Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by two major pathological hallmarks, amyloid plaques and neurofibrillary tangles. The accumulation of amyloid-β protein (Aβ) is an early event associated with synaptic and mitochondrial damage in AD. Therefore, molecular pathways underlying the neurotoxicity and generation of Aβ represent promising therapeutic targets for AD. Recent studies have shown that actin severing protein, Cofilin plays an important role in synaptic remodeling, mitochondrial dysfunction, and AD pathogenesis. However, whether Cofilin is an essential component of AD pathogenesis and how Aβ induced neurotoxicity impinges its signals to Cofilin are unclear. In my dissertation studies, we found Aβ oligomers bind with intermediate activation conformers of β1-integrin to induce the loss of surface β1-integrin and activation of Cofilin via Slingshot homology-1 (SSH1) activation. Specifically, conditional loss of β1-integrin prevented Aβ induced Cofilin activation, and allosteric modulation or activation of β1-integrin significantly reduced Aβ binding to neurons and mitigated Aβ42-induced reactive oxygen species (ROS) generation, mitochondrial dysfunction, synaptic proteins depletion, and apoptosis. Furthermore, we found that SSH1 reduction, which mitigated Cofilin activation, prevented Aβ-induced mitochondrial Cofilin translocation and apoptosis, while AD brain mitochondria contained significantly increased activated/oxidized Cofilin. In mechanistic support in vivo, we demonstrated that APP transgenic mice brains contain decreased SSH/Cofilin and SSH1/14-3-3 complexes which indicates that SSH-Cofilin activation occurred by releasing of SSH from 14-3-3. We also showed that genetic reduction in Cofilin rescues APP/Aβ-induced synaptic protein loss and gliosis, as well as impairments in synaptic plasticity and contextual memory in vivo. Our lab previously found that overexpression of the scaffolding protein RanBP9 increases Aβ production in cell lines and in transgenic mice, while promoting Cofilin activation and mitochondrial dysfunction. However, how endogenous RanBP9 activates cofilin and whether endogenous RanBP9 accelerates Aβ-induced deficits in synaptic plasticity, cofilin-dependent pathology, and cognitive impairments were unknown. In my dissertation studies, we found that endogenous RanBP9 positively regulates SSH1 levels and mediates A-induced translocation of Cofilin to mitochondria. Moreover, we demonstrated that endogenous RanBP9 mediates A-induced formation of Cofilin-actin rods in primary neurons. Endogenous level of RanBP9 was also required for Aβ-induced collapse of growth cones in immature neurons and depletion of synaptic proteins in mature neurons. In vivo, we also found APP transgenic mice exhibit significantly increased endogenous RanBP9 levels and that genetic reduction in RanBP9 rescued APP/Aβ-induced synaptic protein loss, gliosis, synaptic plasticity impairments, and contextual memory deficits. These findings indicated that endogenous RanBP9 not only promotes Aβ production but also meditate Aβ induced neurotoxicity via positively regulating SSH1. Taken together, these novel findings implicate essential involvement of β1-integrin–SSH1/RanBP9–Cofilin pathway in mitochondrial and synaptic dysfunction in AD pathogenesis.

Mitochondria are essential for the viability of mammalian cells and provide a compartment for specific chemical reactions. Cellular respiration -- the main mitochondrial function -- is tightly connected with ROS production: the mitochondrial electron transport chain complexes I and III are the main ROS sources in mammalian cells. It has been reported that complex I and complex III activities are essential for cell cycle, apoptosis and stem cell differentiation (Spitkovsky et al., 2004; Varum et al., 2009; Lee et al., 2011; Ma et al., 2011; Tormos et al., 2012). In our work, we aimed to investigate the role of mitochondrial electron transport chain activity in the regulation of the differentiation potential and to unravel signaling pathways that could participate in this regulation. As a model, we used the P19 pluripotent stem cell line that can be easily differentiated into trophoblasts, expressing intermediate filaments cytokeratin 8/18, and neurons, which express cytoskeleton protein beta-III-tubulin. We first showed that both trophoblast and neural differentiation of P19 cells were accompanied by activation of cellular respiration. The analysis of respiratory chain complexes and supercomplexes, however, showed that undifferentiated P19 cells, as well as their differentiated derivatives did not differ in their respiratory machinery, including functional respirasomes. While undifferentiated cells did not use respiration as the main energy source, cellular respiration was activated during differentiation, indicating that oxidative metabolism was important for efficient differentiation. To investigate the potential role of mitochondrial electron transport chain activity we monitored the influence of a disrupted electron flow on the differentiation of P19 cells. We found that the activity of complex I and complex III influenced the differentiation potential of the pluripotent P19 cell line: the presence of complex I and complex III inhibitors rotenone, antimycin A, or myxothiazol increased the amount of cytokeratin 8/18+ cells during trophoblast differentiation, but almost completely prevented the formation of neuron-like beta-III-tubulin+ cells during neuron differentiation. Moreover, a low oxygen level (1 % O2 vs 21 % O2 in atmosphere) - the final electron acceptor - had the same effect on differentiation. These data suggest that mitochondrial electron transport chain activity contributes to the regulation of differentiation. The presence of complex I and complex III inhibitors, as well as oxygen scarcity, increase ROS production. We suggested that increased ROS level could explain the observed effects. By visualizing mitochondrial superoxide production with a specific dye – MitoSox - we confirmed that rotenone, antimycin A, myxothiazol, as well as low oxygen conditions, increased the superoxide level. These results suggest that the observed changes of the differentiation potential of P19 cells are associated with ROS production. To prove this idea, we differentiated P19 cells in presence of paraquat – a known ROS inducer. In line with our hypothesis paraquat promoted trophoblast differentiation. The received results suggest that the mitochondrial electron transport chain activity regulates differentiation through the ROS level. ROS are secondary messengers that participate in numerous processes including cell proliferation and differentiation. We aimed to predict the signal pathway that connects ROS level in stem cells and their differentiation potential. For this purpose, we performed a microarray analysis and compared the gene expression profiles of cells grown under hypoxia or in the presence of the complex III inhibitor myxothiazol with untreated control cells. The expression analysis revealed p53 as a transcriptional factor that impacts the differentiation potential in treated cells. p53 is a known redox-sensing molecule (Bigarella et al., 2014) that influences the differentiation potential through cell cycle control (Maimets et al., 2008). This observation is in line with our results and suggests that p53 may regulate the differentiation potential of P19 cells. We are planning to investigate the role of p53 signaling in the regulation of cell cycle and differentiation potential of P19 cell line.

The basking shark, Cetorhinus maximus, has historically been a target of international fisheries, leading to well-documented declines in parts of its global distribution. Currently, the basking shark is listed as globally ‘Vulnerable’ and regionally ‘Endangered’ (North Pacific and Northeast Atlantic) on the IUCN Red List of Threatened Species, rendering the species an international conservation priority. Here, we assessed the global matrilineal genetic population structure and evolutionary history of the basking shark by completing the first whole mitochondrial genome sequence level survey of animals sampled from three globally widespread geographic regions: the western North Atlantic (n = 11), the eastern North Atlantic (n = 11), and within New Zealand territorial waters (n = 12). Despite the relatively large amount of sequence data assessed (~16,669 bp per individual), whole mitogenome analyses showed no evidence of population differentiation (ΦST = -0.047, P > 0.05) and very low nucleotide diversity (π = 0.0014 ± 0.000) across a global seascape. The absence of population structure across large distances and even between ocean basins is indicative of long-dispersal by this species, including an ability to cross known biogeographic barriers known to differentiate populations of other highly vagile pelagic fishes. Notably, evolutionary analyses of the mitogenome sequences revealed two globally sympatric but evolutionary divergent lineages, with a Bayesian framework estimated coalescence time of ~2.46 million years ago. Coalescent-based Bayesian skyline analysis uncovered subtle evidence of Pleistocene demographic flux for this species, including a potential decline in female effective population size. Thus, historical population changes may be responsible for the occurrence of the two highly divergent, yet sympatric lineages, as population declines may have resulted in the loss of intermediate haplotypes and resulted in an overall loss of genetic diversity. This work supports the recognition of basking sharks as a single matrilineal global population, and as such requires the application of a cooperative multiagency and international approach to fisheries management to conserve this highly vulnerable and ecologically unique species.

Abstract Neurological features are common in mitochondrial diseases because tissues depending upon oxidative phosphorylation bear the brunt of the pathogenesis. The 3243A>G mutation in the MTTL1 gene in mitochondrial DNA is regarded as the most frequent mitchondrial point mutation and classically presents with mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS). Myopathy and peripheral neuropathy have been documented in patients with mitochondrial diseases, but not properly characterised in patients with the 3243A>G mutation. We have previously determined the prevalence of patients with this mutation in a defined population in northern Finland. The clinical spectrum and molecular aspects of myopathy and peripheral neuropathy are analysed here in a population-based cohort of patients with 3243A>G. Fifty patients were examined neurologically in order to define the frequency of myopathy and its histological, ultrastructural and clinical features. The frequency and phenotypic variability of peripheral neuropathy were determined in 32 patients and muscle computed tomography findings recorded in 24 patients. Finally, variations in mutation heteroplasmy were analysed in 10 patients using single muscle fibre PCR analysis. The frequency of peripheral neuropathy was 22% (95% confidence interval (CI), 9–40%) and that of clinical myopathy 50% (95% CI, 36–64%). Moderate limb weakness was the most common myopathic feature, but mild weakness and external ophthalmoplegia were also present. CT scans revealed myopathic changes in 54% of the patients (95% CI, 33–76%), most frequently in the pelvic muscles. The incidence of myopathy was highest in the fifth decade of life, and higher age and male gender increased the risk of neuropathy. Muscle histology was abnormal in 72% of the cases examined (95% CI, 55–86%). The presence of intramitochondrial crystals and COX-negative fibres and variations in the size and shape of mitochondria were more common in the muscle of myopathic patients. Single muscle fibre analysis pointed to a correlation between the mutation load in ragged red fibres and in adjacent histologically normal fibres, and the proportion of 3243A>G in histologically normal muscle fibres showed a pattern compatible with random genetic drift. The results indicate that myopathy and peripheral neuropathy are common in patients with the 3243A>G and that myopathy is highly variable in presentation. Segregation of 3243A>G in individual muscle fibres showed a complex process with random and non-random elements.

AIF fait partie des protéines mitochondriales inductrices de mort mais possède aussi un rôle vital nécessaire à la respiration cellulaire. Les recherches menées lors de cette thèse portent sur ces deux fonctions. D'une part, j'ai approfondi l'étude de la nécrose régulée induite par un agent alkylant de l'ADN. J'ai découvert l'importance de RIP1 dans cette voie de mort cellulaire et ainsi conduit à la définir comme nécroptose. J'ai aussi mis en évidence le rôle de BID, BH3-only de la famille BCL-2, dans la libération d'AIF des mitochondries. J'ai montré que les protéases calpaïnes clivaient BID permettant à sa forme tronquée de relocaliser aux mitochondries et d'y activer le facteur pro-apoptotique BAX. Cette étude contribue à replacer le rôle des BH3-only dans des voies de mort cellulaire au delà de l'apoptose. D'autre part, j'ai étudié le rôle d'AIF dans l'hématopoïèse grâce à un modèle murin invalidé pour AIF dans ce système. J'ai observé un blocage de différenciation thymique et le développement d'une pancytopénie sévère. J'ai démontré que cette dernière est associée à la perte des cellules souches hématopoïétiques dont j'ai testé les capacités ex vivo et in vivo. Pour comprendre les raisons de ce défaut, j'ai caractérisé les conséquences associées à la perte d'AIF : perte du complexe I de la chaine respiratoire, diminution d'activité de phosphorylation oxydative, diminution de la production d'ATP, augmentation des espèces réactives de l'oxygène. Cette deuxième étude démontre l'importance d'une phosphorylation oxydative fonctionnelle et de mitochondries saines pour une hématopoïèse normale et particulièrement pour le maintien des cellules souches hématopoïétiques. / AIF is one of the cell death effectors released from mitochondria but it also possess a vital role by regulating the cellular respiration. Throughout this thesis work, I have focused my studies on these two functions. On one hand, I have performed a deeper characterization of the DNA alkylating agent induced regulated necrosis. I have identified RIP1 as a crucial determinant of this cell death pathway, hence linking it to necroptosis. I have also highlighted the role of BID, a BH3-only member of the BCL-2 family, in the mitochondrial release of AIF. I have shown that calpains proteases cleave BID into tBID which relocalize to mitochondria where it helps activating the pro-apoptotic factor BAX. This study contributes to reconsider the role of BH3-only proteins in cell death pathways beyond apoptosis. On the other hand, I have studied AIF role in hematopoiesis thanks to a mouse model with hematopoietic lineage-specific deletion of AIF. I have observed a block in T-cell development and the rapid development of severe pancytopenia. I have demonstrated that this pancytopenia is associated with the loss of hematopoietic stem cells whom capacities were tested both ex vivo and in vivo. In order to understand the underlying determinants of these defects, I have characterized the cellular consequences related to AIF deletion : loss of the respiratory chain complex I, decrease of the oxidative phosphorylation capacity, decreased levels of ATP, increased levels of reactive oxygen species. This second study reveals the importance of a proper oxidative phosphorylation system combined with healthy mitochondria for a normal hematopoiesis and hematopoietic stem cells maintenance.

Abstract The 3243A>G mutation in mitochondrial DNA (mtDNA), the most common cause of the syndrome of mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes, is also associated with many other phenotypes such as hearing loss, diabetes mellitus, epilepsy, cognitive decline, myopathy and cardiomyopathy. The prevalence of the mutation has been shown to be 16.3/100 000 adults in Northern Finland. The present study was performed to estimate the frequency and progression of cardiac abnormalities and to examine causes of death in patients with 3243A>G. Left ventricular hypertrophy (LVH) was found in echocardiography in 56% of patients with 3243A>G and in 15% of age and sex-matched controls. The median thickness of the diastolic interventricular septum or posterior wall was 14 mm in the patients with LVH. The prevalence of LVH determined by echocardiography increased from 40% to 56% in 25 patients with 3243A>G during three years of follow-up, this trend being especially marked among the diabetic patients. The ultra-low-frequency (ULF) and very-low-frequency (VLF) components of the spectral analysis of heart rate variability (HRV) were lower among the patients with 3243A>G than in matched controls (p = 0.02 in ULF and p = 0.04 in VLF), and the short-term fractal scaling exponent in detrended fluctuation analysis of HRV was lower in the patients with 3243A>G (1.16 ± 0.18 vs. 1.28 ± 0.13) (p < 0.01). Survival analysis of a birth cohort from pedigrees with 3243A>G revealed excess mortality before the age of 50 years. Neurological and cardiovascular diseases accounted for 32% of all the underlying causes of death in families with 3243A>G. Death was sudden and unexpected in 31% of cases in which 3243A>G was considered to be involved in the cause of death. The results show that cardiac abnormalities are frequent and progressive in patients with the 3243A>G mtDNA mutation and that cardiac autonomic regulation is disturbed. Patients with the 3243A>G mutation and their first degree maternal relatives died younger than was presupposed by their life expectancy at birth or at 15 years. The most common causes of death were neuropsychiatric and cardiovascular diseases.

Abstract The impact of hand-rearing on the morphology and physiology of captive and wild grey partridges (Perdix perdix) and capercaillies (Tetrao urogallus) was studied in three feeding trials conducted under laboratory conditions, and two comparative studies between wild and captive birds. Finally, wild and hand-reared grey partridges from several localities in Europe were sampled and the control region 1 of mitochondrial DNA was sequenced to reveal genetic variation between populations, as well as to compare wild and captive stocks. Wild capercaillies had heavier pectoral muscles, hearts, livers and gizzards, longer small intestines than hand-reared ones, and a higher cytochrome-c oxidase activity in muscle and heart. Invertebrates were essential to the growth, primary and temperature regulation development in grey partridge chicks. Fish was not sufficient to replace invertebrates in the diet. A change in diet from commercial to natural decreased the assimilation efficiency in the grey partridge. It also increased the mass of gizzard reflecting the need for greater grinding ability. Of hepatic P450 enzymes used in this study 7-ethoxyresorufin-0-deethylase and 7-pentoxyresorufin-0-deethylase differed between wild and hand-reared birds. Coumarin-7-hydroxylase activity was higher in grey partridges than capercaillies. Diet differences may have caused these differences. Quebracho tannin added to the diet lowered nitrogen concentration in caecal feces, and elevated the level of excreted tannin. Otherwise its effects were slight. Mitochondrial control region revealed 14 variable sites between two main lineages detected. Nucleotide and haplotype diversities varied greatly between populations. The markedly deep divergence between the two lineages indicated most probably post-glacial recolonisations from geographically isolated refuges. In Finland, wild birds represented the eastern lineage, while the farmstock represented the western lineage. Surprisingly little trace, contrary to expectations, from the large-scale releasing of imported partridges could be seen in the European populations.

Eastern part of the African Sahel, connecting sub-Saharan Africa with North and East Africa, play an important role as a bidirectional corridor for vertically and horizontally migrations of populations. It is the strategic region to study human genetic diversity due to the presence of ethnically, linguistically, culturally and geographically diversity. This work is focused on the analysis of HVS-I and HVS-II segments of mtDNA. The work provides new information about genetic structure and migration activity of this region by analysis twelve populations belonging to three African linguistic families and different subsistent strategies. Analysis of mtDNA revealed the higher diversity of the populations of east Sudan and Horn of Africa, which is connected with the spreading of populations along the Nile River. It seems, that in this region linguistic factors have bigger impact on genetic diversity then the geografic ones. The opposite situation is observed in populations of Chad, where populations with similiar geografic location and different linguistic affilation revealed low genetic differentiation. The intra-population analysis shows the significant influence of genetic drift on the pastoralists living on the Red Sea Coast - Beja and Rashaida. In Beja is probably due to decrease of size of...

Advancements in molecular sequencing have revealed unexpected cryptic genetic diversity and contrasting evolutionary histories within genes and between genomes of many organisms; often in disagreement with recognized taxonomy. Incongruent patterns between the mitochondrial and nuclear evolutionary history can have several plausible explanations, but widespread systematic conflict inevitably challenges our conceptions of species boundaries when there is discordance between coevolving and coinherited genomes. It is unknown to what degree mitonuclear conflict drives the process of divergence, or how ubiquitous these patterns are across the tree of life. To understand the evolutionary relevance of intergenomic discordance we must identify the conflicting patterns that exist in natural systems by generating robust estimates of the underlying species history, quantify support for alternative hypotheses of lineage formation, and describe patterns of genetic variation present in robust nuclear genomic datasets. Empirically testing correlations between mitonuclear genomic conflict and reduced gene flow at the organism level will contribute toward a better understanding of lineage boundaries and how intergenomic interactions shape the process of divergence. Mitochondrial introgression has been inferred in many salamander systems with limited perspective from nuclear sequence data. Within dusky salamanders (Desmognathus), these patterns have been observed between morphologically and geographically disparate populations. I sequenced regions throughout the nuclear genome to reconstruct species trees, performed population-level analyses testing concordance between the mitochondrial, nuclear datasets, and nuclear genes with mitochondrial functions with the expectation that coevolutionary interactions among genomes are more likely to manifest in these regions. I also estimated migration rates between populations that may have experienced historical mitochondrial introgression to evaluate phylogeographic patterns. Using these data we definitively reject species models in which genetic boundaries are based solely on mitochondrial data, favoring geographic models instead. Furthermore, analyses soundly reject current taxonomic models based on morphological characteristics, suggesting there is greater lineage diversity than is currently recognized. I also used empirical assays of pre-zygotic reproductive mating behavior within and among populations containing diverse mitochondrial lineages to test metrics of reproductive isolation, and to determine if introgression shapes the evolution of complex traits directly influencing rates of divergence. These results may explain incongruent patterns observed between the mitochondrial and nuclear data as a function of inheritance and population dynamics rather than directly functioning to suppress nuclear gene flow. This research builds upon recent studies suggesting that speciation is a highly complex and often non-bifurcating process in which introgression can have a profound and lasting signature on the nuclear evolutionary history. Mechanisms responsible for divergence with gene flow challenge evolutionary biologists to reevaluate our notions and definitions of species boundaries to accommodate seemingly conflicted genomic patterns within and between genomes.

>Magister Scientiae - MSc / The primary objective of this Masters project was to investigate the maternal ancient substructure of the Griqua population in South Africa. Genetic ancestry was determined by investigating ancestry informative single nucleotide polymorphisms. These are located in the control region of the mitochondrial genome. The auxiliary aim was to test the validity of the UWC 10plex system in relation to a sample group of Griqua males. This short tandem repeat multiplex targets specific mutations confined to paternal lineages. The Khoi Khoi or Hottentots were the first inhabitants in the Cape. Indigenous Khoi Khoi female slaves had offspring with the European settlers in the 1800s which resulted in the Griqua population group. The incorporated European paternal ancestry is what set the Griqua apart from the native population groups at that time. Colonisation events from the mid-17th to 19th Century and the apartheid regime resulted in land dispossession of the native population and an extensively mixed gene pool in South Africa. One hundred and seventy six (N=176) male and female Griqua people were collectively sampled in Kokstad (2012), Vredendal (2012 and 2013) and at the Griqua National Conference in Ratelgat (2013). All 176 samples were analysed using mtDNA control region Sanger sequencing. The sample group (N=176) was separated based on birthplace (Origin sample group and post-colonial sample group). The origin sample group consists of individuals whose ancestors were not part of the Griqua Trek to Northern regions of South Africa and were less likely to be exposed to colonial influences. Mutations within the hypervariable segments of the mtDNA control region were used to infer haplogroups with geographic-specific population data. In this way one can plot the extent of ancient Khoisan (L0d) and Bantu influences (L1-L5) as well as the influence of East (M, A, B, E) and West (N, R, J, H) Eurasian haplogroups in the maternal ancestry of the Griqua population group. The origin sample group showed 91% African ancestry (76.8% L0d) while the post-colonial group had 78% African ancestry (60% L0d). The origin sample group had 2% East Eurasian and 7% West Eurasian ancestry, while the post-colonial group contained 20% Eurasian ancestry. There is greater admixture in the post-colonial group which can be attributed to the integration of surrounding populations during settlement periods in parts of the Northern Cape and KwaZulu-Natal. The UWC 10plex STR kit was tested to see if it could discriminate between male individuals of this admixed sample group (N=91 males). The markers for this multiplex were selected according to their ability to differentiate between individuals of African descent. It proved to be a viable Y chromosome short tandem repeat testing tool, displaying a statistically significant discrimination capacity value of 0.966 and only having 3 shared haplotypes in the sample group of 91 Griqua males. / National Research Foundation (NRF)