Mitochondrial DNA (mtDNA) has played a major role in human population studies over the past decade due to its maternal inheritance and negligible recombination (Macaulay, 1999). The mtDNA control region has been the focus of these studies due to the highly polymorphic nature of this non-coding region. Forensic scientists also use mtDNA to help determine the identity of missing individuals when nuclear DNA is not present. However, when skeletal remains are unclaimed, identification becomes near impossible. Therefore, mtDNA can play a valuable role in identification in terms of population affiliation, especially in conjunction with morphological analysis. The goals of this research were two-fold: 1) to determine population affiliation of unknown skeletal samples using phylogenetics and 2) to find a method of extraction that leaves a majority of the remains intact. This research depended on the donation of samples from sixteen skeletal remains from the Hillsborough County Medical Examiners office. Mitochondrial DNA from ten of these cases were extracted, amplified, and sequenced in order to determine population affiliation via phylogenetic analysis of hypervariable region I (HVR I). These sequences were aligned and compared to that of sequences in a pre-existing mtDNA control region database (Handt, 1998). The crania of the skeletal remains were measured and subsequently analyzed by the forensic anthropology program FORDISC 2.0 to morphologically determine population affiliation. A secondary morphological analysis included input of the measurements into SPSS, a statistical program package, as a separate discriminant function assessment. This analysis was dependent on a database of craniometrics from known individuals (Jantz, and Moore-Jansen, 2000). The results were compared to those of FORDISC 2.0 as well as that of the phylogenetic tree constructed using molecular techniques. This study will determine whether phylogenetic analysis is a legitimate way to determine population affiliation of unknown individuals, thereby benefiting future forensic studies.

Melongena corona and closely related congeners are a conspicuous part of the marine intertidal benthic communities of Florida and southeastern Alabama. Significant genetic differentiation among adjacent populations has been conjectured based on variation in shell morphology, habitat discontinuity, low levels of adult motility, and the presence of an aplanic lecithotrophic larval stage. Furthermore, studies of the highly variable shell morphology often have resulted in confusing specific and subspecific definitions of these gastropods, which are often referred to as the "corona complex". Variation in shell morphology may indicate local adaptation or environmentally induced phenotypic plasticity. In this study I utilized mitochondrial DNA sequences in order to reconstruct the phylogenetic relationships of crown conchs, and nuclear microsatellite loci to investigate the patterns of relatedness within and among populations inhabiting the southeastern United States. Approximately 500 individuals from 20 populations throughout the known range of the Crown Conch were genotyped at eight microsatellite loci. Additionally, a 1200bp portion of the cytochrome oxidase subunit I gene was sequenced along with a 490bp fragment of the 16s ribosomal gene from individuals representing all known species and subspecies of the genus Melongena. Phylogenetic analyses completed with these data provide no support for current taxonomic designations within this group and these genetic data indicate that the corona complex is composed of a single polymorphic species. Furthermore, microsatellite data reveal population structure consistent with restricted gene flow between extant populations and phylogeography heavily influenced by historical sea-level fluctuations during the Late Pleistocene.

Le cancer de la prostate est un véritable problème de santé publique qui se situe au premier rang des cancers incidents chez l’homme. Les cellules tumorales ont un métabolisme différent des cellules normales, et cibler le métabolisme des cellules cancéreuses est devenu une stratégie thérapeutique prometteuse. La metformine est un médicament couramment prescrit contre le diabète de type II, qui possède des propriétés anti-tumorales et affecte le métabolisme des cellules cancéreuses. L'augmentation de la lipogenèse est observée dans nombreux cancers dont le cancer de la prostate. Nous montrons que la metformine inhibe la lipogenèse dans les cellules cancéreuses de prostate via un déficit énergétique cellulaire. En effet, l’ATP est diminuée de façon dose dépendante par la metformine et cette diminution est significativement corrélée avec l'inhibition de la lipogenèse. De plus, la metformine induit un gonflement des mitochondries et une désorganisation des crêtes mitochondriales dans les cellules cancéreuses de prostate. De façon intéressante, nous observons que la metformine provoque une augmentation des flux calciques et un relargage du calcium du réticulum endoplasmique. Nous émettons l'hypothèse que ce calcium s'accumule dans la mitochondrie ce qui pourrait générer un gonflement de celles-ci. En réponse à ces signaux calciques ou à la diminution de la fonctionnalité des mitochondries, la metformine stimule la biogenèse mitochondriale dans les cellules cancéreuses de prostate. En conclusion, cette étude a permis de mieux comprendre les mécanismes moléculaires et cellulaires induits par la metformine dans le cancer de la prostate. / Prostate cancer is a major public health problem. Tumor cells have a different metabolism than normal cells, and targeting cancer cells metabolism becomes a promising therapeutic strategy. Metformin is a commonly prescribed anti-diabetic drug which has anti-tumor properties. Increased lipogenesis is a common feature of cancer cells including prostate cancer. We show that metformin effect on lipogenesis is due to a cellular energy deficit. Lipogenesis requires ATP and the decrease in ATP induced by metformin is significantly correlated with the inhibition of lipogenesis. Furthermore, we demonstrate that metformin induces mitochondrial swelling and disruption of cristae in prostate cancer cells. Interestingly, we show that metformin triggers a calcium flux and the release of calcium from the endoplasmic reticulum. We hypothesize that the accumulation of calcium into the mitochondria generates its swelling. In addition, we show that metformin stimulates mitochondrial biogenesis in prostate cancer cells. In conclusion, this study allowed to better understand the molecular and cellular mechanisms induced by metformin in prostate cancer.

Abstract not available

Freshwater systems offer important opportunities to investigate the consequences of intrinsic biological and extrinsic environmental factors on the distribution of genetic variation, and hence population genetic structure. Drainages serve to isolate populations and so preserve historical imprints of population processes. Nevertheless, dispersal between and within drainages is important if the biology of the species confers a good dispersal capability. Knowledge of the population genetic structure or phylogeographic patterns of Australia's freshwater fish fauna is generally depauperate, and the present study aimed to increase this knowledge by investigating patterns of genetic diversity in three Australian species of freshwater fish. I was interested in the relative importance of dispersal capability, the hierarchical nature of stream structure and the consequences of earth history events on patterns of genetic diversity among populations. I examined three species from three families of Australian freshwater fish, Pseudomugil signifer (Pseudomugilidae), Craterocephalus stercusmuscarum (Atherinidae) and Hypseleotris compressa (Gobiidae). These species are abundant, have wide overlapping distributions and qualitatively different dispersal capabilities. I was interested in attempting to unravel how the biological, environmental and historical factors had served to influence the patterns and extent of genetic diversity within each species, thereby inferring some of the important evolutionary processes which have affected Australia's freshwater fauna. I used allozyme and 500-650bp sequences from the ATPase6 mitochondrial DNA (mtDNA) gene to quantify the patterns of genetic variation at several hierarchical levels: within populations, among populations within drainages and among drainages. I collected fish at several spatial scales, from species wide to multiple samples within drainages; samples were collected from the Northern Territory, Queensland and New South Wales. The species with the highest potential for dispersal, H. compressa, exhibited the lowest levels of genetic differentiation as measured at several allozyme loci (H. compressa: FST=0.014; P. signifer FST=0.58; C. stercusmuscarum FST=0.74). Populations of H. compressa also had low levels of mtDNA differentiation, with many recently derived haplotypes which were widespread along the coast of Queensland. This suggested either considerable gene flow occurs or recent demographic change in the populations sampled. As there was no relationship between geographic distance and genetic differentiation, the populations appeared to be out of genetic drift - gene flow equilibrium, assuming the two-dimensional stepping stone model of gene flow. Estimating contemporary gene flow was thus difficult. It was apparent that there has been a recent population expansion and / or contraction of H. compressa populations. It was concluded that there has been considerably more connectivity among populations of H. compressa in the recent past than either of the other study species. Populations of P. signifer showed considerable genetic subdivision at different hierarchical levels throughout the sampled range, indicating gene flow was restricted, especially between separate drainages. Two widely divergent regional groups which had high ATPase6 sequence divergence and approximately concordant patterns at allozyme loci were identified. Interestingly, the groups mirrored previous taxonomic designations. There was also significant subdivision among drainages within regional groups. For example, the adjacent Mulgrave-Russell and Johnstone drainages had individuals with haplotypes that were reciprocally monophyletic and had large allozyme frequency differences. This allowed me to examine the patterns of genetic differentiation among populations within drainages of two essentially independent, but geographically close systems. There was as much allozyme differentiation among populations within subcatchments as there was between subcatchments within drainages, and significant isolation by distance among all populations sampled within a drainage. This suggested that the estuarine confluence between subcatchments was not a barrier to P. signifer, but that distance was an important component in the determination of the distribution of genetic diversity within drainages in P. signifer. There were three main areas of investigation for C. stercusmuscarum: comparing upland and lowland streams of the drainages in north Queensland, investigating the consequences of eustasy on coastal margin populations and examining the intriguing distribution of the two putative sub species, C. s. stercusmuscarum and C. s. fulvus in south east Queensland. First, as populations in upland areas of east coast flowing rivers are above large discontinuities in the river profile, their occurrence is presumably the result of gene flow to and / or from lowland areas, or the result of invasions via the diversion of western flowing rivers. Concordant patterns at both genetic markers revealed that the latter possibility was the most likely, with fixed allozyme differences between upland and lowland populations, and large mtDNA sequence divergence. Indeed, it appeared that there may have been two independent invasions into the upland areas of rivers in North Queensland. Second, lowland east coast populations also had large, although not as pronounced, levels of population subdivision. Lack of isolation by distance, but with a concomitant high level of genetic differentiation among many comparisons, was consistent with a scenario of many small, isolated subpopulations over the range. Interestingly, widespread populations in central Queensland coastal populations (drainages which receive the lowest rainfall) were relatively genetically similar. This was consistent with the widest part of the continental shelf which at periods of lower sea level apparently formed a large interconnected drainage, illustrating the effect of eustatic changes on populations inhabiting a continental margin. Third, putative C. s. fulvus in lowland coastal Queensland drainages were genetically more similar to a population of C. s. fulvus collected from a tributary of the Murray-Darling (western flowing) than they were to adjacent putative C. s. stercusmuscarum. This implied that populations in south east Queensland, north to approximately the Burnett River, appeared to be derived from western flowing streams, and not via dispersal from other lowland east coast populations. Determining the relative importance of intrinsic and extrinsic factors to the development of population genetic structure is a difficult task. The present study demonstrated that the species with the highest dispersal potential had the lowest levels of genetic differentiation, waterfalls can limit gene flow, eustasy acts to join and separate populations leading to complex genetic patterns and that drainage rearrangements are important in determining the distribution of genetic diversity of populations now inhabiting isolated drainages. A difficulty with generalising about population genetic structure in obligate freshwater animals is the unique history of not only each drainage, but also the streams within that drainage and the idiosyncratic biological dynamics of the populations inhabiting those drainages.

S100A8 is a highly-expressed calcium-binding protein in neutrophils and activated macrophages, and has proposed roles in myeloid cell differentiation and host defense. Functions of S100A8 are not fully understood, partly because of difficulties in generating S100A8 knockout mice. Attempts to silence S100A8 gene expression in activated macrophages and fibroblasts using RNA interference (RNAi) technology were unsuccessful. Despite establishing validated small interfering RNA (siRNA) systems, enzymaticallysynthesized siRNA targeted to S100A8 suppressed mRNA levels by only 40% in fibroblasts activated with FGF-2+heparin, whereas chemically-synthesized siRNAs suppressed S100A8 driven by an S100A8-expression vector by ~75% in fibroblasts. Suppression of the gene in activated macrophages/fibroblasts was low, and some enzymatically-synthesized siRNAs to S100A8, and unrelated siRNA to GAPDH, induced/enhanced S100A8 expression in macrophages. This indicated that S100A8 may be upregulated by type-1 interferon (IFN). IFN-β enhanced expression, but did not directly induce S100A8. Poly (I:C), a synthetic dsRNA, directly induced S100A8 through IL-10 and IFN-dependent pathways. Induction by dsRNA was dependent on RNA-dependent protein kinase (PKR), but not cyclooxygenase-2, suggesting divergent pathways in LPS- and dsRNA-induced responses. New mechanisms of S100A8 gene regulation are presented, that suggest functions in anti-viral defense. S100A8 expression was confirmed in lungs from influenza virus-infected mice and from a patient with severe acute respiratory syndrome (SARS). Multiple pathways via mitochondria mediated S100A8 induction in LPS-activated macrophages; Generation of reactive oxygen species via the mitochondrial electron transport chain and de novo synthesis of ATP may be involved. This pathway also regulated IL-10 production, possibly via PKR. Extracellular ATP and its metabolites enhanced S100A8 induction. Results support involvement of cell stress, such as transfection, in S100A8 expression. A breast tumor cell line (MCF-7) in which the S100A8 gene was silenced, was established using micro RNA technology; S100A8 induction by oncostatin M was reduced by >90% in stably-transfected cells. This did not alter MCF-7 growth. The new approach to investigate the role of S100A8 in a human tumor cell line may assist in exploring its functions and lead to new studies concerning its role in cancer.

La diatomée Haslea ostrearia a longtemps été considérée comme le seul organisme apte à produire un pigment surnuméraire bleu nommé marennine, connu pour son rôle dans le verdissement des branchies des huîtres affinées dans les bassins ostréicoles. Certains des mécanismes et facteurs influençant l'entrée de cette diatomée en phase de reproduction sexuée (auxosporulation) ont été mis en évidence, tels la concentration cellulaire, la qualité de l'éclairement incident, ou le préconditionnement des algues. La découverte dans le cadre d'un projet européen, de populations de diatomées apparentées à H. ostrearia en divers points du globe a conduit à la description et l'identification de trois nouvelles espèces de diatomées bleues : Haslea silbo sp. nov. des îles Canaries, Haslea karadagensis sp. nov., provenant de Mer Noire et Haslea provencialis sp. nov. de Méditerranée Occidentale. La première phylogénie moléculaire de ces espèces de diatomées bleues, ainsi que d'autres espèces de diatomées appartenant au genre Haslea, a été réalisée en utilisant trois marqueurs génétiques, la cassette ribosomale ITS1-5,8S-ITS2, le gène chloroplastique rbcL ainsi qu'un fragment du gène mitochondrial cox1. Ces trois marqueurs moléculaires montrent que les diatomées bleues forment un clade distinct au sein du genre Haslea. De plus, l'existence de deux populations d'H. ostrearia originaires des côtes françaises et suédoises sexuellement compatibles a permis d'étudier la variabilité génétique intraspécifique, en mettant en évidence quelques différences au niveau de la séquence du gène cox1. Ces différences ont également permis d'étudier chez la progéniture obtenue par croisements de ces populations, la répartition et l'héritabilité de l'ADN mitochondrial. Par ailleurs, la spectophotométrie UV-visible et la spectométrie Raman ont été utilisées pour poursuivre la caractérisation physico-chimique des pigments bleus de ces diatomées. L'existence de pigments distincts chez les nouvelles espèces de diatomées bleues a permis de proposer une première classification chimiotaxonomique. Enfin, les activités biologiques de la marennine et du pigment de l'espèce ukrainienne, H. karadagensis, ont été étudiées grâce à la détermination de leurs propriétés antibactériennes et antivirales.

La bioénergétique moléculaire des systèmes est une nouvelle direction de recherche scientifique qui s'inscrit dans la Biologie des Systèmes. Elle étudie et décrit le métabolisme énergétique intégré cellulaire non seulement comme un réseau de réactions mais aussi décrit ses aspects spatiaux (organisation) et temporels (dynamique). La bioénergétique moléculaire des systèmes considère l'organisation spatiale intracellulaire comme un processus dynamique dont la topologie elle-même renferme des informations. Ce projet tend à mettre l'accent sur une approche expérimentale décrivant les réseaux de phospho-transferts intracellulaire. Le principal objectif de ce travail fut la description comparative de l'hétérogénéité de la compartimentation des nucléotides adényliques et la complexité structurale et fonctionnelle des communications entre la mitochondrie et d'autres structures ou processus intracellulaire (cytosquelette, glycolyse) dans les cellules nerveuses (synaptosomes) et cardiaques (cardiomyocytes adultes et lignée cancéreuse de cellules HL-1). Les résultats de ce projet ont démontré 1/ la régulation de la perméabilité de la membrane externe mitochondriale par le facteur X (association de la tubuline hétérodimérique avec la porine voltage dependent anion channel), 2/ le couplage fonctionnel entre la creatine kinase mitochondriale et l'adénine nucléotide translocase , 3/ les mécanismes de régulation de la respiration mitochondriale in vivo qui ont permis d'établir un schéma de l'Interactosome Mitochondrial, 4/ les variations de régulation métabolique associées au cancer et 5/ l'hétérogénéité de la diffusion des nucléotides adényliques et leur micro- voire nano-compartimentation après activation des créatines kinases (par spectroscopie à corrélation de fluorescence).

<p>Genetic variation within and among populations is a result of past and ongoing processes. Among the most important of such processes are dispersal, habitat fragmentation and selection. This thesis use neutral genetic variation as a tool to investigate these processes in three bird species.</p><p>In the Siberian jay, the timing of dispersal is dependent on social dominance among siblings. Mark-recapture data, radio-tracking and genetic variation was used to investigate whether timing of dispersal had an effect on dispersal distance. The results show that early dispersing individuals also disperse longer. In the same species, genetic correlation between neighbours was used to find areas with high production of philopatric individuals, which could be indicative of high habitat quality.</p><p>Great snipe populations in northern Europe have a breeding range divided into two regions. A Q_ST-F_STapproach was applied to study variation in selection between regions. Differentiation between the regions in neutral molecular markers was low, indicating high gene flow, or short time available for neutral divergence. Morphological divergence between the regions was high, and Q_ST > F_ST, which indicates divergent selection. Thus, neutral genetic markers can be misleading in identifying evolutionary significant units, and the Q_ST-F_ST approach might be valuable to identify targets for conservation.</p><p>Rock ptarmigan, or its ancestors, originated in Beringia, and spread throughout the Holarctic region. Their distribution has subsequently been affected by glaciations, most likely leading to withdrawals and re-colonisations. Neutral genetic variation among five populations around the northern Atlantic was investigated. There was strong genetic structure among the populations, and evidence that Scandinavian rock ptarmigan has been isolated from other populations for considerable time. Rock ptarmigan in Svalbard showed slightly lower genetic variation than others, and comparisons with other studies suggested an eastern colonisation route to Svalbard.</p>

<p>The Ras superfamily of GTPases embraces six major branches of proteins: the Ras, Rab, Ran, Arf, Rho and Miro subfamilies. The majority of GTPases function as binary switches that cycle between active GTP-bound and inactive GDP-bound states. This thesis will focus primarily on the biological functions of the Rho and Miro proteins. The Rho GTPases control the organization of the actin cytoskeleton and other associated activities, whereas the Miro GTPases are regulators of mitochondrial movement and morphology. </p><p>A diverse array of cellular phenomena, including cell movement and intracellular membrane trafficking events, are dependent on cytoskeletal rearrangements mediated by Rho GTPases. Although human Rho GTPases are encoded by 20 distinct genes, most studies involving Rho GTPases have focused on the three representatives RhoA, Rac1 and Cdc42, which each regulate specific actin-dependent cellular processes. In an effort to compare the effects of all Rho GTPase members in the same cell system, we transfected constitutively active Rho GTPases in porcine aortic endothelial (PAE) cells and examined their effects on the organization of the actin cytoskeleton. We identified a number of previously undetected roles of the different members of the Rho GTPases. Moreover, we demonstrated that the downstream effectors of Rho GTPases have a broader specificity than previously thought. </p><p>In a screen for novel Ras-like GTPases, we identified the Miro GTPases (Mitochondrial Rho). In our characterization of Miro, we established that these proteins influence mitochondrial morphology and serve functions in the transport of mitochondria along the microtubule system. Additionally, we provided evidence that Miro can be under control of calcium signaling pathways. Mitochondria are highly dynamic organelles that undergo continuous change in shape and distribution. Defects in mitochondrial dynamics are associated with several neurodegenerative diseases. In conclusion, our findings have contributed to a deeper understanding of the biological roles of Rho and Miro GTPases.</p>