The production of antibiotics, antifungal, enzymes and anti-tumoral agents of economical importance in Streptomyces lividans occurs during the copperdependant morphological switch step of its distinct lifecyle. However, copper can be toxic to the cell if it is not well regulated, affecting copper homeostasis. The regulation of the concentrations of copper is performed by CsoR, a Cu(I)-metalloregulator of the CsoR/RcnR family, on upon Cu(I) binding, it dissociates from its own csoR regulon. This event leads to Cu(I) to be trafficked outside the cytosol via a CopZ chaperoning system. Although Cu(I)-bound structures of CsoR/RcnR family members have been solved, its still unclear how CsoR dissociates from DNA upon Cu(I) binding and how promiscuous its metal ion binding site is, i.e., if it other metals bind and trigger a similar allosteric response as Cu(I) does. Through a structural and kinetic approach, these questions were explored on this work, in order to give insights at atomic and mechanistic level in this metalloregulator family. A novel CsoR structure at pH 6 revealed a striking quasi-Cu(I) bound state, which provides important information on how CsoR may bind to DNA. A mechanism of metal binding to Cu(I) and a non-cognate metal, Ni(II) is proposed, with novel insights on metal selectivity and specificity in this poorly understood family of bacterial metalloregulators.

Giardiasis, cause by the parasitic protozoan Giardia duodenalis, is one of the most common infectious gastrointestinal diseases in humans worldwide. However, its true population burden and epidemiology and in particular its zoonotic transmission potential are still poorly understood. Furthermore, G. duodenalis is not a uniform parasite but a complex of seven genetic assemblages or cryptic species (named A to G) that infect humans and a variety of domesticated and wild animals, and that can only be distinguished using molecular genotyping methods. Although there is some evidence that the two Giardia assemblages infecting humans (namely A and B) may differ in their virulence and major transmission routes, data are still scarce. In the UK, several studies suggested that giardiasis is considerably under-diagnosed and a few data are available on the genetic diversity of the parasite causing infection and disease in this country. We investigated the burden, clinical outcomes, risk factors and molecular diversity of giardiasis in North West England using both a descriptive and analytical approach. In Chapter 2, we analysed the self-reported clinical and exposure data collected over four years from clinical cases of giardiasis in Central Lancashire, as part of an enhanced surveillance program on the illness. The resulting average disease rate of 22.5 cases/100,000 population was high when compared to the available national figures. Giardiasis was particularly abundant in adults in their 30s and children under five, and the disease rate in males was significantly higher than in females. Furthermore, the clinical picture of the cases confirmed the high morbidity associated with this infection particularly in terms of the length of illness and severity of symptoms. Only 32% of the cases reported foreign travel during the exposure window. The results suggested the presence of a hidden burden of disease in adults and males, and indicated that local transmission of Giardia can be more common than expected. In Chapter 3, we performed a case-control study to determine the significant risk factors for symptomatic giardiasis in North West England, by recruiting clinical cases of Giardia and age and sex matched controls from Central and East Lancashire and Greater Manchester. The multivariable logistic regression analysis done on 118 cases and 226 controls revealed that overall travelling abroad (particularly to developing countries) was an important risk factor for the illness (OR 9.59). Following the exclusion of participants that reported foreign travel, four risk factors were significant for the acquisition of giardiasis: going to a swimming pool (OR 2.67), changing nappies (OR 3.38), suffering irritable bowel syndrome (OR 3.66) and drinking un-boiled water from the tap (OR 8.17). The results indicated the important role of swimming pools and contact with children in nappies for the transmission of the parasite. In Chapter 4, whole faecal DNA was extracted from the faecal samples of the cases part of the surveillance and case-control studies and the Giardia assemblages and sub-assemblages causing infection were determined using PCR amplification and DNA sequencing of up to four parasite genes (beta-giardin, glutamate dehydrogenase, triose-phosphate isomerase and small-subunit ribosomal RNA). The majority of infections (64%) were caused by assemblage B, followed by assemblage A (33%), whereas mixed-assemblage infections were rare (3%). The majority of the assemblage A isolates belonged to the sub-assemblage AII and showed completed identity with previously described isolates, and six multi-locus genotypes were identified. The level of genetic sub-structuring as revealed by phylogenetic analysis was significantly higher in assemblage B isolates compared with A isolates: a higher proportion of novel assemblage B sequences was detected compared to what was observed in assemblage A isolates. A high number of assemblage B sequences showed heterogeneous nucleotide positions that prevented the unambiguous assignment to a specific sub-assemblage. Up to 17 different assemblage B multi-locus genotypes were found. The molecular genotyping results showed that Giardia assemblage B was responsible for the majority of the clinical infections and confirmed the occurrence of a high diversity of parasite multi-locus genotypes. In Chapter 5, we integrated the epidemiological and the molecular data generated by the enhanced surveillance and case-control studies and we studied the clinico-epidemiological differences between cases infected with Giardia assemblage A or B. Our results showed a difference in the age prevalence between the two assemblages, with assemblage A being more common in older cases. Cases infected with assemblage B reported a series of symptoms more frequently than cases infected with assemblage A, as well as reporting a longer illness. Although the exposure profile of the cases largely overlapped between the two assemblages, two different types of exposures were reported more frequently in the two groups of cases: keeping a dog in assemblage A cases and the presence in the household of children and children at nursery in assemblage B cases. The results suggested that assemblage A could have a major zoonotic reservoir, whereas assemblage B could be transmitted more commonly via the human-to-human route.

53BP1 is a DNA damage responsive protein that plays a crucial role in checkpoint activation and DNA repair. In addition to its involvement in the cellular response to DNA damage, it has been suggested that 53BP1 can also function to regulate gene expression. 53BP1 was originally identified in a yeast two-hybrid screen for novel modulators of p53 transcriptional activity. Despite this, the role of 53BP1 in transcriptional regulation remains poorly understood. To investigate the effect of 53BP1 on cellular transcription, a microarray approach was utilised to study the gene expression patterns in cells treated with and without 53BP1 siRNA, before and after ionising radiation. Microarray analysis identified numerous genes whose expression was regulated by 53BP1 in the absence and presence of DNA damage. These data suggest that 53BP1 functions as a transcriptional regulator. In support of this, in vitro and in vivo studies have shown that 53BP1 binds to the transcriptional co-activators, CBP and p300. These findings indicate that the binding of 53BP1 to CBP and p300 may be facilitating its role as a regulator of transcription.

Flow cytometry (FCM) was utilized for monitoring the physiology of \(E.\) \(coli\) cells in orange juice (OJ) as well as a model orange juice (MOJ). Compared to FCM, plate counts highly underestimated the true number of viable cells in OJ. As a part of this study, the effects of the change in major components of OJ on viability of the cells in OJ and MOJ was investigated using FCM. Increase in ascorbic acid and amino acid concentrations of MOJ improved both the culturability and FCM viability of the cells. FCM was also employed for studying the effects of OJ clarification on viability of \(E.\) \(coli\) in OJ. Although, reduction in cloud content of OJ increased the number of healthy cells, however, the removal of cloud particles of larger than 0.7 μm appeared to increase the antimicrobial efficacy of particles of smaller than 0.7 μm. The effects of washing E. coli cells with available chlorine, H\(_2\)O\(_2\) and organic acids on their subsequent viability in OJ was also investigated. While increase in concentration of sanitizers resulted in a significant reduction in healthy populations, the total number of viable cells either remained constant or increased particularly in case of H\(_2\)O\(_2\)-washed cells.

Advances in DNA sequencing technologies, particularly the advent of next generation sequencing (NGS) platforms, have revolutionised the field of metagenomics and allowed great progress to be made in the way that microbial communities are analysed. However, the wealth of data now available thanks to these advancements has made the possibilities far more numerous than just the obvious applications, with a wide variety of novel and diverse studies conceivable. The technologies themselves have also created further areas for research as better methods of handling the, often overwhelming in quantity and misleading in content, data are sought. The analysis carried out in this thesis demonstrates the wide range of study possible stemming from two experiments involving the sequencing of meiofauna DNA. The first of these involves community analysis of marine benthic meiofauna with particular emphasis on diversity and distribution. The second experiment involves the sequencing of pooled nematode samples in order to investigate the effects of sample richness and species relatedness on the generation of chimeric reads in sequencing data. It is shown that the data generated from these two experiments can be used to help formulate an algorithm to simulate PCR and therefore assist the generation of realistic noisy NGS data. These data can, in turn, be used to generate a simulated in silico microbial community for analysis, the results of which reveal insights into the accuracy of chimera detection software and the reliability of metagenetic community analyses. Worryingly, these results suggest that findings from similar in vitro studies are not as reliable as originally perceived. The same experimental data may also be used to investigate interactions between meiofauna species based on the incidental presence of prey species highlighted from the sequencing of individual meiofauna organisms. It is shown that these data can be used to accurately predict a nematode’s feeding type without having to examine the organism directly. It is also shown that there is no correlation between this method of inferring interactions between species and other methods which have been used in the past. This suggests that the earlier methods are inadequate when used for the detection of feeding interactions.

Trypanosoma brucei is the protozoan parasite causing African trypanosomiasis, a neurological disease that affects humans and farm-stock in the impoverished sub-Saharan areas where tsetse fly transmission vector is endemic. Although it has great impact on public health and local economies, it has been neglected in drug discovery for almost a century. Current treatments are either toxic or of difficult administration, besides having serious risks of inducing resistance. Protein kinases are the primary set of signaling proteins in eukaryotes, including Trypanosoma brucei. Their druggability has been widely exploited in cancer research, and has been established in the parasite too. A recent kinome-wide RNAi screen with 176 individual cell lines of mammalian infective bloodstream forms of Trypanosoma brucei identified protein kinases required for proliferation in vitro. In order to investigate which protein kinases are also essential virulence factors in vivo, lines were pooled, inoculated into mice and screened for loss of fitness after 48 hours RNAi compared to uninduced controls. The presence of trypanosomes in the bloodstream was assessed using RNAi target sequencing (RITseq) and compared to an in vitro control. This revealed 49 protein kinases with a significant loss of fitness in vivo in two independent experiments, and a strong correlation between in vitro and in vivo loss of fitness for the majority. However, depletion of nine protein kinases affected more pronouncedly the growth in vivo than in vitro. Amongst these protein kinases were several with putative functions related with stress responses mediated through the PI3K/TOR or MAPK signaling cascades including CK2A2, a promiscuous protein kinase whose activity can be stress-induced; two MAP3Ks, involved in cell integrity upon osmotic shock; VPS15, component of the PI3K complex with roles in autophagosome formation and vesicular trafficking; BUD32, transducer of the PI3K/TOR pathway involved in translational regulation; and FAZ20, a parasite-specific pseudo-kinase localizing to the flagellum attachment zone. The other three have been implicated in repair of alkylation-induced cellular damage: SRPK1, a stress response RNA splicing regulator; AUK2, which acts during mitosis; and CAMKL, an AMPK with calcium-binding domains putatively involved in metabolic regulation. Identification of these virulence-associated protein kinases provides new insights in T. brucei-host interaction and reveals novel potential drug targets for protein kinase inhibitors. This RNAi screen revealed that the evolutionary divergent NEK kinase Repressor of Differentiation Kinase 2 (RDK2) has severe loss of fitness both in vitro and in vivo. Depletion of RDK2 had been shown previously to promote differentiation from bloodstream to procyclic-like forms causing the parasite’s death. Further investigation showed RDK2 to be an active protein kinase capable both of phosphorylating a substrate and to autophosphorylate. Protein kinase activity could be ablated by mutation of lysine 70 to methionine. Mutation of both serine residues (195 and 197), identified as sites of phosphorylation by phosphoproteomics, to alanine or glutamic acid, preventing and mimicking phosphorylation respectively, had no effect on protein kinase activity, suggesting they do not have a direct regulatory role on protein kinase activity. Introducing in the RNAi line a recoded RDK2 whose transcript eluded interference, permitted to some extent the rescue of the induced phenotype, while introducing a recoded inactive mutant did not. This may suggest that the lack of kinase activity was responsible for the RNAi phenotype and not depletion of the protein alone. RDK2 RNAi-differentiated cells could be maintained in conditioned procyclic form media for more than a week. However, they were unable to proliferate. Overexpression of RDK2 blocked the differentiation mediated by sequential treatment with 8-pCPT-cAMP and citrate/cis-aconitate (CCA). RNAi experiments in combination with known differentiation cues, suggested that when differentiation is triggered by the CCA signalling pathway, RDK2 inactivation happens downstream of the phosphatase TbPTP1. Differentiation caused by RDK2 inactivation could be traced in flow cytometry by the detection of EP procyclin expression. This was exploited in a cell-based mechanism-directed phenotypic screen for RDK2 inhibitors. A preliminary run with 518 drug-like molecules that had shown protein kinase inhibition, trypanocidal activity and/or activation of the EP procyclin promoter, unveiled 6 compounds triggering EP procyclin expression and parasite death in the low micromolar range. These compounds can be investigated further to assess whether RDK2 is their in vivo target.