The wild populations of the Scarlet Macaw subspecies native to southern Mexico and Central America, A. m. cyanoptera, have been drastically reduced over the last half century and are now a major concern to local governments and conservation groups. Programs to rebuild these local populations using captive bred specimens must be careful to reintroduce the native A. m. cyanoptera, as opposed to the South American nominate subspecies (A. m. macao) or hybrids of the two subspecies. Molecular markers for comparative genomic analyses are needed for definitive differentiation. Here I describe the isolation and sequence analysis of multiple loci from 7 pedigreed A. m. macao and 14 pedigreed A. m. cyanoptera specimens. The loci analyzed include the 18S rDNA genes, the complete mitogenome as well as intronic regions of selected autosomally-encoded genes. Although the multicopy18S gene sequences exhibited 10% polymorphism within all A. macao genomes, no differences were observed between any of the 21 birds whose genomes were studied. In contrast, numerous polymorphic sites were observed throughout the 16,993 bp mitochondrial genomes of both subspecies. Although much of the polymorphism was observed in the genomes of both subspecies, subspecies-specific alleles were observed at a number of mitochondrial loci, including 12S, 16S, CO2 and ND3. Evidence of possible subspecies-specific alleles were also found in three of four screened nuclear loci. Collectively, these mitochondrial and nuclear loci can be used as the basis to distinguish A. m. cyanoptera from the nominate subspecies, A. m. macao, as well as identify many hybrids, and most importantly will contribute to further reintroduction efforts.

Conservation genetics of the grey wolf in Czech Republic and adjacent regions is studied in the present thesis. Analyses of twenty-one microsatellite loci, one sex-determining amelogenin gene and mitochodrial control region were used to verify species determination, identify individuals and estimate relationships between them, analyse population structure and estimate demographic trends based on samples collected between 2014 and 2021. Genetic detection of red fox and dog samples incorrectly assigned to wolves illustrates the hurdles of field monitoring of grey wolf. Direct evidence for the occurrence of F1 hybrids was not found. Wolves from Bohemia showed lower values of allelic richness in comparison to the ones from Western Carpathians, probably as a consequence of recent expansion. Geographic distances between detection sites of identical individuals were relatively small or moderate in this study, suggesting regular movements of animals within their home ranges. Only two long-distance dispersal events exceeding 300 km were detected. Results of parental analysis provided evidence of pack distribution within the studied area. Most relationships were detected between wolves in the northern region of Czech Republic where the first recolonizing wolf pack in 2014 was registered. Within the studied...

Abstract Various genetic aetiologies — including mitochondrial diseases, chromosomal disorders, and other monogenic diseases — are involved in status epilepticus (SE), a common neurologic emergency occurring in children and adults that exhibits high rates of morbidity and mortality. The exact frequency of mitochondrial SE is currently undefined. Furthermore, patients with pathogenic variants of POLG1 encoding mitochondrial DNA polymerase gamma have an increased risk of acute liver failure (ALF) induced by the common antiepileptic drug, valproic acid (VPA), which is problematic due to these patients also often experiencing drug-resistant seizures. Overall, the role of liver transplantation (LT) in VPA-ALF due to mitochondrial disease has been controversial. In the present work, large retrospective cohort studies were conducted for two main purposes: (1) to determine the genetic aetiologies of SE among Finnish paediatric and adult patients by specifically focusing on the common mitochondrial genetic defects associated with an increased risk of SE and (2) to examine whether common POLG1 p.Q1236H and p.E1143G variants are connected to liver or pancreatic toxicity upon exposure to VPA monotherapy. This thesis also describes the characteristics of VPA-ALF associated with the pathogenic POLG1 variant p.W748S and the prognosis of LT in a retrospective case series. Mitochondrial diseases explained 4.5% of SE cases in the study cohort. Patients with mitochondrial SE suffered from refractory SE significantly more often than patients with other forms of genetic or non-genetic SE. Additionally, mortality rates were higher in patients with mitochondrial or chromosomal disorders compared with the other groups, reflecting the severity of the underlying condition and the higher frequency of refractory SE. POLG1 variants p.Q1236H and p.E1143G could not be identified as risk factors for VHT or pancreatic toxicity, implying that VPA treatment might be suitable for patients harbouring these variants when other pathogenic variants are absent. Finally, the homozygous status of the pathogenic POLG1 variant p.W748S and older age of the patient during the presentation of VPA-ALF seem to be associated with higher survival rates following LT, which should be considered in the management of VPA-ALF. / Tiivistelmä Useita perinnöllisiä syitä, kuten mitokondriotauteja, kromosomihäiriöitä ja muita geenimuutoksia on tunnistettu status epilepticuksen (SE) eli pitkittyneen epileptisen kohtauksen taustalla. SE on yleinen neurologinen hätätilanne, johon liittyy merkittävää oheissairastavuutta ja kuolleisuutta sekä lapsilla että aikuisilla. Mitokondriotauteihin liittyvän SE:n tarkkaa esiintyvyyttä ei tiedetä. Potilailla, joilla on patogeenisia variantteja mitokondrioiden DNA-polymeraasia koodaavassa tuman POLG1-geenissä, on todettu kohonnut riski yleisesti käytetyn epilepsialääkkeen valproaatin (VPA) aiheuttaman akuutin maksavaurion kehittymiselle. Tämä tekee lääkehoidon valinnasta ongelmallista, koska näillä potilailla on usein epilepsialääkkeille resistenttejä kohtauksia. Maksansiirron merkitys akuutin maksavaurion hoidossa mitokondriotauteja sairastavilla potilailla on ollut kiistanalainen. Tutkimuksen tavoitteena oli selvittää SE:n perinnöllisiä syitä suomalaisilla lapsi- ja aikuispotilailla retrospektiivisesti kerätyssä laajassa potilasaineistossa. Tutkimuksessa keskityttiin yleisimpiin mitokondriaalisiin perinnöllisiin muutoksiin, joiden on aiemmin todettu liittyvän SE:n lisääntyneeseen riskiin. Tutkimuksen toisena päätavoitteena oli selvittää väestössä yleisten POLG1-geenin muutosten eli varianttien p.Q1236H ja p.E1143G yhteyttä maksatoksisuuteen tai haimatoksisuuteen VPA-monoterapian aikana. Lisäksi tutkittiin VPA:n aiheuttaman maksavaurion kliinisiä erityispiirteitä patogeeniseen POLG1-varianttiin p.W748S liittyen sekä mutaatiostatuksen vaikutusta maksansiirron jälkeiseen ennusteeseen. Mitokondriotaudit selittivät 4,5 % SE-tapauksista tämän väitöskirjatyön potilasaineistossa ja näillä potilailla SE pitkittyi hoitoresistentiksi tai erittäin resistentiksi merkitsevästi muita potilasryhmiä useammin. Kuolleisuus oli suurin potilailla, joilla todettiin mitokondriotauti tai kromosomihäiriö, liittyen todennäköisimmin vakavaan taustasairauteen ja hoitoresistentin SE:n suurempaan esiintyvyyteen. Tutkittuja POLG1-variantteja p.Q1236H ja p.E1143G ei voitu tunnistaa maksa- tai haimatoksisuuden riskitekijöiksi, mikä tarkoittaa, että VPA-hoito voisi sopia näille potilaille, mikäli muita patogeenisiä variantteja ei todeta. Patogeenisen POLG1-variantin p.W748S homotsygoottisuus ja nuoruusikä tai varhainen aikuisikä maksavaurion ajankohtana ovat maksansiirron ennustetta parantavia tekijöitä, mikä tulisi ottaa huomioon hoitopäätöksiä tehtäessä.

No description available.

Trypanosoma brucei and related protists are distinguished from all other eukaryotes by an unusual mitochondrial genome known as kinetoplast DNA (kDNA) that is a catenated network composed of minicircles and maxicircles. Replication of this single nucleoid involves a release, replicate, and reattach mechanism for the thousands of catenated minicircles and requires at least three DNA polymerase (POLIB, POLIC and POLID) with similarity to E. coli DNA polymerase I. Like other proofreading replicative DNA polymerases, POLIB has both an annotated polymerase domain and an exonuclease domain. Predictive modelling of POLIB indicates that it has the canonical right hand polymerase structure with a unique and large 369 amino acid insertion within the polymerase domain (thumb region) homologous to E. coli RNase T. The goal of this study was to evaluate whether the polymerase domain is necessary for the essential replicative role of POLIB. To study the structure-function relationship, an RNAi-complementation system was designed to ectopically express POLIB variants in T. brucei that has endogenous POLIB silenced by RNAi.Control experiments expressing an ectopic copy of POLIB wildtype (IBWTPTP) or polymerase domain mutant (IBPol-PTP) in the absence of RNAi did not impact fitness in procyclic cells despite protein levels being 5 - 8.5 fold higher than endogenous POLIB levels. Immunofluorescence detection of the tagged variants indicated homogenous expression of the variants in a population of cells and negligible changes in kDNA morphology. Lastly, Southern blot analyses of cells expressing the IBWTPTP or IBPol-PTP variants indicated no changes in free minicircle species. A dually inducible RNAi complementation system was designed and tested with the IBWTPTP and IBPol-PTP variants. Inductions of POLIB RNAi accompanied by ectopic expression of either variant using the standard 1 mg/ml tetracycline resulted in low protein levels of both variants while knockdown of the endogenous POLIB mRNA was greater than 85%. Increasing the tetracycline concentration to 4 mg/ml improved expression levels of both variants. However, levels of the ectopically expressed variants never exceeded that of endogenous POLIB. Using the 4 mg/ml induction conditions, complementation with IBWTPTP resulted in a partial rescue of the POLIB RNAi phenotype based on fitness curves, quantification of kDNA content and Southern blot analysis of free minicircles. IBWTPTP complementation resulted in gradual increase of IBWTPTP protein levels over the 10 day induction, and a small kDNA phenotype instead of the progressive loss of kDNA normally associated with POLIB RNAi. Additionally, the loss of free minicircles was delayed. Complementation with the IBPol-PTP variant produced more consistent levels of IBPol-PTP protein although still below endogenous POLIB levels. Loss of fitness was similar to POLIB RNAi alone. However, a small kDNA phenotype emerged early after just four days of complementation and persisted for the remainder of the induction. The majority of the IBRNAi + IBPol-PTP population (70%) contained small kDNA compared to the parental POLIB RNAi or IBWTPTP complementation that had only 45% and 50% small kDNA, respectively. Lastly, the pattern of free minicircle loss closely resembled POLIB RNAi alone. Together, these data suggest that the dually inducible system results in a partial rescue with the IBWTPTP variant. Rescue with IBPol-PTP variant results in a noticeably different phenotype from either POLIB RNAi alone or IBWTPTP complementation indicating that the POLIB polymerase domain is likely essential for the in vivo role of POLIB during kDNA replication.

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.

The aim of this thesis is to investigate how discussions in archaeology and genetics influence the consensus on human origins and migrations in the South Pacific. By analyzing the genetic research on chicken- and sweet potato-DNA, I present a general overview of how genetics and archaeology shape the understanding of how humans have colonized the Pacific. By deconstructing a review on how the Pacific was settled based on aDNA, I analyze a geneticist’s perspective on archaeological problems. Through this analysis I suggest how archaeology should be approached on a theoretical level in order to be relevant in understanding human migrations in the Pacific. I propose that archaeology’s strength lie in interpreting material culture through an agency perspective in order to reach a dimension not obtainable by biological perspectives / Syftet med denna uppsats är att undersöka hur diskussioner i arkeologi och genetik påverkar hur vi ser på mänskliga migrationer i Oceanien. Genom att analysera den genetiska forskning som gjorts på kyckling och sötpotatis ges en övergripande bild av hur genetik och arkeologi formar den förståelse som finns för hur människan koloniserat Söderhavet. Genom att dekonstruera en sammanställning av den genetiska forskning som gjorts på mänskligt DNA i Oceanien analyseras en genetikers synsätt på arkeologiska problemställningar. Genom analysen i denna uppsats föreslår jag hur arkeologi borde arbeta på ett teoretiskt plan för att vara relevant i hur vi förstår Oceaniens migrationsmönster. Jag föreslår att arkeologins styrka ligger i att tolka den materiella kulturen genom ett agency-perspektiv för att komma åt en dimension av migrationsproblematiken som inte går att nås genom biologiska perspektiv.

The mitochondrion is a tiny organelle that is the power supplier of the cell and vital to the functioning of the body organs. Additionally it contains a small circular genome of about 16 kb, present in many copies which makes the mitochondrial DNA more viable than nuclear DNA. Mitochondrial DNA is also maternally inherited and thus provides a direct link to maternal relatives. These two properties are of particular use for forensic samples, which only contain limited or degraded amounts of DNA, and for historical samples (ancient DNA). This thesis presents work on the mitochondrial DNA in the hypervariable regions (HV) I and II, in both contemporary and historical samples. Forensic genetics makes use of mitochondrial DNA analysis in court as circumstantial evidence, and population databases are used for the calculation of evidence value. Population samples (299) across Sweden have been analysed in order to enrich the EDNAP mtDNA database (EMPOP) (paper I). The application of mitochondrial DNA analysis allowed for analysis of historical skeletal remains: Copernicus, 1473-1543 (paper II), Karin Göring, 1888-1931 (paper III) and Medieval bones, 880-1000 AD, from a mass grave found in Sigtuna, Sweden (paper IV). The thesis also includes analyses of bones and teeth from the shipwrecked crew of the Vasa warship, 1628, samples from the Vasa museum, Stockholm, Sweden (paper V). Overall, the varying age of the samples and the different conservation environments (soil and water) accounted for variations in quality, but still allowed for successful DNA analysis.

L'acarien rouge de la tomate Tetranychus evansi (Tetranychidae) est considéré comme une espèce invasive à fort impact économique sur les cultures de solanacées. Il a été découvert pour la première fois en 1954 au Brésil, d'où il est probablement originaire. Historiquement, T. evansi a d'abord été signalé en Afrique et plus récemment en Europe et en Asie. L'objectif de cette thèse était de reconstruire les routes de colonisation de T. evansi et de dégager le scénario évolutif décrivant le mieux l'histoire de l'invasion. Nous avons d'abord analysé des échantillons collectés dans son aire actuelle de distribution, à l'aide des séquences d'un fragment du gène codant pour la sous-unité I de la Cytochrome Oxydase (COI) de l'ADN mitochondrial et de la région ITS1-5,8S-ITS2 de l'ADN nucléaire ribosomique. Les données soutiennent l'hypothèse d'une origine sud américaine de cette espèce et ont révélé que des événements d'invasions multiples et cryptiques ont eu lieu lors de la colonisation de l'Europe. L'invasion résulte de deux lignées génétiquement divergentes et originaires de deux régions géographiques distantes au Brésil. Ces deux lignées semblent avoir des potentiels invasifs contrastés. Elles s'hybrident au laboratoire ainsi que dans la nature. Grâce à 16 locus microsatellites que nous avons développés et utilisés comme marqueurs, nous avons déterminé les zones géographiques de cette hybridation. Nous avons également pu estimer des paramètres historiques de l'invasion et confronter différents scénarios d'introduction, par la comparaison de la composition génétique des populations récemment introduites avec celles de l'aire d'origine de T evansi, et par l'utilisation de la méthode d'inférence bayésienne (Approximate Bayesian Computation, ABC). Les résultats ABC contredisent partiellement le scénario d'invasion basé uniquement sur des données historiques. Ils suggèrent que T. evansi serait d'abord arrivé en Europe dans le sud de l'Espagne (en Andalousie) bien avant les signalements historiques. Ainsi, l'Andalousie semble avoir servi de source de colonisation pour des nouvelles zones en Afrique, d'autres régions méditerranéennes et d'Asie. Les résultats de cette thèse ouvrent des perspectives d'étude visant à comprendre pourquoi certaines populations d'une espèce allochtone réussissent à s'établir et à envahir un nouvel écosystème / The red tomato spider mite Tetranychus evansi (Tetranychidae) is regarded as an invasive species having an important economic impact on solanaceous crops. It was first discovered in Brazil in 1954, where it probably originated. Based on historical records, T. evansi was first reported in Africa and more recently in Europe and Asia. This work aims at reconstructing the colonization routes of T. evansi and identifies the scenario that best describes the evolutionary history of the invasion. To do this, we first analyzed samples collected from most parts of the world where the mite is currently known to occur. We used sequence variation of a fragment of the mitochondrial Cytochrome Oxidase I (COI) sub-unit I gene and the ITS1-5.8S-ITS2 of the nuclear ribosomal DNA. Our results were consistent with the hypothesis of a South American origin of this species. They also suggested that the invasion of south Europe resulted from multiple cryptic introductions from two genetically divergent lineages originated from two distant geographical regions in Brazil. These two lineages seem to have a differential invasive potential. Despite the high genetic divergence, crosses between mites stemming from the two lineages do occur both in the laboratory and in nature. Second, we used 16 microsatellite loci that we developed for this study and in association with Approximate Bayesian Computation (ABC) methods; we reconstructed the historical events of the cryptic invasion of the pest. ABC results challenge the invasion scenario captured by historical data only. They suggest that T. evansi first arrived to Europe in Southern Spain (Andalusia) long before historic records. Thus, Andalusia seems to have served as a source for colonization of new areas in Africa and other Mediterranean regions. The results obtained in this thesis provide an interesting framework to further study and understand why some populations of an exotic species might become invasive.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.