Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Sex influences the severity and evolution of various inflammatory conditions. Women exhibit better clinical courses and increased survival compared to men in acute inflammatory processes, yet worse prognosis in several chronic inflammatory diseases, probably due to the higher inflammation observed in females. This higher inflammation in females may contribute to better pathogen clearance during the early inflammatory response, but also to enhanced tissue damage during prolonged inflammatory response. Many X-linked genes are involved in the immune response and the mechanisms underlying these sex-dependent differences are multiple and probably involve both hormonal and genetic factors. To evaluate the sex differences in the immune response and the role of the X chromosome relatively to the sex hormones, we studied acute inflammatory response in prepubertal children, whose sex hormones levels are very low, as well as women at different phases of the menstrual cycle and subjects with various X/Y sex chromosome ratios. In children with severe sepsis, we observed, in vivo, higher inflammation and lower pH, in girls compared to boys. In vitro stimulation of certain immune functions depending on X-linked genes showed specific profiles of inflammatory cytokine production and leucocyte migration markers expression in males and subjects carrying only one X chromosome but phenotypically females (Turner patients), compared to females and subjects carrying two X chromosomes but phenotypically males (Klinefelter patients), in favor of a role for the X chromosome. Our work highlighted important sex differences in terms of in vivo acute inflammatory response and in vitro activation of certain X-linked genes. These differences cannot be explained by the sex steroid levels, thus supporting the hypothesis of a preponderant role of sex chromosomes in inflammatory response. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished

Selfish genes that bias their own transmission during gametogenesis can spread rapidly in populations, even if they contribute negatively to the fitness of their host. Driving X chromosomes provide a clear example of this type of selfish propagation. These chromosomes, which are found in a broad range of taxa including plants, mammals, and insects, can have important evolutionary and ecological consequences. In this thesis, I report a new case of X chromosome drive (X drive) in a widespread woodland fly, Drosophila testacea. I show that males carrying the driving X (SR males) sire 80-100% female offspring, and that the majority of sons produced by SR males are sterile and appear to lack a Y chromosome. This suggests that meiotic defects involving the Y chromosome may underlie X drive in this species. Abnormalities in sperm cysts of SR males reflect that some spermatids are failing to develop properly, confirming that drive is acting during gametogenesis. Further, I show that SR males possess a diagnostic X chromosome haplotype that is perfectly associated with the sex ratio distortion phenotype. Phylogenetic analysis of X-linked sequences from D. testacea and related species strongly suggests that the driving X arose prior to the split of D. testacea and its sister species, D. neotestacea and D. orientacea. Suppressed recombination between the XST and XSR due to inversions on the XSR likely explains their disparate evolutionary histories. By screening wild-caught flies using progeny sex ratios and a diagnostic X-linked marker, I demonstrate that the driving X is present in wild populations at a frequency of ~10% and that autosomal suppressors of drive are segregating in the same population. Both SR males and homozygous females for the driving X have reduced fertility, which helps to explain the persistence of the driving X over evolutionary timescales. The testacea species group appears to be a hotspot for X drive, and D. testacea is a promising model to compare driving X chromosomes in closely related species, some of which may even be younger than the chromosomes themselves. / Graduate / 2019-04-16

Recently, long non-coding RNAs (lncRNAs) emerged as important regulators of many cellular functions. Many nuclear lncRNAs regulate the expression of geomically proximal or overlapping protein coding genes. Less clear is whether intergenic lncRNAs can regulate transcription by modulating chromatin at genomically distant loci in an RNA-dependent manner. This thesis investigated molecular functions of Dali, an intergenic central nervous system expressed lncRNA conserved in therian mammals. Dali is transcribed from a locus 50 kb downstream of the Pou3f3 transcription factor gene and performs both genomically local and distal RNA-dependent roles. Its depletion disrupts the differentiation of neuroblastoma cells. Locally, Dali regulates transcription of the Pou3f3 locus. Distally, it preferentially binds near to and regulates active promoters across the genome, including by physically associating with the POU3F3 transcription factor. Dali also interacts with the DNMT1 DNA methyltransferase in mouse and human and regulates CpG island-associated promoters by modulating their DNA methylation levels in trans. This work is the first to demonstrate that a lncRNA can regulate the DNA methylation of CpG island-associated promoters in trans and one of the first large scale studies to identify direct transcriptional targets of a lncRNA genome-wide. It also provides a more detailed molecular dissection of the extended Pou3f3 locus and a framework for the prioritisation and comprehensive functional characterisation of nuclear lncRNAs.

Le raccourcissement télomérique est la source majeure de l'instabilité chromosomique (CIN) au cours de la progression tumorale. Nous avons montré que les cellules humaines embryonnaires de rein (cellules HEK) ayant traversé une période de CIN subissent des vastes changements dans l'expression des microARNs, ce qui induit une transition épithélio-mésenchymateuse (TEM), un processus permettant aux cellules cancéreuses épithéliales migrer et envahir de nouveaux tissus et former des métastases. Notre travail a aussi suggéré que les cellules ayant subi une TEM étaient capables de former des tumeurs dans un microenvironnement sénescent. De surcroît, cette évolution dans la capacité tumorale était associée à une dérégulation supplémentaire des microARNs et à l'acquisition des propriétés des cellules souches. Afin d'étudier comment ce potentiel est mis en place au cours de l'instabilité chromosomique et au contact avec le microenvironnement sénescent, nous avons modulé les niveaux d'expression de miR-145 et avons démontré que la répression de miR-145 était nécessaire pour le développement des caractéristiques des cellules souches. Afin de mieux comprendre l'impact de CIN sur le programme génétique des cellules épithéliales, nous avons utilisé des approches de haut débit et avons caractérisé les changements des paysages chromatiniens et leur mise en place dans les cellules ayant traversé une période de CIN. Nos résultats révèlent pour la première fois que l'instabilité télomérique modifie profondément la distribution des marques d'histones en conduisant aux changements d'expression des gènes et au processus de transformation des cellules épithéliales pré-tumorales. / Telomere shortening is a major source of chromosome instability (CIN) at early stages during carcinogenesis. However, the mechanisms through which telomere-driven CIN (T-CIN) contributes to the acquisition of tumor phenotypes remain uncharacterized. We have shown that human epithelial kidney (HEK) cells undergo massive microRNA deregulation upon CIN, in particular a miR-200-dependent epithelial-mesenchymal transition (EMT), which is thought to enable epithelial cancer cells to migrate and invade other tissues to form metastases. Our work also indicated that CIN+ cells that underwent EMT were able to form tumors in a senescent microenvironment. Notably, this progression in tumor capacity was associated with further microRNA deregulation and the manifestation of enhanced stem-like properties. To investigate how stem-like properties are acquired in CIN+ cells in the contact with senescent microenvironment we adapted knockdown and overexpression approaches to modulate miR-145 expression, and demonstrated that enhanced stem-like properties depended on miR-145 repression. To fully apprehend the impact of CIN on the genetic program of epithelial cells, we used an unbiased approach to characterize the chromatin state of HEK CIN+ cells and uncover genome wide redistributions that were in direct correlation with gene expression changes. Our results reveal for the first time that T-CIN profoundly modifies the chromatin landscape genome-wide thereby fueling the transformation process of pre-tumor epithelial cells.

>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)

Epigenetic differences between maternally inherited and paternally inherited chromosomes, such as CpG methylation, render the maternal and paternal genome functionally inequivalent, a phenomenon called genomic imprinting. This functional inequivalence is exemplified with imprinted genes, whose expression is parent-of-origin specific. The dosage of imprinted gene expression is disrupted in cells with uniparental disomy (UPD), which is an unequal parental contribution to the genome. I have derived mouse embryonic stem (ES) cell sub-lines with maternal UPD (mUPD) for mouse chromosome 6 (MMU6) to characterize regulation and maintenance of imprinted gene expression. The main finding from this study is that maintenance of imprinting in mitotic UPD is extremely variable. Imprint maintenance was shown to vary from gene to gene, and to vary between ES cell lines depending on the mechanism of loss of heterozygosity (LOH) in that cell line. Certain genes analyzed, such as Peg10, Sgce, Peg1, and Mit1 showed abnormal expression in ES cell lines for which they were mUPD. These abnormal expression levels are similar to that observed in ES cells with meiotically-derived full genome mUPD (parthenogenetic ES cells). Imprinted CpG methylation at the Peg1 promoter was found to be abnormal in all sub-lines with mUPD for Peg1. Two cell sub-lines which incurred LOH through mitotic recombination showed hypermethylation of Peg1, consistent with the presence of two maternal alleles. Surprisingly, a cell sub-line which incurred LOH through full chromosome duplication/loss showed hypomethylation of Peg1. The levels of methylation observed in these sub-lines correlates with expression, as the first two sub-lines showed a near-consistent reduction of Peg1, while the latter showed Peg1 levels close to wild-type. Altogether these results suggest that certain imprinted genes, like Peg1 and Peg10, have stricter imprinting maintenance, and as a result show abnormal expression in UPD. This strict imprint maintenance is disrupted, however, in UPD incurred through full chromosome duplication/loss, possibly because of the trisomic intermediate stage which occurs in this mechanism. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

The Saccharomyces cerevisiae iron-responsive transcription factor, Aft1p, has a well established role in regulating iron homeostasis through the transcriptional induction of iron-regulon genes. However, recent studies have implicated Aft1p in other cellular processes independent of iron-regulation such as chromosome stability. In addition, chromosome spreads and two-hybrid data suggest that Aft1p interacts with and co-localizes with kinetochore proteins, however the cellular implications of this have not been established. Here, we demonstrate that Aft1p associates with the kinetochore complex through Iml3p. Furthermore, we show that Aft1p, like Iml3p, is required for the increased association of cohesin with the pericentromere and that aft1Δ cells display sister chromatid cohesion defects in both mitosis and meiosis. Our work defines a new role for Aft1p in the sister chromatid cohesion pathway.

Polycyclic aromatic hydrocarbons (PAHs) are produced via the incomplete combustion of organic matter. They are ubiquitously present in the environment, and human exposures typically involve complex PAH mixtures in complex matrices (e.g., soil, urban air). Many PAHs are genotoxic carcinogens; exposures can augment cancer risk and reliable risk assessment of PAH mixtures is a regulatory concern. There is a paucity of in vivo genotoxicity information for most PAHs and PAH mixtures. Risk assessment of PAH mixtures assumes dose addition (i.e., additive, incremental contributions from each PAH); however, there is a lack of evidence to support this assumption. This thesis assessed the in vivo genotoxicity of 9 PAHs and 6 PAH mixtures following sub-chronic oral exposure of transgenic Muta™Mouse (i.e., adduct and lacZ mutant frequency across 5 tissues). The results revealed that PAHs and PAH mixtures induce significant levels of genetic damage; the mixtures induced very high levels of damage and mutations. Differences in the nature and magnitude of the effects in individual tissues appear to be related to the processes that govern PAH metabolism and the processing of genetic damage (e.g., repair and translesion synthesis). Scrutiny of the dose addition assumption revealed more-than-additive effects in tissues proximal to the exposure route (i.e., intestine, liver), but less-than-additive effects in distal tissues (i.e., bone marrow); however, discrepancies between the experimentally-observed and predicted responses were typically small (i.e., within 5-fold). Comparisons of cross-tissue patterns in adduct and mutant frequencies revealed that the frequency of the former is generally inversely related to that of the latter. This appears to be related to the experimental design, and the influence of repair and replication on adduct and mutant frequency. The BMD approach was employed to estimate genotoxic (i.e., adduct) potency and mutagenic (i.e., lacZ mutant) potency for all agent-tissue combinations. The results demonstrate that the mutagenic potency of PAHs and PAH mixtures is empirically related to genotoxic potency; moreover, that there is cross-tissue and cross-compound congruence in the processing of PAH-induced damage. The results obtained significantly advance existing knowledge regarding the genotoxic hazards of PAHs and PAH mixtures; moreover, the empirical relationships between genetic toxicity endpoints.

This thesis describes the alignment of cloned DNA with the genetic map, and the identification of coding elements within the aligned DNA. The region of study was the dpy-5 unc-29 interval from chromosome I of the nematode Caenorhabditis elegans, with an emphasis on the region surrounding the gene dpy-14. The objectives of this thesis were: 1) to align the physical and genetic maps .of the region; 2) to identify and characterize the coding elements in the vicinity of dpy-14; and 3) to cross-hybridize the identified C. elegans coding elements to mammalian DNA in an attempt to identify evolutionarily conserved genes. Six polymorphisms from the dpy-5 unc-29 interval were mapped with respect to the free duplication sDp2. The polymorphisms hP5, sPl, and hP9 were found to.be inside the region spanned by sDp2 while the polymorphisms hP4, hP6, and hP7 were found to be outside this interval. In addition, these six polymorphisms were mapped with respect to visible markers from the dpy-5 unc-29 interval. These analyses demonstrated the genetic order to be dpy-5, hP5, unc-37, (dpy-14, sPl), hP9, unc-13, hP7, (hP4, hP6), unc-29. Lambda phage containing the hP5, sPl, and hP6 sites identified and anchored cosmid contigs to the genetic map. The interval from the left of hP5 to the right of unc-13 is contained in a single contig of approximately 1400 Kb. The amount of DNA in Kb across the hP5 and unc-13 interval was compared to the genetic distance in map units. The DNA per map unit value was found to vary in this interval with the greatest value found between hP9 and unc-13. Seven cosmids representing 173 Kb of N2 genomic DNA near the gene dpy-14 were isolated. Using cross-species hybridization to C. briggsae DNA ten conserved regions were identified within these seven cosmids. The ten conserved fragments were used to identify seven cDNAs, six of which also identified RNAs on Northern blots. The relative abundance of the isolated cDNAs varied 250 fold with the most abundant having a level similar to that found for actin. The first comprehensive survey of mammalian homologies in a contiguous set of ten coding regions found three coding elements to be. conserved. One was demonstrated to be the small nuclear RNA gene U1. Another shared sequence similarities with the gene S-adenosyl-L-homocysteine hydrolase. No detectable homologies were identified with the third. A formaldehyde-induced mutation that failed to complement the genes unc-37, unc-87, dpy-14, let-83 and let-86 was isolated. This mutation appeared to be the result of a DNA rearrangement which had one breakpoint within the cosmid C14A12. Using the conserved elements identified in this thesis together with the rearrangements and mapped genes from the region, a detailed physical and genetic map in the vicinity of dpy-14 was constructed. / Medicine, Faculty of / Medical Genetics, Department of / Graduate