In this thesis I aimed to provide base-line data to inform conservation of neotropical otters (Lontra longicaudis ) at both the range-wide and local (Mexico) scale. In Chapter 2, I compared three commonly used preservation methods for faecal DNA in order to identify the best method for neotropical otter faeces under challenging field conditions and long-term storage: 1) ambient-temperature drying, 2) a two-step protocol involving incubation in 95% ethanol and posterior silica desiccation, and 3) RNAlater. The results of this experiment showed that that RNAlater provides the highest mtDNA amplification success. In Chapter 3, I looked into the demographic history, genetic diversity and genetic structure of L. Longicaudis in Mexico using mtDNA. I found high genetic structure among North and South regions of the country, potentially due to geographic formations. Analyses of demographic history in Mexico indicated a recent expansion coinciding with the end of the Pleistocene. Given that recent evidence supports the existence of three subspecies of L. longicaudis across its range, I combined mtDNA haplotypes identified in this study with available Central and South American haplotypes in order to examine phylogeographic patterns; as a result, a distinct lineage distributed in North and Central America (NCAM) was identified. Due to the monophyly of this lineage, I propose to consider it a distinctive Evolutionary Significant Unit (ESU). In Chapter 4, I used landscape genetics to identify landscape features that affect otter geneflow in Mexico by means of microsatellites. I looked into the effect of elevation, slope, river networks and land cover on geneflow at a country-wide scale and two regional scales (North and South Pacific). I used Bayesian clustering to examine country-wide genetic structure. In terms of landscape genetics, elevation and slope were the only variables that explained genetic distance among individuals at the range-wide and North Pacific scale, respectively. The results of Bayesian clustering indicated two population clusters roughly distributed in the North and South of Mexico. The results of this thesis suggest that non-invasive methods can be applied to inform conservation efforts for Neotropical otters. I suggest that the NCAM lineage should be considered a distinct Evolutionary Significant Unit (ESU) throughout the range of L.longicaudis. Within Mexico, it is recommended to plan conservation corridors for the species where naturally low elevations and slopes allow genetic connectivity.

Doxorubicin (DOX) is one of the most potent anthracycline antibiotics used for treatment of multiple tumor types including breast cancer treatment. However, its efficacy is limited by fatal toxicities associated with DOX therapy causing damage to healthy tissues and organs. In this project, a high molecular weight gelatin-doxorubicin (GDOX) conjugate was synthesized and purified. Gelatin was linked to DOX in this conjugation via glygly spacer and an acid labile hydrazone bond. GDOX was characterized for molecular weight distribution of gelatin by size exclusion chromatography. Drug load on the conjugate was measured by UV-visible spectroscopy. DOX release from conjugate was measured at three different pH: 4.8, 6.5 and 7.4. A minimal DOX release of 9% ± 3 % was observed at systemic pH (7.4) and maximum release of 49 ± 1.8 % was observed at tumor microenvironment at pH 4.8. GDOX was then evaluated in an in vitro model consisting of two breast cancer cell lines: MCF7 cells and a triple negative breast cancer (TNBC) MDA-MB-231 cells for intracellular trafficking with emphasis on lysosomal targeting. Cell uptake and localization of an equivalent concentration of 10 µM DOX in GDOX was determined with fluorescence microscopy in these cells and fluorescence quantification was performed with Fiji software. Whole cell fluorescence showed localization of GDOX in both cell lines with higher conjugate accumulation in MDA-MB-231 cells. Lysosomal membrane permeabilization (LMP) was studied using a fluorescent labelled dextran. After 24 h, GDOX triggered 100% LMP in TNBC cells but surprisingly no LMP was observed in MCF7 cells. Finally, to evaluate GDOX induced DNA damage in these cells, TUNEL assay was performed which labels DNA strands breaks using a fluorescent tag. Images were captured on a fluorescent microscope and evaluated for percent apoptotic cell count. A similar extent of apoptosis was seen in both the cell lines and was comparable to free DOX. Overall, these results suggest that conjugate has potential of inducing toxicity to TNBC cells via lysosomal pathway which may induce apoptosis and thus has a potential for treatment of TNBC tumors. However, MCF7 cell studied results indicates a different pathway of toxicity in inducing apoptosis.

In Guinea-Bissau (GB) the Guinea baboon (Papio hamadryas papio) is threatened by hunting pressure. Along with local extinctions, these practices may be inducing long-term genetic changes and disrupting underlying social structure. In this study, the bushmeat trade in GB was evaluated for the first time and the effect of hunting practices on the genetic diversity and population structure was investigated. By following the bushmeat trade at urban markets, we found baboons to be the third most traded primate species. Male baboon carcasses were sold at a price 60% higher than any other primate due to their larger body mass. Semi-structured interviews conducted with hunters revealed a preference towards male baboons and recent difficulty in finding this primates species. Non-invasive DNA sampling in southern GB and two different genetic markers (fourteen microsatellite loci and a fragment of the mitochondrial control region) suggested substantial levels of genetic diversity and recent genetic contact between different populations. However, geographic distances had a weak effect on population structure and the genetic discontinuities found were not related with landscape features. A contact zone was identified. Here, gene flow seems to be unidirectional and admixed individuals were in higher proportion. Hunting pressure may have induced recent contact between genetically differentiated individuals, which now co-exist in the same social unit. Additionally, the sex-specific patterns of gene flow and the composition of social units were compared with a non-hunted Guinea baboon population, using a molecular sex determination protocol and thirteen microsatellite loci. GB displayed a lower ratio of males within social units, which are formed in some cases by unrelated individuals. The clear female-biased dispersal pattern displayed in Senegal was less intense in GB, where gene flow seems to be mediated through both sexes. The aforementioned contact zone resulted from male immigration. Male baboon dispersal in GB could be the result of flight behaviour or a consequence of an altered sex ratio induced by hunting practices. The GB baboons displayed signs of a disrupted population and its future conservation requires specific actions to reduce or eliminate this activity.

This thesis explores the potential of new substrates for ancient DNA studies and addresses novel questions that can now be asked. It also highlights an additional use of ancient DNA extracted from a traditional source.

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.

The banteng (Bos javanicus lowi) is an endangered wild bovid that is endemic to the island of Borneo. Within their last stronghold, the Malaysian state of Sabah, their population is believed to be less than 500 individuals, which are threatened with extinction by habitat loss and hunting. The banteng is highly elusive and rarely seen, and their preference for dense and remote tropical forest habitat makes them a highly challenging species to study. No extensive quantitative surveys have been undertaken in Sabah, and there is little information available to underpin their conservation and management. This thesis provides the first baseline data on the Bornean banteng in Sabah using ecological and molecular techniques. In Chapter 2, I created the first extensive natural history account of the banteng, which will help further the knowledge of this species. This compilation helped identify gaps in the knowledge, which were then addressed by this thesis. In Chapter 3, I test non-invasive survey techniques and individual identification, and estimate the population size in two forest reserves. In Chapter 4, I demonstrate that logged forests undergo dramatic changes in structure and ambient temperature, and that banteng mitigate these changes by altering their behaviour to avoid thermal-stress. Chapter 5 presents new information of the population genetic structure of banteng in four forest reserves in Sabah. Using mitochondrial markers I show that the ancestral lineage of the Bornean banteng reinforces the suggestion that they should be recognised as a separate subspecies to the Burmese and Javan banteng. I also show that the banteng experienced a population expansion following their colonisation of Borneo, and that the present genetic diversity indicates the population may be managed as two geographically-distinct units. Chapter 6 summarises the main findings of this thesis and the implications for the conservation of the Bornean banteng in Sabah.

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.

There are some contradictory theories on how tolerant honey bees are of pesticides. Since the honey bee genome has been published (Honey bee Genome Sequencing Consortium, 2006), more is known about their metabolic systems, especially the detoxification pathways for potential xenobiotics. Bioassay and biochemical data from various studies have shown that both P450s and carboxylesterases are responsible for pesticide metabolism in honey bees. Here, those metabolic enzymes that confer primary defence to different classes of insecticides in honey bee were validated. Metabolic enzymes are characterised regarding their ability to interact with the insecticide. Synergist bioassay results with PBO and EN 16/5-1 suggest that detoxification mechanism(s) play an important role in protecting honey bees from selected insecticide toxicity. No binding was found between honey bee esterases and tested insecticides, whilst inhibition of P450 activity sensitised the honey bees to these chemicals. Metabolism of tau-fluvalinate and thiacloprid in honey bees is reportedly due to P450 activity, but this metabolism may not be the only reason for the relatively benign action of this insecticide on bees. Honey bees are less sensitive to neonicotinoids containing a cyanoimino pharmacophore than to those with a nitroimino group, however the specific enzymes involved in detoxification remain to be characterised. In this work, pre-treatment of honey bees with a sub-lethal dose of an insecticide induced protection to the same compound. Transcriptome profiling, using microarrays, identified a number of genes encoding detoxification enzymes that were overexpressed significantly in insecticide-treated bees compared to untreated controls.

Mutations of the WFS1 gene are responsible for most cases of Wolfram syndrome (WS), a rare, recessively inherited neurodegenerative disorder characterised by juvenile-onset non-autoimmune diabetes mellitus and optic atrophy. Variants of WFS1 are also associated with non-syndromic hearing loss and type-2 diabetes. Understanding the function of the WFS1 protein-product wolframin, would enable developments in targeted therapy for WS patients and important insights to its possible contribution to type-2 diabetes pathogenesis. This study was aimed at expanding the spectrum of WS-associated genetic mutations and clinical data, and investigating the molecular mechanisms responsible for phenotypic variation associated with WFS1-mutation. The mutational and phenotypic spectrum of WS is broadened by our report of novel WFS1 mutations and a case of WFS2-associated WS. New perspectives on the molecular mechanisms linking mutation to disease manifestation are also taken by characterisation of representative WFS1 mutations specific to phenotype, identification of potentially novel WFS1 interacting partners, and the first evidence linking WFS1 with the exocrine pancreas. Our data suggests that some WFS1-mutations may allow residual protein function and these findings lay the groundwork for future functional investigation of mutated wolframin to explore this hypothesis further.

Tolerance of the semi-allogeneic fetus presents a significant challenge to the maternal immune system. The effect of pregnancy on maternal cellular immunity was established by assessing maternal effector and regulatory T-cell subsets during human pregnancy. This demonstrated that an increase in maternal peripheral regulatory T-cells or a shift from a Th1 to Th2 phenotype was not a requirement for normal pregnancy. We also determined the profound impact of maternal Cytomegalovirus seropositivity on maternal T- cell dynamics. T-cells with specificity for fetal epitopes have been detected in women with a history of pregnancy but it has been thought that such fetal specific cells were deleted during pregnancy. We identified, using MHC-peptide multimers, fetal-specific CD8 T-cells in half of all pregnancies. The fetal-specific response increased during pregnancy and persisted in the post natal period. Fetal-specific cells demonstrated an effector memory phenotype and retained functional potential. These data show that the development of a fetal-specific adaptive cellular immune response is a normal consequence of human pregnancy. Women with recurrent miscarriage were found to have abnormal T-cell function, with increased IFN\(\gamma\) and Il-17 production. Fetal specific T-cells were also detected in this cohort and progesterone attenuated their function, which may have therapeutic implications.