Relating to risk : sexual behaviour and risk perception among men who have sex with men

Background: Men who have sex with men (MSM) are a key population for HIV worldwide. Overall HIV incidence is declining but over the past years the proportion of HIV diagnoses attributed to sex between men has remained high in Europe. MSM account for 40% of the reported HIV diagnoses in Europe (2016) and more than 47% in Sweden (cases reported transmitted in Sweden, 2017) despite the notion that HIV is preventable through treatment and effective public health measures. A matrix of social, legal, biological, epidemiological and behavioural causes contribute to the disproportionate vulnerability for HIV infection among MSM in general and to greater extent among certain sub-groups of MSM. Aim: The overall aim of this thesis is to explore and analyse risk factors and risk behaviour for HIV transmission among MSM. Methods: This research project employed quantitative (I-III) and qualitative (V) methods. Studies I-III were undertaken in Sweden and based on data collected with stratified sampling among men in a web community for LGBT persons. The cross-sectional survey, MSM2013, reached 2 751 MSM residing in Sweden. Study IV was based on semi-structured in-depth interviews with 15 MSM recruited through respondent driven sampling and analysed with content analysis. Results: In study I, a weighted Latent Class Analysis with covariates and distal outcomes was conducted to identify four sub-groups of MSM sharing similar sexual practice characteristics: experimentals, bottoms, riskreducers and clubbers. Experimentals clearly stood out from the other classes with its broad sexual repertoire and high self-rated HIV preventive knowledge. In study II, we found that many MSM (40%) test for HIV regularly and that outreach and promoting initiatives contribute to testing. Still, factor analysis revealed that fear, anxiety, low risk perception and inaccessible test services remain barriers for testing. In study III and IV, we concluded that MSM of different HIV status meet in gay-oriented venues both in their home countries and when travelling abroad. We documented that MSM having anal intercourse without condom with casual partners abroad are high sexual risk-takers with broad sexual repertoire. The in-depth interviews in study IV captured a priority target group for HIV prevention: a highly mobile and highly sexually active subgroup of MSM with high HIV and STI risk taking behaviour regardless of context. Berlin provides venues and spaces for sexual liberation greatly appreciated by this subgroup of MSM. Conclusions: This thesis has shown that the diversity among MSM, based on similar sexual practice patterns, should be considered when designing HIV preventive interventions. The finding, that sub-groups of MSM have different sexual practice patterns can provide a base for screening-instruments, medical and behavioural history forms and counselling guides within healthcare and social work, as well as a base for allocation of resources for prevention. This thesis has also highlighted that preventive interventions focusing solely on increasing knowledge may not be an effective method to reach and decrease HIV risk behaviour among MSM with broad sexual repertoire and high numbers of sex partners. These men may specifically benefit from a HIV and STI testing routine that includes counselling. Healthcare professionals providing care for MSM who test for HIV regularly should remember that this group is reachable and a priority for HIV preventive interventions. Healthcare professionals and prevention workers meeting MSM should have MSM competency and be comfortable and competent to deliver easily accessible test services, including outreach test promotion/testing. Within HIV prevention and HIV test promotion in settings where MSM from different countries and contexts meet there is a need to further prioritise a needs-based approach to risk reduction measures

An RNA-binding compound that stabilizes the HIV-1 gRNA packaging signal structure and specifically blocks HIV-1 RNA encapsidation.

Background NSC260594, a quinolinium derivative from the NCI diversity set II compound library, was previously identified in a target-based assay as an inhibitor of the interaction between the HIV-1 () stem-loop 3 (SL3) RNA and Gag. This compound was shown to exhibit potent antiviral activity. Here, the effects of this compound on individual stages of the viral lifecycle were examined by qRT-PCR, ELISA and Western blot, to see if its actions were specific to the viral packaging stage. The structural effects of NSC260594 binding to the HIV-1 gRNA were also examined by SHAPE and dimerization assays. Results Treatment of cells with NSC260594 did not reduce the number of integration events of incoming virus, and treatment of virus producing cells did not affect the level of intracellular Gag protein or viral particle release as determined by immunoblot. However, NSC260594 reduced the incorporation of gRNA into virions by up to 82%, without affecting levels of gRNA inside the cell. This reduction in packaging correlated closely with the reduction in infectivity of the released viral particles. To establish the structural effects of NSC260594 on the HIV-1 gRNA, we performed SHAPE analyses to pinpoint RNA structural changes. NSC260594 had a stabilizing effect on the wild type RNA that was not confined to SL3, but that was propagated across the structure. A packaging mutant lacking SL3 did not show this effect. Conclusions NSC260594 acts as a specific inhibitor of HIV-1 RNA packaging. No other viral functions are affected. Its action involves preventing the interaction of Gag with SL3 by stabilizing this small RNA stem-loop which then leads to stabilization of global packaging signal region (psi or ψ). This confirms data, previously only shown in analyses of isolated SL3 oligonucleotides, that SL3 is structurally labile in the presence of Gag and that this is critical for the complete psi region to be able to adopt different conformations. Since replication is otherwise unaffected by NSC260594 the flexibility of SL3 appears to be a unique requirement for genome encapsidation and identifies this process as a highly specific drug target. This study is proof of principle that development of a new class of antiretroviral drugs that specifically target viral packaging by binding to the viral genomic RNA is achievable.This work was supported by grants from the Biomedical Research Centre (RCCT.EFPO to JK and AML) and the Medical Research Council (RCAG/565 to AML)

A scale-free analysis of the HIV-1 genome demonstrates multiple conserved regions of structural and functional importance

HIV-1 replicates via a low-fidelity polymerase with a high mutation rate; strong conservation of individual nucleotides is highly indicative of the presence of critical structural or functional properties. Identifying such conservation can reveal novel insights into viral behaviour. We analysed 3651 publicly available sequences for the presence of nucleic acid conservation beyond that required by amino acid constraints, using a novel scale-free method that identifies regions of outlying score together with a codon scoring algorithm. Sequences with outlying score were further analysed using an algorithm for producing local RNA folds whilst accounting for alignment properties. 11 different conserved regions were identified, some corresponding to well-known cis-acting functions of the HIV-1 genome but also others whose conservation has not previously been noted. We identify rational causes for many of these, including cis functions, possible additional reading frame usage, a plausible mechanism by which the central polypurine tract primes second-strand DNA synthesis and a conformational stabilising function of a region at the 50 end of env.Work in the University of Cambridge Department of Medicine is supported by the Biomedical Research Centre (cambridgebrc.nihr.ac.uk) and the Clinical Academic Reserve. This work was supported by the National Institute for Health Research (www.nihr.ac.uk) to JPS (grant number ACF-2015-14-002). The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. CKI is supported by a Medical Research Council (mrc.ukri.org) Confidence in Concept grant (grant number RCAG/655)

Expression of Herpes Simplex Virus Thymidine Kinase/Ganciclovir by RNA Trans -Splicing Induces Selective Killing of HIV-Producing Cells

Antiviral strategies targeting hijacked cellular processes are less easily evaded by the virus than viral targets. If selective for viral functions, they can have a high therapeutic index. We used RNA trans-splicing to deliver the herpes simplex virus thymidine kinase-ganciclovir (HSV-tk/GCV) cell suicide system into HIV-producing cells. Using an extensive in silico bioinformatics and RNA structural analysis approach, ten HIV RNA trans-splicing constructs were designed targeting eight different HIV splice donor or acceptor sites and were tested in cells expressing HIV. Trans-spliced mRNAs were identified in HIV-expressing cells using qRT-PCR with successful detection of fusion RNA transcripts between HIV RNA and the HSV-tk RNA transcripts from six of ten candidate RNA trans-splicing constructs. Conventional PCR and Sanger sequencing confirmed RNA trans-splicing junctions. Measuring cell viability in the presence or absence of GCV expression of HSV-tk by RNA trans-splicing led to selective killing of HIV-producing cells using either 3′ exon replacement or 5′ exon replacement in the presence of GCV. Five constructs targeting four HIV splice donor and acceptor sites, D4, A5, A7, and A8, involved in regulating the generation of multiple HIV RNA transcripts proved to be effective for trans-splicing mediated selective killing of HIV-infected cells, within which individual constructs targeting D4 and A8 were the most efficient.The reagent pNL4-3 was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH from Dr. Malcom Martin. This work was supported by the National University of Singapore (NUSCambridge Start-Up grant number R-182-000-163-646), Medical Research Council grant (G0800142), Medical Research Council Confidence in Concept award, the Biomedical Research Centre, and a Higher Education Funding Council for England award via the Stevenage Bioscience Catalyst

Duplex formation between the template and the nascent strand in the transcription-regulating sequences is associated with the site of template switching in SARS - CoV-2.

Funder: Clinical Academic ReserveFunder: NUS Department of MedicineFunder: Biomedical Research CentreRecently published transcriptomic data of the SARS-CoV-2 coronavirus show that there is a large variation in the frequency and steady state levels of subgenomic mRNA sequences. This variation is derived from discontinuous subgenomic RNA synthesis, where the polymerase switches template from a 3' proximal genome body sequence to a 5' untranslated leader sequence. This leads to a fusion between the common 5' leader sequence and a 3' proximal body sequence in the RNA product. This process revolves around a common core sequence (CS) that is present at both the template sites that make up the fusion junction. Base-pairing between the leader CS and the nascent complementary minus strand body CS, and flanking regions (together called the transcription regulating sequence, TRS) is vital for this template switching event. However, various factors can influence the site of template switching within the same TRS duplex. Here, we model the duplexes formed between the leader and complementary body TRS regions, hypothesizing the role of the stability of the TRS duplex in determining the major sites of template switching for the most abundant mRNAs. We indicate that the stability of secondary structures and the speed of transcription play key roles in determining the probability of template switching in the production of subgenomic RNAs. We speculate on the effect of reported variant nucleotide substitutions on our models

Individually Modified Saliva Delivery Changes the Perceived Intensity of Saltiness and Sourness

Individuals vary largely in their salivary flow and composition, and given the importance of saliva on perception of taste, this might influence how the tastant stimuli are perceived. We therefore hypothesise that altering the individual salivary flow rates has an impact on the perceived taste intensity. In this study, we investigated the role of saliva amount on the perceived taste intensity by excluding parotid saliva and adding artificial saliva close to the parotid duct at preset flow rates. Significant decreases in perception with increasing salivary flow rates were observed for citric acid and sodium chloride. This can partially be explained by a dilution effect which is in line with previous studies on detectable concentration differences. However, since the bitterness and sweetness remained unaffected by the salivary flow conditions and the dilution effect was comparable to that of saltiness, further explanation is needed. Furthermore, we investigated whether the suppression of taste intensity in binary mixtures (taste–taste interactions) could possibly be caused by the increased salivary flow rate induced by an additional taste attribute. The results show, however, that suppression of taste intensity in binary mixtures was not affected by the rate of salivation. This was more likely to be explained by psychophysics

Evaluation of CD46 re-targeted adenoviral vectors for clinical ovarian cancer intraperitoneal therapy

Ovarian cancer accounts for >140 000 deaths globally each year. Typically, disease is asymptomatic until an advanced, incurable stage. Although response to cytotoxic chemotherapy is frequently observed, resistance to conventional platinum-based therapies develop rapidly. Improved treatments are therefore urgently required. Virotherapy offers great potential for ovarian cancer, where the application of local, intraperitoneal delivery circumvents some of the limitations of intravenous strategies. To develop effective, adenovirus (Ad)-based platforms for ovarian cancer, we profiled the fluid and cellular components of patient ascites for factors known to influence adenoviral transduction. Levels of factor X (FX) and neutralizing antibodies (nAbs) in ascitic fluid were quantified and tumor cells were assessed for the expression of coxsackie virus and adenovirus receptor (CAR) and CD46. We show that clinical ascites contains significant levels of FX but consistently high CD46 expression. We therefore evaluated in vitro the relative transduction of epithelial ovarian cancers (EOCs) by Ad5 (via CAR) and Ad5 pseudotyped with the fiber of Ad35 (Ad5T*F35++) via CD46. Ad5T*F35++ achieved significantly increased transduction in comparison to Ad5 (P<0.001), independent of FX and nAb levels. We therefore propose selective transduction of CD46 over-expressing EOCs using re-targeted, Ad35-pseudotyped Ad vectors may represent a promising virotherapy for ovarian cance

<i>ABCB1</i> (MDR1) induction defines a common resistance mechanism in paclitaxel- and olaparib-resistant ovarian cancer cells

BACKGROUND: Clinical response to chemotherapy for ovarian cancer is frequently compromised by the development of drug-resistant disease. The underlying molecular mechanisms and implications for prescription of routinely prescribed chemotherapy drugs are poorly understood. METHODS: We created novel A2780-derived ovarian cancer cell lines resistant to paclitaxel and olaparib following continuous incremental drug selection. MTT assays were used to assess chemosensitivity to paclitaxel and olaparib in drug-sensitive and drug-resistant cells±the ABCB1 inhibitors verapamil and elacridar and cross-resistance to cisplatin, carboplatin, doxorubicin, rucaparib, veliparib and AZD2461. ABCB1 expression was assessed by qRT-PCR, copy number, western blotting and immunohistochemical analysis and ABCB1 activity assessed by the Vybrant and P-glycoprotein-Glo assays. RESULTS: Paclitaxel-resistant cells were cross-resistant to olaparib, doxorubicin and rucaparib but not to veliparib or AZD2461. Resistance correlated with increased ABCB1 expression and was reversible following treatment with the ABCB1 inhibitors verapamil and elacridar. Active efflux of paclitaxel, olaparib, doxorubicin and rucaparib was confirmed in drug-resistant cells and in ABCB1-expressing bacterial membranes. CONCLUSIONS: We describe a common ABCB1-mediated mechanism of paclitaxel and olaparib resistance in ovarian cancer cells. Optimal choice of PARP inhibitor may therefore limit the progression of drug-resistant disease, while routine prescription of first-line paclitaxel may significantly limit subsequent chemotherapy options in ovarian cancer patients

The DNA damage response to adeno-associated virus : centrosome overduplication and induction of cell death independently of the spindle checkpoint

Summary: Adeno-associated virus type 2 (AAV2) is a small virus containing single-stranded DNA of approximately 4.7kb in size. Both ends of the viral genome are flanked with inverted terminal repeat sequences (ITRs), which serve as primers for viral replication. Previous work in our laboratory has shown that AAV2 DNA with ultraviolet radiation-generated crosslinks (UV-AAV2) provokes a DNA damage response in the host cell by mimicking a stalled replication fork. Infection of cells with UV-AAV2 leads to a p53-and Chk1-mediated cell cycle arrest at the G2/M border of the cell cycle. However, tumour cells lacking the tumour suppressor protein p53 cannot sustain this arrest and enter a prolonged impaired mitosis, the outcome of which is cell death. The aim of my thesis was to investigate how UV-inactivated AAV2 kilts p53-deficient cancer cells. I found that the UV-AAV2-induced DNA damage signalling induces centriole overduplication in infected cells. The virus is able to uncouple the centriole duplication cycle from the cell cycle, leading to amplified centrosome numbers. Chk1 colocalises with centrosomes in the infected cells and the centrosome overduplication is dependent on the presence of Chk1, as well as on the activities of ATR and Cdk kinases and on the G2 arrest. The UV-AAV2-induced DNA damage signalling inhibits the degradation of cyclin B 1 and securin by the anaphase promoting complex, suggesting that the spindle checkpoint is activated in these mitotic cells. Interference with the spindle checkpoint components Mad2 and BubR1 revealed that the UV-AAV2-provoked mitotic catastrophe occurs independently of spindle checkpoint function, This work shows that, in the p53 deficient cells, UV-AAV2 triggers mitotic catastrophe associated with a dramatic Chk1-dependent overduplication of centrioles and the consequent formation of multiple spindle poles in mitosis. Résumé Le virus associé à l'adénovirus type 2 (AAV2) est un petit virus contenant un simple brin d'ADN d'environ 4.7kb. Des expériences antérieures dans notre laboratoire ont montré que les liens intramoléculaires sur l'ADN de AAV2 provoqués paz l'irradiation aux ultraviolets (UV) ressemblent à une fourche de réplication bloquée, ce qui provoque une réponse aux dommages à l'ADN dans la cellule hôte. L'infection des cellules avec UV-AAV2 résulte en un arrêt du cycle cellulaire à la transition G2/M entraîné par les protéines ATR et Chk1. Cependant, les cellules tumorales auxquelles il manque le suppresseur de tumeur p53 ne peuvent pas tenir cet arrêt et entrent dans une mitose anormale et prolongée qui se terminera par la mort cellulaire. Le but de ma thèse était d'étudier comment l'AAV2 inactivé par l'irradiation UV tue les cellules cancéreuses n'ayant pas p53. Je montre ici que le signal de dommages à l'ADN induit par UV-AAV2 génère une surduplication des centrioles dans les cellules infectées. Le virus est capable de dissocier le cycle de duplication du centriole du cycle cellulaire ce qui crée un nombre amplifié de centrosomes. Chk1 est co-localisé avec le centrosome dans les cellules infectées et la swduplication du centrosome est dépendante de la présence de Chk1, de l'activité des kinases ATR et Cdk et de l'arrêt en G2 de la cellule. Le signal d'ADN endommagé induit par UV-AAV2 réprime la dégradation des protéines cycline B1 et securine par le complexe promoteur de l'anaphase (APC), ce qui suggère que le point de contrôle du fuseau mitotique est activé dans ces cellules en mitose. L'étude d'interférence avec des éléments du point de contrôle du fuseau mitotique, Mad2 et BubR1, a révélé que la catastrophe mitotique provoquée paz UV-AAV2 survient indépendamment du point de contrôle du fuseau mitotique. Ce travail montre que dans les cellules déficientes en p53, UV-AAV2 induit une catastrophe mitotique associée à une surduplication des centrioles dépendant de Chk1 et ayant pour conséquence dramatique la formation de multiples fuseaux mitotiques dans la cellule en mitose

Ramism, Rhetoric and Reform : An Intellectual Biography of Johan Skytte (1577–1645)

This thesis is an intellectual biography of the Swedish statesman Johan Skytte (1577–1645), focusing on his educational ideals and his contributions to educational reform in the early Swedish Age of Greatness. Although born a commoner, Skytte rose to be one of the most powerful men in Sweden in the first half of the seventeenth century, serving three generations of regents. As a royal preceptor and subsequently a university chancellor, Skytte appears as an early educational politician at a time when the Swedish Vasa dynasty initiated a number of far-reaching reforms, including the revival of Sweden’s only university at the time (in Uppsala). The contextual approach of the thesis shows how Skytte’s educational reform agenda was shaped by nationally motivated arguments as well as by a Late Renaissance humanist heritage, celebrating education as the foundation of all prosperous civilizations. Utilizing a largely unexplored source material written mostly in Latin, the thesis analyzes how Skytte’s educational arguments were formed already at the University of Marburg in the 1590s, where he learned to embrace the utility-orientated ideals of the French humanist Petrus Ramus (1515–1572). Moreover, the analysis shows that the expanding Swedish state administration in the early seventeenth century was in urgent need of educated civil servants, and that this basic demand favored an ideology based on education, skill and merit. It is shown that Skytte skillfully combined a Ramist and patriotic rhetoric with narratives of individual merit and rewards, conveying not least himself as an example. The thesis argues that Skytte’s rhetoric reflects the formation of a new professional category in the Swedish society, one that was distinguished from the royal courtier, the clergyman, the merchant, the warrior, and the scholar. This category is the professional civil servant whose identity was dependent on skills and education