The freshwater pearl mussel (FPM) is an iconic bivalve mollusc whose presence in rivers is assumed to indicate a healthy, bio-diverse ecosystem, capable of providing a range of goods and services. However, excessive sedimentation has been shown consistently to have detrimental effects on FPM, at both the juvenile and adult life stages. As a major source of diffuse river pollution, previous studies have shown erosion rates rising with increasing precipitation, suggesting also increased risk under wetter, future climates. So far, however, most erosion studies have been at the small plot scale and hence it is not possible to make predictions at the catchment scale where risk assessments for FPM are most relevant. Furthermore, little research has focussed on how work to remediate sediment delivery might affect public appreciation of rivers as highly valued landscape features. This research focussed on three typical FPM rivers in the UK: the Ehen catchment in Northern England; the Conwy in North Wales and the Dee in East Scotland and asked 1) How will climate change predictions for the period 2010 – 2039 affect soil erosion at the catchment scale? 2) What factors influence public attitudes towards rivers, the FPM, and mitigation measures to control sediment movement? and 3) Can habitat management for FPMs take into account climate-driven environmental change and social values when constructing conservation goals? In respect of the first aim, the Pan-European Soil Erosion Risk assessment model, PESERA, showed that whilst soil erosion rates increased with rising precipitation, land cover was a more dominant driver of erosion rates over the period studied (2010-2039). Despite being flatter, arable land had higher erosion rates than those from forested portions of each catchment, which were in regions of steeper topography. Secondly, based on a mixture of qualitative focus groups and quantitative surveys, the majority of people had positive attitudes toward rivers, both in a general and local sense. The FPM was not a well-known aquatic species but information about possible human or ecological beneficiaries of mitigation to control sediment delivery into rivers did not affect how acceptable these measures were perceived to be. Factors increasing acceptability of mitigation measures included natural looking scenes that were accessible. In contrast, concerns about impacts on agriculture and food production led to lower levels of acceptability. Finally, this research highlighted crossovers between FPM habitat needs and ideal river scenes from a public perspective and concluded that social values of riverscapes can be included in habitat management plans for the FPM, without compromising conservation goals. A case study exemplifying the methodology used to do this, using the Dee catchment, Scotland and future scenarios from the National Ecosystem Assessment showed that conservation measures in aid of the FPM can accommodate different land management priorities and societal needs. As one of the first studies to assess interactions between evidence from physical sciences, ecology and public perception for an iconic species, this research is expected to have far reaching consequences for public policy, land management practices and river conservation. At a policy level, this includes the ways in which environmental practices can accommodate the social values identified within this research to allow a more holistic approach to ecosystem management; for on the ground practitioners, this research will influence how ecologically important but socially unfamiliar species are managed and how the impacts of land management are assessed both temporally, (to include the impacts of future climate change), spatially, (to take account of catchment wide effects) and socially (to examine social acceptability of different management options).

This research was conducted to provide empirical evidence to supplement advice available to the forensic community for the collection of muscle tissue for forensic analysis. This type of collection is normally carried out to determine the identity of individuals following mass disasters, such as plane crashes or natural disasters. DNA degradation was assessed in two model organisms, pig and rabbit (with human DNA as a control), over various time points. Rabbit recombination activating gene (RAG 1) was aligned to identify conserved regions in pig, rabbit and human. Primers were designed and optimised to create a 4-plex PCR multiplex that can amplify 70 bp, 194 bp, 305 bp and 384 bp in three species. The 4-plex multiplex was found to work efficiently in all three species down to 0.3 ng of DNA template. The multiplex was used to assess whether DNA degradation can be predicted by accumulated degree-days (ADD), which provides a measure of both time and temperature. A series of field studies were performed to assess DNA persistence in pig and rabbit soft muscle tissues using a combination of whole animals, suspended muscle tissues (insect activity free) and muscle fragments. Field studies were carried out in: August-September 2009; February-May 2010; May-June 2010; June-July 2010 and September-November 2010. Soft muscle tissue samples were collected at different ADD. 4-plex multiplex results showed that DNA was more persistent in pig tissues compared to rabbit tissues. In the September 2010 experiments, full multiplex amplification was obtained from rabbit until 137 ADD (whole carcases) and 210 ADD (body fragments and suspended tissues), while in the August 2009 experiments, full multiplex amplification was obtained until 112 ADD (whole carcases and body fragments) and until 141 ADD (suspended tissues). In the June 2010 experiments, full multiplex amplification was possible until 64 ADD. Pig whole carcases which were placed in the field in February 2010, showed multiplex amplification until day 90 (603 ADD), followed by September 2010 (until day 44 (490 ADD)) and May 2010 (until day 27 (338 ADD)). During the September 2010 project, body fragments produced full amplification until muscles were collected (342 ADD), while in case of whole carcases and suspended tissues; the amplification was possible until 490 ADD. There was complete failure of amplification of 305 bp and 384 bp in pig whole carcases after 342 ADD, while in suspended tissues, the amplification of 305 bp and 384 bp was possible until 420 ADD. The statistical analysis showed that amplification success of larger amplicons (194 bp, 305 bp and 384 bp) reduces with increase in ADD in pig and rabbit whole carcases, body fragments and suspended tissues while 70 bp was more persistence. The results showed that there was no significant difference in DNA persistence between whole carcases verses suspended tissues (Z=0.57, p>0.05) and whole carcases verses body fragments (Z=1.71, p>0.05), There was however a significant difference (Z=2.31, p<0.05) in DNA persistence in suspended tissues and body fragments with increase in ADD. The results from field experiments suggested that muscle tissues, if available, should be collected for DNA profiling, since even if degraded, a profile can be obtained. The results also suggested that the isolation of tissues from insect activity as quickly as possible (even if immediate storage is not possible) may be beneficial for DNA persistence. Seasonal variation in DNA persistence was observed due to maggot mass growth which increases carcase decomposition and ultimately effect on DNA persistence. Controlled incubation experiments were also performed at 27 °C, 37 °C and 47 °C until 21 days to assess DNA persistence, as these temperatures were not available under field conditions. The results showed that the amplification of 70 bp was more persistent compared to larger amplicons (194 bp, 305 bp and 384 bp). The drop-out in amplification of larger amplicons occurred more rapidly in samples incubated under laboratory conditions compared to the field samples. The statistical analysis showed species, ADD and temperature have strong effect (p<0.05) on DNA persistence under controlled conditions. The appearance of 70 bp amplicons in all samples collected from field and in most samples from controlled incubation experiments suggested that soft muscle tissues exposed to different environments can be used to perform SNP analysis. The full 4-plex multiplex amplification obtained from rabbit and pig preserved and dehydrated samples suggested that 96% ethanol, cell lysis solution (with and without 1% sodium azide) and dehydration can be used to preserve fresh and partially decomposed soft muscle tissues at room temperature for one year. The drop-out in amplification of larger amplicons in tissues preserved in 10% buffered formalin suggested that formalin was not suitable for long term storage. This system should therefore be considered as an additional method during Disaster victim identification (DVI) work to preserve fresh and partially decomposed samples. This study also suggested that the developed multiplex (4-plex) can be used to assess DNA persistence in human decomposing bodies and in experimental studies.

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.

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.

Prostate cancer is one of the most prevalent cancers worldwide. The early stages of prostate cancer (PCa) are highly dependent on the androgen receptor (AR) pathway and hence therapies target this signalling axis. This approach is successful initially but invariably fails and the tumours progress to castration resistant prostate cancer (CRPC), for which few therapeutic options exist. Therefore, there is a great need to identify and characterize novel therapeutic targets for this stage of the disease. Cancer cells undergo alterations that allow them to survive and proliferate, and metabolic reprogramming is one of the most important manifestations in cancer progression. Therefore, targeting tumour metabolism is an attractive approach to treat cancer. Screening for novel metabolic targets was performed using an siRNA library. 26 metabolic factors were identified to affect proliferation and/or migration, and these were found to be involved in essential pathways including lipogenesis, heme-biosynthesis, redox homeostasis, and glycolysis. The lead targets were validated in a range of cell lines and additional assays performed to investigate the effect upon cell cycle and cell death. UROS, the fourth step of heme synthesis, was further investigated and depletion of this enzyme significantly inhibited prostate cancer proliferation and migration, promoted cell cycle arrest and induced cell death. Further, inhibition of heme synthesis using the inhibitor succinylacetone was found to significantly induce caspase-independent cell death and to sensitise cells to ROS. Importantly, the inhibitory activity of succinylacetone in combination with ROS showed specificity for cancer cell lines. Targeting heme synthesis therefore represents a novel targeted treatment option for prostate cancer and further work is needed to develop this into a therapeutic strategy.

Throughout this body of work low amplitude high frequency (500 Hz – 5000 Hz) mechanical stimulation and its effect to induce osteogenesis on bone marrow derived MSCs has been investigated. Due to the nanolevel amplitudes of these high frequency vertical vibrations the term nanokicking appeared to be appropriate and was subsequently used throughout this thesis to refer to these high frequency sinusoidal stimulations provided by the bioreactor. In the first instance this work was performed in 2D and biological analyses to determine osteogenesis were carried out at a transcript (mRNA), protein and mineralisation level. Affirmative results for osteogensis were observed from genes and proteins (RUNX2, osteocalin, osteopontin) related to the osteoblast phenotype by qRT PCR, in cell western, and immunostaining. To determine the prescence of inorganic osseous minerals, more specific techniques such as Raman spectroscopy, micro computed tomography and histological stainings (Von Kossa/Alizarin Red) were further employed. The results observed remained in line with previously published material (Gentleman et al., 2009) drawing the conclusion that calcium phosphate (Ca10(PO4)6, through nanokicking,was formed in vitro. The natural progession of this research meant that a novel vibrational bioreactor was conceived and designed, through the use of Lean and Six Sigma principles (Andrew Thomas, 2004; Caldwell, 2006), in order to assess the potential of nanokicking in 3D. Here collagen was employed as a biomimetic scaffold and affirmative results for osteogenesis were observed. The bioreactor was unique in that long term (up to 46 days) sterile culture was achieved, it was easy to use and there was no requirement for osteogenic media, growth factors or complex chemistries (e.g. dexamethasone, rhBMP2) in order to induce osteogenesis. The cost of use and maintence was relatively cheap compared to available commercial bioreactors (Rauh et al., 2011b). It is envisaged that this technology may one day have real world use for ossesous tissue regeneration and care in a GMP and clinical setting, or for the preparation of autologous tissue for medical testing in the burgeoning field of personalised medicine.

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.