京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第22605号 / 生博第438号 / 新制||生||58(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 杉田 昌彦, 教授 藤田 尚志, 教授 朝長 啓造 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

>Magister Scientiae - MSc / The viral Integrase (IN) protein is an essential enzyme of all known retroviruses, including HIV-1. It is responsible for the insertion of viral DNA into the human genome. It is known that HIV-1 is highly diverse with a high mutation rate as evidenced by the presence of a large number of subtypes and even strains that have become resistant to antiretroviral drugs. It remains inconclusive what effect this diversity in the form of naturally occurring polymorphisms/variants exert on IN in terms of its function, structure and susceptibility to IN inhibitory antiretroviral drugs. South Africa is home to the largest HIV-1 infected population, with (group M) subtype C being the most prevalent subtype. An investigation into IN is therefore pertinent, even more so with the introduction of the IN strand-transfer inhibitor (INSTI) Dolutegravir (DTG).

L’intégrase (IN) du VIH-1 est une enzyme clé qui catalyse l’insertion stable du génome viral dans celui de la celluleinfectée. D’autre part, l’IN participe également à de nombreuses étapes du cycle viral telles que la transcriptioninverse ou la maturation virale. La compréhension des mécanismes impliqués dans la régulation de l’intégrationcellulaire au cours de l’infection est un enjeu important. L’IN fait partie du complexe de préintégration composé defacteurs cellulaires et viraux. La dynamique des interactions au sein de ce complexe régule les activités catalytiquesmais également non catalytiques de l’IN. C’est dans ce contexte de recherche de nouveaux cofacteurs de l’intégraseque nous avons identifié une interaction entre l’IN et la protéine Kinase GCN2.Mon travail de thèse s’est orienté sur trois questions autour de l’étude du rôle de cette interaction IN/GCN2.- Dans un premier temps, le rôle de la protéine kinase cellulaire GCN2 au cours du cycle viral a été étudié. Nousavons pu montrer que l’infection par le VIH-1 provoque un stress activant GCN2. Cette activation aboutit à uneinhibition de la traduction dès les premières heures de l’infection.- GCN2 est capable de phosphoryler l’IN du VIH-1 sur deux positions : les sérines en position 24 et 255. L’étude durôle de la phosphorylation de l’IN par GCN2 a permis de montrer que l’absence de phosphorylation de l’IN entraîneune stimulation de l’infection. GCN2 via la phosphorylation de l’IN a donc un effet restrictif sur l’infection par le VIH-1.- L’étude du domaine d’interaction entre l’IN et GCN2 a permis d’identifier un résidu essentiel de l’IN, l’acideglutamique en position 85 (E85). En effet, la mutation E85A de l’IN entraîne la production de virus non infectieux,suite à un défaut de maturation / HIV-1 integrase (IN) catalyzes the integration of the viral DNA into the cellular genome. Besides, IN is also involved inother steps of the viral life cycle like the reverse transcription or the viral maturation. Our group is interested in themechanisms involved in the regulation of cellular integration during the infection. IN is part of a pre-integrationcomplex composed of cellular and viral proteins. The dynamic of these interactions regulates IN activities but also noncatalytic activities such as nuclear import and tethering of the complex to the integration site. During theidentification of news partners of IN, an interaction between IN and the kinase GCN2 was characterizedMy PhD project is composed of 3 topics:- The role of the cellular kinase GCN2 was studied during the viral cycle. We showed that GCN2 is activated uponHIV-1 infection. This activation leads to a general decrease of cellular translation during the first hours of infection.-GCN2 is able to phosphorylate IN on two positions: the serines in position 24 and 255. The absence of INphosphorylation causes a stimulation of HIV-1 infection. We demonstrate that GCN2 affects the viral cycle via thephosphorylation of IN.- Binding domain analysis between IN and GCN2 led to the identification of a critical residue of IN, the glutamicacid in position 85 (E85). Mutations E85A of IN impair the viral production by inhibiting the viral maturation.

The study involved retrospective data analysis using statistical methods to re-analyse data collected during a long-term study in a Human Immunodeficiency Virus infected population of South African National Defence Force employees and their dependents, where different parameters related to treatment and disease status of HIV infected patients were collected. This study attempted to identify possible predictors of both early and late occurrence of first-line antiretroviral therapy virologic failure, potential predictors of first-line antiretroviral virologic failure and for switching to second line therapy were identified. / Dissertation (MSc)--University of Pretoria, 2018. / HJF- Phidisa project / Pharmacology / MSc / Unrestricted

Current epidemiological data showing that the use of the injectable contraceptive progestin Depotmedroxyprogesterone acetate (DMPA) is associated with increased HIV-1 acquisition is controversial. However, animal and ex vivo data reveal plausible biological mechanisms whereby MPA may increase HIV-1 acquisition. Relatively high levels of endogenous progesterone (P4) found in the luteal phase of the menstrual cycle have also been linked to increased HIV-1 acquisition in animal, clinical and ex vivo models. One of the central hypotheses of the present study was that the mechanism of MPA-induced increase in HIV-1 infection occurs via a different mechanism to that of the luteal phase. Furthermore, MPA has been shown to activate both the glucocorticoid receptor (GR) and its target, the progesterone receptor (PR) isoform B (PR-B), which are both transcription factors and regulate genes involved in immune function. Both the GR and PR are expressed in the cervix, the primary site of heterosexual HIV-1 infection. PR is regulated by endogenous estrogen (E2), of which the concentrations fluctuate throughout the menstrual cycle, and GR expression also varies in response to stress hormones, leading to conditions of varied relative levels of GR/PR. The immune-related consequences of changing the relative levels of GR and PR-B are not well understood. Therefore another hypothesis of this study was that changing the relative levels of GR/PR-B modulates HIV-1 infection and immunomodulatory gene expression in response to the GR/PR agonist, MPA. Since GR and PR-B recognize similar DNA target sequences and may regulate the same genes at the same time, the final hypothesis of the present study was that GR and PR-B reciprocally modulate each other’s activity, through possible association. To investigate the effects of exogenous hormones on HIV-1 infection and mechanisms thereof, peripheral blood mononuclear cells (PBMCs) and TZM-bl cervical cells were used as model systems for HIV-1 infection. These cells were stimulated with P4 and E2 at concentrations mimicking the menstrual cycle phases or with levels of MPA at the upper range of peak serum levels detected in DMPA users. Cells were infected with the R-tropic HIV-1 infectious molecular clone, HIV-1Bal_Renilla and luciferase assays were used to measure HIV-1 infection. Levels of HIV-1 CD4 receptor and CCR5 co-receptor protein or mRNA were measured by flow cytometry or qPCR, respectively, while activation of CD4+ T cells using the activation marker CD69 was measured by flow cytometry in PBMCs. To investigate the effects of changing GR/PR-B levels on HIV-1 infection and immune gene regulation, GR/PR levels were altered in End1/E6E7 immortalized endocervical and HeLa/TZM-bl cervical carcinoma cells by GR siRNA knockdown with or without the simultaneous over-expression of PR-B, and cells were stimulated with MPA or the GR agonist Dexamethasone. mRNA expression iii of key immunomodulatory genes in End1/E6E7 and HeLa cells was measured by qPCR. The modulation of GR activity by PR-B was assessed by promoter-reporter assay in COS1 and U2OS cells over-expressing GR and PR and stimulated with GR- and/or PR-specific ligands. Association of GR and PR-B was measured by co-immunoprecipitation in COS1 and MCF-7 cells, while co-localization of GR and PR-B was measured by confocal microscopy and super-resolution structured illumination microscopy in COS1 cells. MPA significantly increased HIV-1 infection in both PBMCs and TZM-bl cells, while luteal phase hormones did so to a lesser extent. However, MPA but not luteal phase hormones increased the ratio of CD4+/CD8+ T cells in PBMCs. MPA but not luteal phase hormones also increased CCR5 protein expression on CD4+ T cells in PBMCs and total CCR5 mRNA expression in TZM-bl cells. In addition, MPA but not luteal phase hormones increased activation of CD4+ T cells in PBMCs. Using a GR antagonist or GR siRNA, it was shown that the GR but not PR-B is required for MPA-, but not luteal phase hormone-induced increased HIV-1 infection in PBMCs and TZM-bls. The presence of PR-B altered the anti-inflammatory, GR-mediated regulation of some key immunomodulatory genes, including GILZ and IL-6, in End1/E6E7 and HeLa cells in response to MPA. In general, basal (unliganded) expression of immunomodulatory genes exhibited a pro-inflammatory profile in the presence of PR-B. Co-immunoprecipitation assays showed that GR and PR-B appeared to associate. Confocal microscopy suggested GR and PR co-localized in the nucleus in response to GR- and/or PRspecific ligands, while super-resolution microscopy showed that co-localization occurred in select regions within the nucleus. Taken together, MPA increases HIV-1 infection in a manner different from that of luteal phase hormones, most likely involving increased CD4+ T cell frequency (CD4+/CD8+ ratio), activation and increased expression of CCR5 on CD4+ T cells, and requiring the GR. Furthermore, PR-B modulates GR-mediated immune function gene regulation, via potential association and region-specific nuclear co-localization. This suggests that the relative levels of GR/PR may play an important role in determining the inflammatory and immune responses and HIV-1 infection in HIV-1 target cells, both in DMPA users and women not using hormonal contraception.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18881号 / 医博第3992号 / 新制||医||1009(附属図書館) / 31832 / 京都大学大学院医学研究科医学専攻 / (主査)教授 小柳 義夫, 教授 松岡 雅雄, 教授 朝長 啓造 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(エネルギー科学) / 甲第19905号 / エネ博第337号 / 新制||エネ||67(附属図書館) / 32982 / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 片平 正人, 教授 森井 孝, 教授 木下 正弘 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM

The Human Immunodeficiency Virus (HIV) which causes Acquired Immunodeficiency Syndrome (AIDS) has caused a global scare with mainly poor African countries suffering the greatest burden. Treatment of HIV is more of palliation rather than cure such that there is no room for treatment interruption if treatment goals are to be met. Antiretroviral treatment is associated with short term and long term side effects which have the potential to negatively impact on the high levels of adherence to treatment that is required to maintain virological suppression and may eventually lead to development of drug resistance and treatment failure. This research aims to identify the extent to which these side effects, through possible poor adherence, impact on treatment successes by measuring the risk that side effects contribute towards treatment failure. Methods Secondary data analysis was conducted on a cohort of patients who initiated ART between 2004 and 2010 at a large tertiary facility in Johannesburg. Patients who were switched to second line ART due to treatment failure were identified. Assessment of side effects on adherence was done. The hazards of side effects among patients switching and not switching to second line were calculated using Cox proportional hazards regression adjusting for other socio-demographic and clinical predictors for treatment failure. Interaction between side effects, gender, age and that of side effects and adherence was investigated. Time dependent covariates were also investigated. Confounding was controlled using multivariate Cox regression analysis. Results There were 5285 patients in the baseline cohort with multiple entry points who contributed 16035 person-years of follow up. The cohort consisted of 63.2% females and 36.8% males. Of these 85.9% were initiated on stavudine (d4T)- based regimen, 7.1% on tenofovir (TDF), 6.3% on zidovudine (AZT)-based regimen and 0.7% on other regimens. The median and mean time at risk per subject was 2.2 and 2.3 years respectively. A total of 770 episodes of side effects due to first line ART were experienced with some patients recording multiple side effects at different time points. Adherence data were found to be missing and incoherent in some of the regimen dosages and could not be used to objectively compare patients. There were 430 patients who were switched to second line ART due to treatment failure. Relative to the group of no side effects, the adjusted hazard ratios for mild, moderate and severe side effects were 1.40 (95% CI=0.94-2.09) p=0.10; 1.72 (95% CI=1.35-2.20) p<0.01 and 1.24 (95% CI=0.65-2.35) p=0.52 respectively. Therefore, overally side effects did not seem to play a role in the time to switch to second line ART. Sex, baseline CD4 cell count, the period during which ART was initiated and the time between date of testing HIV positive and date of initiating were significantly associated with the time to switching to second line ART. Conclusion The study informs that side effects overally may not play a significant role in switching patients from first line to second line ART with the exception of moderate side effects. However, patients who experience side effects should be closely monitored and adequately counselled to help them cope with the side effects so that optimal adherence levels are maintained. Availability of adherence scores or additional information on pills that should have been taken on periods during which pills were reported to have been missed would have made the research more valuable by allowing objective comparison of adherence among patients.

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