No description available.

No description available.

No description available.

No description available.

Malaria is a vector-borne disease that is still responsible for high human morbidity and mortality. Of the five Plasmodium species that can cause malaria in humans, Plasmodium falciparum is regarded the most virulent species. The most fundamental component of sustained control and eradication efforts is the development of effective drugs for malaria treatment and prophylaxis. Plasmodium falciparum’s sexual stages (gametocytes) are not associated with malarial pathogenesis or the clinical symptoms, but they are responsible for the transmission of the disease from human hosts to mosquitos. As such, the development of gametocytocidal interventions that targets the transmission stage to break the disease’s lifecycle forms the basis of efforts towards malaria elimination and eradication. However, despite the importance of this developmental stage, the biology and pharmacology of gametocytes are still very poorly understood. This thesis has set out to gain a better understanding of the identity of gametocyte-active antimalarials and a deeper understanding of the mechanisms underpinning the activity. Using a newly generated luciferase-reporting transgenic line, pharmacodynamic gametocyte studies could be performed to help characterise the activity of selected known reference antimalarials, new potential gametocyte inhibitors in pre-clinical development as well as newly developed fully synthetic compounds designed against the sexual stages. This novel assay revealed that the efficacy of active tested compounds is highly stage-specific. Of all the tested reference antimalarial drugs, MB and DHA were the most potent antimalarial across all gametocyte stages and importantly they were active at clinically relevant levels. These observations were progressed further, developing a time- dependent killing assay that was performed with different concentrations of targeted drug over discrete time intervals to determine the drug’s kill rate. These parameters were then used to simulate the PK/PD relationship of the drug in order to estimate gametocyte clearance profiles during the human treatment period (Chapter 3 and 4). A main focus of the thesis was conducted to better understand the mechanism of drug activity of the 8-aminoquinolines against gametocytes. The ability of a series of 8-aminoquinolines (primaquine as the parent drug, synthesised metabolites in chapter 5, three novel analogues and tafenoquine in (chapter 6) to interact with CYP2D6 was tested by measuring their ability to specifically inhibit the metabolism of fluorescently-tagged tracer substrate by recombinant human CYP 2D6. Reaction products from the CYP metabolites and HLM were then used to test firstly their ability to kill gametocytes, and then to establish their ability to generate hydrogen peroxides and finally measure their haemolytic toxicity. At 10 µM, primaquine CYP metabolites showed activity against the gametocytes that was higher than that of the parent drugs, with the exception of tafenoquine which, interestingly, demonstrated good activity and haemolytic toxicity as a parent drug. These analyses are presented and discussed in the context of strategies that aim at the discovery and development of new transmission-reducing antimalarial drugs.

To understand the mechanisms of insecticide resistance in Anopheles stephensi, DDTand pyrethroid-resistant lines were selected in the laboratory. Selection process showed fixation of L1014S-kdr mutation in DDT- and pyrethroid resistant colonies. Similar fixation was recorded in unselected mosquitoes (control) as well, suggesting low fitness cost of L1014S in laboratory conditions. Synergistic assays using piperonyl butoxide (PBO), a mixed function oxidase and esterase inhibitor, led to reversal of resistance in pyrethroid resistant mosquitoes but no reversal in DDT resistant mosquitoes was recorded. Elevated GST activity in laboratory strains led us to characterize the DDT resistance implicated GST epsilon genes. Quantitative-PCR showed elevated expression of AsGSTe2 and AsGSTe4 in DDT-resistant mosquitoes in both laboratory selected DDT-resistant strain and field caught mosquitoes. These observations prompted further investigations to molecularly characterize these genes examining mutational changes and the possible roles of allelic variation. Cloning and sequencing of the full genes revealed polymorphism which resulted in four variants in AsGSTe2 and three variants in AsGSTe4. Of the four variants of AsGSTe2, two variants (AsGSTe2.1 and AsGSTe2.2) found in DDT-resistant individuals were expressed in vitro in E.coli. Recombinant expression and DDTase assays of AsGSTe2.1 and AsGSTe2.2 showed them to efficiently metabolise DDT. DDTase activity examined for recombinant AsGSTe4 (AsGSTe4.1 and AsGSTe4.2 and AsGSTe4.3) showed that they did not metabolise DDT. Enzyme thermostability tests showed AsGste2 variants to be highly unstable compared to the orthologues in An. gambiae, Aedes aegypti and its corresponding AsGSTe4 variants. Further examination into the GST epsilon array provided evidence of tandem coduplication of AsGSTe2 and AsGSTe4 together in the GST-epsilon array of the laboratory DDT-resistant colony.

Background: Severe bacterial infections and sepsis are a major global cause of mortality and morbidity and with the number of antibiotic resistance cases on the rise. Despite the introduction of treatment guidelines such as those implemented by the surviving sepsis campaign mortality remains high and new therapies are desperately needed. One new therapy, which may be of benefit, is P4 therapy the combination of the immunomodulating peptide P4 and intravenous immunoglobulin (IVIG). The ability of P4 to augment phagocytosis in models of pneumococcal infections, decreasing bacterial burden and improving survival has previously been shown. This thesis goes on to investigate the efficacy of P4 therapy in models of Gram-negative infections, with and without antibiotics and in ex vivo studies from patients with severe community acquired pneumonia (CAP). Methods: Murine models of Escherichia coli and Klebsiella pneumoniae infection were used to evaluate the efficacy of P4 peptide with IVIG in the treatment of severe Gram-negative infections. Flow cytometry and ELISA were used to assessed immune responses to infection with P4 treatment. Neutrophils from patients with severe CAP were isolated and their responses to P4 assessed with ex vivo phagocytosis assays and flow cytometry. In vivo and ex vivo studies were performed with naïve mice and tissue culture cell lines to evaluate the effect of P4 on neutrophil receptor expression and the binding of P4 peptide to cells. Results: Treatment with P4 and IVIG in combination with antibiotics led to significant improvements in survival and bacterial burden in Klebsiella pneumoniae infection. Treatment of Escherichia.coli infection with P4 and IVIG in combination with antibiotics showed no benefits over treatment with antibiotic with IVIG, this was likely due to the infection being too severe. In neutrophils from CAP patients increases in bacterial killing when treated with P4 in phagocytosis assays were seen in 60% of patient. Patients who did not respond to the P4 treatment showed higher levels of IL-8 and IL-10 in their serum and higher disease severity scores. Conclusions: P4 treatment showed efficacy in the treatment of Klebsiella infection but data from E. coli infections and ex vivo treatment of CAP patient neutrophils suggest that infection severity and levels of IL-8 and IL-10 may effect treatment success. P4 could be a potential new treatment option for patients with severe bacterial infections but further studies are needed to better establish which patients would benefit from this treatment and the influence of host immune status on treatment efficacy.

Malaria remains a global health and economic issue affecting nearly half of the world's population. In the past decade, effective chemotherapy and vector control have been the major interventions used to control and reduce the burden of malaria. However, resistance to antimalarial drugs and insecticides is compromising the control and treatment strategies and the goal to eliminate malaria. In most malaria endemic countries, artemisinin combination therapies (ACTs) are the first line treatment for uncomplicated Plasmodium falciparum malaria, the most lethal cause of malaria. Despite the widespread use of artemisinin-based therapies, the mechanism of action of this class of drug remains elusive. Emergence of resistance to ACTs in South East Asia is a global concern for drug efficacy. In this thesis, a click chemistry coupled with mass spectrometry (MS) proteomics approach was used to identify the molecular targets of artemisinin in various stages of P. falciparum strain 3D7 and extensively applied to the candidate trioxolanes, a new class of fully-synthetic artemisinin-like drugs. Using artemisinin activity-based probes, a number of biological targets were identified, these targets derive from key biological pathways/process that include; haemoglobin metabolism, glycolysis, nucleic acid and protein biosynthesis, antioxidant defence and oxidative stress response. The identified fingerprint of biological targets was similar between semi synthetic artemisininin and fully synthetic next generation artemisinins. Identified biological targets were enriched with glutathionylated proteins, indicating that these proteins are vulnerable to endoperoxide antimalarial inhibition and loss of function. The shared protein targets or protein pattern of semisynthetic artemisinin and fully synthetic trioxolane suggest that they might share similar mechanism or action and, possibly, mechanism of resistance. This raises the concern of cross resistance between them. The ring stage parasites which showed the least sensitivity to artemisinins and associated with resistance to artemisinins have much less proteins identified, including the absence of proteins in haemoglobin metabolism and reduction in proteins of major pathways. These findings support the working hypothesis that artemisinin is most effective against later stages of the parasite in line with the activity of haemoglobin digestion, the main activator of artemisinins and other endoperoxides. The reduced sensitivity during the ring stage is possibly due to less activation of artemisinin. A whole genome sequence comparative approach was undertaken with parasites displaying phenotypic artemisinin resistance (slow clearance phenotype) derived from an experimental in vivo model of infection. Parasite genes that we correlated to the slow clearance phenotype included genes involved in the unfolded protein response pathway consistent with recent models of parasite resistance to artemisinins. The results presented in this thesis using chemical biology and omics technologies, have contributed to our understanding of the mechanism of action and resistance of endoperoxides and offer future research directions to study this important class of antimalarials.

Recent years have seen the emergence of two serious coronavirus pathogens with the emergence of the MERS-CoV still an ongoing concern. In addition, the recent unprecedented Ebola outbreak has claimed more than 10,000 lives and affected the lives of countless more. All three of these viruses have been linked through differing strands of research to the second largest mammalian family, Chiroptera, the bats. Bats are among the most diverse a widespread of all mammalian species and have become subject of intensive research in recent years as various bats species have been linked to a number of severe viral outbreaks. In the studies described in this thesis attempts were made to develop, pseudotyped viruses (PV) bearing the glycoprotein of a number of highly pathogenic viruses including MERS-CoV, Ebolavirus, Marburgvirus and SARS-CoV coupled with envelope-defective Human Immunodeficiency Virus and envelope-defective Murine Leukemia Virus. These tools were then used to examine the potential for cross reactivity among related coronaviruses and a number of computational tools were employed to investigate the phenomenon and attempt to develop a better understanding of the antigenic regions that are responsible for the observed cross reactivity. The next stage in the thesis involved attempts to develop a novel form of multiplex assay that made use of PV to attempt to make serological screening of bat specimens more feasible and efficient. The novel bat-borne influenza A haemagglutinin H17 was then successfully incorporated into the PV system and screening of this PV against a number of cell lines led to improved understanding of the viral tropism and the role protease plays in this tropism. The final set of experiments carried out in this thesis involved a combination of computational biology and PV based protocols to both predict patterns of viral evolution through selection analysis and to then test these predictions in the PV framework. This study lead to the generation of a number of mutant MERS-CoV spike proteins and Ebolavirus glycoproteins. These were then incorporated into the PV system and the effects of these mutations of PV production and serum neutralization were investigated.

Campylobacter jejuni is the most prevalent cause of bacterial diarrhoea worldwide and is frequently associated with severe post-infectious complications such as the Guillain-Barre syndrome. Despite the serious health burden caused by the bacterium disease pathogenesis remains ill defined. Human (3-defensins (hBDs)), a family of epithelial antimicrobial peptides, are a major component of host innate defence at mucosal surfaces. In the present study we investigated the effect of C. jejuni on intestinal epithelial innate responses. Up-regulation of IL-8, hBD-2 and hBD-3 gene and peptide expression was observed in Caco-2 and HT-29 cell-lines in response to C. jejuni strains 11168H and 81-176. Furthermore, recombinant hBDs were found to exhibit potent bactericidal activity against C. jejuni suggesting a major role for these peptides in disease pathogenesis. Secondly, we aimed to identify host receptor(s) involved in sensing of C. jejuni and initiating innate defence. Given the invasive nature of infection, we investigated the potential role of cytoplasmic nucleotide-binding oligomerisation domain (NOD) proteins. Using small interfering (si) RNA targeting NODI and transfection of NOD2 overexpression plasmids, we identified NODI as a major pattern recognition receptor involved in mediating innate host defence to C. jejuni while NOD2 was found to play a minor role. Additionally, reduced NODI expression resulted in an increased number of intracellular C. jejuni thus highlighting a critical role for NODI mediated antimicrobial defence in limiting infection. In the final part of the study an ex-vivo model of C. jejuni infection using human intestinal biopsies was developed. Additionally, a vertical diffusion chamber system was utilised to improve culture conditions in C. jejuni infection models. In conclusion, this study highlights the important role of intestinal innate host defence to C. jejuni. The development of new and improved models of infections has the potential to provide previously unavailable opportunities to study C. jejuni disease pathogenesis.