The role of homologous recombination deficiency (HRD) in lower grade glioma (LGG) has not been elucidated, and accurate prognostic prediction is also important for the treatment and management of LGG. The aim of this study was to construct an HRD-based risk model and to explore the immunological and molecular characteristics of this risk model. The HRD score threshold = 10 was determined from 506 LGG samples in The Cancer Genome Atlas cohort using the best cut-off value, and patients with highHRDscores had worse overall survival. A total of 251 HRD-related genes were identified by analyzing differentially expressed genes, 182 of which were associated with survival. A risk score model based on HRD-related genes was constructed using univariate Cox regression, least absolute shrinkage and selection operator regression, and stepwise regression, and patients were divided into high- and low-risk groups using the median risk score. High-risk patients had significantly worse overall survival than lowrisk patients. The risk model had excellent predictive performance for overall survival in LGG and was found to be an independent risk factor. The prognostic value of the riskmodel was validated using an independent cohort. In addition, the risk score was associated with tumor mutation burden and immune cell infiltration in LGG. High-risk patients had higher HRD scores and “hot” tumor immune microenvironment, which could benefit from poly-ADP-ribose polymerase inhibitors and immune checkpoint inhibitors. Overall, this big data study determined the threshold of HRD score in LGG, identified HRD-related genes, developed a risk model based on HRD-related genes, and determined the molecular and immunological characteristics of the risk model. This provides potential new targets for future targeted therapies and facilitates the development of individualized immunotherapy to improve prognosis.

No description available.

<p>DNA double-strand breaks pose a serious threat to genomic integrity. Double-strand breaks can cause chromosomal rearrangement, leading to uncontrolled cell proliferation, or even cell death. However, mammalian systems have in place the non-homologous end-joining pathway for repair of DNA double-strand breaks, which requires a core group of proteins to function: Ku70/80, DNA-PKcs, and Artemis for recognition, protection, and processing of the DNA ends, and XLF, XRCC4, and DNA LigaseIV for ligation of the DNA break. The work presented here focuses on the specific roles of XLF and XRCC4 within non-homologous end-joining. Initially, the structure of the N-terminal 224 residues of XLF was determined and found to consist of a head and tail domain, structurally homologous to XRCC4. Furthermore, L115 of XLF and K63, K65 and K99 of XRCC4 were identified as essential for an interaction between both proteins. This interaction was then shown to be required for stimulating ligation of mismatched DNA ends. To further understand how XRCC4 and XLF enhance LigaseIV activity, an XRCC4-XLF complex was crystallized. Truncated XRCC4 (1-157) was co-crystallized with truncated XLF (1-224), grown under conditions of decreasing temperature and increasing dehydration. The resulting structure at 3.94Å confirmed the necessity of L115 (XLF) and K63, K65 and K99 (XRCC4) to the XRCC4-XLF interaction, but also illustrated that XRCC4-XLF exists as an extended helical filament. DNA binding regions in both XRCC4 and XLF were also identified and used to construct a structural XRCC4-XLF-DNA binding model. Interestingly, XRCC4-DNA binding occurs in the same region of XRCC4 required for homo-tetramerization and binding to LigaseIV. These results culminate in a proposed model of non-homologous end-joining where XRCC4-XLF is involved not only in ligation of the double-strand break, but also in initial protection of the DNA ends.</p> / Doctor of Philosophy (PhD)

Information fusion aims to develop intelligent approaches of integrating information from complementary sources, such that a more comprehensive basis is obtained for data analysis and knowledge discovery. Our Protein Biological Unit (ProtBuD) database is the first database that integrated the biological unit information from the Protein Data Bank (PDB), Protein Quaternary Server (PQS) and Protein Interfaces, Surfaces and Assemblies (PISA) server, and compared the three biological units side-by-side. The statistical analyses show that the inconsistency within these databases and between them is significant. In order to improve the inconsistency, we studied interfaces across different PDB entries in a protein family using an assumption that interfaces shared by different crystal forms are likely to be biologically relevant. A novel computational method is proposed to achieve this goal. First, redundant data were removed by clustering similar crystal structures, and a representative entry was used for each cluster. Then a modified k-d tree algorithm was applied to facilitate the computation of identifying interfaces from crystals. The interface similarity functions were derived from Gaussian distributions fit to the data. Hierarchical clustering was used to cluster interfaces to define the likely biological interfaces by the number of crystal forms in a cluster. Benchmark data sets were used to determine whether the existence or lack of existence of interfaces across multiple crystal forms can be used to predict whether a protein is an oligomer or not. The probability that a common interface is biological is given. An interface shared in two different crystal forms by divergent proteins is very likely to be biologically important. The interface data not only provide new interaction templates for computational modeling, but also provide more accurate data for training sets and testing sets in data-mining research to predict protein-protein interactions. In summary, we developed a framework which is based on databases where different biological unit information is integrated and new interface data are stored. In order for users from the biology community to use the data, a stand-alone software program, a web site with a user-friendly graphical interface, and a web service are provided. / Computer and Information Science

Argonaute proteins play substantial roles in post-transcriptional regulation of gene expression within RNA interference (RNAi) pathways, making them crucial subjects for research, aimed at understanding their interactions with small non-coding RNAs (ncRNAs) and other RNAi components. This study focuses on investigating these properties of Argonaute proteins, particularly Argonaute protein A (AgnA), in the social amoeba Dictyostelium discoideum that is renowned for its broad genetic toolbox and unique life cycle. While previous studies have examined the disruption of three Argonaute genes (agnB, agnC, agnE) and their effect on mRNA levels and small ncRNA expression, this study extends to agnA gene, which remains less studied. Key questions surrounding the influence of AgnA on the cellular processes such as the cell growth rate, development, gene expression, as well as potential targets and small ncRNA binding, remain unanswered. A well-established approach that could provide the necessary answers is the disruption of the gene through traditional homologous recombination, by insertion of a drug-resistance cassette flanked by homology arms complementary to the target locus. However, the emerging CRISPR/Cas9 gene editing tool on contrary offers straightforward protocols for disruption of gene expression through efficient induction of genomic knockouts, point mutations and deletions. In this study, both approaches were applied in parallel to knockout the agnA gene, enabling comparison of knockout efficiency and further study of the growth rate, development and gene expression in the knockout strains. Moreover, important information regarding the growth patterns of both wild-type and agnE knockout strains were also elucidated, complementing the previous growth rate analyses. The obtained data from this research could provide valuable insights for future studies ofthe RNAi machinery components and particularly the function of Argonaute proteins in D. discoideum.

Yes / Abstract The budding yeast genome contains regions where meiotic recombination initiates more frequently than in others. This pattern parallels enrichment for the meiotic chromosome axis proteins Hop1 and Red1. These proteins are important for Spo11-catalyzed double strand break formation; their contribution to crossover recombination remains undefined. Using the sequence-specific VMA1-derived endonuclease (VDE) to initiate recombination in meiosis, we show that chromosome structure influences the choice of proteins that resolve recombination intermediates to form crossovers. At a Hop1-enriched locus, most VDE-initiated crossovers, like most Spo11-initiated crossovers, required the meiosis-specific MutLγ resolvase. In contrast, at a locus with lower Hop1 occupancy, most VDE-initiated crossovers were MutLγ-independent. In pch2 mutants, the two loci displayed similar Hop1 occupancy levels, and VDE-induced crossovers were similarly MutLγ-dependent. We suggest that meiotic and mitotic recombination pathways coexist within meiotic cells, and that features of meiotic chromosome structure determine whether one or the other predominates in different regions.

La famille des Flexiviridae a été créée en 2004 et regroupe plusieurs genres viraux affectant particulièrement des espèces ligneuses dont des arbres fruitiers. Grâce à diverses approches plusieurs nouveaux Flexiviridae ont été partiellement caractérisés au cours de ces dernières années. En revanche la position taxonomique précise de certains d’entre eux et leur contribution à des pathologies particulières restent encore incertaines du fait de difficultés inhérentes à l’étude de ces agents. Dans le présent travail, nous avons obtenu les séquences génomiques complètes pour quatre agents proches de l’Apricot latent virus. Ceci a permis de préciser l’organisation génomique de ces virus et d’en déterminer la position taxonomique. Cette étude a également permis de montrer que la partie C-terminale de la capside et la protéine TGBp1 sont soumises à une pression sélective particulièrement forte. Dans un second volet de ce travail, plusieurs approches permettant l’obtention simple et rapide d’ADNc infectieux, sous forme clonée ou non ont été développées. Travaillant sur plusieurs Flexiviridae, dont le virus des taches foliaires chlorotiques du pommier (Apple chlorotic leaf spot virus, ACLSV), nous avons mis au point l’amplification d’ADNc génomiques complets en une seule étape à partir d’extraits d’acides nucléiques totaux obtenus à partir de plantes infectées. Des amplifiats comportant l’ADNc viral sous le contrôle du promoteur 35S du CaMV ou du promoteur de la RNA polymérase du phage T7 ont été obtenus et utilisés pour infecter des plantes directement par biolistique (promoteur 35S) ou pour obtenir des ARN infectieux par transcription in vitro (promoteur T7). Ces données ont mis en évidence des différences importantes dans le comportement de deux hôtes de l’ACLSV, Chenopodium quinoa et Nicotiana occidentalis 37B. Nous avons également utilisé le système de recombinaison homologue de la levure Saccharomyces cerevisiae simplifier le clonage d’ADNc complets amplifiés par PCR ou pour réaliser en une seule étape la construction d’un vecteur navette ternaire levure-E. coli-A. tumefaciens et l’obtention d’un clone ADNc de l’ACLSV inoculable par agroinfiltration. Ces différentes stratégies devraient trouver une large application, en particulier pour tester plus rapidement des hypothèses d’étiologie pour les virus de plantes réputés "difficiles", tels que ceux infectant des hôtes ligneux. / The Flexiviridae family was created in 2004 and contains several viral genera affecting in particular woody hosts, including fruit trees. Using various strategies several new Flexiviridae have been partially characterized in the past few years. However, due to difficulties inherent in studying these agents, the precise taxonomic position of some of them and their contribution to particular diseases are still uncertain. In the present work, the complete genomic sequences of four Prunus-infecting Apricot latent virus (ApLV) like isolates have been determined. This has allowed to determine the genomic organization and the taxonomic position of these viruses. The results obtained also indicate that the C-terminal half of the coat protein and the TGBp1 are the genomic regions under the strongest purifying selection pressure. In the second part of this work, a set of approaches to simplify and streamline the construction of cloned or uncloned infectious full-length viral cDNAs were developed. working with several Flexiviridae and, in particular, with the Apple chlorotic leaf spot virus (ACLSV), we have developed protocols allowing the one-step amplification from total nucleic acids extracts of full-length cDNAs. under the control of the CaMV 35S or phage T7 RNA polymerase promoters. Successful inoculation of plants with these uncloned amplification products was obtained by biolistic bombardment (35S promoter) or using in vitro synthesized RNA transcripts (T7 promoter). Results obtained showed significant differences in the behavior of the two ACLSV hosts, Chenopodium quinoa and Nicotiana occidentalis 37B. We also used the yeast homologous recombination system for the efficient cloning of full-length cDNAs and for the simultaneous one-step construction of a ternary yeast-E. coli-Agrobacterium tumefaciens shuttle vector and generation of an agroinfiltrable infectious ACLSV construct. These various strategies should find broad applications, in particular for the validation of etiological hypotheses in the case of “difficult” plant viruses, such as those infecting woody hosts.

Homologous recombination (HR) is essential for the repair of DNA doublestrand breaks (DSBs) and damaged replication forks. However, HR can also cause gross chromosomal rearrangements (GCRs) by producing crossovers (COs), resulting in the reciprocal exchange of sequences between non-sister chromatids. Therefore, HR-mediated GCRs are suppressed via the promotion of HR pathways that favour noncrossover (NCO) formation, such as the synthesis-dependent strand annealing (SDSA) and dissolution pathways, which are modulated by Mph1 and Sgs1 helicases, respectively. The mismatch repair (MMR) pathway is intricately associated with HR via its roles in repairing mismatches on heteroduplex DNA that can arise during HR and in preventing homeologous recombination. Using a plasmid break-repair assay, we have revealed a novel, MMR-independent role of MutSα in promoting the formation of a subset of COs that is specifically supressible by Mph1, during HR between two completely homologous sequences. In contrast, the MMR-dependent function of MutSα, together with Mph1 and Sgs1, was shown to be required for the suppression of CO formation during homeologous recombination. These data indicate that Mph1 can both antagonise and promote the functions of MutSα during DSB repair, depending on the levels of homology between the two recombining sequences. COs are generated by the resolution of Holliday junction (HJ) intermediates formed at the terminal stages of HR. Several S.cerevisiae proteins such as Yen1, Mus81, Slx1 and Rad1 have been implicated in HJ resolution. However, the in vivo roles of these proteins in HJ resolution remain to be confirmed. To directly and quantitatively monitor in vivo HJ resolution in S.cerevisiae, a transformation-based HJ resolution assay using a plasmid-borne HJ substrate has been developed. Using this system, we have demonstrated an in vivo HJ resolution function of Yen1, which acts redundantly with Mus81. Moreover, these redundant activities of Yen1 and Mus81 are essential for survival during replication stress, but are dispensable for DSB repair. An Slx4 and Rad1-dependent in vivo HJ resolution activity was also observed in the absence of Yen1 and Mus81 that was suppressed by presence of Slx1. Models describing how the nucleases interact to process HJs in vivo will be discussed.

Neisseria meningitidis causes 500,000 cases of meningitis and septicaemia annually worldwide, with a mortality rate of approximately 10%. Most disease in developed countries is caused by serogroup B infection, against which there is no universal vaccine. Opa proteins are major meningococcal outer membrane proteins, and a limited number of Opa variants have been associated with hyperinvasive serogroup B meningococci, suggesting their use as a potential novel vaccine. Immunisation of mice with recombinant Opa elicited high levels of meningococcal-specific serum bactericidal antibody (SBA), demonstrating proof in principle of this approach. Opa proteins mediate bacterial adherence to host cells and modulate human cellular immunity, and there are conflicting data regarding their effects on CD4⁺ T cells. opa genes from N. meningitidis strain H44/76 were cloned into the plasmid vector pBluescript, disrupted using antibiotic resistance cassettes and transformed into H44/76 to sequentially disrupt the four opa genes. This produced a unique panel of 15 isogenic Opa-deficient strains, including an Opa-negative strain, which enabled investigation of the immunomodulatory role of surface-expressed Opa proteins. There was no consistent effect of Opa expressed on the surface of OMVs and inactivated bacteria on CD4⁺ T cells, with significant heterogeneity of responses between individuals. The rate of Opa phase variation was between 10^-3 and 10^-4, and increased 180-fold following transformation of bacteria with unrelated DNA. These data support further investigation of Opa as a potential meningococcal vaccine component, and highlight the importance of host and bacterial factors in the development of OMV vaccines.

During early embryonic development oocytes are arrested in prophase I of the first meiotic division, in which they can persist for years. After reaching sexual maturity and the luteinizing hormon surge resumption of meiosis and meiotic maturation occur. Oocytes are arrested again at metaphase of the second meiotic division. At this stage they are ovulated and waiting for a fertilisation. Oocytes are during their development exposed to factors that cause DNA damage, of which DNA double-strand breaks (DSBs) are the most serious threat. The maintaining of genome integrity is crucial for quality of oocytes, fertility and proper embryonic development. The mechanism of the oocyte response to DSBs presence is not fully understood and it seems to differ from somatic cells. We assume that DSBs are repaired during meiotic maturation probably by a mechanism of homologous recombination (HR). In this thesis we focuse on essencial recombinase RAD51, which participates in the repair by HR. We found that RAD51 inhibition leads to an increase of segregation errors in anaphase I. Using high resolution live cell imaging we observed chromosomal fragments and anaphase bridges. Immunofluorescence detection of DSBs-marker γH2AX showed increased amount of DSBs in prophase I and MII stage after RAD51 inhibition. Our data...