Alternative splicing occurs in more than 90% of human genes and is particularly abundant in the nervous system. It has been recognized that toxicity can be caused at the level of pre-mRNA processing and potentially lead to age-dependent neurodegeneration upon low-dose chronic exposure. ELAV (Embryonic Lethal Abnormal Visual system)/Hu family proteins are prototype RNA binding protein and gene specific regulators of alternative mRNA splicing in the nervous system. Analysis of mutants in ELAV family proteins shows overlapping and distinct functions during development and age-dependent neurodegeneration. Overexpression of ELAV family proteins further revealed that cytoplasmic localization of ELAV family proteins in associated with enhanced neurotoxicity. Intriguingly all Drosophila ELAV family proteins and mammalian Hu proteins can regulate neuron-specific alternative splicing of Drosophila neuroglian gene- a known ELAV target. The blood brain barrier (BBB) and efficient excretion are protective mechanisms making delivery of many drugs to the brain difficult in vivo. Therefore, I analyzed the roles of a number of key Organic Anion Transporter Protein (OATP) and Multi- Drug Resistance (MDR) proteins and established a sensitized genetic background for CNS drug delivery. To assess if xenobiotics can interfere with ELAV function leading to neurodevelopmental/neurodegenerative defects, I assessed ELAV regulation of its major target erect wing (ewg) using an ewg fluorescent reporter, which recapitulates endogenous ELAV-mediated splicing and allows rapid visualization of potential modulators. From a compound screen in a sensitized genetic background, I identified a number of xenobiotics that cause changes in ewg splicing, indicating interference with ELAV function. Importantly, these compounds also phenocopy specific characteristics of ELAV mutants. My approach demonstrates the potential for using Drosophila in drug screening and neurotoxicity assessments.

Trypanosoma brucei, the eukaryotic parasite that causes human African trypanosomiasis in humans, evades the immune system through antigenic variation. T. brucei antigenic variation involves the periodic switching of the variant surface glycoprotein (VSG) coat to an antigenically distinct variant. A single VSG is expressed on the cell surface at any one time, but the T. brucei genome contains a vast number of silent VSGs. To be expressed, a VSG must be located in a specialised VSG blood stream form expression site (VSG BES). Silent VSGs are copied into VSG BES by homologous recombination. Several proteins have been demonstrated to be involved in this process but how VSG switching is initiated remains unclear. Four putative DNA repair factors were identified in T. brucei, whose eukaryotic homologues play a range of roles in DNA repair and other aspects of genome maintenance. These were two RecQ-like helicases, a Mus81 endonuclease and a Pif1 family helicase (PIF6). To examine whether these factors play a role in DNA repair and VSG switching, mutants were generated in blood stream form T. brucei cells. Analysis of RecQ1 by RNAi knockdown revealed it to be an essential gene in bloodstream form T. brucei, possibly involved in nuclear DNA replication. Phenotypic analysis of recq2 mutants suggests that RECQ2 is involved in the repair of a range of DNA damaging agents. Furthermore, analysis of survival following DSB induction suggests RECQ2 is involved in the repair of DNA DSBs, including those in the active VSG BES. VSG switching analysis showed that recq2-/- mutants have an elevated VSG switching rate and increase in recombination events upstream of the active VSG. These analyses suggest that RECQ2 suppresses VSG switching in T. brucei by suppressing recombination events near the active VSG. Analysis of mus81 mutants showed mus81-/- mutants to be sensitive to agents inducing replication stalling and DNA breaks, and that MUS81 is important in the repair of DSBs. PIF6 appears to be a complicated DNA repair factor, different from MUS81 and RECQ2. pif6+/- and pif6-/- mutants appear to be more resistant to MMS than wild type cells, though more sensitive to the replication stalling agent hydroxyurea. pif6 mutants do not appear to be more sensitive to DSBs than wild type cells and may even be more resistant. It is unclear whether PIF6 is involved in VSG switching and more work is required on this factor to attempt to understand its DNA repair and VSG switching function in T. brucei. These analyses shed light on the DNA repair functions of four previously uncharacterised T. brucei proteins. In particular, observations that RECQ2 is deficient in repairing DSBs upstream of the active VSG and mutants exhibit an elevated VSG switching rate cannot be reconciled with current thinking that direct formation of DSBs in this location initiates VSG switching. This suggests that the initiation of VSG switching is more complex than currently thought and requires careful further study and consideration of the relevance of using direct DSBs in this location to model VSG switching.

In commercial barley cultivars meiotic crossover (CO) distribution is skewed to the distal regions of the paired chromosomes. This restricts recombination to these regions thereby reducing the potential genetic variation that can be exploited in plant breeding programs. The aim of this project was to develop experimental strategies that will enable the frequency and distribution of meiotic crossovers to be modified in order to generate progeny with novel gene combinations. Treatment with the histone deacetylase inhibitor trichostatin A, led to significant modifications in crossover frequency in a concentration-dependent manner with lower concentrations not greatly impacting fertility, allowing for the extraction of fertile seeds. The genetic screening of a treated marker population at The James Hutton Institute (JHI), demonstrated subtle but significant shifts in the distribution of meiotic recombination, indicating that modifying recombination through chemicals applied via the transpiration stream is indeed feasible in barley and hence, possibly in other cereals. The cytological study of a barley desynpatic mutant \(des8\) in collaboration with JHI revealed that synapsis is normal despite reduced chiasma frequency. Genetic mapping studies are in progress to identify the mutant gene responsible for this phenotype which will help us to improve our current knowledge of meiosis in barley.

Two major reasons for the failure of central nervous system axon regeneration are (i) lack of neurotrophic factors available to CNS neurones and (ii) the presence of molecules that inhibit the growth of axons. In this study a gene therapy approach using adeno-associated virus 8 (AAV8) was used to manipulate these two factors. The following major aims were addressed: (i) confirm the bioactivity of transgenes that would be packaged into the AAV8 vector; (ii) assess the cellular tropism of AAV8 in the dorsal root ganglion (DRG); (iii) evaluate the inflammatory responses of the nervous system to AAV8 after intra-DRG and intrathecal injection; (iv) determine the axon regenerative effect of AAV8-mediated delivery of nt-3 (a neurotrophic factor) and shRNA\(_{RhoA}\) (a disinhibitory therapy) to dorsal root ganglion neurones after spinal cord injury in the rat. Delivery of the nt-3 transgene in vitro resulted in production of high levels of NT-3 protein. Transfection of shRNA\(_{RhoA}\)-containing plasmids into cell lines resulted in a marked decrease in the amount of RhoA detectable in cell lysates. AAV8 was found to preferentially transduce large diameter, proprioceptive DRG neurones (DRGN) but in the context of a significant inflammatory response after intra-DRG injection 28d following intra-DRG injection. Axon regenerative effects of AAV8-mediated transgene delivery before lesioning were ambiguous and further work need to be undertaken to clarify this matter.

Synucleinopathies are a group of diseases characterised by the presence of insoluble aggregated forms of α-synuclein. The most common of these diseases is Parkinson’s disease (PD) which affects approximately 1% of the UK population over the age of 60. Alpha-synuclein has also been linked to the disease through familial mutations and genome wide association studies as well as by its presence in sporadic cases. Although solid evidence exists for a role of α-synuclein in PD, it remains unclear as to how this protein exerts its toxicity on neurons and exactly how this leads to the cell death characteristic of this neurodegenerative disease. Alpha-synuclein belongs to a family of three proteins which also includes β- and γ-synuclein. These three proteins are highly homologous and evolutionarily conserved, however none of them have a well defined function. Evidence suggests a role for these proteins in synaptic vesicle dynamics but a more specific function remains to be unveiled. However, due to the considerable degree of homology across these three proteins, knockout models have been considered to allow functional compensation of the missing synuclein protein through one of the remaining family members. This has hindered studies from elucidating not only the role of α-synuclein but also β- and γ-synuclein. To overcome this problem triple synuclein knockout mice have been produced and characterised, as described in this thesis. As expected studies of these animals revealed no alterations in the number of dopaminergic neurons in either the substantia nigra pars compacta or ventral tegmental area. Despite this, a significant deficit in striatal dopamine concentrations was detected, regardless of the fact that the levels and function of tyrosine hydroxylase being normal. As well as this triple synuclein null mice were demonstrated to be hyperdopaminergic through various behavioural tests. Work employing physcostimulants and, through a collaboration, using fast scan cyclic voltametry suggested a role for these proteins in normal dopamine release dynamics at the level of the synaptic vesicle. A previous body of work has indicated that the loss of α- and/or γ-synuclein is able to provide a degree of resistance against the toxic affects of the dopaminergic neurotoxin MPTP. It was therefore hypothesised that the triple synuclein null animals would also display resistance to this toxin. However, these animals were shown to be more sensitive than wild type controls. Importantly it was apparent that animals lacking β-synuclein alone or in combination with other synucleins were the most sensitive to this toxin. Further work revealed a significant deficit in the ability of triple synuclein null mice to store dopamine in their synaptic vesicles. This may explain the sensitivity to MPP+, the active metabolite of MPTP, due to the fact it cannot be efficiently stored in synaptic vesicles, which restricts the toxins access to the mitochondria where it normally inhibits complex I, thus leading to cell death. When recombinant β-synuclein was reintroduced the deficit in synaptic vesicle dopamine uptake could be restored. However, β-synuclein can not do this alone and requires incubation with cytosolic factors, suggesting it acts as a chaperone in this role. This may explain why lines of synuclein null mice that specifically have the absence of β-synuclein apparently fair least well when exposed to MPTP. Finally, in order to assess the extent to which a loss of function role of α-synuclein leads to pathological alteration at the synapse an entirely novel conditional α-synuclein knockout mouse model was produced. Currently no ideal model exists to answer this question as conventional knockout models are based on the knockout of the protein in development. This may allow functional compensatory mechanisms to be established which can be overcome with a conditional knockout approach. As well as this it is important to assess this loss in an aged nervous system, as PD is a disease of aging. It is likely that, as α-synuclein forms insoluble Lewy bodies and undergoes abnormal posttranslational modifications, the amount of normally functioning protein at the synapse is depleted, therefore allowing a loss of function effect to develop. It is hoped this model will allow new insight into the early disease process. Overall this work further contributes to a body of evidence that suggests the synucleins play an important role in synaptic dopamine handling, particularly at the synaptic vesicle level. It is hoped that the newly established conditional α-synuclein knockout model will produce a new perspective on the loss of function role of α-synuclein in early disease development, an avenue that has yet to be fully explored.

Under genotoxic stress conditions, the genome of any organism may become compromised thus undermining cellular functions and the high fidelity transmission of the genome. Should integrity become compromised, cells have evolved a plethora of pathways to monitor, assess and direct the removal or bypass of genomic lesions. Collectively, this response is known as the DNA damage response (DDR). At the forefront of the DDR are specialised enzymes known as protein kinases (PKs), which act to co-ordinate many aspects of this response. In the kinetoplastid parasite Trypanosoma brucei, the role of PKs in other processes, such as the control of the cell cycle and during differentiation between the mammalian and insect lifecycle stages, have already been investigated using mutant cells lines, RNA interference (RNAi) and genome wide and kinome focused screens. However, virtually no work has examined the role of PKs in the context of genome repair. To this end, two RNAi targeted screens (RITseqs), one to examine the genome as a whole and the other to focus directly on the kinome compliment, were performed by others in the mammalian infection stage of T. brucei, searching for genes whose loss sensitises the cells to the presence of the DNA alkylating agent Methyl methanesulfonate (MMS). To validate both screens here, putative DNA damage associated PKs were examined by RNA interference (RNAi). Across both screens, a total of eleven PKs, whose loss sensitised cells to MMS, were validated in this study by monitoring their proliferation. Amongst these novel PKs were a pseudokinase (Tb6560) and an aurora kinase (TbAUK2), whose functions were investigated using disruption mutants generated in BSF T. brucei cells combined with immunolocalisaton. Analysis of the Tb6560 mutants revealed the pseudokinase to be non-essential in vitro and uncovered a potential role for Tb6560 during endocytosis or internal trafficking. Analysis of the TbAUK2 mutants also revealed this PK to be non-essential in vitro. However, when exposed to a variety of genotoxic agents, the growth of the mutants was significantly reduced suggesting TbAUK2 is required for parasite survival under DNA damage conditions. Further phenotypic analysis revealed a potential role for TbAUK2 in the maintenance of the parasite’s nuclear genome. Additionally, the roles of two PKs central to the DDR in other organisms were examined by RNAi. The atypical PKs Ataxia Telangiectasia Mutated (ATM) and the related PK ATM and Rad3 related (ATR) are activated in response to double stranded break (DSB) lesions or replicative lesions, respectively, in other eukaryotes. RNAi of TbATR was associated with perturbation of proliferation, extensive nuclear defects and sensitivity to genotoxic agents. Further analysis also revealed a potential role for this kinase in maintaining transcriptional silencing of bloodstream VSG expression sites (BESs). Conversely, RNAi of TbATM did not disrupt cellular proliferation in vitro and nor were TbATM deficient cells sensitised to alkylating or replication blocking agents, suggesting TbATM is not required for T. brucei survival or for maintenance of growth in these conditions. Collectively, these data reveal a diverse array of PKs required for genome maintainance, in BSF parasites, under genotoxic stress conditions. An indepth characterisation of three DNA damage associated PKs uncovered a variety of putative roles ranging from nuclear and chromosomal segregation to cell cycle regulation, proliferation and endocytosis. These data in particular highlight the complexity underpinning a cell’s response when threatened with genomic instability. Furthermore, depletion of one kinase, TbATR, was also associated with loss of transcriptional silencing within BESs suggesting that PK activity could operate during antigenic variation, a process vital for the persistence of this parasite within a host.

Plant performance under reduced water availability has traditionally been assessed as drought resistance and more recently as water use efficiency (WUE). An extensive body of work has been established over the past 15 years where the natural variation of water use efficiency has been studied in the model species Arabidopsis thaliana (Arabidopsis). At the same time, a substantial degree of criticism has arisen with respect to the use of drought resistance and WUE as measures of plant performance, due to the lack of relatedness of these parameters to reproductive performance, i.e. yield. The work in this thesis is centered on understanding the physiological and genetic basis of water use and water productivity as alternative measures of plant performance under the context of reduced water availability. The first part of this study describes an extensive assessment of the natural variation of water use and water productivity in Arabidopsis in relation to numerous key physiological, phenological, and developmental parameters. Furthermore, this work concisely relates plasticity of key traits to historical climatic variation. A fundamental aspect of this work was the clarification that it is possible to estimate long term water use to a high degree of accuracy based on short term water use, i.e. soil drying rate, and flowering time. Flowering time was demonstrated to be the predominant driver of vegetative performance and water use, however it appeared to be genetically uncoupled from reproductive performance. This is in contrast to previous work that suggests WUE, measured as the ratio of C12 to C13 isotopes (δ13C), is positively associated with flowering time. Additionally, it was demonstrated that multiple commonly employed proxies of reproductive performance including total biomass, WUE, and flowering time, were not sufficient at predicting seed yield in Arabidopsis across multiple environments. The second part of this study involved the genetic dissection of water use and productivity related traits in Arabidopsis through a quantitative trait loci (QTL) mapping study and a genome wide association study (GWAS). QTL mapping using a recombinant inbred line (RIL) population developed from the ecotypes Col-0 and C24 revealed two key flowering time genes, FLOWERING LOCUS C (FLC) and FRIGIDA (FRI), as key regulators of water use. It was demonstrated that a combination of non-functional alleles of both FLC and FRI reduced long term water use via a shorted life cycle, which is again in contrast to previous work relating to the genetic dissection of WUE in Arabidopsis. Crucially, it was observed that reduced water use mediated in this fashion did not detrimentally impact upon reproductive performance. GWAS was employed subsequent to the QTL mapping in order to identify candidate genes underlying the variation for productivity as a unique trait and also as a factor of water use, i.e. water productivity. GWAS identified multiple promising candidate genes that potentially underlie the heritable genetic variation for flowering time, water use, and water productivity.

Animal colouration is associated with a multitude of ecologically adaptive traits known to drive biological diversification, from predator avoidance to physiological regulation. As such, it is an ideal system in which to study the evolutionary patterns and processes that generate and maintain biological diversity. Within the terrestrial vertebrates, amphibians display some of the greatest complexity and variation in terms of colour patterning, with the salamander genus Salamandra particularly renowned for its colour diversity. Typically, Salamandra species present bright, highly variable yellow-black patterns consisting of spots and/or stripes, which are thought to hold an aposematic (warning) function related to their toxic secretions. In addition to this, individual species and populations have evolved melanic, fully yellow and fully brown colourations, with gradations seen in-between. Importantly, there are also indications of parallel colour pattern evolution, making Salamandra an attractive system for studying the repeated evolution of adaptive phenotypes. However, the genus currently lacks phylogenetic resolution, and the molecular mechanisms underlying amphibian colouration are poorly understood. In this thesis, I aim to fill both of these knowledge gaps through the use of next-generation sequencing (NGS) techniques, which offer both unpredicted opportunities to resolve systematically challenging relationships and allow us to study the genetic basis of ecologically adaptive phenotypes in wild non-model organism. In Chapter 2 we reconstruct the controversial interspecies phylogeny of Salamandra using three largely independent phylogenomic data sets. First, using restriction site associated DNA sequencing (RAD-Seq), I genotyped representatives of all six currently recognised Salamandra species (and two outgroup species from its sister genus Lyciasalamandra). This was combined with nuclear protein-coding sequences derived from RNA-Seq and full mitochondrial genomes. Analyses of concatenated RNA-Seq and RAD-Seq data retrieved well supported, fully congruent topologies that placed: (1) S. infraimmaculata as sister to all other species in the genus; (2) S. algira sister to S. salamandra; (3) these two species sister to a clade containing S. atra, S. corsica and S. lanzai; and (4) the Alpine species S. atra and S. lanzai as sister taxa. The phylogeny inferred from mitochondrial genomes differed from this in its placement of S. corsica, as did species tree analyses of RNA-Seq and RAD-Seq data. However, the general congruence among topologies recovered from the RNA-Seq and RAD-Seq data sets gives us confidence in our methodologies and results. In Chapter 3, I perform more in-depth phylogenomic analyses, using RAD-Seq to genotype 231 salamanders from across the taxonomic and geographic breadth of Salamandra. Both Bayesian and maximum likelihood based analyses of concatenated RAD-loci (comprising 187,080–294,300 nt of sequence data) returned well-supported, largely congruent topologies that supported the monophyly of all six currently recognised species. However, the placement of S. corsica was again unclear, and data filtering parameters were found to have a great impact on downstream analyses. Further, I identified undescribed diversity within the North African species (S. algira) and find that 43% of S. salamandra subspecies do not meet a criterion of monophyly. Following this, I use the phylogenetic hypothesis generated to assess the parallel evolution of reproductive (parity) mode and two colour phenotypes (melanism and stripe formation) through ancestral state reconstruction analyses. I find that pueriparity (giving birth to fully metamorphosed juveniles) has independently arisen in at least four lineages, melanism in at least five, and a striped phenotype in least two, all from a common yellow-black spotted larviparous (larvae depositing) ancestor. Finally, in Chapter 4, I leverage and highly colour-variable lineage of the European fire salamander (S. salamandra bernardezi) to identify genetic associations with colour, test for selection on colouration, and test the relationship between colour phenotype and toxicity (the functional basis of aposematism). I show that, within a geographically restricted region, colour phenotypes form a gradient of variation, from fully yellow to fully brown, through a yellow-black striped pattern. Population genetic analyses suggest a sympatric evolutionary origin for this colour variation, and I found no association between a salamanders colour pattern and the metabolomic profile of its toxic secretions, which calls into question the adaptive significance attributed to these striking colourations. Following this, I identified significantly differentially expressed genes between skin colours using transcriptomic (RNA-Seq) analyses and genomic loci associated to representative colour phenotypes (yellow, brown and striped) using RAD-Seq approaches. I also found signals of selection on genomic loci between representative colour phenotypes, several of which overlap with genomic analyses. Overall, my results provide greater phylogenetic resolution for the genus Salamandra than ever before, revealing the need for taxonomic revisions and confirming the convergent (or parallel) evolution of both reproductive and colour phenotypes. My data also represents a significant contribution to our understanding of the genetic basis of amphibian colouration, providing a valuable resource for future comparative research on vertebrate colour evolution.

Redshank breeding biology is examined and reviewed in relation to other sandpiper studies. "Primarily, general aspects are described. The mean clutch size was 3.82 eggs. Fourth eggs laid were significantly smaller and more likely to hatch last. Nest hatching success varied annually from 30-741, with tidal flooding the main cause of nest failure. In successful nests, hatch success was less variable. Chick size at hatch was positively correlated with egg size. Pre-fledging mortality is discussed in relation to overall fledging success. Surviving adults generally remained mate faithful. Experienced birds were found to nest earlier in the season and to produce larger eggs. In all years, female age correlated positively with egg volume. Inexperienced birds laid smaller eggs later in the season. Older birds were more successful at hatching eggs. Experienced breeders were more likely to return to their former breeding grounds than were younger birds, particularly when they were successful the previous year. Birds unsuccessful in the previous year were more likely to divorce. Divorced females were less site faithful than males and were more likely to disperse. Natal philopatry is discussed in relation to pre and postfledging mortality. Philopatry is non sex biased and is estimated at being very high. Chick growth and development is considered and an age determining formula devised for ageing chicks from their weight and bill length. Growth rates were constant between and within years. Annual adult survival (0.75), life expectancy (3.48 years), and study area population are calculated. An estimated 175 pairs breed in the restricted area (168 pairs/km ) and approximately 500 pairs on the Nature Reserve. A model of population dynamics estimates pre-fledging mortality at between 66-74%. Timing of breeding in other waders and a comparison with the related Greenshank are reviewed in the Appendices.

No description available.