Decreased mitochondrial function is associated with aging and is an early step in Alzheimer's disease (AD). Autophagy also declines with age and is required for degradation of dysfunctional mitochondria but it is not known whether autophagosomal formation is overactive and/or degradation of autophagosomes is inhibited in AD. Although two-thirds of the 5 million Americans diagnosed with AD are women, without a clear mechanism. Since the role of gender and autophagy in AD is unclear, we wanted to know whether: A) decreased mitochondrial biogenesis precedes brain plaque formation, neuronal death and memory deficits B) autophagosomal formation is overactive and/or degradation of autophagosomes is inhibited in AD and C) deficits in mitochondrial biogenesis and autophagy occur earlier in life in the hippocampus/cortex of female AD-Tg, than male AD-Tg mice. To answer these questions, mitochondrial DNA (mtDNA), TFAM (transcription factor A mitochondrial) and two stages in autophagy, autophagosomal formation and degradation were measured in the hippocampus/cortex of non-transgenic (non-Tg) and the APP(Swe, DI)/NOS2-/- AD mouse model (AD-Tg) from 2 mo. through 12 mo. of age, when memory deficits develop. Male and female mice were evaluated for gender differences. By measuring mitochondrial gene copy number relative to nuclear gene copy number by qPCR, we found female specific decreases in mtDNA and TFAM protein levels in AD-Tg hippocampus/cortex, before 12 mo., when memory deficits develop. The male AD-Tg mice did not show any decline in the levels of mtDNA or TFAM protein through 12 mo. of age, indicating a decline in mitochondrial biogenesis earlier in life in female AD-Tg mice, than males. To determine whether autophagosomal formation is overactive and/or autophagosomal degradation is inhibited in AD, non-Tg and AD-Tg mouse neurons from 2-12 mo. of age were cultured and transfected with an adenovirus expressing the dual fusion protein, mCherry-GFP-LC3B. This system enables the distinction of early (autophagosomal formation) from late (autophagosomal degradation) stages of autophagy. Autophagosomal formation increased in female AD-Tg mice, while male AD-Tg neurons have decreased formation of autophagosomes, compared to non-Tg. Neurons from AD-Tg mice show deficits in degradation of autophagosomes by 2 mo. Our results show increased autophagosomal formation in female neurons with age and in our AD-like model without corresponding increases in degradation of autophagosomes. All together, our data indicates that the presence of familial APP mutations and knockout of NOS2 increases deficits in both mitochondrial biogenesis and autophagy in female mice, compared to males. These deficits occur before amyloid plaque formation and memory deficits, suggesting that mitochondrial and autophagic deficits may play a role in the higher incidence of AD in females.

Mitochondrial dysfunction causes moderate to profound hearing loss both in isolation and as a feature of multi-systemic mitochondrial disease. The m.1555A > G mitochondrial DNA (mtDNA) variant is associated with a predisposition to aminoglycoside ototoxicity and maternally inherited non-syndromic deafness. However, the reasons for the highly variable penetrance of the associated hearing loss have not yet been fully resolved. Aminoglycosides are a recognised modifier factor of the hearing loss, but cannot account for all hearing impaired carriers in multi-generational pedigrees, implicating additional co-segregating genetic factors. By identifying and characterising the c.3G > A SSBP1 variant as a nuclear modifier of m.1555A > G the work detailed in this thesis extends our understanding of mitochondrial-nuclear interactions in human disease. To ascertain the frequency of the m.1555A > G variant in patients with suspected mitochondrial hearing loss we surveyed the laboratories within the United Kingdom that undertake genetic testing for this variant. We determined that the variant was not found more frequently in patients with known hearing impairment providing further evidence that m.1555A > G does not cause hearing loss in isolation. These results strengthened the case for nuclear genetic modifiers as important contributors to m.1555A > G pathogenesis. We next identified a multi-generational family that transmitted the m.1555A > G variant with variable clinical penetrance of hearing loss. In addition, a cohort of sporadic individuals carrying m.1555A > G was used to test the hypothesis that a conserved genetic mechanism accounted for the phenotype in all carriers. To this effect, we undertook whole exome sequencing in selected familial and sporadic carriers of m.1555A > G, identifying a heterozygous start loss mutation in the core mtDNA replisome protein gene, SSBP1, that co-segregated with the m.1555A > G variant and the phenotype in the family. The SSBP1 variant lead to a perturbation of mtDNA metabolism, and was associated with multiple mtDNA deletions and mtDNA depletion in skeletal muscle. Fibroblasts from these patients also showed mitochondrial network fragmentation and reduced intra-mitochondrial protein synthesis in keeping with the co-existing m.1555A > G variant, leading to reduced proliferation rates under conditions of forced mitochondrial respiration. Our findings provide an explanation for the variable clinical penetrance of the disorder within these m.1555A > G carriers and highlight the importance of trans-acting modifiers in mitochondrial disease.

The Francolins constitute the largest genus in the Galliform family Phasianidae. There is little accord concerning the taxonomic classification of its members. In the past, information on this group has been provided by morphological and palaeontological evidence. An investigation into the molecular history of this group is presented, using mitochondrial DNA (mtDNA) as an evolutionary tool. A comparison of mtDNA restriction fragment lengths has been used to help define the phylogenetic relationships between 13 southern African Francolin species and a selected outgroup, the Japanese Quail. Both cladistic and distance-based analytical methods have been used to construct phylogenies from the molecular fragment data. The trees relating the Francolins are in general agreement with the traditional classification based on morphological, behavioural and morphometric studies, but differ in the branching order of two species, F. levaillantii and F. hartlaubi. A recent proposal for the partitioning of the genus into two monophyletic assemblages of quail-like "partridges" and pheasant-like "francolins" is supported by mtDNA fragment data, with the exception of the two aberrant taxa. On the basis of the initial fragment size comparison, F. hartlaubi and F. levaillantii constitute part of an unresolved quadrichotomy at the base of the tree. A restriction endonuclease site mapping approach has been utilized to provide a deeper resolution for the molecular phylogeny. Detailed mtDNA restriction endonuclease maps of F. levaillantii, F. hartlaubi, two species representing the "partridge" and "francolin" monophyletic groups respectively, and also of the Madagascar Partridge, have been constructed. Phylogenetic analysis of this data has helped to resolve the problematic placement of the two aberrant taxa by showing an early separation of F. levaillantii from the "partridge" lineage, and of F. hartlaubi from the "francolin" lineage. The Madagascar Partridge was anticipated to be a likely sister-taxon to the whole group, but instead appears to have close relationships within the "partridge" lineage.

Mutations affecting the mitochondrial electron transport chain cause numerous neurodegenerative disorders in humans and affect longevity in other organisms. A natural model system to study the relationship between mitochondrial function and aging within an evolutionary or population genetic context has been lacking. Natural populations of Caenorhabditis briggsae nematodes were recently found to harbor mitochondrial genetic variation with likely functional consequences for aging. Specifically, C. briggsae isolates containing high frequencies of a deletion mutation affecting the mitochondrial NADH dehydrogenase 5 (ND5) gene were found to have reduced reproductive fitness and lifespan and elevated levels of mutagenic superoxide. Here, rates of growth and aging and aerobic respiratory capacity were evaluated in several isolates spanning the range of mitochondrial genetic variation in this species. There is considerable variation among isolates for all measured traits, although the observed relationships between isolate-specific trait means and ND5 deletion frequency did not always conform to my expectations. In an effort to determine whether the among-isolate phenotypic variation is due to mitochondrial rather than to nuclear genetic variation, inter-population hybrids of C. briggsae were created and compared to the progenitor isolates. Surprisingly, evidence for paternal mitochondrial inheritance was detected in many of these hybrid lines. Where mitochondrial genomes were maternally inherited as expected, intergenomic epistasis appears to contribute to fitness, longevity, and aging in this species.

No description available.

No description available.

<P> The study of apoptosis is a rapidly growing field due to its relevance not only to development, but also its relationship to several diseases such as cancer. The Bcl-2 family of proteins function at the mitochondrial membrane, where many apoptotic stimuli converge. There are three main theories on the regulation of the Bcl-2 family proteins, supported by several methods of in vitro and cell-based studies. </p> <p> Mitochondria isolated from wild type and Bak-/-C57BL6 mouse liver are free of Bax, Bci-XL and Bid as determined by immunoblotting. A comparison of the two membranes and the use of recombinant proteins demonstrated that tBid activated Bak or Bax to permeabilize the membrane in this system, and lower concentrations of tBid were required for membrane permeabilization in the presence of both Bak and Bax. Recombinant Bci-XL inhibited this process, indicated by a decrease in cytochrome c release. Mutant recombinant proteins demonstrated that Bci-XL inhibits cytochrome c release through interactions with Bax/Bak and tBid, and by a third protein-independent mechanism. Together, this supports the Embedded Together Model. </p> <p> The mitochondrial system also functions as an intermediate in the study of inhibitors of the Bcl-2 family of proteins. Potential inhibitors 3e and 3e-D2 previously demonstrated to bind Bci-XL through fluorescence polarization were shown to have a Bcl-2 protein-independent method of membrane permeabilization that has not yet been determined. 3e also functioned as an activator of Bax and Bak. Similar to fluorescence polarization experiments, the dimeric compound 3e-D2 was more potent than monomeric 3e. </p> <p> A comparison of membranes in the presence and absence of Bak provides a robust system in which to study multiple facets of apoptosis, including but not limited to regulation of Bcl-2 proteins and the development of proteinspecific inhibitors. </p> / Thesis / Master of Science (MSc)

Mitochondrial diseases are a group of heterogenous disorders that share common cellular consequences resulting from mitochondrial dysfunction: (i) decreased ATP production; (ii) increased reliance on alternative anaerobic energy sources; and (iii) increased production of reactive oxygen species. Objective: We evaluated the effect of a combination (COMB) therapy comprising creatine monohydrate, coenzyme Q1 and lipoic acid to target the above mentioned consequences using a randomized, double-blind, placebo-controlled, crossover study design in patients with mitochondrial cytopathies. Results: Compared with placebo, the COMB therapy resulted in lower resting plasma lactate concentrations, lower urinary 8-isoprostane excretion and attenuated the decline of peak dorsiflexion strength in all patient groups. Improved body composition was only observed in patients in the MELAS group. Interpretation: These results suggest that combination therapies targeting multiple final common pathways of mitochondrial dysfunction favorably influence surrogate markers of cellular energy dysfunction. Future therapies should be designed to target specific mitochondrial diseases to provide the greatest therapeutic benefits for those patients. In addition, future studies employing larger sample sizes in homogeneous groups of patients will be required to determine whether such combination therapies will influence function and quality of life. / Thesis / Master of Science (MS)

With the aging of the baby boom population and an increased life expectancy, individuals aged 65 years and older are the fastest growing segment of our population. Aging brings about changes in skeletal muscle such as reduced muscle strength and mass, as well as cellular deficits such as increased production of reactive oxygen species (ROS), and mitochondrial DNA (MtDNA) deletions and mutations. Muscle mass declines at a rate of 1-2% each year after the age of 50, leading to muscle weakness, functional impairments, loss of independence, and an increase in falls. Additional declines in muscle mass and reduced muscle strength may result in a lower resting metabolic rate, reduced lipid oxidative capacity, increased adiposity, and insulin resistance. The rising number of individuals aged 65+ will increase demands on health care and health care costs, possibly leading to inadequate public resources and less care for the aged. This large societal impact, coupled with the aging of our population, suggests a clear need for methods that will improve the aging phenotype to enhance functionality, quality of life, and overall health for our aging population. This investigation aspires to delve into a relatively unexplored area of aging research and evaluate potential means that could help improve the aging phenotype. The associated mitochondrial impairments, mitochondrial mediated apoptosis, and mitochondrial DNA (MtDNA) deletions and mutations that accompany aging lead to a decline in physical fitness and oxidative capacity, and exercise has been shown to reverse or help prevent many of these disturbances. Resistance exercise training (RT) is currently the most effective known strategy to stimulate skeletal muscle hypertrophy and increase strength. Strength gains after RT lead to an improvement in activities of daily living and quality of life. There is some evidence suggesting that RT may lead to increased antioxidant enzyme capacity, decreased ROS production and increased electron transport chain (ETC) function in older individuals. The present study will lay a foundation for future research and further developments in the area of RT, mitochondrial function and aging. / Ph. D.

Academic abstract The millipede order Polydesmida, or flat-backed millipedes, is the most species rich among the class Diplopoda. At present, the order does not have a phylogenetic systematic classification, and the relationships of the 29 families are unknown. Whole genome DNA sequencing technologies and bioinformatic tools for the analysis of systematic data have improved over the last couple of decades, and have facilitated analysis of phylogenetic relationships. Here I sequenced the whole genomes of 82 species of the order Polydesmida and assembled a set of 281 orthologous genes using aTRAM. These genes were then used to estimate a maximum likelihood phylogeny. The families Paradoxosomatidae, Sphaeriodesmidae, Rhachodesmdidae, Platyrhacidae, Xystodesmidae, Polydesmidae, and Dalodesmidae were recovered as monophyletic groups. Some other families such as Trichopolydesmidae, Chelodesmidae and Macrosternodesmidae now appear to be polyphyletic groups, and a more comprehensive study of their molecular and morphological characters is needed. Phylogenetic trees can be used as maps to trace the evolution of characters in a group. Using the estimated tree for Polydesmida, I mapped morphological and molecular characters to understand their evolution. Traditional taxonomy in millipedes primarily used the morphology of the male gonopods to differentiate species. In my phylogeny, the morphological characters associated with the gonopod were less variable between specimens of the same family than non-gonopodal morphological characters, which showed a great diversity between individuals of the same family. A set of molecular characters that I studied were the gene order of the mitochondrion, as they may be a source of important evolutionary information. I found an inversion of half of the mitochondrial genome, which was consistent for the entire order Polydesmida. The majority of the changes in gene order were observed in the tRNA's, some of which can be considered synapomorphies for some families. Finally, I described three new species of millipedes in the superfamily Platyrhacoidea from southwestern Colombia: Aphelidesmus carcharodus and Pycnotropis svanae in the family Aphelidesmidae, and Barydesmus peineta in the family Platyrhacidae. I present diagnoses, descriptions, color habitus photographs and illustrations for these new species which contribute to the knowledge of millipedes of Colombia. / Doctor of Philosophy / Millipedes are a diverse group of soil dwelling animals that fulfill an important ecological role as decomposers. The Polydesmida, commonly known as flat-backed millipedes, is the millipede order with the greatest number of described species. Despite its richness, the evolutionary relationships of the families of the Polydesmida are not currently known. I extracted and sequenced DNA from the genomes of 82 polydesmidan species to construct an evolutionary tree. My results showed that many families, such as Paradoxosomatidae, are well defined groups. The evolutionary tree was then used as a map to determine how some morphological characters have evolved in the group through time. Traditionally in the study of millipede classification, the male sexual organs or gonopods are used to differentiate species. Here I found that the gonopod morphology is more useful to understand the relationships between families than other morphological characters as the shape of the body that is greatly variable and do not show consistent evolutionary patterns. I found that the genetic information encoded by the mitochondria of millipedes is useful to understand their evolution. Mitochondria have a single chromosome with genes that change in position during evolution, and those changes are consistent amongst individuals of the same family. Lastly, I described three new species of polydesmidan millipedes from the Private Nature Reserve "Finca Merenberg" in the southwestern of Colombia, and I named these new species: Aphelidesmus carcharodus, Pycnotropis svanae, and Barydesmus peineta.