MITOCHONDRIAL GENE EXPRESSION IN HUMAN MONONUCLEAR CELLS By Monika D. Ruchała, M.S. A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Virginia Commonwealth University. Virginia Commonwealth University, 2014. Director: Dr. James P. Bennett Jr, M.D., Ph.D., Bemiss Professor Departments of Neurology, Psychiatry and Physiology and Biophysics Adult neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), have been intensively studied in recent years in pursuit of mechanisms responsible for origin and progression. One emerging theme is mitochondrial energetic deficiency as a mechanism of neuronal death. Recent descriptions of protocols to generate induced pluripotent stems cells (iPSCs) from living patients offer the potential to create unique disease models. This model can potentially lead to crucial advances in developing treatment options for a wide variety of neurodegenerative diseases. In this thesis, we attempt to induce iPSCs from mononuclear cells (MNC) in peripheral blood acquired from patients with ALS and healthy control (CTL) subjects, and analyze their mitochondrial genomes. The reprogramming of MNC to yield iPSC was done by nucleofection of an episomal plasmid pEB­ C5, expressing OriP sequences of the Epstein­Barr and five reprogramming transgenes Oct4, Sox2, Klf4, c­Myc and Lin28. We investigated the expression of mitochondrial DNA genes, ND2, ND4, COXIII and 12s rRNA in the ALS and CTL MNC before and after their culturing. The results implicate deregulated mitochondrial bioenergetics as a characteristic of ALS. Future work will establish whether these abnormalities in mitochondrial bioenergetics persist in iPSC’s and iPSC-derived neurons from ALS subject

A subset of cyclophilins have been implicated in mechanisms of neuroinflammation and neurodegeneration that contributes to the pathogenesis of Multiple Sclerosis. Mitochondrial dysfunction leading to mitochondrial permeability transition plays a pivotal role in axonal damage and disease progression in Multiple Sclerosis. Cyclophilin D (CypD) is a crucial regulatory component of the mitochondrial permeability transition pore and it was demonstrated that the cyclophilin D knockout animals showed reduced experimental allergic encephalomyelitis (EAE) clinical disease severity and axonal injury. We investigated the effect of N-methyl-4-isoleucine-cyclosporin (NIM811), a non-immunosuppressive and non-selective cyclophilin inhibitor, on the course and severity of EAE. EAE mice treated with NIM811 showed a significant reduction in clinical disease severity compared to vehicle treated mice. FACS analysis performed with the dissociated thoracolumbar spine showed that NIM811 treatment was associated with a reduction in CNS macrophages but does not alter T-helper lineage frequencies. In addition, we demonstrated NIM811’s effect on crude mitochondrial fraction obtained from brain and liver homogenates of both wild type and CypD knockout mice in order to determine drug specificity. Benefits observed from the pharmacological inhibition of cyclophilin may lead to a novel MS therapy but NIM811’s exact mechanism of action has yet to be elucidated.

To further facilitate mitochondrial DNA (mtDNA) sequence analysis for human identity testing, a better understanding of its mutation rate is needed. Prior to the middle 1990's the mutation rate applied to a forensic or evolutionary analysis was determined by phylogenetic means, This method involved calculating genetic distances as determined by amino acid or DNA sequence variability within or between species. The mutation rate as determined by this method ranged from 0.025-0.26 nucleotide substitutions/ site/ myr (million years). With the recent advent of mtDNA analysis as a tool in human identity testing an increased number of observations have recently come to light calling into question the mutation rate derived from the phylogenetic method. The mutation rate as observed from forensic analysis appears to be much higher than that calculated phylogenetically. This is an area that needs to be resolved in human identity testing. Mutations that occur within a maternal lineage can lead to a possible false exclusion of an individual as belonging to that lineage. A greater understanding of the actual rate of mutation within a given maternal lineage can assist in determining criteria for including or excluding individuals as belonging to that lineage. The method used to assess the mutation rate in this study was to compare mtDNA sequences derived from the HVI and HVII regions of the D-loop from several different maternal lineages. The sequence information was derived from five unrelated families consisting of thirty-five individuals. One intergenerational mutational event was found. This derives to approximately 1.9 nucleotide substitutions/ site/ myr. This mutation rate was very consistent with several other similar studies. This increased mutation rate needs to be considered by forensic testing laboratories performing mtDNA sequence analysis prior to formulating any conclusive results.

Four populations of nine-banded armadillos, Dasypus novemcinctus, were investigated in the south-central United States in order to assess genetic variation in an isolated population (Electric Island, Lake Hamilton, Garland County, Arkansas); a semi-isolated population (Arkansas Post, Arkansas County, Arkansas), and two free ranging populations (southern Arkansas and central Texas). A 233 basepair sequence of the D-loop region of mitochondrial DNA was sequenced in individuals from each population. Individuals and populations were compared to assess relatedness among populations and individuals. Higher sequence diversity was detected in the semi-isolated population, while lower sequence diversity was observed in the isolated and free ranging populations. Overall, all populations exhibited low genetic variation when compared to genetic variation for other mammals. The results support the hypothesis that rapid range expansion combined with the organism's unique reproductive strategies have promulgated low genetic variation in the North American populations of nine-banded armadillos.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

De nos jours, l'apport de polyphénols s'avère être bénéfique dans la lutte contre les dommages oxydatifs liés au stress oxydant. Cependant, leur biodisponibilité est limitée par leur polarité. Pour contourner ce problème, une des stratégies consiste à ajuster la lipophilie de l'antioxydant par des réactions de lipophilisation mettant en jeu un vecteur lipidique. Ainsi, des précédentes études ont montré que l'activité antioxydante était directement liée à la longueur de la chaîne alkyle greffée et ont également révélé un effet non linéaire dit effet « cut-off ». L'objectif de notre étude a donc été de comprendre l'effet non linéaire de la lipophilisation sur l'activité antioxydante de l'acide rosmarinique. Ainsi, des rosmarinates d'alkyle et de diacylglycérol ont été synthétisés, et leurs propriétés antioxydantes évaluées sur des fibroblastes surexprimant une quantité d'EROs. L'effet « cut-off » a été à nouveau observé avec ces phénolipides. En effet, le rosmarinate de décyle et le 1-rosmarinoyl-2,3-dilauroyl glycérol sont les antioxydants les plus actifs. Cependant, la série des rosmarinates d'alkyle étant la plus efficace, l'influence du vecteur lipidique a été étudié uniquement sur cette dernière. Il a été ainsi constaté que la lipophilisation de l'acide rosmarinique avec une chaîne alkyle de 4 à 10 atomes de carbone améliore son transport au sein de la cellule, alors que les esters à 16 et 18 atomes de carbone s'organisent sous forme d'agrégats extracellulaires limitant ainsi leur internalisation. De plus, alors qu'une partie de ces esters est localisée dans le cytoplasme, tout comme l'acide rosmarinique et le rosmarinate de butyle, le rosmarinate de décyle est en revanche situé dans la mitochondrie. Cette propriété en fait donc une molécule très prometteuse pour la conception de nouveaux antioxydants à activité mitochondriale ciblée. Néanmoins, sa cytotoxicité joue un rôle majeur dans son mécanisme d'action. A fortes concentrations, il affecte la viabilité cellulaire, tandis qu'à faibles concentrations, aucun effet cytotoxique notable n'est mis en évidence. / Nowadays, polyphenols supply has proved to be beneficial against oxidative damages linked to oxidative stress. However, their bioavaibility is limited by their polarity. To solve the issue, one strategy consists in adjusting the antioxidant hydrophobicity by lipophilization reactions with lipidic carriers. Indeed, previous studies showed that antioxidant activity was directly linked to the alkyl chain length grafted and also revealed a “cut-off” effect. The objective of this study is to understand this nonlinear effect of the lipophilization of rosmarinic acid on antioxidant activity. Thus, alkyl rosmarinate and diacylglycerol rosmarinate were synthesized and their antioxidant activity was evaluated into fibroblasts cells overexpressing radical oxygen species. The “cut-off” effect was again observed with these phenolipids. In fact, decyl rosmarinate and dilauroyl rosmarinate were shown to the best antioxydants. The lipophilization of rosmarinic acid with an alkyl chain from 4 to 10 carbon atoms improve its cell uptake whereas esters of 16 and 18 carbon atoms assemble in extracellular aggregate limiting their cell uptake. Furthermore, one part of these esters is located into cytoplasm like rosmarinic acid and butyl rosmarinate. On the contrary, the decyl rosmarinate (R10) is located into mitochondria. This property allows targeting mitochondria. Nevertheless, its cytotoxicity is involved in mechanism. At high concentration, R10 affects cell viability, while at low concentration, no cytotoxic effect is observed.

The Hap2/3/4/5 complex is a heme-activated, CCAATT binding, global transcriptional activator of genes involved in respiration and mitochondrial biogenesis in the yeast species Saccharomyces cerevisiae. Hap4 is the regulatory subunit of the complex and its levelsdetermine the activity of the complex. Hap4 is known to play a signaling role in response toenvironmental conditions; however, little is known about the regulation of Hap4 levels or how it responses to a cell’s functional state. The activity of the Hap2-5 complex is known to be reduced in respiratory-deficient cells. In Liu Lab, it has previously been found that a link between Hap4 stability, mediated through 26S proteasome-dependent degradation, and dependence on mitochondrial functional state plays a regulatory role on downstream targets of the Hap complex. However, the mechanism behind this regulation is still largely unknown. In normally functioning yeast cells, Hap4 is a highly unstable protein with a half-life of ~10 min. We have observed that loss of mitochondrial DNA in respiratory deficient rho 0 cells has a role in the further destabilization of Hap4 to a half-life of ~4 min through the ubiquitin-proteasome pathway. Through the screening of a collection of mutants defective in E2 ubiquitin-conjugating enzymes, we show that Hap4 is greatly stabilized in ubc1Δubc4Δ double mutant cells. We also show that Hap4 stabilization in the ubc1Δubc4Δ mutant leads to increased activity of the Hap2-5 complex, indicating that mitochondrial biogenesis in yeast is regulated by the functional state of mitochondria through ubiquitin/proteasome-dependent degradation of Hap4. Furthermore, studies on Hap4 mutants involving two highly conserved cysteine residues led to a proposed mechanism behind the regulation of Ubc4 activity towards Hap4 in response to changes in the cellular redox state.

The mitochondrial control region (MCR) has played an important role as a population genetic marker in many taxa but sequencing of complete eukaryotic genomes has revealed that nuclear integrations of mitochondrial DNA (numts) are abundant and widespread across many taxa. If left undetected, numts can inflate mitochondrial diversity and mislead interpretation of phylogenetic relationships. Comparative analyses of complete genomes in humans, orangutans and chimpanzees, and preliminary studies in gorillas have revealed high numt prevalence in great apes, but rigorous comparative analyses across taxa have been lacking. The present study aimed to systematically compare the evolutionary dynamics of MCR numts in great apes. Firstly, an inventory numts derived from the region containing the MCR subdomains was carried out by genomic BLAST searches. Secondly, presence/absence of each candidate numt was determined in great ape taxa to estimate numt insertion rate. Thirdly, alternative mechanisms of numt insertion, either through direct mitochondrial integration or post-insertional duplications, were also assessed. Fourthly, the effect of nuclear and mitochondrial environment on patterns of nucleotide composition and substitution was assessed through sequence comparisons of nuclear and mitochondrial paralogous sequences. Finally, numts in the gorilla genome were identified through two experimental methods and their use as polymorphic genetic markers was then evaluated in a sample of captive gorillas from U.S. zoos. A deficit of MCR numts covering two particular mitochondrial subdomains was detected in all three apes examined, and is largely attributed to rapid loss of mitochondrial and nuclear sequence identity in the mitochondrial genome. Insertion rates have varied during the great ape evolution and exhibit substantial differences even between related taxa. The most likely mechanism of numt insertion is direct mitochondrial integration through Non-Homologous-End-Joining Repair. Transition/transversion ratios differed significantly between both mitochondrial and nuclear sequences and between numts from coding and non-coding mitochondrial regions. A previously documented upward bias in the GC content of the primate mitochondrial genome was confirmed and the extent of this bias relative to the corresponding numt sequences increased with numt age. Five gorilla-specific numts were isolated, including three exhibiting insertional polymorphisms that will be used in future population genetic studies in free-range gorilla.

Thermotolerance limit in aquatic organism is set by the ability to sustain aerobic scope to sudden temperature shifts. This study tested the genetic and plastic differences in thermotolerance of Daphnia that can be explained by the differences in the ability to retain mitochondrial integrity at high temperatures. Five genotypes with different biogeographic origins were acclimated to 18ᵒC and 25ᵒC. We developed a rhodamine 123 in-vivo assay to measure mitochondrial membrane potential and observed higher fluorescent in heat damaged tissues as the disruption of the mitochondrial membrane potential. Significant effects on temperature tolerance were observed with CCCP and DNP but not with NaN3. Effects of toxins were significant in temperature sensitive genotype and high concentration of lactate was observed in 18ᵒC acclimated genotype only. We conclude that genetic and physiological differences are intricately linked to the ability of sustaining aerobic respiration at high temperatures which sets limit to the thermotolerance.

Aging is a biological process that has many detrimental effects due to the accumulation of oxidative damage to key biomolecules due to the action of free radicals. Methionine sulfoxide reductase (Msr) functions to repair oxidative damage to methionine residues. Msr comes in two forms, MsrA and MsrB, each form has been shown to reduce a specific enantiomer of bound and free oxidized methionine. Effects of Msr have yet to be studied in the major developmental stages of Drosophila melanogaster despite the enzymes elevated expression during these stages. A developmental timeline was determined for MsrA mutant, MsrB mutant, and double null mutants against a wild type control. Results show that the Msr double mutant is delayed approximately 20 hours in the early/mid third instar stage while each of the single mutants showed no significant difference to the wild type. Data suggests that the reasoning of this phenomenon is due to an issue gaining mass. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2013. / FAU Electronic Theses and Dissertations Collection