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.

Cette thèse de doctorat est un projet franco-polonais en co-tutelle entre le Département du Fonctionnement des Ecosystèmes Marins (l'Université de Gdansk, Pologne) et le laboratoire DIMAR (l'Université de la Méditerranée, France). La variation physiologique et la structure génétique des populations d’une espèce lagunaire, le bivalve, Cerastoderma glaucum, ont été étudiées. C. glaucum est une espèce sessile avec une phase larvaire planctonique. Elle est présente surtout dans les bassins isolés ou semi-isolés sans marée, comme les lagunes, les estuaires, les baies et les lacs. Ces habitats sont extrêmes de par des changements de conditions environnementales à court terme plus importants que dans le milieu marin. Ils sont souvent isolés, le flux de gènes est limité et des adaptations locales sont attendues. C. glaucum a révélé une forte variabilité des paramètres morphométriques et physiologiques entre les 3 populations de la Mer Baltique, la Mer du Nord et la Méditerranée. Les coques de la Mer Baltique sont beaucoup plus petites que celles des autres populations et leur taux de respiration est le plus élevé, ce qui est probablement provoqué par le stress osmotique. Les populations de l’Europe du nord ont un pattern de la reproduction monocyclique, tandis que la population de la Méditerranée peut se reproduire tout au long de l’année. Les changements saisonniers dans la composition biochimique sont corrélés avec les changements de condition trophique et avec le cycle reproductif. / This PhD is a Polish-French project between the Department of Marine Ecosystems Functioning (University of Gdansk, Poland) and DIMAR laboratory (Université de la Méditerranée, France). Physiological variation and genetic structure of the populations of lagoon specialist, the bivalve, Cerastoderma glaucum, have been studied. C. glaucum is a sessile species with a planctonic larval stage. It inhabits mainly non-tidal areas, like lagoons or brackish lakes. Those habitats are extreme, because they are much more subject to short-term variations in environmental conditions than marine habitats. They are also often isolated and as a consequence the gene flow among populations is limited and local adaptations are expected. The lagoon cockle revealed a strong interpopulation variability of morphometric and physiological parameters among 3 populations studied: from the Baltic Sea, the North Sea, and the Mediterranean Sea. The cockles from the Baltic Sea were much smaller than those from other populations and they exhibited the highest respiration rate, probably due to osmotic stress. The populations from the northern Europe had a monocyclic reproductive pattern, whereas the Mediterranean population seemed to reproduce throughout the year. Seasonal changes in biochemical components contents appeared to be correlated with changes in trophic conditions and the reproductive cycle. High respiration rates in populations from the northern Europe in spring and autumn could have resulted from gamete development (in spring) and phytoplankton blooms (in spring and autumn).

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Alcohol abuse is a growing crisis within South Africa, with severe health and socio-economic implications. Alcohol compromises the function of the blood-brain barrier (BBB), and thus its ability to regulate the homeostatic environment of the CNS is interrupted. In this study, an in vitro model of the BBB was utilized to study the effects of selected concentrations of alcohol (25mM-200mM) and the ameliorating effects of fermented rooibos (Aspalathus linearis) (0.003125%-1%), in an attempt to reverse the harmful oxidative effects of alcohol. The literature clearly states that alcohol (ethanol) compromises the BBB by reactive oxygen species (ROS) production and, therefore, rooibos, a shrub high in antioxidants and widely utilized nationally, was added to alcohol-exposed mouse brain endothelial (bEnd5) cells with the view to reverse the alcohol-induced effects on the BBB model. Alcohol-treated (25mM-400mM) bEnd5 monolayers expressed no toxicity, however, cell numbers were significantly suppressed (P<0.0274). To validate this finding, the activity of the mitochondria was investigated in order to understand if the cell’s metabolism was related to the decrease in cell division. Results showed that for both acute and chronic exposure there was a decrease in mitochondrial activity (MA) for a period of 24-48 hours, thereafter, the MA of the bEnd5 cells returned to normality. However, in experiments which chronically (600mM and 800mM) exposed cells to alcohol over a period of 96 hours, MA was suppressed and did not return to normal. Fermented rooibos caused a biphasic response to cellular proliferation at 24-72 hours, where the lower concentrations (0.0625-0.125 %) caused an increase in cellular proliferation and the higher concentrations (0.5-1%) resulted in a relative decrease in cellular proliferation. The long-term effect, after acute exposure, however, resulted in cell suppression at 96 hours (P<0.0073). With respect to the MA, bEnd5 cells exposed to fermented rooibos showed that lower concentrations (0.003125-0.0125%) were suppressed at 24 hours and was elevated at 48 hours and96 hours for all concentrations. The exception being the highest concentration (0.1%), which showed a depression in MA (P<0.05). Treating cells with both alcohol and rooibos, resulted in exacerbated suppressing of the MA. The physiological function of the BBB model was investigated by monitoring the permeability using transendothelial electrical resistance (TEER) studies and the in vitro model used in this study was endorsed for the first time using high resolution scanning electron microscopy. TEER indicated incidental changes in the permeability, only at 24 hours, for both acute and chronic exposure to alcohol and rooibos. A novel finding, within this study, was the increase in electrical resistance across the formation of the cell monolayer, after treatment with alcohol. The data lead to the hypothesis for the effect of ROS on resistivity and provides a rationale to explain the effects of combinatory treatments that were expected to ameliorate the negative effect of alcohol, however, this study showed synergistically negative effects on the bEnd5 cells. In summary the main findings in this study were: (a) alcohol was not toxic on bEnd5cells, (b) alcohol increased the permeability across monolayers of bEnd5 cells and(c) rooibos did not significantly reverse the ROS-induced effects of alcohol, but exacerbated the effects. Rooibos treatment caused the following: (i) biphasic effect on cellular proliferation, (ii) an increase in MA, and (iii) a cyclic effect in TEER studies.

No description available.

The cell possesses a number of vital mechanisms to respond to different stressors. Mitochondria are dynamic organelles which undergo constant changes in length, transport and inner membrane structure and curvature. Invaginations of this inner membrane, cristae, have been known to respond to the energetic state of mitochondria, but the regulation of these changes as well as the consequences thereof remain undetermined. We find that Optic Atrophy 1 (OPA1), a protein involved in inner membrane fusion and cristae maintenance during cell death, can respond to the energetic state of mitochondria and the cell. Moreover, OPA1-dependent changes in cristae structure are required for resistance to starvation induced cell death, proper functioning of the electron transport chain, for growth in galactose media and for maintenance of ATP synthase assembly. Interestingly, we demonstrate that select members of the mitochondrial solute carriers (SLC25A) interact with OPA1 and affect the response of OPA1 to substrate levels. Taken together, we propose an SLC25A-dependent role for OPA1 in sensing energy substrate availability and responding to alter cristae, bioenergetics and cellular survival. We also identified KIAA0664 as a novel OPA1-interacting protein, describe its subcellular localization and investigate its role in mitochondrial fusion and in mitochondrial localization. Finally, since both known carriers of mitochondrial glutathione were demonstrated to interact with OPA1, we investigated the role of OPA1 in cellular glutathione redox. OPA1 depleted cells demonstrated both increased total cellular glutathione and a shift in redox to its reduced form. The role of OPA1 in glutathione levels and redox ratios required GTPase activity, but surprisingly not fusion. Since glutathione is a master regulator of reactive oxygen species detoxification, these findings may shed light on the role of OPA1 in ROS-induced cell death pathways.

In addition to ATP generation, mitochondria are essential in various cellular processes ranging from biosynthetic pathways, apoptosis, cell cycle progression, and calcium buffering. Studies in living cells have now firmly established that mitochondria exist as a dynamic network sculpted by fission and fusion reactions, rather than separated, individual organelles. Not surprisingly, mutations in genes involved in mitochondrial dynamics and quality control lead to human diseases such as Charcot-Marie-Tooth disease type 2A, Optic atrophy, and autosomal recessive Parkinson disease. I have designed a high-throughput protocol to permit genome-wide screening for novel genes that are required for normal mitochondrial morphology. I have executed a genome-wide RNA interference screen and identified several novel genes required for mitochondrial dynamics in addition to known regulators of mitochondrial dynamics. A detailed high-throughput genome-wide screening protocol is presented. I have shown that TID1, a gene identified from the screen, has a dual-role in maintaining the integrity of mitochondrial DNA and preventing the aggregation of complex I subunits. My analysis of the mitochondrial role of TID1 supports the existence of a TID1- mediated stress response to ATP synthase inhibition. The genome screen also identified the novel gene ROMO1 as essential for normal mitochondrial morphology. I have shown that ROMO1 may have an additional role in maintaining mitochondrial spare respiratory capacity, possibly by affecting cellular substrate availability. Finally, in a collaborative effort, we have shown that homozygous mutations in the mitochondrial fusion gene MFN2 lead to multiple symmetric lipomatosis (MSL) associated with neuropathy. Mechanistically, this mutation reduces MFN2 homocomplex formation. Taken together, these results show the utility of genome-wide screening in identifying genes involved in mitochondrial quality control.

Siberia is an ideal region for exploring population histories from a molecular anthropological perspective given the diverse human populations, in terms of linguistic affiliation and lifestyle, currently inhabiting this geographically large region. As such, this thesis explores new methodologies for the investigation of the genetic histories of Siberian populations. While previous genetic work in this area of the world was able to provide detailed insights into paternal histories based on Y chromosomal data, it was not as successful on the maternal side. There existed difficulties in exploring the complex maternal demographic histories due to high levels of sequence identity between individuals in different populations when using only a very small region of the mitochondrial DNA (mtDNA), known as the hypervariable region I (HV1). This realization led to the initial focus of this dissertation which was to identify and test improved methods of sequencing entire mtDNA genomes. This was necessary because the mtDNA genomes that were published for human Siberian populations and across the globe prior to the work described here were chosen based on specific sub-sample selection criteria that introduced an ascertainment bias rendering them unusable for population-wide analyses. After testing multiple next generation DNA sequencing methods, I helped develop a sequencing library preparation method based on multiplexing and hybridization enrichment of mtDNAs for sequencing by synthesis that has since become widely used in labs across the globe. Comparing the same samples sequenced by both the traditional and new methods for five ethnolinguistic populations showed that these new methods were robust and could lead to different inferences about population histories while avoiding a sampling bias. Based on the results of this thesis it is now recommended for researchers to sequence complete mtDNA genomes for all relevant samples within a collection. By applying these methods to additional Siberian populations it was possible to better describe maternal population contact and identify demographic changes over time. This additional information allowed for the identification of putative drops in the maternal effective population sizes in the Siberian populations examined here. When examining the potential migrations and population contact between Turkic-speaking Yakuts and the Tungusic-speaking Even and Evenks, there exists a differential sharing of haplotypes suggesting that the Tungusic speaking populations herein were already in the northern region and split prior to the expansion of the Yakuts into their territory. The putative origin of the Yakuts as being around Lake Baikal was given additional support from the analyses included in this study and the origins of the Dolgans were shown to predominately include the admixture of Yakuts and Evenks.

The rampant growth of obesity worldwide has stimulated explosive research into human metabolism. Metabolic rate has been shown to be altered by diets differing in macronutrient composition, with low-carbohydrate, ketogenic diets eliciting a significant increase over other interventions. The purpose of this study was to determine the effects of the ketone β-hydroxybutyrate (βHB) on mitochondrial respiration and coupling status in adipose tissue. To explore this, we employed three distinct systems, namely cell, rodent, and human models. In every model, βHB robustly increased mitochondrial respiration. Furthermore, in cultured adipocytes and rodent adipose, we quantified the expression of genes involved in mitochondrial biogenesis and coupling status. We observed that genes involved in mitochondrial biogenesis and uncoupling were significantly higher in models exposed to ketone treatments. In conclusion, ketones increase mitochondrial respiration in cells and mammalian adipose tissue, but not ATP production, indicating greater mitochondrial uncoupling. These findings may partly explain the increased metabolic rate evident in states of elevated ketones and may facilitate the development of novel obesity interventions in the future.

Bupivacaine is one of the amide type local anesthetics and is widely used for epidural anesthesia and blockade of nerves. Bupivacaine administration locally could result in neuron injury showing transient neurologic symptoms. Dexamethasone is a synthetic glucocorticoid and may exert cytoprotective properties against damage induced by some stimuli. In the present study, we evaluated the effects of dexamethasone on bupivacaine-induced toxicity in mouse neuroblastoma N2a cells. N2a cells were exposed to bupivacaine in the presence or absence of dexamethasone. After treatment, the cell viability, nuclear condensation, and lactate dehydrogenase levels were evaluated. Mitochondrial potential and Akt (threonine-serine protein kinase B) activation were also examined. In a separate experiment, we examined the effect of Akt inhibition by triciribine on cell viability following dexamethasone treatment. We also investigated whether dexamethasone could prevent lidocaine-induced neurotoxicity. Treatment of N2a cells with bupivacaine resulted in significant cell injury as evidenced by morphological changes, LDH leakage, and nuclear condensation. Pretreatment of the cells with dexamethasone significantly attenuated bupivacaine- and lidocaine-induced cell injury. Dexamethasone treatment prevented the decline of mitochondrial potential caused by bupivacaine and increased the levels of Akt phosphorylation. Importantly, pharmacological inhibition of Akt abolished the protective effect of dexamethasone against bupivacaine-induced cell injury. Our data suggest that pretreatment of neuroblastoma cells with dexamethasone exerts a protective effect on bupivacaine-induced neuronal cell injury. The mechanisms involve activating the Akt signaling pathway.