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.

Thesis (Ph. D.)--Massachusetts Institute of Technology, 1980. / Supervised by Robert R.L. Guillard. Includes bibliographical references (p. 208-227).

Thesis (Ph. D.)--Massachusetts Institute of Thechnology and Woods Hole Oceanographic Institution, 1980. / Grant no.: OCE 77-10876, OCE 78-08858, OCE 79-03621. Photocopy of typescript. Bibliography: p. 208-227.

No description available.

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.

The under-reporting of cases of infectious diseases is a substantial impediment to the control and management of infectious diseases in both epidemic and endemic contexts. Information about infectious disease dynamics can be recovered from sequence data using time-varying coalescent approaches, and phylodynamic models have been developed in order to reconstruct demographic changes of the numbers of infected hosts through time. In this study I have demonstrated the general concordance between empirically observed epidemiological incidence data and viral demography inferred through analysis of foot-and-mouth disease virus VP1 coding sequences belonging to the CATHAY topotype over large temporal and spatial scales. However a more precise and robust relationship between the effective population size (N_e) of a virus population and the number of infected hosts (or 'host units') (N) has proven elusive. The detailed epidemiological data from the exhaustively-sampled UK 2001 foot-and-mouth (FMD) epidemic combined with extensive amounts of whole genome sequence data from viral isolates from infected premises presents an excellent opportunity to study this relationship in more detail. Using a combination of real and simulated data from the outbreak I explored the relationship between N_e, as estimated through a Bayesian skyline analysis, and the empirical number of infected cases. I investigated the nature of this scaling defining prevalence according to different possible timings of FMD disease progression, and attempting to account for complex variability in the population structure. I demonstrated that the variability in the number of secondary cases per primary infection R_t and the population structure greatly impact on effective scaling of N_e. I further explored how the demographic signal carried by sequence data becomes imprecise and weaker when reducing the number of samples are described, including how the extent of the size and structure of the sampled dataset impact on the accuracy of a reconstructed viral demography at any level of the transmission process. Methods drawn from phylodynamic inference combine powerful epidemiological and population genetic tools which can provide valuable insights into the dynamics of viral disease. However, the strict and sensitive dependency of the majority of these models on their assumptions makes estimates very fragile when these assumptions are violated. It is therefore essential that for these methods to be applied as reliable tools supporting control programs, more focused theoretical research is undertaken to model the epidemiological dynamics of infected populations using sequence data.