No description available.

Previous findings showed that mild chronic restraint stress causes motor impairments in rats. These behavioural impairments might be related to molecular changes in brain areas that regulate motor functions, such as the cerebellum. Little is known about the role of the cerebellum in stress-induced behavioural alteration. We hypothesized that alteration in animal behaviour after chronic restraint stress is due to brain-specific changes in miRNA and proteins encoding gene expression. Our results revealed that expression of three miRNAs and 39 mRNAs was changed significantly after two weeks of stress. Furthermore, we verified one putative target for one of the changed miRNAs and expression of four randomly selected genes. Changes in gene expression disappeared after two weeks of recovery from stress. These findings provide a novel insight into stress-related mechanisms of gene expression underlying altered behavioural performance. The observations bear implications for the prevention and treatment of stress-related disorders and disease. / xii, 109 leaves. : ill. ; 29 cm

Abiotic stresses are one of the major limiting factors of plant growth and thus crop productivity. Exposure to these stresses, including temperature and UV, cause physiological and epigenetic changes in plants. Such changes may be inherited in the progeny of stressed plants, and may change their ability to respond to stress. To understand the ability of plants to inherit an epigenetic stress memory as well as the physiological manifestations of such a memory, we propagated both stressed and control plants and compared the progeny under both normal and stressed conditions. In addition to wild-type plants we used Dicer-like mutants dcl2, dcl3 and dcl4, as Dicers have been linked to RNA-directed DNA methylation, a form of epigenetic memory. These studies revealed that leaf number decreases in the progeny of stressed plants, and bolting occurs earlier in the progeny of temperature stressed plants but later in the progeny of UV-C stressed plants. Transposons were also re-activated in the progeny of stressed plants. While heat shock transcription factor 2A increased expression in the progeny of heat stressed plants, many genes involved in DNA repair and histone modifications decreased. DCL2 and DCL3 appeared to be more important in transgenerational stress memory than DCL4. However, all dcl plants were generally not significantly different than wild-type plants, indicating that a single DCL deficiency may be compensated for by another DCL. / xiv, 246 leaves : ill. ; 29 cm

Endocrine tumors arise from endocrine glands. Most endocrine tumors are benign but malignant variants exist. Several endocrine neoplasms display loss of parts of chromosome 11 or 18, produce hormones and responds poorly to conventional chemotherapeutics. The multiple endocrine neoplasia syndromes are mainly confined to endocrine tumors. This opens the question if there exists a single or several endocrine tumor genes. The aim of the study was to describe genetic derangements in endocrine tumors. Paper I: Investigation of mutational status of SDHAF2 in parathyroid tumors. SDHAF2 is located in the proximity of 11q13, a region that frequently displays loss in parathyroid tumors. We established that mutations in SDHAF2 are infrequent in parathyroid tumors. Paper II: Study of SDHAF2 gene expression in a cohort of benign pheochromocytomas (PCC) (n=40) and malignant PCC (n=10). We discovered a subset of  benign PCC (28/40) and all malignant PCC (10/10) with significantly lower SDHAF2 expression. Benign PCC with low SDHAF2 expression and malignant tumors consistently expressing low levels of SDHAF2 were methylated in the promoter region. SDHAF2 expression was restored in vitro after treatment with 5- aza-2-deoxycytidine. Paper III: HumanMethylation27 array (Illumina) covering 27578 CpG sites spanning over 14495 genes were analyzed in a discovery cohort of 10 primary small neuroendocrine tumors (SI-NETs) with matched metastases. 2697 genes showed different methylation pattern between the primary tumor and its metastasis. We identified several hypermethylated genes in key regions. Unsupervised clustering of the tumors identified three distinct clusters, one with a highly malignant behavior. Paper IV: Loss of chromosome 18 is the most frequent genetic aberration in SI-NETs. DNA from SI-NETs were subjected to whole exome capture sequencing and high resolution SNP array. Genomic profiling revealed loss of chromosome 18 in 5 out of 7 SI-NETs. No tumor-specific somatic mutation on chromosome 18 was identified which suggests involvement of other mechanisms than point mutations in SI-NET tumorigenesis. Paper V: The cost for diagnostic genetic screening of common susceptibility genes in PCC is expensive and labor intensive. Three PCC from three patients with no known family history were chosen for exome capture sequencing. We identified three variants in known candidate genes. We suggest that exome-capture sequencing is a quick and cost-effective tool.

No abstract available. / Thesis (Ph.D.)-University of KwaZulu-Natal, Piertermaritzburg, 2010.

Fluctuations, or "noise", can play a key role in determining the behaviour of living systems. The molecular-level fluctuations that occur in genetic networks are of particular importance. Here, noisy gene expression can result in genetically identical cells displaying significant variation in phenotype, even in identical environments. This variation can act as a basis for natural selection and provide a fitness benefit to cell populations under stress. This thesis focuses on the development of new conceptual knowledge about how gene expression noise and gene network topology influence drug resistance, as well as new simulation techniques to better understand cell population dynamics. Network topology may at first seem disconnected from expression noise, but genes in a network regulate each other through their expression products. The topology of a genetic network can thus amplify or attenuate noisy inputs from the environment and influence the expression characteristics of genes serving as outputs to the network. The main body of the thesis consists of five chapters: 1. A published review article on the physical basis of cellular individuality. 2. A published article presenting a novel method for simulating the dynamics of cell populations. 3. A chapter on modeling and simulating replicative aging and competition using an object-oriented framework. 4. A published research article establishing that noise in gene expression can facilitate adaptation and drug resistance independent of mutation. 5. An article submitted for publication demonstrating that gene network topology can affect the development of drug resistance. These chapters are preceded by a comprehensive introduction that covers essential concepts and theories relevant to the work presented.

Les protéines à bromodomaines (BCPs) sont notamment impliquées dans la régulation de la transcription de gènes et la signalisation cellulaire. Leur dérégulation conduit au développement pathologies, telles que les maladies inflammatoires, cardiovasculaires et plus particulièrement les cancers. Les BCPs sont capables de reconnaitre les lysines acétylées de protéines histones via leur module BromoDomaine(s) (BDs). Parmi les huit familles de BCPs, mon projet de thèse s’intéresse à la famille « BET ». Celle-ci, comprend quatre protéines constituées de deux BDs en tandem, formant deux sous-familles BD1 et BD2. L’architecture de la cavité centrale des BDs, qualifiée de « druggable », a permis l’émergence de ces protéines en tant que nouvelles cibles épigénétiques prometteuses. À ce jour, une vingtaine d’essais cliniques ont été initiés pour des molécules « pan-BET », inhibant l’ensemble des membres de cette famille. Cependant, l'inhibition « pan-BET » est problématique au niveau clinique puisqu’elle impacte de nombreuses voies de transcription et engendre l’apparition de cellules résistantes. Mon projet de thèse s’intègre au challenge actuel qui est de développer des inhibiteurs plus sélectifs, par exemple envers l’une des sous-familles BD1 ou BD2 ou plus idéalement envers un seul BD de la famille BET. Le développement de « sondes épigénétiques sélectives » ciblant des BDs de la famille BET, devrait permettre de décrypter leur rôle et leur mécanisme d’action dans les divers processus biologiques. L’identification de « candidat médicament » devrait aboutir à de nouvelles thérapies ciblées et de pallier les résistances liées à l’utilisation de molécules pan-BET inhibitrices. / Bromodomain-containing proteins (BCPs) are especially involved in the regulation of gene transcription and cell signalling. Their dysregulation lead to the development of pathologies, such as inflammatory, cardiovascular diseases, and more particularly cancers. BCPs involved in the recognition of acetylated lysine of the histone tails, through their BromoDomain(s) module(s) (BDs). Among the eight families of BCPs, my thesis project focuses on the “BET” family. This family comprises four proteins which are composed of a tandem of two BDs each belonging to the BD1 or the BD2 subfamily. The architecture of the central cavity of the BDs, qualified as "druggable", allows the emergence of these proteins as new promising epigenetic targets. To date, about twenty clinical trials targeting different types of cancer have been initiated for "pan-BET" molecules that target all the members of this family. However, "pan-BET" inhibition is clinically problematic because it impacts many transcriptional pathways and causes the appearance of resistant cells. My thesis project is part of the current challenge is to develop more selective inhibitors, for example towards the BET-BD1 subfamily or the BET-BD2 subfamily or ideally towards one single BD inside the BET family. The development of such "selective epigenetic probes" targeting BET family BDs should allow deciphering their role and mechanism of action in various biological processes. Identifying "drug candidates" should lead to new targeted therapies and overcome the resistances related to the use of pan-BET molecules.

Epigenetic control of gene expression by histone post-translational modifications (PTMs) is a complex process regulated by proteins that can 'read', 'write' or 'erase' these PTMs. The histone lysine demethylase (KDM) family of epigenetic enzymes remove methyl modifications from lysines on histone tails. The Jumonji C domain (JmjC) family is the largest family of KDMs. Investigating the scope and mechanisms of the JmjC KDMs is of interest for understanding the diverse functions of the JmjC KDMs in vivo, as well as for the application of the basic science to medicinal chemistry design. The work described in this thesis aimed to biochemically investigate the inhibitor and substrate diversity of the JmjC KDMs, it led to the identification of new inhibitors and substrates and revealed a potential combinatorial dependence between adjacent histone PTMs. Structure-activity relationship studies gave rise to an n-octyl ester form of IOX1 with improved cellular potency and selectivity towards the KDM4 subfamily. This compound should find utility as a basis for the development of JmjC inhibitors and as a tool compound for biological studies. The rest of this thesis focused on the biochemical investigations of potential substrates and inhibitors for KDM3A, a JmjC demethylase with varied physiological functions. Kinetic characterisation of reported KDM3A substrates was used as the basis for evaluations of novel substrates and inhibitors. Further studies found TCA cycle intermediates to be moderate co-substrate competitive inhibitors of KDM3A. Biochemical investigations were carried out to study potential protein-protein interactions of KDM3A with intraflagellar transport proteins (IFTs), non-histone proteins involved in the formation of sperm flagellum. Work then addressed the exploration of novel in vitro substrates for KDM3 (KDM3A and JMJD1C) mediated catalysis, including: methylated arginines, lysine analogues, acetylated and formylated lysines. KDM3A, and other JmjC KDMs, were found to catalyse novel arginine demethylation reaction in vitro. Knowledge gained from studies with unnatural lysine analogues was utilised to search for additional novel PTM substrates for KDM3A. These results constitute the first evidence of JmjC KDM catalysed hydroxylation of an Nε-acetyllysine residue. The H3 K4me3 position seems to be required for acetyllysine substrate recognition, implying a combinatorial effect between PTMs. Preliminary results provide evidence that JMJD1C, a KDM3 protein previously reported to be inactive, may catalyse deacetylation in vitro. An additional novel reaction, observed with both KDM3A and JMJD1C, is deformylation of N^ε-formyllysine residues on histone H3 fragment peptides. Interestingly, H3 K4 methylation was also observed to enhance the apparent deformylation of both KDM3A and JMJD1C catalysed reactions. Overall, findings in this thesis suggest that the catalytic activity of JmjC KDMs extends beyond lysine demethylation. In a cellular context, members of the KDM3 subfamily might provide a regulatory link between methylation and acylation marks. Such a link will further highlight the complex relationships between histone PTMs and the epigenetic enzymes that regulate them. The observed dependency of H3 K9 catalysis on H3 K4 methylation adds another layer of complexity to the epigenetic regulation by histone PTMs.

Though antibodies as therapeutics are limited to extracellular targets, their repertoire of molecular interactions has particular relevance to the many intracellular cellular proteins for which small molecule screening has reached impasse. For such proteins there is little recourse to theory, since molecular recognition is, in practical terms, still not well understood. Here I apply antibody discovery to the lysine demthylases KDM4D and PHF8, two proteins difficult to inhibit selectively due to the similarity of their binding pockets to those of the larger family. With a selective, picomolar affinity antibody, dependent on residues distal to the KDM4D active site, I present what is likely the first example of allosteric inhibition of a KDM4 lysine demethylase, demonstrating that there is opportunity outside active sites oversubscribed with pan inhibitors. Antibody discovery for PHF8, however, was plagued by a familiar problem: antibodies that bound when their antigen was immobilised directly to a surface, but barely bound at all when it was free in solution. The common explanation is that the partial denaturation that accompanies immobilisation reveals epitopes unavailable in solution, but examining the problem in detail for the Plant Homeodomain of PHF8 revealed a connection to its rarely sampled conformations. The prominence these antibodies in the immune responses to PHF8, and to some extent KDM4D, motivates two hypotheses on their origin: either the states are very immunogenic or there is a connection between states of irreversible damage and those sampled reversibly, but rarely, by a protein in solution.

The discovery of RNA in 1868 by Friedrich Miescher was meant to be the prologue to an exciting new era in Biology full of scientific breakthroughs and accomplishments. Since then, RNAs have been proven to play an indispensable role in biological processes such as coding, decoding, regulation and expression of genes. In particular, the discovery of small non-coding RNAs and especially miRNAs, in C. elegans first and thereafter to almost all animals and plants, started to fill in the puzzle of a complex gene regulatory network present within cells. The aim of this thesis is to shed more light on the features and functionality of small RNAs. In particular, we will focus on the function and biogenesis of miRNAs and piRNAs, across multiple species, by employing advanced computational methods and machine learning. We first introduce a novel method (Chimira) for the identification of miRNAs from sets of animal and plant hairpin precursors along with post-transcriptional terminal modifications that are not encoded by the genome. This method allows the characterisation of the prevalence of miRNA isoforms within different cell types and/or conditions. We have applied Chimira within a larger study that examines the effect of terminal uridylation in RNA degradation in oocytes and cells in either embryonic or adult stage. This study showed that uridylation is the predominant transcriptional regulation mechanism in oocytes while it does not retain the same functionality on mRNAs and miRNAs, both in embryonic and adult cells. We then move on to a large-scale analysis of small RNA-Seq datasets in order to identify potential modification signatures across specific conditions and cell types or tissues in Human and Mouse. We extracted the full modification profiles across 461 samples, unveiling the high prevalence of modification signatures of mainly 1 to 4 nucleotides. Additionally, samples of the same cell type and/or condition tend to cluster together based on their miRNA modification profiles while miRNA gene precursors with close genomic proximity showed a significant degree of co-expression. Finally, we elucidate the determinant factors in strand selection during miRNA biogenesis as well as update the miRBase annotation with corrected miRNA isoform sequences. Next, we introduce a novel computational method (mirnovo) for miRNA prediction from RNA-Seq data with or without a reference genome using machine learning. We demonstrate its efficiency by applying it to multiple datasets, including single cells and RNaseIII deficient samples, supporting previous studies for the existence of non-canonical miRNA biogenesis pathways. Following this, we explore and justify a novel piRNA biogenesis pathway in Mouse which is independent of the MILI enzyme. Finally, we explore the efficiency of CRISPR/Cas9 induced editing of miRNA targets based on the computationally predicted accessibility of the targeted regions in the genome. We have publicly released two web-based novel computational methods and one on-line resource with results regarding miRNA biogenesis and function. All findings presented in this study comprise another step forward within the journey of elucidation of RNA functionality and we believe they will be of benefit to the scientific community.