Les défauts de génome mitochondrial provoquent des maladies neuromusculaires, pour lequel aucun traitement efficace n'a été mis au point. La plupart des mutations mitochondriales sont hétéroplasmique, ce qui signifie que l'ADN mitochondrial (ADNmt) de type sauvage et muté coexistent dans la même cellule, et le changement de proportion entre deux types d'ADNmt pourrait rétablir les fonctions mitochondriales. Le but du projet était le développement du système pour cibler l'ARN thérapeutique dans les cellules humaines vivantes. Au cours de ma thèse j'ai synthétisé une série de nouveaux ARN anti-réplicatifs contenant modifications chimiques pour augmenter leur stabilité dans la cellule, et mis au point la nouvelle méthode de synthèse chimique des molécules d'ARN contenant cholestérol fixé par l'intermédiaire d'un pont biodégradable. Ces ARN étaient capable de pénétrer dans les cellules humains, d'être adressées dans les mitochondries et de diminuer la proportion d' ADNmt muté. / Defects in mitochondrial genome cause neuromuscular diseases, for which no efficient therapy has been developed. Since most mitochondrial mutations are heteroplasmic, wild type and mutated mitochondrial DNA (mtDNA) coexist in the same cell, and the shift in proportion between two mtDNA types could restore mitochondrial functions. The aim of the project was development of carrier-free system for targeting the therapeutic mitochondrially importable RNA into living human cells. During my PhD study, I have synthesized a set of new anti-replicative RNAs containing various chemical modifications, aiming to increase their stability in the cell, and developed a new method for the chemical synthesis of RNA molecules containing cholesterol attached through a biodegradable bridge. Cholesterol containing antireplicative RNAs were characterised by efficient cellular uptake, partial colocalisation with mitochondria and ability to decrease the proportion of mutant mtDNA.

Abstract Many waders are in decline, and the number of endangered species and populations is increasing. Their protection and management requires knowledge of both ecological and genetic state of the populations. In this thesis, I studied the distribution-wide genetic variation, structure and phylogeography of the Temminck’s Stint (Calidris temminckii) and Terek Sandpiper (Xenus cinereus) using microsatellites and sequence data from the mitochondrial control region and cytochrome oxidase I gene. I compared these regionally endangered species to other waders with varying evolutionary histories, breeding systems and habitat preferences to examine the levels of genetic variation and structure at different spatial scales. In addition, I studied the genetic structure of the endangered Baltic population of the Southern Dunlin (Calidris alpina schinzii) with microsatellites. I used genetic information in all three study species to determine units for conservation. The Temminck’s Stint and Terek Sandpiper, both not restricted to the Arctic, had low distribution-wide structuring. They also had quite low levels of variation when compared to other species breeding at similar latitudes, indicating reductions in population sizes during past climate changes. Especially the peripheral breeding populations were differentiated and showed signs of inbreeding and genetic drift when compared to the main range. The Temminck’s Stint populations at the Bothnian Bay and Yakutia, and Terek Sandpiper populations in Finland and Belarus, should be treated as separate management units. The broader interspecific comparison of waders suggests that habitat availability, mating system and the extent of philopatry may affect the genetic composition of species. The genetic analyses of the Southern Dunlin indicated strong effects of philopatry and inbreeding throughout the range. Local subpopulations at the Bothnian Bay and in Sweden need to be considered as separate management units. Management efforts at the Baltic should be focused on increasing connectivity and providing large enough breeding habitats for potential immigrants and recruits. / Tiivistelmä Useat kahlaajapopulaatiot ovat pienentyneet ja uhanalaistuneet maailmanlaajuisesti. Lajien ja populaatioiden ekologiaa ja genetiikkaa on tunnettava, jotta suojelutoimia voidaan kohdistaa oikein. Tutkin väitöskirjassani lapinsirrin (Calidris temminckii) ja rantakurvin (Xenus cinereus) geneettistä rakennetta, muuntelua ja fylogeografiaa levinneisyysalueen laajuisesti mikrosatelliittien ja mitokondrion kontrollialueen ja sytokromioksidaasi I -geenin sekvenssien avulla. Tutkin, mitkä tekijät vaikuttavat geneettisen rakenteeseen ja muunteluun vertaamalla näitä lajeja muihin kahlaajiin, joilla on erilaisia lisääntymisstrategioita, jotka pesivät vaihtelevissa ympäristöissä ja joista monet eroavat toisistaan myös fylogeografialtaan. Lisäksi tutkin Itämeren rannalla pesivän etelänsuosirrin (Calidris alpina schinzii) geneettistä populaatiorakennetta mikrosatelliittien avulla. Käytin geneettistä tietoa hyväksi luonnonsuojeluyksikköjen määrittämisessä kaikille kolmelle tutkimuslajilleni. Lapinsirrin ja rantakurvin fylogeografinen historia oli samankaltainen. Geneettisen muuntelun määrä oli vähäisempää verrattuna muihin, samankaltaisissa ympäristöissä pesiviin kahlaajiin. Molemmat lajit ovat todennäköisesti kärsineet historiallisten ilmaston-muutosten aiheuttamasta populaatioiden pienenemisestä. Erityisesti levinneisyysalueen reunoilla pesivät populaatiot olivat erilaistuneita, ja niissä näkyi sukusiitoksen ja geneettisen satunnaisajautumisen merkkejä. Perämeren ja Jakutian lapinsirri- sekä Valko-Venäjän ja Suomen rantakurvipopulaatioita tulee kohdella erillisinä suojeluyksiköinään. Vertailu muihin kahlaajiin osoitti, että niin pesimä- ja talvehtimisalueiden laajuus kuin lisääntymisstrategiat ja paikkauskollisuus voivat vaikuttaa lajien geneettiseen koostumukseen. Etelänsuosirrin geneettiset analyysit paljastivat merkkejä sukusiitoksesta, joita paikkauskollisuus ja populaatioiden pienuus ovat voimistaneet. Perämeren ja Ruotsin populaatioita tulee kohdella erillisinä suojeluyksiköinään. Suojelutoimet on kohdistettava tarpeeksi suurien, hyvälaatuisten pesimäpaikkaverkostojen ylläpitämiseen.

Magister Scientiae - MSc / In many forensic cases, such as mass disasters reconstruction cases, the recovered DNA is highly degraded. In such incidences, typing of STR loci has become one of the most powerful tools for retrieving information from the degraded DNA. However, as DNA degradation proceeds, three phenomena occur consecutively: loci imbalance, allele dropout and no amplification. To solve the problem of degraded DNA, redesigned primer sets have been developed in which the primers were positioned as close as possible to the STR repeat region. These reduced primer sets were called Miniplexes. Unfortunately, a few of the CODIS STR loci cannot be made into smaller amplicons. For this reason non-CODIS miniSTRs have been developed. The present study was undertaken for the population genetic analysis of microsatellite variation in four South African populations; Afrikaner, Xhosa, Mixed Ancestry and Asian Indian using eight non-CODIS miniSTR loci. These miniSTRs loci were characterized within the populations by estimating the levels of diversity of the markers, estimating the population genetic parameters, and studying the inter-population relationships. All of the miniSTRs were amplified successfully and the genetic variability parameters across all loci in Afrikaner, Mixed Ancestry, Asian Indian and Xhosa were estimated to be in the range of 3 (D4S2364) to 12 (D9S2157) alleles, the total number of alleles over all loci ranged from 100 to 204, the allelic richness ranged from 3.612 to 10.307 and the heterozygosity ranged from 0.4360 to 0.8073. Genetic distance was least between Afrikaner and Asian Indian and highest between Xhosa and Mixed Ancestry. Deviations from Hardy-Weinberg equilibrium were not observed for most of the loci. The low mean FIS (-0.027) and FIT (-0.010) and FST (0.017) values across the populations indicated low level of inbreeding within (FIS) and among (FST) the populations. The Asian Indian population showed higher levels of the inbreeding coefficient, indicating less gene exchange between it and other populations. These 8 markers can be used for genetic investigations and assessing population structure. The study contributed to the knowledge and genetic characterization of four South African populations. In addition, these MiniSTRs prove to be useful in cases where more genetic information is needed. / South Africa

<p>Sagledavanje ukupnog genetičkog i fenotipskog diverziteta i evolucionog potencijala vrste E. tenax izvr&scaron;eno je analizom jedinki poreklom sa 42 područja Evrope, Australije, Severne i Južne Amerike i laboratorijske kolonije iz &Scaron;panije. Analiza intraspecijske varijabilnosti vrste E. tenax izvr&scaron;ena je kvantifikovanjem varijacija u veličini i obliku krila 1653 jedinke i obojenosti abdomena 936 jedinki. Analiza genetičkog diverziteta na nivou polimorfizma nukleotidnih sekvenci mtDNK je izvr&scaron;ena kod 58 jedinki, dok je analiza alozimske varijabilnosti obuhvatila 821 jedinku prirodnih populacija i laboratorijske kolonije vrste E. tenax. Rezultati su ukazali da inbriding i stohastički procesi utiču na redukciju genetičkog diverziteta i da &ldquo;oslobađaju&rdquo; skrivenu genetičku varijabilnost koja je povezana sa fenotipskom diferencijacijom. Vremenska distribucija fenotipskog diverziteta vrste E. tenax je kvantifikovana analizom jedinki četiri alohrona uzorka poreklom sa lokaliteta Venac, Fru&scaron;ka gora. Mali stepen genetičke i fenotipske diferencijacije između durmitorskih uzoraka vrste E. tenax ukazuje na odsustvo prostorne substruktuiranosti i njihovu međusobnu povezanost intenzivnim protokom gena. Analiza konspecifičkih populacija vrsta E. tenax ukazala je na odsustvo jasne međupopulacione diferencijacije na osnovu parametrara krila i molekularnih markera (alozimski lokusi i COI mtDNK), te se može zaključiti da postoji intenzivan protok gena koji elimini&scaron;e razlike između populacija. Registrovan je polni dimorfizam u veličini i obliku krila i obojenosti abdomena. Uočeno je da mužjaci u proseku imaju manja i uža krila i svetlije obojene abdomene od ženki. Analizom fenotipske diferencijacije u karakterima abdomena na uzorcima vrste E. tenax sakupljenim duž geografskog gradijenta Evrope je utvrđeno odsustvo promena po tipu kline. Dobijeni rezultati omogućavaju preciznije sagledavanje intra- i interpopulacione varijabilnosti ovog takona i ukazuju da vrsta E. tenax ima visok evolucioni potencijal za adaptacije na sredinske promene</p> / <p>This paper examines molecular and phenotypic variability in the widely spread hoverfly species Eristalis tenax. We compared 42 samples from Europe, Australia North and South America, with the aim of obtaining insights into the temporal and spatial variations and sexual dimorphism in the species. Additionally, wild specimens from Spain were compared with a laboratory colony reared on artificial media. The integrative approach was based on allozyme loci, cytochrome c oxidase I mitochondrial DNA, morphometric wing parameters (shape and size) and abdominal colour patterns. Our results indicate that the fourth and eighth generations of the laboratory colony show a severe lack of genetic diversity compared to the figures observed in natural populations. Reduced genetic diversity in subsequent generations of the laboratory colony was found to be linked with phenotypic divergence. The distribution of genetic diversity at polymorphic loci indicated genetic divergence among collection dates from Fru&scaron;ka Gora Mt, and landmark-based geometric morphometrics revealed significant wing shape variation throughout the year. Phenotypic differentiation in abdominal pattern of the E. tenax populations along latitudinal gradient Europe has not been established. Consistent sexual dimorphism was observed, indicating that male specimens had lighter abdomens and smaller and narrower wings than females. It is reasonable to assume high mobility of the dronefly and high rate of gene flow reflected the similarity of genetic and phenotypic diversity of otherwise geographically distinct populations. Hence, the present study expands our knowledge of the genetic diversity and phenotypic variability of E. tenax. The quantification of such variability represents a step towards the evaluation of the adaptive potential of this species of medical and epidemiological importance.</p>

In addition to the interaction of evolutionary forces, the population history of the African Sahel and Arabia has been influenced by the spread of Neolithic cultural innovations. The reflection of these processes today is a very complex structured diversity of the current populations, which is presented here through the analysis of several genetic markers. The aim is to provide a comprehensive view of the history of demographic processes in the Sahel and Arabia, by combining genetic, linguistic, subsistence and geographical data obtained from local populations. A study of a large dataset of mtDNA sequences showed that Arabia was a major crossroads in gene flow, and although it was colonized by anatomically modern humans from East Africa, today's differentiation from Africa is greater than the differentiation between local populations in these regions. Even the Sahel was an important biocorridor in the past. Today, we encounter populations of various subsistence strategies (nomadic pastoralists and settled farmers), between which gene flow has been severely restricted. A comparison of uniparently inherited loci in both groups points to different migratory activity in the eastern and western parts of the Sahel. Analyzes of Alu elements, which indicated the inclination of West African herders (Fulbs)...

Culicoides (Diptera: Ceratopogonidae) are small (<3mm) blood feeding flies. These flies are biological vectors of viruses, protozoa and filarial nematodes affecting birds, humans, and other animals. Among the viruses transmitted those causing bluetongue (BT), African horse sickness (AHS) and epizootic haemorrhagic disease (EHD) are of major veterinary significance. Culicoides (Avaritia) imicola Kieffer, a proven vector of both AHS and BT viruses, is the most abundant and wide spread livestock-associated Culicoides species in South Africa. Field isolations of virus and oral susceptibility studies, however, indicated that a second Avaritia species, C. bolitinos Meiswinkel may be a potential vector of both BT virus (BTV) and AHS virus (AHSV). Differences in oral susceptibility, which are under genetic control, of populations from different geographical areas to viruses may be an indication of genetic differences between these populations, which may be the result of limited contact between these populations. A good knowledge of the distribution, spread and genetic structure of the insect vector is essential in understanding AHS or BT disease epidemiology. In the present study, an effort was made to gather field specimens of both C. imicola and C. bolitinos from different areas within their natural distribution in South Africa. The aim was to partially sequence two mitochondrial genes from these specimens and to analyse the sequence data making use of phylogenetic trees to clarify the genetic relationships between individuals or groups collected from geographically distinct sites. The two species were collected from four geographically separated areas in South Africa viz. Gauteng Province, Eastern Cape Province, Western Cape Province as well as the Free State Province. DNA was extracted from a total of 120 individual midges of the two Culicoides species using DNA extraction kits. Extracted DNA was analysed using PCR, sequencing as well as phylogenetic methods. A total of 117 mitochondrial DNA COI and 104 mitochondrial 16S ribosomal RNA Culidoides</i. sequences were analysed. DNA sequence polymorphism and phylogenetic relationships of various groups of C. imicola and C. bolitinos midges were determined. The results of the phylogenetic analysis of Culicoides populations using mitochondrial COI gene fragment showed that, at least one subpopulation of C. imicola and two distinct genotypes of C. bolitinos species do exist in South Africa, and further analysis is necessary. This study showed that COI has the potential to separate Culicoides midges based on their geography / Dissertation (MSc)--University of Pretoria, 2010. / Veterinary Tropical Diseases / unrestricted

Les mitochondries sont généralement transmises de façon strictement maternelle. Chez les animaux, il existe une seule exception à ce mode de transmission mitochondriale : la transmission doublement uniparentale (DUI). La DUI est retrouvée uniquement chez certaines espèces de bivalves. Les mâles possèdent dans leurs gamètes le génome mitochondrial paternel, alors que les femelles ont dans leurs oeufs le génome mitochondrial maternel. Ces génomes possèdent respectivement m-orf ou f-orf, un cadre de lecture supplémentaire (outre les 13 codant pour les protéines mitochondriales de référence) potentiellement codant. La présence de ces ORF étant liée au sexe de l’animal, l’hypothèse a été avancée que ces protéines pourraient jouer un rôle dans le maintien de la DUI ou dans le déterminisme sexuel chez ces espèces. Ce projet consiste donc à mieux cerner les fonctions potentielles de ces orfs chez la moule bleue Mytilus edulis. Pour caractériser leur expression, nous avons procédé à des tests d’immunobuvardage sur des lysats de tissus gamétiques et somatiques mâles et femelles, ainsi qu’à des tests d’immunofluorescence sur des cultures cellulaires des deux sexes. Aussi, nous avons effectué des co-immunoprécipitation et des essais pull-down pour préciser les fonctions des protéines par l’entremise des partenaires d’interaction. Nous avons pu observer la présence de M-ORF dans les gonades mâles uniquement, plus particulièrement dans les mitochondries des spermatozoïdes et dans l’acrosome, et ce, uniquement durant la saison de reproduction des moules. F-ORF, cependant, était produite dans tous les tissus à tous les moments de l’année, encore une fois dans les mitochondries des cellules. Les deux protéines ont de nombreux partenaires d’interactions possibles, dont plusieurs sont liés à des processus spécifiques au sexe ou encore aux acides nucléiques. Les protéines M-ORF et F-ORF sont donc bien fonctionnelles. Leurs partenaires potentiels sont multiples, et d’autres essais doivent être effectués afin de préciser les fonctions des protéines. La présence dans l’acrosome de M-ORF est toutefois d’un grand intérêt en lien avec son rôle potentiel dans le DUI et le déterminisme sexuel. / Mitochondria are usually transmitted by strict maternal inheritance. In animals, there is only one exception to this: doubly uniparental inheritance (DUI). DUI can be found only in some bivalve species. Males have in their sperm a paternal mitochondrial genome whereas females have in their eggs the maternal mitochondrial genome. Both genomes possess an orf (other than the 13 coding for annotated mitochondrial proteins) that can potentially code for a protein, called respectively m-orf and f-orf. These genes are sex-specific in gametes, which brought the possibility that there is a link between the orfs and the maintenance of DUI or with sex determination in DUI species. Therefore, this project aims to have a better understanding of the potential functions of these proteins in the blue mussel Mytilus edulis. To demonstrate the proteins' existence, we did Western blot assays on gametic and somatic tissues from males and females, along with immunohistochemistry on cellular cultures of both sexes. To look for possible interaction partners, we did co-immunoprecipitation assays and pull-downs assays. Our results show expression of M-ORF in the male mantle only, more specifically in sperm mitochondria and acrosome. This is found only during the reproductive season of Mytilus edulis. However, F-ORF is expressed in all tissues all year in both sexes, in cells mitochondria. Both proteins have numerous possible interaction partners. Several are linked to sex-specific processes or to interactions with nucleic acids. Both M-ORF and F-ORF are expressed. Potential partners are multiple, and other assays have to be done to further ascertain these proteins' functions. However, the presence of M-ORF in acrosome is of great interest toward a potential function in DUI or in sex determination.

No description available.

Maintenance of genomic integrity and stability is of prime importance for the survival of an organism. Upon exposure to different damaging agents, DNA acquires various lesions such as base modifications, single-strand breaks (SSBs), and double-strand breaks (DSBs). Organisms have evolved specific repair pathways in order to efficiently correct such DNA damages. Among various types of DNA damages, DSBs are the most serious when present inside cells. Unrepaired or misrepaired DSBs account for some of the genetic instabilities that lead to secondary chromosomal rearrangements, such as deletions, inversions, and translocations and consequently to cancer predisposition. Nonhomologous DNA end joining (NHEJ) is one of the major DSB repair pathways in higher organisms. Mitochondrial DNA (mtDNA) deletions identified in humans are flanked by short directly-repeated sequences, however, the mechanism by which these deletions arise are unknown. mtDNA deletions are associated with various types of mitochondrial disorders related to cancer, aging, diabetes, deafness, neurodegenerative disorders, sporadic and inherited diseases. Compared to nuclear DNA (nDNA), mtDNA is highly exposed to oxidative stress due to its proximity to the respiratory chain and the lack of protective histones. DSBs generated by reactive oxygen species, replication stalling or radiation represents a highly dangerous form of damage to both nDNA and mtDNA. However, the repair of DSBs in mitochondria and the proteins involved in this repair are still elusive. Animals deficient for any one of the known Classical-NHEJ factors are immunodeficient. However, DSB repair (DSBR) is not eliminated entirely in these animals suggesting evidence of alternative mechanism, ‘alternative NHEJ’ (A-NHEJ/A-EJ). Several lines of evidence also suggest that alternative and less well-defined backup NHEJ (B-NHEJ) pathways play an important role in physiological and pathological DSBR. We studied NHEJ in different tissue mitochondrial protein extracts using oligomeric DNA substrates which mimics various endogenous DSBs. Results showed A-EJ, as the predominant pathway in mitochondria. Interestingly, immunoprecipitation (IP) studies and specific inhibitor assays suggested, mitochondrial end joining (EJ) was dependent on A-EJ proteins and independent of C-NHEJ proteins. Further, colocalization studies showed A-EJ proteins localize into mitochondria in HeLa cells. More importantly knockdown experiments showed the involvement of DNA LIGASE III in mitochondrial A-EJ. These observations highlight the central role of A-EJ in maintenance of the mammalian mitochondrial genome. By using oligomeric DNA substrates mimicking various endogenous DSBs, NHEJ in different cancer cell lines were studied. We found that the efficiency of NHEJ varies among cancer cells; however, there was no remarkable difference in the mechanism and expression of NHEJ proteins. Interestingly, cancer cells with lower levels of BCL2 possessed efficient NHEJ and vice versa. Removal of BCL2 by immunoprecipitation and protein fractionation using size exclusion column chromatography showed elevated levels of EJ. Most importantly, the overexpression of BCL2 in vivo or the addition of purified BCL2 in vitro led to the downregulation of NHEJ in cancer cells. Further, we found that BCL2 interacts with KU proteins both in vitro and in vivo using immunoprecipitation and immunofluorescence, respectively. Hence, NHEJ in cancer cells is negatively regulated by the anti-apoptotic protein, BCL2, and this may contribute towards increased chromosomal abnormalities in cancer. In summary, our study showed that the efficiency of EJ in cancers could be regulated by the antiapoptotic protein BCL2. However, it may not affect the mechanistic aspect of EJ. BCL2 instead may interfere with EJ by sequestering KU and preventing it from binding to DNA ends. This may help in better understanding towards increased chromosomal abnormalities in cancer. Study of mitochondrial DSBR in mammalian system highlights the central role of microhomology-mediated A-EJ in the maintenance of the mammalian mitochondrial genome and this knowledge will helpful for the development of future therapeutic strategies against variety of mitochondria associated diseases.

We have previously shown that chronic Hepatitis C virus (HCV) infection can induce DNA damage and immune dysfunctions with excessive oxidative stress in T cells. Furthermore, evidence suggests that HCV contributes to increased susceptibility to metabolic disorders. However, the underlying mechanisms by which HCV infection impairs cellular metabolism in CD4 T cells remain unclear. In this study, we evaluated mitochondrial mass and intracellular and mitochondrial reactive oxygen species (ROS) production by flow cytometry, mitochondrial DNA (mtDNA) content by real-time qPCR, cellular respiration by seahorse analyzer, and dysregulated mitochondrial-localized proteins by Liquid Chromatography-Mass Spectrometry (LC-MS) in CD4 T cells from chronic HCV-infected individuals and health subjects. Mitochondrial mass was decreased while intracellular and mitochondrial ROS were increased, expressions of master mitochondrial regulators peroxisome proliferator-activated receptor 1 alpha (PGC-1α) and mitochondrial transcription factor A (mtTFA) were down-regulated, and oxidative stress was increased while mitochondrial DNA copy numbers were reduced. Importantly, CRISPR/Cas9-mediated knockdown of mtTFA impaired cellular respiration and reduced mtDNA copy number. Furthermore, proteins responsible for mediating oxidative stress, apoptosis, and mtDNA maintenance were significantly altered in HCV-CD4 T cells. These results indicate that mitochondrial functions are compromised in HCV-CD4 T cells, likely the deregulation of several mitochondrial regulatory proteins.