An Exploration of the Relationships between Markers of Social Status and Position and HIV Risk Behaviours in African, Caribbean, and Other Black Populations

African, Caribbean, and other Black (ACB) people are a priority group for HIV prevention in Canada, but little is known about the epidemiology of HIV risk in this population. The overall goal of this thesis is to guide HIV prevention interventions for ACB communities. It focuses on social factors that impact HIV vulnerability. This research used data from the Black, African, and Caribbean Canadian Health Study—a mixed methods study that used 30 semi-structured interviews, and a cross-sectional survey using a structured, self-administered quantitative questionnaire to collect information about HIV and health from 188 ACB people. The first manuscript compares risk perceptions to the social epidemiology of HIV risk. ACB people generally perceived their personal HIV risk to be low and they focused on sexual risks. Service providers’ perceptions about HIV risk behaviours were sometimes inconsistent with ACB people’s experiences. Quantitative results confirmed that HIV risk was mainly sexual. There were few gender-based differences in risk behaviours. Those living in poverty were more likely to be abstinent and use condoms. Born Canadians had the highest prevalences of forced sex, mixing alcohol or drugs with sex, and past STI diagnoses. Stable employment was associated with higher prevalences of not using condoms and past STI diagnoses. The second manuscript identified social and proximate determinants of HIV testing in the past year. Approximately 20% of ACB people had tested for HIV in the past year. Testing for HIV was independently associated with higher education, stable immigration classes, living in Canada foryears, and gender and ethnicity combined. Proximate determinants mediating these relationships included: lower English language proficiency, greater HIV knowledge, and higher numbers of lifetime and past-year sex partners. The third manuscript ascertains social and proximate determinants of the frequency of condom use. About 20.5% of sexually active ACB adults used condoms consistently. Male gender, wealth, unstable immigration classes, and less secure employment statuses status were independently associated with the frequency of condom use. Proximate determinants mediating these relationships included: not having a cohabiting regular partner, not disliking condoms, having one lifetime sex partner, and having a history of unwanted sex

Preparation and topology of the Mediator middle module

Mediator is the central coactivor complex required for regulated transcription by RNA polymerase (Pol) II. Mediator consists of 25 subunits arranged in the head, middle, tail and kinase modules. Structural and functional studies of Mediator are limited by the availability of protocols for the preparation of recombinant modules. Here, we describe protocols for obtaining pure endogenous and recombinant complete Mediator middle module from Saccharomyces cerevisiae that consists of seven subunits: Med1, 4, 7, 9, 10, 21 and 31. Native mass spectrometry reveals that all subunits are present in equimolar stoichiometry. Ion-mobility mass spectrometry, limited proteolysis, light scattering and small-angle X-ray scattering all indicate a high degree of intrinsic flexibility and an elongated shape of the middle module. Protein–protein interaction assays combined with previously published data suggest that the Med7 and Med4 subunits serve as a binding platform to form the three heterodimeric subcomplexes, Med7N/21, Med7C/31 and Med4/9. The subunits, Med1 and Med10, which bridge to the Mediator tail module, bind to both Med7 and Med4

Minimal components of the RNA polymerase II transcription apparatus determine the consensus TATA box

In Saccharomyces cerevisiae, multiple approaches have arrived at a consensus TATA box sequence of TATA(T/A)A(A/T)(A/G). TATA-binding protein (TBP) affinity alone does not determine TATA box function. To discover how a minimal set of factors required for basal and activated transcription contributed to the sequence requirements for a functional TATA box, we performed transcription reactions using highly purified proteins and CYC1 promoter TATA box mutants. The TATA box consensus sequence is a good predictor of promoter activity. However, several nonconsensus sequences are almost fully functional, indicating that mechanistic requirements are not the only selective pressure on the TATA box. We also found that the effect of a mutation at a certain position is often dependent on other bases within a particular TATA box. Although activators and coactivators strongly influence TBP recruitment and stability at promoters, neither Mediator, the activator Gal4-V16, nor TFIID specifically compensate for the low transcription levels of the weak TATA boxes. The addition of Mediator to purified transcription reactions did, however, increase the functional selectivity for certain consensus TATA sequences. Transcription in whole-cell extracts or in vivo with these TATA box mutants indicated that factors, other than those in our purified system, may help initiate transcription from weak TATA boxes

Med5(Nut1) and Med17(Srb4) Are Direct Targets of Mediator Histone H4 Tail Interactions

The Mediator complex transmits activation signals from DNA bound transcription factors to the core transcription machinery. In addition to its canonical role in transcriptional activation, recent studies have demonstrated that S. cerevisiae Mediator can interact directly with nucleosomes, and their histone tails. Mutations in Mediator subunits have shown that Mediator and certain chromatin structures mutually impact each other structurally and functionally in vivo. We have taken a UV photo cross-linking approach to further delineate the molecular basis of Mediator chromatin interactions and help determine whether the impact of certain Mediator mutants on chromatin is direct. Specifically, by using histone tail peptides substituted with an amino acid analog that is a UV activatible crosslinker, we have identified specific subunits within Mediator that participate in histone tail interactions. Using Mediator purified from mutant yeast strains we have evaluated the impact of these subunits on histone tail binding. This analysis has identified the Med5 subunit of Mediator as a target for histone tail interactions and suggests that the previously observed effect of med5 mutations on telomeric heterochromatin and silencing is direct

MC EMiNEM Maps the Interaction Landscape of the Mediator

The Mediator is a highly conserved, large multiprotein complex that is involved essentially in the regulation of eukaryotic mRNA transcription. It acts as a general transcription factor by integrating regulatory signals from gene-specific activators or repressors to the RNA Polymerase II. The internal network of interactions between Mediator subunits that conveys these signals is largely unknown. Here, we introduce MC EMiNEM, a novel method for the retrieval of functional dependencies between proteins that have pleiotropic effects on mRNA transcription. MC EMiNEM is based on Nested Effects Models (NEMs), a class of probabilistic graphical models that extends the idea of hierarchical clustering. It combines mode-hopping Monte Carlo (MC) sampling with an Expectation-Maximization (EM) algorithm for NEMs to increase sensitivity compared to existing methods. A meta-analysis of four Mediator perturbation studies in Saccharomyces cerevisiae, three of which are unpublished, provides new insight into the Mediator signaling network. In addition to the known modular organization of the Mediator subunits, MC EMiNEM reveals a hierarchical ordering of its internal information flow, which is putatively transmitted through structural changes within the complex. We identify the N-terminus of Med7 as a peripheral entity, entailing only local structural changes upon perturbation, while the C-terminus of Med7 and Med19 appear to play a central role. MC EMiNEM associates Mediator subunits to most directly affected genes, which, in conjunction with gene set enrichment analysis, allows us to construct an interaction map of Mediator subunits and transcription factors

Comparative genomics supports a deep evolutionary origin for the large, four-module transcriptional mediator complex

The multisubunit Mediator (MED) complex bridges DNA-bound transcriptional regulators to the RNA polymerase II (PolII) initiation machinery. In yeast, the 25 MED subunits are distributed within three core subcomplexes and a separable kinase module composed of Med12, Med13 and the Cdk8-CycC pair thought to control the reversible interaction between MED and PolII by phosphorylating repeated heptapeptides within the Rpb1 carboxyl-terminal domain (CTD). Here, MED conservation has been investigated across the eukaryotic kingdom. Saccharomyces cerevisiae Med2, Med3/Pgd1 and Med5/Nut1 subunits are apparent homologs of metazoan Med29/Intersex, Med27/Crsp34 and Med24/Trap100, respectively, and these and other 30 identified human MED subunits have detectable counterparts in the amoeba Dictyostelium discoideum, indicating that none is specific to metazoans. Indeed, animal/fungal subunits are also conserved in plants, green and red algae, entamoebids, oomycetes, diatoms, apicomplexans, ciliates and the ‘deep-branching’ protists Trichomonas vaginalis and Giardia lamblia. Surprisingly, although lacking CTD heptads, T. vaginalis displays 44 MED subunit homologs, including several CycC, Med12 and Med13 paralogs. Such observations have allowed the identification of a conserved 17-subunit framework around which peripheral subunits may be assembled, and support a very ancient eukaryotic origin for a large, four-module MED. The implications of this comprehensive work for MED structure–function relationships are discussed

HIV Risk Perceptions and the Distribution of HIV Risk among African, Caribbean and Other Black People: Mixed-Methods Results from the BLACCH Study

BACKGROUND: African, Caribbean and other Black people (ACBP) are a priority group for HIV prevention efforts in Canada. ACBP and service providers’ (SP) perceptions about HIV risk impact the uptake and delivery of prevention messages. These perceptions may not reflect actual risk among ACBP, so it is important to assess them and identify groups for which they may be valid. Emerging evidence from Sub-Saharan Africa shows that social determinants of health (SDOH) impact the distribution of HIV risk. However, SDOH are context-specific, and to date, virtually no research has explored their impact on HIV risk among ACBP in North America. OBJECTIVE: Compare ACBP’s and SP’s perceptions about HIV risk among ACBP to a corresponding quantitative risk profile. METHODS: Using a community-based approach, a purposive sample of eight SPs and 22 ACBP were recruited for qualitative interviews. They were asked questions about HIV, and their responses are being analyzed using qualitative content analysis to identify themes. Following the interviews, a convenience sample of 188 ACBP completed a quantitative questionnaire covering HIV-related risk behaviours. Quantitative data were analyzed to describe the distribution of HIV risk behaviours according to the following SDOH: gender, poverty status, educational attainment, immigration experience, ethnicity and employment status. Qualitative and quantitative data were compared using concurrent triangulation. RESULTS: Interview participants mainly discussed sexual behaviours related to HIV risk, and the survey results showed that much of the community’s risk is sexual in nature. Risk behaviours acknowledged by ACBP and SP that showed statistically significant variation according to the SDOH include: forced sex, condom use, sex while drunk or high, and abstinence. It appears as though individuals with higher social status (based on SDOH) are at higher risk than those of lower social status. DISCUSSION: Further analyses including statistical weights need to be performed before these results can be finalized

The Black, African and Caribbean Canadian Health (BLACCH) Study: Laying the Foundation for Conducting HIV Epidemiologic Studies with Ethno-Racial Minority Communities in Understudied Urban-Rural Locales

Challenge: London reportedly has Ontario’s third-highest HIV infection rate, and a small (2.2%) but growing African, Caribbean and other Black (ACB) population. These diverse communities historically faced racism, exploitation and social exclusion, which have lasting impacts. Although a target population for HIV prevention, they are difficult to reach for research and programming, especially in London and similar urban-rural locales with few ACB-specific resources. In ACB communities, HIV is most commonly spread through heterosexual contact, yet cultural and religious norms often discourage discussions about sex and sexuality, whether normative or non-normative. Homophobia, racism and HIV-related stigma discourage ACB persons from seeking information about HIV/AIDS. Additionally, few local researchers have worked with ACB communities; service providers are seldom researchers; trust is lacking between ACB communities and service providers; and there are inter- and intra-ethnic separations in ACB communities. Approach: The Black, African and Caribbean Canadian Health Study is an interdisciplinary, mixed-methods, community-based epidemiologic project about health and HIV in London’s ACB communities. This project involved: networking with ACB community members, service providers, and academic researchers; learning about ACB communities through semi-ethnographic work; immersing a multi-disciplinary team in health research; and interviewing community members and service providers. Team members represent AIDS service organizations, a settlement agency serving ethno-racial minority communities, and a university. ACB persons comprise over half of the research team. Discussion: Community-based research is unusual in epidemiology but necessary for conducting good-quality epidemiologic studies in communities like London’s ACB communities. It helps build capacities of service providers, community members and academic researchers to undertake research. Taking our approach, we identified: relevant epidemiologic survey topics; methods for recruiting respondents; and appropriate question formats. The team was able to: build relationships with community members and service providers; promote the project; identify individuals to help recruit respondents; and learn about norms in different communities