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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.

Les maladies mitochondriales regroupent un ensemble de pathologies liées à un déficit de la chaînerespiratoire mitochondriale. Au laboratoire, nous focalisons notre intérêt sur les mitochondriopathies liées à undéfaut de stabilité de l’ADN mitochondrial (ADNmt), qui se traduit par des délétions multiples et/ou unedéplétion (diminution du nombre de copies). Ces pathologies sont caractérisées par une importantehétérogénéité clinique et génétique et sont secondaires à des mutations dans des gènes nucléaires codantpour des protéines impliquées dans le maintien de l’ADNmt. De nos jours, la recherche des gènesresponsables d’instabilité de l’ADNmt s’avère négative chez plus de 70% des malades, d’où un grand intérêtpour améliorer les techniques d’identification des mutations et la recherche de nouveaux gènes impliquésdans ces pathologies / Non communiqué

Mitochondrial DNA (deoxyribo nucleic acid) is typically used in forensic casework when small quantities of high molecular weight quality DNA is not expected to be present thus negating the chances of obtaining usable nuclear DNA. Typical samples that utilized mitochondrial DNA analysis are: hair, bones, teeth, ancient remains (samples or remains that are at least 100 years old) or very old samples (samples that are less than 100 but greater than 10 years old). The current method used to evaluate mitochondrial DNA is Sanger sequencing. Although robust, it is also time consuming and labor intensive, on the other hand pyrosequencing is a nonelectrophoretic, rapid, reliable, and sensitive sequencing method which can be easily automated. Therefore pyrosequencing could enable the widespread use of mitochondrial DNA in forensic casework and reduce the amount of time spent on each sample without compromising quality. The aim of this study is to evaluate the efficacy of pyrosequencing for forensic DNA applications, in particular mitochondrial DNA. Two dispensation orders, cyclic and directed, were examined to determine if there is any effect on the sequence generated. The accuracy of pyrosequencing was evaluated by sequencing samples of known sequence provided by the FBI. The sensitivity of pyrosequencing was evaluated by sequencing samples at different DNA concentrations and inputs. Experiments were conducted to determine the ability of pyrosequencing to detect mixtures and heteroplasmy. Additionally, the ability of pyrosequencing to sequence damaged/degraded DNA was evaluated using blood, semen, and saliva samples that were subjected to three different environmental conditions. A blind study will be conducted to confirm the accuracy of pyrosequencing. Finally, a comparison study will be conducted in which pyrosequencing will be compared to Sanger sequencing.

No / The DNA content of hair [1.] and [2.] is typically low compared to other tissues, as hair cells undergo dehydration and catabolic breakdown of nucleic acids and organelles during keratinisation [3]. As a consequence, ancient hair specimens have not been widely used as a source of ancient DNA. However, mitochondrial DNA (mtDNA) has been extracted from degraded and old hair samples, including burnt specimens [4], 100-year-old Native American samples [5], and wool from a 9,400 year old Bighorn sheep [6]. We have investigated the potential of hair as an aDNA source by analyzing DNA survival in 12 samples which range from 60 to >64,800 years of age and their susceptibility to contamination with modern DNA. mtDNA was successfully amplified, cloned, and sequenced from 10 of the 12 hair samples following decontamination procedures (Table 1). DNA was quantified using Quantitative Real-Time PCR in a subset of the samples (Table 1). The survival of high copy numbers of 16S DNA from the 3,000 year-old Pazyryk horse hairs is consistent with the observation that DNA survives longer at sub-zero temperatures [7]. Of greater surprise was the persistence of high numbers of 16S and Control Region DNA molecules in hairs sampled from a bison mummy 14C dated to >64,800 years. This result was independently replicated and extends the time frame from which authentic DNA has been retrieved from hair by at least seven-fold, placing it on a par with the oldest authentic DNA retrieved from bones and teeth [8]. No nuclear DNA could be amplified from the bison hair, consistent with observations of modern hair samples [1.] and 9. M.R. Wilson, D. Polanskey, J. Butler, J.A. DiZinno, J. Replogle and B. Budowle, Extraction, PCR amplification and sequencing of mitochondrial DNA from human hair shafts, Biotechniques 18 (1995), pp. 662¿669.[9.]. It is probably significant that the bison hairs are exceedingly well preserved ¿ the atomic carbon to nitrogen ratio (3.47) is similar to modern mammal hair [10.] and [11.] and histological analysis of the specimen demonstrates the only structural modifications to be slight cuticular loss and adherent deposits (Supplemental data).

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The Yellow Warbler (Dendroica petechia) is a highly polytypic bird species with a vast breeding range in the Americas. To assess the level of population structuring within the northern part of its range, I surveyed the nucleotide variation present in a 344bp segment of the mitochondrial DNA (mtDNA) control region I (CR-I) from 155 breeding individuals. These birds were caught at seven locations in Canada and Alaska. Fifty-nine haplotypes were observed in this sample, with pairwise distances between haplotypes ranging from 0.29 to 4.35%. The number of nucleotide sites with multiple hits indicates a high rate of evolution in this region. A homologue to the CR-I was also identified and likely originated from a paralogous duplication event, as suggested by the comparison of sequences from the two regions. Significant population structuring in Yellow Warblers across North America was revealed by analyses of nucleotide diversity and molecular variance, which demonstrated the existence of a major subdivision between eastern (Manitoba to Newfoundland) and western (Alaska and British Columbia) warbler populations. This finding provides evidence for very low levels of gene flow between these two groups. Fifteen out of 21 pairs of populations differ significantly in their genetic composition, indicating further structuring at a smaller geographic scale. Within the eastern group of populations, both the high mutation rate of the CR-1 and an isolation-by-distance process seem to be responsible for differences between locations. Phylogenetic analyses indicate that western birds form a monophyletic group whereas eastern birds are paraphyletic with respect to the western ones. However this conclusion remains hypothetical because of a lack of statistical support for the monophyly of western haplotypes. Nevertheless, this situation is consistent with a historical splitting of warbler populations by a vicariant event, possibly of Pleistocene origin, and provides intraspecific support for vicariance as a mechanism leading to isolation and speciation of western warbler taxa, as hypothesized by Mengel (1964). However, other scenarios, such as a founder event in the west from an eastern stock, cannot be excluded, although they are less likely based on mtDNA data. The absence of phylogeographic structure in the East suggests a recent expansion of Yellow Warbler populations from a restricted geographic range. These findings demonstrate that populations of continentally distributed North American passerine species can show high level of population structuring when assayed with an hypervariable molecular marker such as the mtDNA control region I. / Thesis / Master of Science (MSc)

Mitochondria are intracellular organelles responsible for oxidative phosphorylation. They contain their own DNA which encodes some components involved in oxidative phosphorylation. Mitochondrial DNA is very susceptible to mutations. Mitochondrial abnormalities have been observed in several disorders of muscle and brain. Alzheimer's disease is a form of dementia characterized by the formation of numerous neuritic plaques and neurofibrillary tangles. There is evidence suggesting a possible role for mitochondrial abnormalities in Alzheimer's disease. The goal of this project was to determine if there were quantitative changes in mitochondrial DNA content in large neurons from Alzheimer's disease patients, compared to age-matched control patients. The relative mitochondrial DNA content per unit area was assessed in brain sections from Alzheimer's disease subjects and age-matched control subjects using in situ hybridization to mitochondrial DNA. The results were not conclusive due to technical concerns with the in situ hybridization technique which are discussed. / Thesis / Master of Science (MS)

Dilated cardiomyopathy (DCM), a disease of the myocardium, causes morbidity and premature death in humans and other domestic animals including turkeys. Though DCM results from many different factors including those that are unknown or idiopathic, genetic factor is a major cause of idiopathic DCM. In this study, I assessed the molecular basis of toxin-induced DCM in turkeys by evaluating the association and effect of mutations in candidate genes in the nucleus and mitochondria on the incidence and severity of this disease. Echocardiographic measurements at 3 weeks of age showed that birds on furazolidone-containing diet exhibited a significant DCM phenotype (increased left ventricular end diastolic dimension and left ventricular end systolic dimension) with a marked decrease in the left ventricular shortening fraction. Pathological phenotype confirmed the dilated heart with extended cell necrosis. Two mutations, both in NADH dehydrogenase genes, were found to be associated with DCM. Real-time RT-PCR quantification indicated that mRNA expression of alpha cardiac actin gene (ACTC) were significantly different between control and treatment birds. While ACTC expression increased, though moderately, in control birds from week 1 to 3, it decreased significantly in treatment birds. These findings suggest that the mitochondrial DNA variation and ACTC expression may be associated with the turkey's response to toxin. Therefore, further research is needed to investigate the molecular mechanism of toxin-induced DCM in the turkey. / Master of Science

Thesis (Ph. D.)--University of Tartu, 2004. / Includes reprint of 5 previously published articles. Includes bibliographical references.