With the increasing prevalence of obesity and the increasing proportion of older adults, type 2 diabetes (T2D) is becoming a massive public health problem. T2D is associated with socioeconomic position (SEP) where individuals from lower SEP suffer a disproportionate burden of the disease and older adults may be particularly vulnerable to experiencing socioeconomic inequalities. Furthermore, T2O complications can cause vast increases in the expenditure of healthcare services and possibly detrimental effects on an individual’s health. During this thesis the effect of SEP on the incidence of T2D and outcome from T2D are investigated in older adults. To examine the relationship between SEP and T2D incidence in older adults, a mediator analysis Is implemented. This analysis is conducted to determine characteristics that influence the association in older adults, identifying possible targets for intervention that could reduce the socioeconomic gap in T2D incidence. Of the risk factors investigated, body mass index was identified as the risk factor that explained the greatest proportion of the association. A register based dataset of older adults with T2D is utilised to examine the effects* of SEP on the outcome from T2D. A novel Markov approach is introduced to model the rate that individuals with T2D transition from diagnosis to complication. By interpreting the underlying phases an insight into the position of an individual on the disease to complication pathway can be gained. The Markov model identified two distinct stages of the transition from T2D diagnosis to complication. Finally, a novel approach is presented in order to calculate a predictive time interval from T2D diagnosis until the occurrence of a complication, achieved using a combination of survival tree, parametric modelling and simulation techniques. Lower SEP individuals are consistently predicted to experience a complication of T2D earlier than a similar group of individuals in higher SEP.

No description available.

Diarrhoeal diseases remain a global health threat and are responsible for high levels of morbidity and mortality worldwide, with an estimated 1.7 billion cases every annum. Additionally, according to the World Health Organisation, diarrhoeal diseases are the second leading cause of death in children under 5 years old. Salmonella are one of the most common diarrhoeal pathogens [1] (WHO Accessed 20 February 2015) with serovars Enteritidis, Typhimurium and Typhi playing a major role in outbreaks worldwide. However, Salmonella enterica serovar Weltevreden (S. Weltevreden) has recently attracted a great deal of interest due to increasing reports of its isolation by reference laboratories around the world, with a particular high incidence in South East Asia. However, relatively little is known about the genotypic or phenotypic properties of this understudied serovar. In this study, phylogenetics and comparative genomics based on whole genome sequences were used to define the genetic diversity within a sizeable collection of S. Weltevreden isolates collected from across the globe, with a focus in South East Asia. This phylogenetic analysis confirmed that the S. Weltevreden isolates belong to a monophyletic clade formed of several sub-clades presenting distinct geographical clustering and characteristics. Phenotypic characterisation was performed on selected isolates, with an aim to dissect aspects of host-pathogen interaction during infection, providing a foundation to compare S. Weltevreden with other serovars such as S. Typhimurium. Interestingly, an overall attenuated pathology was observed both invitro (hep 2 cell line) and in-vivo (murine and zebrafish embryos) for S. Weltevreden compared to the S. Typhimurium reference strain. This is the first report of the phylogenetic analyses of S. Weltevreden and of a systematic in-vitro and in-vivo characterisation of the sub-species.

Background: Several studies suggested association between consanguinity and risk of developing Type 2 Diabetes (T2D). Aim: To examine mechanisms by which consanguinity might increase the risk of T2D in a Saudi population. Methodology: 362 adult male participants were recruited, 179 were T2D patients and 183 healthy participants were siblings of recruited patients. Severity was assessed in patients by recording age at diagnosis. Diabetes risk in healthy subjects was inspected by measuring their body mass index (BMI), fasting blood glucose (FBG), and waist circumference. Extended pedigrees were constructed to calculate inbreeding coefficients. 23 Single Nucleotide Polymorphisms (SNPs) incurring higher risk of T2D were genotyped. All subjects were interviewed to complete food frequency and physical activity questionnaires to provide information on environmental variation between participants. Results: Significant inverse association was detected between inbreeding coefficients and age at diagnosis accounting for environmental covariates (β: -0.572 P-value: 0.012). In 42 families, we were able to recruit 2 healthy siblings from each. Pearson’s correlation coefficient of FBG between siblings was 0.317 (P= 0.04). Correlations between siblings’ FBG increased with increased range of consanguinity suggesting a stronger genetic influence leading to lower variation of FBG between siblings. The effect of consanguinity on variation of FBG was further assessed by fitting a regression line and controlling for difference in age, calorific intake, and level of physical activity (β: -0.118 P-value: 0.024). No significant associations were detected between number of loci identical for risk alleles and age at diagnosis, BMI, FBG or waist circumference. An association of marginal significant was detected between age at diagnosis and total number of risk alleles when accounting for parental history of diabetes and inbreeding coefficients (β:-0.399 P: 0.052). Conclusion: Study’s findings suggest consanguinity might increase risk of T2D by earlier development of the disease, and by strengthening possible genetic effect on FBG.

No description available.

Hepatitis C virus (HCV) is a major public health burden. It is thought to currently infect 2-3% of the world population. Treatment options are limited, a situation which is compounded by the abandonment of regimens due to adverse effects as a resuit of the treatment. Furthermore there are genotype dependent outcomes, some genotypes responding better than others. Consequently, a better understanding of virus-host interactions and the virus life-cycle may provide new insights for developing new treatment strategies. HCV induces autophagy. Autophagy ('self-eating') is a homeostatic process in eukaryotic cells in which regions of cytoplasm, damaged or redundant organelles, insoluble protein aggregates, or invading pathogens are engulfed and sequestered by a double membrane. These double membranes form vesicles and are termed autophagosomes. Autophagosomes then fuse with lysosomes to degrade their contents. The replicative success of HCV and the establishment of an infection has been shown to be dependent upon this cellular process. It has been suggested that HCVinduced endoplasmic reticulum (ER)-stress is the induction mechanism. To understand in more detail the mechanisms that HCV might employ to mediate this crucial process, experiments were conducted with infectious virus and subgenomic repliains to elucidate these processes. During the course of these experiments it was found that Hut' could mediate the induction of autophagy in the absence of ER-stress. ER-stress was mediated by the viral glycoproteins, but their absence did not impede autophagy induction. Furthermore, the viral replicase alone was necessary and sufficient for the induction of autophagy. Autophagosomes and viral replication complexes were found not to colocalize. Furthermore, non-replicative, exogenous viral RNA was found to be a potent inducer of autophagy. A number of pathways leading to autophagy were investigated in order to determine how HCV might induce this process. The host cellular signalling pathways mediated by 5'-AMP-activated protein kinase (AMPK) and class I Ptdlns3K-protein kinase B (AKT), modulated by HCV, were found not to play a role in HCV-mediated autophagy induction. The role of B-cell leukaemia/lymphoma 2 (Bcl-2) in HCV-mediated autophagy remained inconclusive, whilst GTPase Rab5 interference did not prevent ongoing HCV-mediated autophagy. With the importance of autophagy to HCV noted, it was attempted to develop a compound screening assay for autophagy inhibition in collaboration with our industrial partners GlaxoSmithKline. Presented here is the work carried out to understand HCV-mediated autophagy induction along with the insights gained during this study.

The search for tools that target malaria vector that resist insecticides and bite outdoors has become a research priority. Such tools will be necessary for managing residual malaria transmission and hastening the eradication of this devastating disease. This study investigated chemicals that potentially affect the oviposition substrate choices of Anopheles gambiae sensu lato (s.l.). It is foreseen that increased knowledge of the oviposition behaviour of this major malaria vectors and chemicals cues that mediate oviposition site-selection can be applied in the development of additional sampling methods and alternative interventions that to trap gravid malaria mosquitoes outdoors. To achieve a reproducible high egg-laying success of An. gambiae sensu stricto (s.s.) and An. arabiensis four factors were evaluated: (1) the time provided for mating; (2) the impact of cage size, mosquito age and female body size on insemination; (3) the peak oviposition time; and, (4) the host source of blood meals. Then four bioassays were optimised for studying oviposition responses of An. gambiae s.s. in the laboratory and semi-field conditions: a WHO-tube bioassay and a wind-tunnel that detected short-range attraction in the laboratory; a two-tier choice egg-count bioassay that compared the relative proportion of eggs laid in substrates in the laboratory; and a modified BG Sentinel mosquito gravid trap that evaluated long-range attraction of gravid females to olfactory cues in the semi-field. Finally, the oviposition responses of gravid An. gambiae s.s. mosquitoes to water vapour, Bermuda grass hay infusion (hay infusion), and putative semiochemicals identified from the hay infusion and a soil infusion previously shown to elicit higher egg deposition compared to filtered Lake Victoria water (lake water) in two choice egg-count bioassays (Herrera-Varela et al. 2014), were evaluated. High oviposition rates [84%, 95% confidence interval (CI) 77-89%] were achieved when 300 male and 300 blood-fed female An. gambiae s.s. were held together in a cage for four days. The chance of oviposition in the mosquitoes dropped when human host source of blood-meal was substituted with a rabbit (Odds ratio (OR) 0.30, 95% CI 0.14-0.66) but egg-numbers per female were not affected. All four optimised oviposition bioassays effectively showed between 15-20% shifts in oviposition substrate choices of mosquitoes with 80% statistical power and 5% significance. Using the WHO-tube bioassay, gravid An. gambiae s.s. were shown to be 2.4 times (95% CI 1.3-4.7 times) more likely to move towards high humidity in still air compared to non-gravid Preamble mosquitoes. This was more pronounced in the airflow olfactometer where the gravid mosquitoes were 10.6 times (95% CI 5.4-20.8 times) more likely to fly into a chamber with water than a dry chamber. Two-choice egg-count bioassays showed that An. gambiae s.s. were less likely to lay eggs in six-day old hay infusion (OR 0.10, 95% CI 0.03-0.33) compared to lake water. Ten putative semiochemicals were identified from the hay infusion using mass spectrometry and published electrophysiology data: 4-hepten-1-ol, 4-ethylphenol, phenylmethanol, 2-phenylethanol, indole, phenol, 3-methylindole, 3-methyl-1-butanol, 4-ethylphenol, and nonanal. Tested in two-choice egg-count bioassays, the first four listed compounds had no effect on egg deposition at the tested concentrations (between 0.01-5 parts per million) but mosquitoes were less likely to lay eggs in at least one concentration of 3-methylindole (OR 0.39, 95% CI 0.21-0.71), indole (OR 0.57, 95% CI 0.37-0.87), 3-methyl-1-butanol (OR 0.32, 95% CI 0.22-0.47), phenol (OR 0.55, 0.32-0.95), 4-methylphenol (OR 0.32, 0.18-0.57) and nonanal (OR 0.66, 95% CI 0.47-0.91) compared to lake water. In contrast to the hay infusion and hay infusion volatiles, An. gambiae s.s. were about two times more likely to lay eggs in cedrol, a sesquiterpene alcohol identified from the soil infusion, compared to lake water (OR 1.84, 95% CI 1.16-2.91). Cedrol attracted twice as many gravid mosquitoes in the semi-field also (OR 1.92, 95% CI 1.63-2.27). In the field, modified BG-Sentinel traps, electrocuting nets and OviART gravid traps with lake water and cedrol were three times more likely to trap malaria mosquitoes compared to traps with water only (OR 3.3, 95% CI 1.4-7.9). In conclusion, water vapour was shown to be a strong, non-specific pre-oviposition attractant for gravid An. gambiae s.s. in still air and moving air. It is probably the long range cue that gravid An. gambiae s.l. use to detect the presence aquatic habitats beyond the range of chemical cues. Evidence showed that An. gambiae s.s. discriminate between potential oviposition substrates and that this selective process is in-part mediated by volatile organic compounds originating from the site. Water vapour leads gravid mosquitoes to aquatic sites but semiochemicals enable the mosquitoes to discriminate and select between potential habitats. It was demonstrated that synthetic equivalents of semiochemicals found to attract gravid mosquitoes such as cedrol can be used to trap malaria mosquitoes outdoors.

The study of infectious diseases and their spreading mechanisms is fundamental to prevent new infections and limit the eventual death toll. Just as not all diseases have the same characteristics, not all individuals have the same probability of contracting the infection and different scenarios lead to different outcomes. Focusing on the heterogeneity of the populations, we create an interdisciplinary and novel approach that relax the assumption of well-mixed populations, typical of compartmental models, by incorporating state-of-the-art results of empirical and analytical pedestrian studies. By knowing the relationship between crowd density and walking velocity we are able to devise a density-dependent contact rate allowing us to estimate the number of contacts between infectious and healthy individuals in crowded and confined situations. The advantages of this method are: (i) the ability of getting more realistic solutions on the number of new infections and (ii) the possibility of studying a scenario a-priori. This means that just by knowing the size of the population and the extension of the environment we are able to infer the number of contacts between individuals and, consequently, the number of new infections. We apply the method to the London Underground network by using data from Transport for London (TfL). We firstly devise a transportation model that evaluates the amount of time passengers spend in each station in order to walk from entrance to platform and vice-versa. After that we infer the density inside the stations and the number of contacts between passengers. As a validation we compare the results with data of infections (reported by general practitioners) in London boroughs and obtain a clear correlation between the use of public transport and the incidence rate of infections. Further studies using this new type of modelling could help evaluate and, eventually, prevent contagion in most crowded environments such as public transport, offices, schools and hospitals.

Klebsiella pneumoniae is a worldwide concern as it is resistant to most antibiotics. It causes a wide range of pathologies like pneumonia, urinary-tract diseases, community-acquired and nosocomial infections. K.pneumoniae has been shown to prevent the fusion between the phagosome and the lysosome in macrophages in a PI3K/AKT-dependent manner (Cano et al., 2015). We then, postulated that it could activate the kinase mammalian target of rapamycin complex 1 (mTORCl) located downstream PI3K/AKT. mTORCl is involved in multiple cellular processes like the growth, biogenesis and the inhibition of autophagy. We demonstrated that K. pneumoniae, by activating mTORCl, inhibits autophagy, thus, promoting its survival. Moreover, K. pneumoniae controls the levels of cytokines IL-12 and IL-10 in a mTORCl and GSK3p-dependent manner. In addition, during K. pneumoniae infection, levels of IkBα are maintained in a mTORCI-dependent manner. Inflammation is also limited during the infection, through the nuclear translocation of Nrf2 and the activation of an antioxydant, HO1. The pathways TLR4-MyD88/MAL, TLR4-TRAM/TRIF, STING, PI3K/AKT/ERK and FAK are involved in the activation of mTORCl during K. pneumoniae infection. Activation of TRAM/TRIF and STING results in production of type 1 IFN, which is essential for the activation of mTORCl. K. pneumoniae activates mTORCl in a OmpA, 0 antigen, and capsule-dependent manner. Therefore, K. pneumoniae manages to manipulate the host immune system through the activation of mTORCl.

Vector competence is a complex characteristic which governs an insect's ability to acquire, support the development and transmit a parasite from one host to another. It influences variation in disease transmission among mosquito populations, hence affecting disease epidemiology. In this project, I have studied some aspects of ecological interactions and genetic factors in a step towards understanding how these affect variation in disease transmission and exploiting these in future disease control programmes. Mosquito gut bacteria affect the development of parasites ingested by mosquitoes. As different bacterial species have different effects, dissimilarities in gut composition could be an important cause of variation in vector populations. The first study investigates the gut microbiome of mosquitoes collected from Kenya. Using 454 pyrosequencing of 16S rRNA, I provide a comprehensive catalogue of the gut composition of 8 species of mosquitoes (Chapter 2). I show that while there is greater variation within host species (fixation index= 0.64), different mosquito species tend to have rather similar gut bacteria. An individual mosquito gut has a low diversity of bacteria with, the microbiota being dominated by a single Operational Taxonomic Unit. This suggests that gut bacteria may be one factor influencing within-species variation in disease transmission, and a minor factor in between-species variation. Wolbachia endosymbionts are able to reduce the intensity and development of RNA viruses and metazoan parasites in their insect hosts, blocking the transmission of such parasites. This makes Wolbachia a likely candidate for control programmes. I extend the investigation of naturally-occurring bacteria to Wolbachia (Chapter 3). Using the gut samples used in Chapter 2, I amplify the Wolbachia surface protein gene to identify Wolbachia infections. I identify Wolbachia in Aedes bromeliae, a vector of yellow fever, and its close relative Aedes metallicus and in Mansonia uniformis and Mansonia africana, which are competent vectors of human bancroftian filariasis. Aedes bromeliae showed the highest prevalence (75%) suggesting that this strain of Wolbachia may be manipulating the host reproduction by cytoplasmic incompatibility. Using a multi locus typing system and accounting for effects of recombination in the construction of bacterial phylogeny, I show that these mosquito Wolbachia strains cluster into supergroups A and B of Wolbachia. The phylogeny also shows significant recombination events indicating horizontal transfer events between taxa. These Wolbachia strains, isolated from the disease vectors, may be reducing parasite intensity and transmission, and could be a better choice for transinfecting other mosquito vectors rather than distantly related strains. Previous studies show that high frequency of susceptibility to Brugia pahangi exists among populations of Aedes aegypti from East Africa, providing an excellent resource for investigating variation in a natural population. I test the frequency of susceptibility of peri-domestic subpopulations of Aedes aegypti collected from Kenya to Brugia malayi (Chapter 4). The results are consistent with previous data with up to 30% of individuals being susceptible. The number of susceptible individuals varied significantly between populations (Fisher's exact: p= 0.03). These populations now provide the resource to identify polymorphisms associated with susceptibility to Brugia and also enable comparison with results obtained from laboratory strains. In Chapter 5, I continue with efforts to identify and map quantitative trait loci (QTL) associated with Brugia susceptibility in Aedes aegypti. However, with the Aedes genome still highly fragmented with many supercontigs having no chromosomal assignments, mapping the gene to a definitive locus is almost impossible. Using an improved DNA-based mapping technology, Restricted-site Associated DNA tags (RADtags), I make novel assignments of 79 supercontigs to the 3 chromosomes of Aedes aegypti. These new assignments account for 122Mb of the genome, increasing the percentage genome mapped to /approx 40%. The technique also identifies potential scaffold misassemblies and misassignments of supercontigs to chromosomes. I also use the same method to prepare libraries for sequencing which will provide more markers and allow mapping and identification of candidate genes which can be evaluated for involvement in susceptibility to Brugia infections. Aedes aegypti and Anopheles gambiae share similarities in their immune proteins, but little is known about the functions of immune proteins in Aedes aegypti. To be able to make functional comparisons between mosquito vectors, I inoculate Sephadex beads into a laboratory strain of Aedes aegypti to investigate the expression of pathogen recognition genes (Chapter 6). Thioester-containing proteins (TEPs) show significant up-regulation (p= 0.03-0.0002) with up to 7-fold increase in gene expression of TEP20 in immune-challenged individuals compared to non-challenged controls. TEP20 is an orthologue of Anopheles gambiae TEP1, emphasising the evolutionary function of TEPs in immune activation. As TEP1 is an important determinant of vectorial capacity in Anopheles gambiae, this indicates that TEPs may also be an important factor influencing variation in susceptibility to pathogens in Aedes aegypti. Generally, this project has contributed to three broad areas of factors that influence variability in diseases transmission by mosquitoes: ecological interactions with bacteria, host genetic background and immune system. The results, resources and techniques used in this thesis can be widely used in further studies in these areas and extended to other mosquito vectors and natural populations.