Around 35 million people are living with HIV-1 infection globally. Untreated infection results in progression to AIDS and mortality. Current antiretroviral treatment is not curative and does not treat the underlying inflammatory processes caused by the presence of HIV-1, a cytotoxic and tissue toxic virus. HIV-1 triggers overwhelming and dysregulated pro-inflammatory cytokine response during infection, including deranged interleukin-1 beta and interleukin-18 levels. We investigate the mechanisms underlying how HIV-1 activates the inflammasome, a multimolecular complex involved in the production of potent inflammatory markers such as interleukin-1 beta and interleukin-18. The inflammasome is often activated in a two-signal process requiring initial sensing by Toll-like receptors to generate signal 1, priming inflammasome components; and signal 2 sensed by NOD-like receptors which recruit and activate the inflammasome. Human leukocytes were stimulated with live HIV-1 or its cognate envelope proteins gp120 or gp41, or transfected with plasmids encoding HIV-1 viroporin Vpu or gp41. NLRP3 knock-down attenuated inflammasome activation and interleukin-1beta/interleukin-18 secretion following HIV-1, gp120 or gp41 stimulation. gp120 induced inflammasome activation also required the presence of TLR2 and TLR4, suggesting a degree of cooperation in effecting signal 1. Dual NLRP3/NLRC4 knockdown reduced the inflammasome response to HIV-1 gp41 stimulation which was found to be dependent on chloride anion flux across integral lipid rafts in the trans-Golgi network. The results imply a critical role for TLR2 and TLR4 in recognising HIV-1 proteins providing signal 1 for inflammasome formation. Ion fluxes in the cell due to HIV-1 infection are the catalyst that triggers inflammasome activation via NLRP3 or NLRC4, and secretion of interleukin-1 beta and interleukin-18. The findings contribute to the general understanding of HIV-1 recognition by the human innate immune system and help to further clarify precisely how HIV-1 causes striking dysregulation in cytokine profiles in people living with HIV-1 infection.

HIV associated lipodystrophy (HIVLD), an adverse effect of combination antiretroviral therapy (cART), further introduces complexity in the management of HIV infection. There is also increased of cardiovascular disease in HIV patients, even in the absence of HIVLD. cART impairs adipogenesis and dysregulates the secretion of adipokines, fat and glucose metabolism in the adipose tissue. This is seen with both protease inhibitors (PIs) and nucleoside reverse transcriptase inhibitors. The aim of this thesis was to examine the pathogenesis of cART-induced metabolic disturbances using in vitro models (adipocytes) and in vivo (gene association) studies. Plasma levels of IL18, an adipokine associated with insulin resistance, are elevated in HIVLD patients. The murine 3T3-F442A cell line was used as an in vitro model to assess the effects of PIs (lopinavir [LPV], ritonavir [RTV], atazanavir [ATV]) and NNRTIs (efavirenz [EFV]) in the presence or absence of telmisartan (TEL; 1-5μM). All antiretrovirals (ARV) resulted in a dose dependent increase in IL18 protein levels by ELISA (LPV, 330pg/ml+2.1 [p=0.008]; ATV, 267.3+5.1 [p=0.0001]; RTV, 265.7+2.2 [p=0.0005] and EFV, 98.6+2.4 [p=0.0004]) as compared to the vehicle control; this also correlated with IL18 gene expression determined by RT-PCR. Sequenom MALDI-TOF was used to genotype 14 single nucleotide polymorphisms (SNPs) in the IL18 gene in ARV-treated patients with (HIVLD+; n=115) and without LD (HIVLD-; n=51), but no association was identified. ARV treatment also resulted in the upregulation of NFATC4 gene expression (LPV, 1.9+0.05 [p=0.0015]; ATV, 2.1+0.1 [p=0.0007]; RTV, 1.4+0.05[ p=0.0002] and EFV, 1.8+0.85 [p=0.0001]) as compared to the vehicle. Co-incubation with TEL partially attenuated ARV-mediated upregulation of IL18 (1.6+0.2 [p=0.01) and NFATc4 (1.2+0.1 [p=0.0001]). siRNA knockdown of NFATc4 in adipocytes resulted in significant upregulation in the levels of adiponectin and PPARgamma and down-regulation in IL6 secretion levels following treatment with ARVs. The data suggest that ARV-induced upregulation of NFATc4 could be a mediator of ARV-induced adipocyte toxicity and potentially insulin sensitivity. This study also observed changes in the regulation of various miRNAs following ARV treatment. Validation experiments confirmed LPV to result insignificant downregulation of miR-103 (0.5+ 0.01[p=0.002]) and miR-107 (0.4+ 0.03[p=0.001]) along with their target gene, Cav-1 (0.6+ 0.01[p=0.01]) during adipogenesis; this was reversed by co-incubation with Tel. A systematic review of the genetic associations with HIVLD was also undertaken – this identified that polymorphisms in mitochondrial DNA and cytokines had been the primary ones studied using a candidate gene approach. The results showed that several SNP associations have been identified which are biologically plausible, but in almost all cases, replication of the findings in different cohorts has either been contradictory or limited. This may be due to several reasons including small sample sizes, different diagnostic criteria used for HIVLD, differences in genetic strategy, ethnic variation in genetic architecture and differences in drugs used across different cohorts. Although further studies in larger patient groups with HIVLD should be undertaken, this may not be possible given its rarity now in clinical practice, and perhaps the emphasis should switch to insulin resistance per se. In summary, PIs and NNRTIs upregulate proinflammatory cytokines with NFATc4 potentially playing a major role in their transcriptional upregulation. Differential regulation of specific miRNAs were also found to be important in ARV-mediated dysregulation ofadipogenesis; further validation work which evaluates their role in adipocyte toxicity in vitro and in vivo is now warranted. Whilst this study did not find any genetic association with HIVLD, future studies using larger sample sizes and better defined phenotypes are now required.

Whether the epidemiological association between antiretroviral therapy and increased blood pressure is of a causal nature remains largely unknown. The transition from association to causation could represent a fundamental step for taking preventive measures against hypertension and its untoward effects. Such preventive measures are especially crucial for people living with HIV in sub-Saharan African countries, where HIV is most widespread, trends in cardiovascular disease risk factors are rising persistently, and antiretroviral treatment coverage rates are rapidly increasing. Aims and objectives • To review the evidence for the epidemiological associations of antiretroviral therapy with increased blood pressure and other cardiovascular disease risk factors, and to obtain comprehensive estimates of the strengths of these associations in people living with HIV worldwide (Evidence synthesis). • To examine the epidemiological association between antiretroviral therapy and increased blood pressure using Hill’s criteria of causation (mini-review). • To investigate a plausible causal link between antiretroviral therapy and increased blood pressure among HIV-infected patients in a sub-Saharan setting using appropriate statistical methods (primary data analysis). Methods Evidence synthesis: A series of systematic reviews with meta-analyses were conducted to examine the associations of antiretroviral therapy with cardiovascular disease risk factors. Studies were sought from electronic databases and cross-references of relevant articles. Studies in which blood pressure and other cardio-metabolic outcomes were compared between HIV-infected adults naïve and exposed to antiretroviral therapy were eligible for the meta-analyses. The data from these studies were combined using random-effects meta-analyses to obtain pooled estimates of the associations between antiretroviral therapy and each cardio-metabolic outcome. Mini-review: Each of Hill’s criteria of causation was examined separately with regards to the association between antiretroviral therapy and increased blood pressure. Primary data analysis: Four hundred and six HIV-infected adults―306 antiretroviral-exposed and 100 antiretroviral-naïve―were recruited from a tertiary HIV clinic in semi-urban Nigeria between August and November 2014 as part of a cross-sectional study. To assess if antiretroviral therapy improves the prediction of hypertension, candidate logistic regression models for predicting hypertension were compared using Nagelkerke’s R2 and parameter estimates. Structural equation models were fitted to determine the indirect effects of antiretroviral therapy on blood pressure through body mass index, waist circumference, blood glucose level, and sleep quality, while controlling for socio-demographic and clinical characteristics. A propensity score matching model was fitted to examine the average treatment effects on the treated (ATT) of antiretroviral therapy on systolic and diastolic blood pressure. Results Evidence synthesis: Ninety-one observational studies comprising data for 83,669 HIV-infected patients were included in the meta-analyses. Cardio-metabolic measures were significantly higher among antiretroviral-exposed patients, compared to their naïve counterparts as shown in the forest plots below: Summary estimates of the pooled associations between antiretroviral therapy and cardio-metabolic outcomes (continuous) ART, antiretroviral therapy; CI, confidence interval; N, number of studies included in the meta-analyses. Summary plot shows ART is significantly associated with increases in all cardio-metabolic outcome measures. Summary estimates of the pooled associations between antiretroviral therapy and cardio-metabolic outcomes (categorical) CI, confidence interval; N, number of studies included in the meta-analyses. With the exception of combined overweight/obesity, ART is significantly associated with increased risk of cardio-metabolic disorders. Mini-review: The association between antiretroviral therapy and increased blood pressure fulfilled all of Hill’s criteria of causation, with the exception of ‘specificity’. Nonetheless, one would not expect the exposure to be specific in the context of a multifactorial condition, such as high blood pressure. Primary data analysis: Of 400 HIV-infected patients with reported hypertension status, 60 (15%) were diagnosed as hypertensive. Other important findings of primary data analysis include the following: • Without considering antiretroviral treatment status, the best parsimonious model for predicting hypertension comprised age, body mass index, family history of hypertension, and sleep quality (R2 = 0.265). The extent to which these variables fit within the predictive model was improved slightly when antiretroviral treatment status was included (R2 = 0.274). • The indirect effects of antiretroviral therapy on systolic (coefficient = 0.94, 95% CI = 0.73 to 2.43) and diastolic blood pressure (coefficient = 0.64, 95% CI = 0.16 to 2.09) through waist circumference remained statistically significant after adjusting for age, sex, lifestyle factors, CD4 cell count, and HIV infection duration. • After propensity score-matching, 25% of the initial population sample were dropped, so that 229 patients on antiretroviral therapy were matched to 74 antiretroviral-naïve patients. In this propensity score-matched sample, the estimated ATT of the effects of antiretroviral therapy on systolic (7.85 mmHg, 95% CI = 3.72 to 15.68) and diastolic blood pressure (7.45 mmHg, 95% CI = 4.99 to 13.61) remained statistically significant after achieving a balanced distribution of baseline covariates between antiretroviral-naïve and exposed patients. Conclusion Overall, antiretroviral therapy is potentially the single most consistent correlate of high blood pressure (and other cardiovascular disease risk factors) in people living with HIV. These findings also suggest a high probability that the epidemiological association between antiretroviral therapy and increased blood pressure may be causal. People living with HIV in sub-Saharan African countries may benefit from regular hypertension screening and other cardiovascular risk assessments after the commencement of antiretroviral therapy. Future studies should identify what phenotypes on the HIV clinical spectrum are most susceptible to the effects of antiretroviral therapy on blood pressure and other cardio-metabolic parameters, as well as the efficacies of targeted iinterventions on these phenotypes.

HIV is a stigmatised chronic condition which affects approximately 100,000 people on the UK. Sexuality, gender and ethnicity and cultural background are some of the factors which give rise to different experiences of living with HIV; not all experience is well documented. Psychological support is an important part of HIV care; knowledge of socio-cultural context can assist in the provision of therapy through building therapeutic relationships based on understanding. This study looks at Black Caribbean MSM living with HIV and using analytical pluralism, explores their experiences beyond their HIV identity. Through the use of IPA it produces accounts of (dis)connection and inner conflict while a psychosocial lens, utilising the dynamics of the research interview space, notes the self-reliance apparent in the three participants today. It reflects on the methodological benefits of pluralism and considers how counselling psychologists can use the findings to refine their practice when working with sexual and ethnic minorities.

Impairments in executive functions (EF), for example planning and impulsivity, are often identified in people with Human-Immunodeficiency Virus-1 (HIV). Previous research has predominantly used ‘traditional tests’ such as the Stroop, which do not have good ecological validity (Bennett et al., 2005). The main aim of this study was to assess EF in people with HIV using a battery approach with good ecological validity, the Behavioural Assessment of the Dysexecutive Syndrome (BADS) (Wilson et al., 1996). The study used a comparison pilot design to compare performance on the BADS within a sample of participants with HIV to the published normative data. A total of 20 participants with HIV were assessed (13 men; 7 women). On average, participants scored significantly lower on the BADS relative to normative data. Further research should develop the use of neuropsychological batteries with good ecological validity to consider EF impairment in people with HIV. Clinical implications include the potential of EF screening for people with HIV.

Background: The introduction of highly active antiretroviral therapies (HAART) in 1996 to treat patients living with the human immunodeficiency virus type 1 (HIV-1), led to dramatic improvements in their mortality and morbidity. However, high levels of adherence to HAART regimens are required and due to the very nature of HIV-1: its high replicative capacity and lack of a proof reading mechanism, drug resistance mutations emerge, which impact on the ability of the drugs to suppress the patient’s circulating viruses. Genotypic resistance testing can determine whether mutations have developed which confer resistance to specific antiretrovirals (ARV) and thus enhance clinical care. Methods: A clinical cohort database was developed to host the demographic, treatment and resistance mutation data for patients living with HIV-1 across the United Kingdom (UK) who had a genotypic resistance test (tests) conducted as part of their clinical care. These data were pooled and interrogated to determine the evolution and dynamics of resistance in targeted sub-groups of patients including treatment-naïve patients; treatment-experienced patients and their potential susceptibility to new ARV drugs; and the evolution of new subtype profiles within the clinical cohort and the impact of this on clinical outcomes. The over-riding aim of each of the studies was to improve the clinical care of patients with HIV-1 infection in the UK. Results: In the treatment-naïve patient cohort (n=380), a resistance prevalence rate of 16.5% was determined. In the treatment-experienced cohort (n=1,786), the resistance prevalence rate was 68.1%. Of those treatment-experienced, 91.3% would be susceptible to the new ARV Etravirine (ETV) and 89.7% to Darunavir (DRV). In the subtype patient cohort (n=1,642), an increase in the prevalence of pure and recombinant non-B subtypes over time was demonstrated and characterised, as well as the identification of polymorphisms specific to non-B subtypes compared to subtype B. Conclusions: The resistance prevalence rate of >10.0% in the treatment-naïve patient cohort supported the need to conduct genotypic resistance tests for all treatment-naïve patients with HIV-1 infection before commencement of HAART in order to ensure the patient was starting on the optimal first-line treatment regimen to control their virus. National and European guidelines were subsequently amended to reflect this requirement. The treatment-experienced patient cohort analyses confirmed the resistance mutations circulating within the treated HIV-1 community which are the source of transmitted resistance to the treatment-naïve patients. Further analyses of the treatment-experienced cohort suggested two new ARVs which were due to be licenced for use with HIV-1 patients would be “theoretically susceptible”, providing further treatment options for these patients with resistance mutations. The subtype patient cohort work determined that subtype characterisation should be introduced as part of clinical care due to the impact of non-B subtypes on the success of genotypic resistance testing, and the different mutational pathways which might occur, leading to resistance in different subtypes. All these studies provided data and evidence of current issues which impacted on the clinical care of patients living with HIV-1 in the UK and influenced changes in guidelines on how best to manage and improve patient care.

Anti-ganglioside antibodies have been implicated in autoimmune neuropathies for several decades. They are thought to elicit injury through binding to sites in the peripheral nervous system, where they activate the complement pathway to induce cell death. Patient serum is therefore regularly screened for these antibodies to aid in the diagnosis of various conditions. Recent work has found that complexes composed of gangliosides and other glycolipids can improve the detection of these antibodies beyond the signals detected to the single ganglioside species. In MMN research, complexes comprised of GM1 and GalC have been found to significantly enhance antibody detection in patient sera. In certain patients, however, antibody binding was only detected against these complexes and not the single antigens. This led some researchers to hypothesise that an unidentified class of antibody may have arisen that binds specifically to a neoepitope formed by the combination of the two glycolipids. It has also been hypothesised that that this complex may be the true target of immune mediated attack in MMN. This thesis sought to address this hypothesis by either cloning these antibodies directly from patient serum or through active immunisations with mice. Analysis of previously generated human monoclonal antibodies indicated that their behaviours were modified by complexes containing particular gangliosides or glycolipids. Furthermore, the antibodies behaviours were found to diverge, when they were screened against complexes comprised of gangliosides and different concentrations of accessory lipids. These findings suggested that the accessory lipids were interacting with the ganglioside headgroups to modify the presentation of different binding epitopes. This indicated that conformational modulation, rather than neo-epitope formation, may be responsible for complex enhancement Cloning antibodies from patient sera was unsuccessful but examination of the screening techniques suggested that the appearance of complex-dependent antibodies may have been an artefact. Attempts to induce complex-specific responses in mice were similarly unsuccessful but several anti-ganglioside and anti-sulfatide antibodies were created. The subsequent chapters focused on the characterisation of these antibodies and indicated that most of them bound well to solid-phase assays, cells and tissue and may therefore be of use in future studies. Taken together, the data from this thesis suggests that complex-dependent antibodies may not exist but are merely low concentrations of anti-ganglioside antibodies that are cis-enhanced by particular lipids. Future work should therefore focus on assessing how the ganglioside microenvironment modifies epitope presentation and how this affects the binding capabilities of antiganglioside antibodies.

Background Papular pruritic eruption (PPE) of human immunodeficiency virus (HIV) infection is common HIV-infected populations who live in tropical and subtropical regions. It is characterized by chronic and intensely itchy papules that are usually more highly concentrated on the extremities, adversely impacting on quality of life. Its aetiology has been postulated to be an altered and exaggerated immunological response to insect bites or stings. It has been reported to diminish in severity or resolve with antiretroviral therapy (ART). Its presence after at least six months of ART has been proposed as one of several clinical markers of failure of antiretroviral treatment. Objectives 1. To systematically summarise the evidence of interventions for PPE 2. To translate, culturally adapt, and test oral administration of a Runyankore-version of Skindex-16 for use in dermatology research in Mbarara, Uganda 3. To determine factors associated with PPE in HIV-infected Ugandan adults receiving ART for at least 15 months 4. To describe the natural history of PPE in HIV-infected Ugandan adults over two years from the time of ART initiation and explore the association between recurrent or persistent PPE and antiretroviral treatment failure Methods Systematic review of interventions for PPE Electronic searches of Medical Literature Analysis And Retrieval System Online (Medline), Excerpta Medica Database (Embase), Cumulative Index To Nursing And Allied Health Literature (CINAHL), Global Health Library, Cochrane Library, World Health Organization (WHO) International Clinical trials registry and National Library Of Medicine (NLM) gateway were carried out from January 1980 to July 2014. Studies of any design were included. The primary outcome measure for this review was resolution of skin disease. The quality of evidence was assessed using the Newcastle-Ottawa quality assessment scale and Grading Of Recommendation, Assessment, Development And Evaluation (GRADE) approach, where appropriate. Two authors carried out data extraction and quality assessment of studies independently. Runyankore-version of Skindex-16 for oral administration in Mbarara, Uganda Skindex-16 in English was translated to Runyankore, and then back-translated to English. The original and back-translated versions of Skindex-16 were compared for fidelity of translation. The Runyankore-version was administered orally to 47 dermatology patients and 47 random hospital visitors. Study participants were also asked about the characteristics of their skin condition including its duration, presence of skin colour change and ease or difficulty of concealment as well as an open question on how their skin condition has affected them. Case control study examining factors associated with PPE in the ART era This is a case–control study nested within a 515-person cohort receiving care at the HIV clinic of a teaching hospital in Mbarara, Uganda. Forty-five cases and 90 controls were enrolled. Cases had received ART for ≥15 months, fulfilled the clinical case definition of PPE and had skin biopsy findings consistent with PPE. Each case was individually matched with two controls for age, sex and ART duration. Cohort study describing the natural history of PPE over two years from ART initiation This is a cohort study of HIV-infected Uganda adults initiating ART and receiving care at the HIV clinic of a teaching hospital in Mbarara, Uganda who fulfilled the clinical case definition of PPE and had skin biopsy findings consistent with PPE. Standardised interviews, clinical photography, HIV viral load, CD4 counts and CD8+ T-cell activation markers were measured at three-month intervals for two years. Results Systematic review of interventions for PPE No randomised controlled trials were identified. Thirteen studies with a total 188 participants were included. ART was associated with resolution of PPE in a prospective observational study that had high loss to follow-up rates. Two observational studies reported positive responses of PPE to oral antihistamines (promethazine and cetirizine). Pentoxifylline was associated with diminished signs and symptoms of PPE in an uncontrolled open trial and superior to dapsone and a combination of antihistamine and topical corticosteroids in a parallel group non-randomised trial. Resolution of PPE was reported with a combination of topical corticosteroids and oral antihistamines in a case report. The efficacy of ultraviolet B (UVB) phototherapy was reported in an observational study with eight participants and three case studies with a total of five participants. Runyankore-version of Skindex-16 for oral administration in Mbarara, Uganda Oral delivery was feasible, taking ≤10 minutes per subject. High Cronbach α values (0.86, 0.88 and 0.85 for Symptoms, Emotions and Functioning subscales, respectively) demonstrated internal consistency reliability. As hypothesised, subjects with reported skin problems, dyspigmentation and difficulty in concealment had higher mean Skindex-16 scores, indicating construct validity. A large proportion (72.4%) of responses to the open-ended question were addressed in Skindex-16, indicating content validity. Case control study examining factors associated with PPE in the ART era Twenty-five of 45 cases (56%) had histological findings consistent with PPE (known as PPE cases). At skin examination and biopsy (study enrolment), a similar proportion of PPE cases and their matched controls had plasma HIV RNA <400 copies/ml (96% vs. 85%, p=0·31). The odds of having PPE increased four-fold with every log increase in viral load at ART initiation (p=0.02) but not at study enrolment. CD4 counts at ART initiation and study enrolment, and CD4 gains and CD8 T-cell activation measured 6 and 12 months after ART commencement were not associated with the presence of PPE. Study participants who reported daily insect bites had greater odds of being cases [odds ratio (OR) 8.3, p<0.001] or PPE cases (OR 8.6, p=0.01). Cohort study of natural history of PPE over 2 years from ART initiation Seventeen (15 female and 2 male) participants with a median age of 29.8 years were enrolled. Median CD4 count and HIV viral load at ART commencement was 108 cells/mm3 and 114,442 copies/ml, respectively. Resolution of PPE occurred in 13 of 17 (76%) study participants at a median time of 8.5 months after ART initiation, although PPE recurrence was observed at seven participants during the study period. Two participants had persistent PPE. Virological failure was not detected in any study participant. HIV RNA was less than 400 copies/ml at a median time of three months from ART initiation in all study participants. Conclusions 1. The evidence base of interventions for PPE is of low quality. There is some evidence of the efficacy of ART in the management of PPE. Pentoxifylline and phototherapy may have a role in its management but are unlikely to be available in resource-limited settings. Oral antihistamines and topical corticosteroids may be helpful in some individuals affected by PPE. 2. The orally administered Runyankore-version of Skindex-16 is reliable, with construct and content validity, and feasible for use in dermatology research in Mbarara, Uganda. 3. PPE in HIV-infected Ugandan adults receiving ART for at least 15 months was associated with reported daily insect bites and greater HIV viraemia at ART commencement, independent of CD4 count. 4. Recurrent or persistent PPE in HIV-infected Ugandan adults observed over two years from initiation of ART was not associated with virological failure in participants of this study.

CD4+ T cells and macrophages are the principal targets of HIV-1. They can be productively infected with the virus and transfer virions to contacting bystander cells. It has been suggested that soon after initial infection, free virions and virus-bearing or infected T cells and macrophages can enter the brain, triggering a cascade of inflammatory signals and recruitment of other immune cells. Chronic inflammation and increased viral antigens in the brain lead to HIV-1 associated neuropathy. Once free virions or infected cells enter the central nervous system, the first type of brain cells that they are likely to encounter are astrocytes, which extend endfeet around the blood vessels. These cells have been observed to contain virions and viral products, but their permissivity to productive infection has not been clearly demonstrated. By contrast, productive infection of resident microglia and perivascular macrophages is well established. Here, I investigate the permissivity of astrocytes to HIV-1 infection and found no evidence of infection by the free route. However, I found that astrocytes intimately contact HIV-1 infected macrophages and CD4+ T cells and, in some cases, extend filopodial membrane toward the infected cell. In astrocyte-T cell contact sites, termed synapses, virions appear to move along the astrocytic filopodia from the T cell to the astrocyte. In this case, the target cell mediated viral transfer across the intercellular gap. HIV-1-infected macrophages released virus that associated with astrocytes, remaining either on the surface of the astrocytes or within intracellular compartments. HIV-1 bound to astrocytes could be transmitted efficiently to permissive cells in trans. However, astrocyte-associated virus was sensitive to inhibitors including proteases and neutralizing antibodies, suggesting a surface-accessible compartment. This work provides insight into mechanisms of HIV-1 spread in the brain from infected CD4+ T cells and macrophages to astrocytes and their potential as virus reservoirs. I also optimized high resolution, correlative focused ion beam scanning electron microscopy technology to answer fundamental biological questions. I demonstrate the application of the technology to study skeletal muscle cell differentiation mechanisms. I combine the power of genetic mapping with structural analysis to qualitatively and quantitatively describe cellular states and functions. Using semi-automatic image processing analysis, I was able to compute high volumes of data and generate statistics that relate quantitative measurements of cellular structures to functions. The toolset developed here will be instrumental in studying cells and tissues in both research and clinical applications.

Despite current improvement in newborn care, infection is still a common cause of neonatal morbidity and mortality. Innate immunity is the first line of defence against pathogens particularly in newborn infants. Quantitative and functional deficit in non-cellular (complement system) and cellular (neutrophils) arms of innate immune system is believed to contribute to neonatal susceptibility to infection. Neutrophil granule subsets contain a variety of proteases, including elastase, cathepsins, MMP-9 and proteinase 3 along with different granule markers and receptors. This thesis demonstrated that normal term neonatal neutrophils express more proteinase 3 and CD177 on their surface while no differences were found in expression of markers CD35, CD66b and CD63 (representing the secretory, secondary and primary granule subsets, respectively). Cord neutrophils contain more PR3 than adult cells but the proportion of PR3 released by cord and adult neutrophils was similar. In contrast, neonatal neutrophils contained only half of the cathepsin G and elastase functional activity of adult neutrophils. Bronchoalveolar lavage fluid studied from preterm infants ventilated for respiratory distress demonstrated higher proteinase 3 concentrations in lavage samples from infants who went on to develop chronic lung disease than in infants with resolved respiratory distress syndrome. Concentration of proteinase 3 in lavage samples was significantly higher than MMP-9 and elastase levels, suggesting that it may have an important role in disease pathogenesis. Complement is an equally important component of the innate immune system that plays a central role in recruiting and activating neutrophils, as well as being directly bactericidal through the terminal lytic pathway. Analysis of neonatal complement system revealed that Complement function and terminal components levels particularly C9 are deficient (except C7) in healthy term newborn infants compared to normal adults. Bactericidal capacity of a selection of neonatal sera was tested along with adult sera against four serovars of Ureaplasma parvum, a potentially important perinatal pathogen. Results showed impaired bactericidal capacity of neonatal serum compared to adult serum especially against SV1. Ureaplasma SV3 was the most serum sensitive serovar whereas killing of the resistant serovars SV6 and 14 could not be induced by supplementation of the deficient components C6, C8 and C9.