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The serotonin-1A (5-HT1A) receptor plays an important role in the regulation of the serotonin (5-HT) system as an autoreceptor on 5-HT neurons. The transcription factor CC2D1A/Freud-1 is a potent repressor of the 5-HT1A promoter in neuronal but not in non-neuronal cells. The clinical relevance of Freud-1 is evident in a naturally occurring mutation, resulting in a truncated form of Freud-1 lacking its C-terminal half, that is associated with non syndromic mental retardation in humans. Thus, it is of interest to clarify the structure and function of Freud-1. As Freud-1 was shown to interact with the transcriptional regulator Brg1 at the 5-HT1A promoter, identification of the structural domains mediating the Brg1/Freud-1 interaction is required to assess the role of Brg1 in Freud-1 repression. In this study, I used pull-down assays with recombinant proteins, co-immunoprecipitation studies and immunofluorescent staining with confocal microscopy to show that Freud-1 interacts directly with the C-terminus of Brg1 and that the C-terminal domain of Freud-1 is required for this interaction.

La problématique de mon travail de recherche de doctorat tourne autour de la question de l'activité du tourisme au service du développement des populations locales en basse Casamance. enclavée au nord par la république anglophone de la Gambie et au sud par la république lusophone de la Guinée Bissao, cette entité méridionale du Sénégal, actuelle région de Ziguinchor constitue l'épicentre de l'axe (Banjul, Bignona, Bissao) de peuples unis par l'histoire, la culture et le cadre naturel. la région considérée comme la Floride du Sénégal regorge des potentialités naturelles et culturelles qui la font un terroir recherché. sa dimension sociologique et linguistique la démarque du reste du pays et suscite la curiosité. en raison de cette admiration et de la beauté de son paysage, la région attire de plus en plus d'acteurs de développement touristique.la diversité de sa richesse constitue un enjeu pour son développement local. le patrimoine est détenu par l'ethnie Diola qui est majoritaire et se caractérise par un particularisme sociolinguistique correspondant aux entités satellites suivantes : les Diola Karone, les Diola du Blouf, les Diola du Fogny et les Diola du Kassa. ces communautés régies par des us et coutumes ont un dénominateur commun le « boekin », le génie protecteur et garant de l'abondance des pluies. elles peuvent regrouper une vingtaine de villages dont les limites territoriales remontent aux premiers occupants des lieux. aujourd'hui avec la croissance rapide du tourisme due aux technologies de l'information et de la communication, les acteurs, les professionnels et les touristes sont à la quête de nouveaux horizons susceptibles de satisfaire leurs attentes. cependant cette croissance ne manque pas d'effets négatifs au nombre desquels la perturbation des structures sociales et la dégradation de l'environnement naturel. face à cette envergure de l'activité du tourisme, notre approche essayera de répondre au thème ; tourisme et population en basse Casamance : enjeux pour un développement local. / Tourism and populations in the Lower Casamance

LTBP-1 is a large extracellular glycoprotein that is a component of the large latent TGF-β complex. The extracellular sequestration of latent TGF-β in the extracellular matrix (ECM) is fundamental to the regulation of TGF-β bioavailability and activity. LTBP-1 is described to contribute to the regulation of TGF-β bioavailability through mediating the extracellular sequestration of newly secreted latent TGF-β with fibrillin microfibrils in the ECM. However it is not well understood how LTBP-1, and thus latent TGF-β, becomes deposited into the ECM. Previous work by our group suggested that LTBP-1 interactions with the glycosaminoglycan heparan sulphate (HS) at the cell-matrix interface might facilitate the association of LTBP-1 with fibrillin microfibrils. Using recombinant LTBP-1 fragments and mutants, LTBP-1 interaction with HS have been fine-mapped. Deposition of a LTBP-1 HS-binding mutant, and of LTBP-1 when HS was depleted, was studied in cell cultures; findings presented here demonstrate that HS may not be critical for the deposition of LTBP-1 into the ECM. Contributions of fibrillin and fibronectin to LTBP-1 deposition were investigated, and data presented here support published findings that fibrillin is not always required for LTBP-1 deposition. In addition, the dependency of LTBP-1 deposition upon fibronectin was suggested to differ between different cell types (epithelial and mesenchymal). How LTBP-1 may be stabilised within the ECM through crosslinking by tissue transglutaminase was investigated using recombinant fragments and cell culture studies. Tissue transglutaminase was found to promote the extracellular incorporation of LTBP-1, and novel cross-links within LTBP-1, and between LTBP-1 and fibrillin-1, but not LTBP-1 and fibronectin, were identified. Additionally, results indicated that LTBP-1 was present in extremely high molecular weight assemblies in the ECM of cultured fibroblasts. Collectively, these results have contributed to current knowledge of how LTBP-1 becomes deposited into the ECM. They indicate that the deposition of LTBP-1 is not underpinned by HS, may be cell type-specific, and that LTBP-1 may potentially self-assemble extracellularly into homotypic structures that may associate with fibrillin microfibrils.

Immunoglobulin-containing and proline-rich receptor-1 (IGPR-1), is a newly identified cell adhesion molecule and is expressed in various cell types including, epithelial and endothelial cell origin. IGPR-1 regulates cell-cell adhesion and promotes angiogenesis, activated by shear stress and mediates endothelial cells response to shear stress. Moreover, IGPR-1 expression is upregulated in colon cancer and supports colon tumor growth. IGPR-1 contains a single extracellular immunoglobulin domain, a transmembrane domain and followed by a cytoplasmic proline-rich C-terminus. We demonstrate that ubiquitin E3 protein ligase neural precursor cell expressed developmentally down regulated 4 (NEDD4) binds to and ubiquitinates IGPR-1. Furthermore, among the four WW domains, the C-terminus WW domain#4 selectively mediates the binding of NEDD4 with IGPR-1. We used in vitro ubiquitination assay and identified UbcH6, as an E2 conjugating enzyme required for NEDD4-mediated ubiquitination of IGPR-1. Taken together, our data identifies NEDD4/Ubc6H ubiquitination system as a major pathway involved in the ubiquitination of IGPR-1. / 2021-06-08T00:00:00Z

Une bonne connaissance des sections efficaces de l'eau légère est importante, car l'eau est le modérateur le plus employé dans les réacteurs à eau pressurisée (REP), qui fonctionnent à des températures et des pressions avoisinant 550 K et 150 bar. Les sections efficaces neutroniques dans le domaine d'énergie thermique dépendent de la structure et de la dynamique du matériau diffusant, décrites par des lois de diffusion thermiques (TSL). Les évaluations des TSL existantes n’ont pas été validées aux hautes températures et pressions, et doivent être revues. Pour produire de nouvelles TSL, des mesures de la diffusion inélastique des neutrons sur l'eau ont été effectuées à l'Institut Laue-Langevin (ILL), à l'aide de deux spectromètres IN4c et IN6. Afin de compléter ces mesures, des simulations de dynamique moléculaire (MD) ont été réalisées en s'appuyant sur deux modèles classiques d'interaction moléculaire, le modèle non polarisable TIP4P/2005f et le modèle polarisable TCPE. Les spectres de fréquence à différentes pressions et températures obtenus grâce aux mesures de temps de vol et aux simulations MD ont été exploités pour développer de nouvelles TSL. Les performances de ces nouvelles bibliothèques ont été testées sur une série de mesures de sections efficaces différentielles, double-différentielles et totales disponibles dans la littérature. Des benchmarks critiques (ICSBEP benchmarks) ont également été utilisés. Les résultats de ces études permettent une meilleure compréhension de l'impact de la température et de la pression sur les TSL dans les applications liées à l'exploitation des REP. / Precise knowledge of light water thermal scattering cross section is important as it is the most widely used moderator in pressurized water reactors (PWRs) which operate at temperature around 550 K and pressure around 150 bar. In the thermal neutron energy region, the cross sections are governed by the structure and dynamics of the scattering material described by thermal scattering law (TSL). There is a need for reviewing the existing TSL evaluations and consequently performing new experiments, to develop new TSL evaluations valid for a large range of temperature and pressure conditions. To generate new TSL for light water, inelastic neutron scattering measurements were carried out at two time-of-flight (TOF) spectrometers, namely the IN4c and IN6, at the Institut Laue-Langevin (ILL), Grenoble, France. A corresponding set of molecular dynamics (MD) simulations were performed to complement the experimental data using two classical interaction models for water namely, a flexible non-polarizable TIP4P/2005f and a rigid polarizable TCPE model. Frequency spectra obtained from both TOF experiment and MD simulations at different temperatures and pressures have been analyzed and new TSL evaluations have been developed. The performance of the newly developed TSL evaluations were tested on a series of differential, double differential and total cross section measurements available in the literature. For further verification and validation of the new TSL data, critical benchmarks available in the ICSBEP Handbook, sensitive to TSL have been used. The outcome of this study leads to a better interpretation of the impact of temperature and pressure on TSL in PWR applications.

archives@tulane.edu / These studies make strides in better understanding retroelement L1 expression and regulation at the locus-specific level using a combination of sequencing technologies. A picture is painted demonstrating tissue specific patterns of L1 expression when identified stringently and confidently with our developed EL-Seq approach. As it was also determined that expressed L1s significantly correlate with regions of open chromatin, these tissue-specific patterns of L1 expression are then most likely explained by tissue-specific chromatin architecture. Evidence is also presented here that L1s in tissues respond differently with genomic stresses and perturbations as is seen in the case of aging indicating that the risk associated with L1 damage and mutagenesis is related to cell type and tissue. This is particularly notable when considering the genesis and promotion of age-related somatic diseases like epithelial cancers. L1s are commonly referred to as the dark matter of the genome, but here we illuminate its biology and regulation to better understand L1-associated damage and risk to human health. / 0 / Tiffany Kaul

Understanding the mechanisms used by HIV-1 to evade antibody neutralisation may contribute to the design of a high-coverage vaccine. This thesis explores the mechanism used by a Tier 3 virus leading to its high antibody neutralisation resistance phenotype. This thesis also describes how the glycans at the base of the V3 loop contribute to (i) breadth and potency in a cohort of unselected HIV-1-infected individuals and (ii) the selective pressures resulting from the V3/glycans shielding the virus from neutralisation and the glycans themselves being targets of broad antibody responses. HIV-1 isolates that are highly resistant to broadly neutralising antibodies could limit the efficacy of an antibody-based vaccine. For this reason, it is important to understand the mechanisms behind high HIV-1 resistance to neutralising antibodies. Chapter 2 and Chapter 3 of this thesis describe virus 253-11, a highly neutralisation resistant virus, which is particularly resistant to commonly-elicited, anti-membrane proximal external region (MPER) antibodies in sera. To further understand its resistance, mutations in the MPER were introduced that are known to delay fusion following CD4-binding and thus increase the time the virus spends in the open conformation. Interestingly, we found that these mutations affect the 253-11 Envelope (Env) spike before CD4-binding by destabilising the closed trimer structure. From these data, we hypothesized that the neutralisation resistance of 253-11 was due to an unusually tight, compact pre-fusion Env trimer that resists transient changes to the open conformation. The open conformation frequently exposes narrowly-neutralising antibody epitopes. Because the unliganded 253-11 Env presumably transitions infrequently into the open conformation, it would be able to evade these responses. 253-11 was sensitive to most but not all of the most potent broadly neutralising antibodies (bnAbs) tested, most likely because those broadly neutralising antibodies can access their epitopes in the pre-fusion Env conformation. To gain further information about the structure of the 253-11 Env, we designed a recombinant 253-11 SOSIP trimer and found it to be stable and predominantly adopt a closed conformation. The crystal structure of the SOSIP trimer revealed structural elements likely responsible for 253-11 Env compactness including the inward disposition of the heptad repeat helices and gp120 protomers towards the trimer axis. Taken together, the data from Chapter 2 and Chapter 3 highlight an underappreciated Env compactness mechanism of HIV-1 resistance to neutralising antibodies and these data may be useful in HIV-1 immunogen design research. Previous candidate HIV vaccines have failed to induce wide-coverage neutralising antibodies capable of substantially protecting vaccinees. A key approach in HIV immunogen development has been to define and model epitopes recognised by anti-HIV bnAbs. Candidate immunogen models identified by bnAbs include the V3/glycans, the V2/apex and the MPER epitopes. Autoreactivity and polyreactivity of anti-V3/glycan and anti-MPER antibodies are thought to pose both direct and indirect barriers to achieving neutralisation breadth. Chapter 4 of this thesis explored which of these bnAb epitopes were associated with breadth and potency in a South African cohort of chronically HIV-infected individuals. The study found that antibodies targeting the V3/glycans were associated with breadth and potency. In contrast, antibodies to the V2/apex were not associated with neutralisation breadth/potency. This suggests that auto/polyreactivity are not critical factors in the development of breadth and potency and that the V3/glycans should remain a high-priority vaccine candidate. Since targeting the V3/glycans was associated with breadth and potency in this cohort, the study continued to look at this epitope to investigate the role of these glycans in neutralisation resistance of Tier 2 viruses. The HIV-1 Env is surrounded by glycans that often prevent antibody neutralisation, leading to the term the "glycan shield", however some bnAbs have evolved to recognise these carbohydrates. Chapter 4 of this thesis describes how the N-linked glycan at position N301 is critical for maintaining neutralisation resistance of one subtype C virus (Du156.12), but not for another subtype-matched virus (CAP45.2.00.G3). Thus, the loss of the N301 glycan may have a substantial antibody-related fitness cost for some viruses but not others. The N301 glycan, as well as glycans at positions 332 and 334, are the primary targets of the anti-V3/glycan class of neutralising antibodies, which may select for loss of the targeted glycan. The evidence presented in Chapter 4 suggests that in some viruses, loss of the N301 glycan may result in evasion of anti-V3/glycan antibody responses while maintaining overall neutralisation resistance. This phenomenon may impair efficacy of passively-infused anti-V3/glycan bnAbs or a therapeutic vaccine.

Abstract The immaturity of the neonatal immune system places infants at an increased risk of infections and also affects the effective induction of protective immune responses by vaccines. In utero sensitisation to infectious pathogens results in immune activation and can establish immunological memory and may influence the immune response to unrelated antigens. In this study, we investigated in early life (birth to 6-10 weeks) the development of interferon-gamma (IFN-g) responses in uninfected infants born to HIV-1 infected mothers (exposed uninfected (EU) infants). Whole blood cell cultures stimulated with phytohaemagglutinin (PHA) or Mycobacterium bovis bacillus Calmette-Guérin (BCG) showed that EU infants have a greater ability to produce IFN-g in response to PHA and BCG at birth compared to control infants (born to HIV-1 uninfected mothers). However, by six weeks of age control infants produced significantly more IFN-g in response to PHA only. These results suggest that responses amongst EU infants establish and mature earlier than in control infants. In fact, over time a greater number of EU infants have a reduced ability or an inability to respond to stimuli such as PHA or BCG compared to control infants (when comparing responses at six weeks of age to responses of the matched birth samples). Full blood counts (FBC) counts were carried out using an automated AcT 5 diff haematology analyser which measures proportions and absolute counts of the five groups of white blood cells, namely, lymphocytes, monocytes, neutrophils, eosinophils and basophils. Importantly, there were no significant differences in the absolute lymphocyte counts of control infants and EU infants either at birth or at six weeks which could account for the IFN-g production differences noted between these infant groups, although the EU infants did exhibit a trend of higher absolute lymphocyte counts at six weeks than control infants. Age-dependent maturational changes in cell counts were observed in both control and EU infant groups, with neutrophils predominating at birth and lymphocytes predominating by six weeks, indicative of immune development with age in both infant groups. Short-course antiretroviral exposure increased basophil counts of infants at birth but did not affect counts by six weeks. Flow Cytometry studies using an Intracellular Cytokine (ICC) assay were conducted to establish which mononuclear cell types are predominantly responsible for producing IFN-g in infants. ICC assays done on whole blood revealed that natural killer cells (NK) were predominantly responsible for the IFN-g produced by 10 week old EU infants, and also at birth (control and EU infants). At birth whole blood cultures of 48% of EU infants already showed BCG-induced IFN-g responses (prior to BCG vaccination); this could be explained by a non-specific response (NK cells) to the antigen but could also involve T cell responses (CD4+ and/or CD8+ T cells) as supported by ICC data obtained from control and EU newborn infants. The infant immune system was clearly unique from that of their mothers. In particular, mothers’ demonstrated significant changes in blood counts from labour to six weeks postpartum indicative that immuno-modulation plays an essential role in a successful pregnancy. The single dose of nevirapine (sdNVP) taken by the mother at the onset of labour did not influence maternal blood counts significantly and maternal CD4+ and CD8+ T cells, and NK cells produced significantly more IFN-g than the CD14+ monocytes and CD19+ B cells, with CD8+ T cells producing the most. Our results have shown that EU infants are distinct from control infants with respect to immunological responses as measured by IFN-g production, and that maturational differences do exist between control and HIV-1 exposed infants. While the clinical importance of these results remains undetermined it is important to establish whether such immunological changes may result in altered susceptibility to infectious diseases in this already vulnerable population, and how this may impact on the induction of protective immune responses by vaccines. It remains important to identify neonatal immune system deficiencies and understand the consequences of exposure to maternal HIV-1 (in the absence of acquiring HIV-1 infection), the understanding of which could contribute to the development of more effective vaccines to HIV-1 and other infectious diseases.

A thesis submitted to the School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 2016 / Human Immunodeficiency Virus Type 1 (HIV-1) infects approximately two million people annually, highlighting the need for a preventative vaccine. An effective HIV-1 vaccine will likely need to elicit broadly neutralizing antibodies (bNAbs), which arise naturally in some infected individuals and recognize the envelopes (Env) of multiple HIV-1 strains. Understanding the molecular events that contribute to bNAb development during infection may provide a blueprint for vaccine strategies. Here we investigated the development of a V1V2-directed bNAb lineage in the context of viral co-evolution in an HIV-1 superinfected participant (CAP256). For this, clonally-related monoclonal antibodies (mAbs), with a range of neutralization breadth, were isolated. We determined their developmental pathway from strain-specificity towards neutralization breadth and identified viral variants responsible for initiating and maturing this bNAb lineage. MAbs were isolated by memory B cell culture or trimer-specific single B cell sorting and extensively characterized by Env-pseudotyped neutralization, cell surface-expressed Env binding, electron microscopy and epitope-predictive algorithms. Antibody next-generation sequencing (NGS) at multiple time-points enabled the inference of the unmutated common ancestor (UCA) of this lineage. Viral co-evolution was investigated using Env single genome amplification and V1V2 NGS. A family of 33 clonally-related mAbs, CAP256-VRC26.01-33, was isolated from samples spanning four years of infection. The UCA of this lineage possessed an unusually long heavy chain complementarity determining region 3 (CDRH3), which resulted from a unique recombination event. Surprisingly, this UCA potently neutralized later viral variants that had evolved from the superinfecting virus, which we termed bNAb-initiating Envs. Viral diversification, which peaked prior to the development of neutralization breadth, created multiple immunotypes at key residues in the V1V2 epitope. Exposure to these immunotypes allowed adaptation of some mAbs to tolerate this variation and thus mature towards neutralization breadth. Based on these data, we proposed a four-step immunization strategy which includes priming with bNAb-initiating Envs to engage rare B cells with a long CDRH3; followed by three sequential boosts (including select V1V2 immunotypes) to drive antibody maturation. In conclusion, this study has generated a testable HIV-1 vaccine immunization strategy through the delineation of mAb-virus co-evolution during the development of neutralization breadth. / MT2017