequencing data from these two loci when examined in the same populations. Four tests of population growth were employed and results indicated that food-producing populations best fit a model of exponential growth for the mtDNA but not the NRY, and hunter-gathering populations best fit a model of constant population size for both mtDNA and the NRY. These results are likely due to sex-specific migration or differences in the effective population sizes of males and females.Next, I examined mtDNA and NRY population structure in these same populations, to assess the relative effects of migration and effective population size on patterns of mtDNA and NRY nucleotide variability. I used an Isolation with Migration (IM) model to disentangle estimates of effective population size and migration. Results indicated that levels of mtDNA population structure are higher than those of the NRY, and female migration tends to be unidirectional while that of males is largely bidirectional. I found that in food-producing populations, male migration rate estimates are in fact higher, not lower, than those of females, while estimates of male effective population size are strikingly small. I infered that males have experienced a period of population size reduction due to replacement, and that this most likely occurred during the Bantu expansions, approximately 5,000 years ago.Finally, I assessed population structure in these populations using a multilocus approach which estimated current and ancestral effective population sizes, migration rates, split times and fraction of the ancestral population that contributed to current populations. Current and ancestral effective population sizes ranged from ~5,000-8,000 individuals. Most populations showed an increase in size relative to the ancestral population. Population split times ranged from 17-142 thousand years (KYR); the Khoisan split times were the oldest and the Niger-Congo speaking populations' split times the most recent. Since the oldest population split times precede the dates for the earliest modern humans outside of Africa, I posited that modern humans likely evolved at a time when structured populations already existed in Africa.

Sequence variation of a 238 bp segment of the mitochondrial control region was analyzed for 1,568 Steller sea lions (2.8% of the estimated species population) sampled from 50 rookeries representing nearly every locality at which Steller sea lions are known to breed in significant numbers. Haplotype diversity (H = 0.9164 ± 0.0035) was high and nucleotide diversity (π = 0.00967 ± 0.00586) was moderate. No evidence was observed for significant genetic bottleneck effects. Rookeries were grouped into regions and stocks to examine structure at different spatial scales. F- and Φ-statistics were computed for all pairwise comparisons of rookeries, regions and stocks. Significant (P<0.05) divergence of eastern stock (southeastern Alaska to California) animals from western stock animals was supported in analyses at all spatial scales. Likewise, rookeries and regions from Asia were found to be significantly different from all other western stock rookeries. This was most clearly demonstrated using Φ-statistics at the regional level. The Commander Islands clearly associate with Alaskan western stock rookeries, not with the Asian rookeries. Within each of the three stocks there is significant isolation by distance among rookeries. This relationship does not hold for inter-stock comparisons indicating that there are important barriers to gene flow among stocks. Mitochondrial DNA analysis supports the recognition of three stocks for appropriate conservation of the species. The currently recognized eastern stock is unaffected, but the western stock is now partitioned west of the Commander Islands yielding a western stock which ranges from Prince William Sound west to the Commander Islands, and an Asian stock including rookeries from the Kamchatka Peninsula, Kuril Islands, and Sea of Okhtosk.

There is strong evidence that the skeletal muscles of pinnipeds are adapted for an aerobic, lipid-based metabolism under the hypoxic conditions associated with breath-hold diving. However, regional variations in mitochondrial density are unknown, and the few fiber typing studies performed on pinniped skeletal muscles are not consistent with an aerobic physiological profile. Thus, the objectives of this study were to (1) reexamine the fiber type distribution throughout the primary locomotory muscles of the harbor seal, and (2) to better understand the density and distribution of mitochondria in the locomotory muscles. Multiple samples from transverse sections of the epaxial muscles and a single sample of the pectoralis muscle of wild harbor seals were analyzed using immunohistochemical fiber typing and electron microscopy. Fiber typing results indicated that harbor seal epaxial muscles are composed of 47.4% type I (slow twitch, oxidative) fibers and 52.8%, IIa (fast twitch, oxidative) fibers. No fast twitch, glycolytic (type IIb) fibers were detected in the epaxial muscles or the pectoralis muscle. Mean volume density of mitochondria [Vv(mt,f)] was 5.6%, which is elevated over what would be predicted for a terrestrial mammal of similar mass. The elevated Vv(mt,f) had a high proportion of intermyofibrillar mitochondria, a trait not normally found in the muscles of terrestrial mammals with elevated Vv(mt,f). These results provide further evidence that the elevated mitochondrial volume density in pinniped muscle decreases the oxygen diffusion distance between myoglobin and mitochondria to facilitate aerobic respiration in working muscles. In addition, analyses of heterogeneity revealed that the regions of the epaxial muscles that were located deep within the muscle showed a significantly higher Vv(mt,f) relative to those regions that were superficially-located. In contrast, there was no significant heterogeneity of fiber type detected in either plane of the epaxial muscles. Thus, there was a fine-scale pattern of spatial heterogeneity of Vv(mt,f) within the epaxial muscles that does not manifest in fiber type distribution, indicating that the fibers have similar oxidative capacities.

The spatial scale at which populations are genetically structured is of immense interest for the understanding of a species’ ecology and evolutionary biology. This can have important implications for management of resources as well as predicting responses to future change. Rainbow smelt (Osmerus mordax) is an anadromous species with a relatively short freshwater residence time compared to other species with similar life-history strategies. Therefore, while they offer the opportunity to sample distinct spawning aggregations, they also offer an insight into the relative roles of contemporary and historical factors shaping connectivity among marine populations, an area of great interest, and for which further understanding is required. With the use of both mitochondrial DNA and nuclear microsatellite markers, I explored the historical and contemporary factors influencing population structure in smelt. While previous phylogeographic work on this species has resolved two mtDNA lineages dating back to previous glacial episodes, I document the discovery of a zone of contact between these lineages in Newfoundland. This is in addition to the established contact zone in the St. Lawrence estuary, and results in a longitudinal distribution of the races with one race predominating on opposite ends of the species distribution, while the other race is geographically intermediate. Patterns of nuclear genetic variation largely mirror the phylogeographic signals in Newfoundland and suggest a more recent colonization of the Avalon Peninsula as well as implicating a remnant historical signal of colonization of the west coast of Newfoundland from the mainland. In addition, contrasting patterns of genetic diversity and levels of differentiation were apparent between the mainland and Newfoundland and suggest differing scales of dispersal within this species. While the population structure within Newfoundland is most consistent with dispersal restricted to within bays, larger scale biogeographic regions were identified in the mainland range, suggesting dispersal is more common and widespread. In addition, sampling of different run times (i.e. ‘early’ vs. ‘late’) demonstrated the potential for isolation by time when spawning events are separated by a break in activity. Overall, these results shed light into the possible roles of both historical and contemporary factors shaping the dynamics and connectivity among populations.

Mitochondrial DNA (mtDNA) was isolated from 8 different natural populations of the rodent species Otomys irroratus (Muridae: Otomyinae) and from one population of the species 0. angoniensis occurring in South Africa. MtDNA samples were cleaved with five different restriction endonucleases, end-labelled with phosphorous-32, separated by electrophoresis on horizontal 1 % agarose gels and the resulting fragment bands were detected by autoradiography. The individual-specific fragment banding patterns were analysed and compared among the various populations. The percent sequence divergence among and between the populations was calculated using the formula of Nei (1979). A matrix of sequence divergence values for all intergenomic pairwise comparisons was subjected to a clustering analysis by the unweighted pair group method with arithmetic means (UPGMA, Sneath and Sokal, 1973), using the computer programme NTSYS (Rohlf: 1988). The results of these analyses allowed for a preliminary identification of phenetic groupings in the data set. A matrix generated by scoring the restriction endonuclease fragments as present or absent was used to generate a phylogenetic dendogram using the BIOSYS (Swofford and Selander, 1989) programme. The overall restriction fragment variation uncovered in this study revealed 15 different mtDNA haplotypes within the 20 individuals examined. This corresponded to a high degree of polymorphism in the populations where more than one specimen was available, as well as within the species 0. irroratus. There were no clones that were shared between any of the populations sampled. The intrapopulation sequence divergence values uncovered in this study were high (range 0.35 % to 5.08 %), but also consistent with some other reports in the literature for intrapopulation variation. The outgroup, 0. angoniensis revealed the highest divergence values when compared to the mtDNA clones found in 0. irroratus. The phenetic and cladistic cluster diagrams revealed overall similarity with one another. There appeared to be little correlation between the topology of the mtDNA haplotype phenograms and the geographic distance of the sample localities. There was, however, a marked congruence between the distribution of mtDNA haplotypes and the distribution of three distinct cytotypes occurring over the species range. A possible polyphyletic evolution of populations of 0. irroratus was inferred from the cladistic analysis. / Thesis (M.Sc.)-University of Natal, 1994.