DNA replication is central to the propagation of life and initiates by the designation of genome sequences as origins, where synthesis of a copy of the genetic material begins once per cell division. Despite considerable progress in understanding mitochondrial (kinetoplast) DNA replication in kinetoplastid parasites, little is known about nuclear DNA replication. The mechanism and machinery of DNA replication initiation is well-conserved among characterised eukaryotes. The six protein origin recognition complex (ORC, Orc1-Orc6), Cdc6, and Cdt1 are recruited sequentially to DNA, and once bound, they load the replicative helicase (MCM, a heterohexamer; subunits Mcm2-7) to form a pre-replicative complex (pre-RC) at potential origins of replication. The largest subunit of ORC, Orc1, is related in sequence to Cdc6, indicative of derivation from a common ancestor. Such an ancestral molecule appears still to function in archaea. These prokaryotes lack Cdc6 and possess a protein named Orc1/Cdc6, which appears to provide all ORC functions, since orthologues of Orcs2-6 are absent. In addition to this, archaeal orthologues of Cdt1 have not been clearly described, though potentially related factor, named WhiP (winged helix initiator protein), has been found. Comparative genome analysis of Trypanosoma brucei and related trypanosomatids (Leishmania major and Trypanosoma cruzi) revealed, remarkably, only a single ORC protein that is equally related to eukaryotic Orc1 and Cdc6 (named here TbORC1/CDC6). In addition, no clear homologue of Cdt1 was found. These observations have been interpreted as suggesting that origin designation in trypanosomatids, although eukaryotic, may be archaeal-like, raising numerous mechanistic and evolutionary questions. To test this hypothesis, and to dissect the process of nuclear DNA replication, a number of experiments are described in this thesis. We used RNA interference (RNAi) to demonstrate that knockdown of TbORC1/CDC6 in procyclic form (PCF) T. brucei cells inhibits nuclear DNA synthesis, as revealed by cell cycle analysis and a BrdU incorporation assay. Immunofluorescence and GFP-tagging showed that in procyclic form (PCF) cells TbORC1/CDC6 is a nuclear protein. In PCF cells, based on the evidence gathered, we confirm that TbORC1/CDC6 acts in nuclear DNA synthesis. In contrast, RNAi knockdown of TbORC1/CDC6 in bloodstream form (BSF) T. brucei cells resulted in the rapid accumulation of cells with more than two nuclei and two kinetoplasts, indicating a deregulation of the cell cycle, which is then followed by cell rapid cell death. This RNAi result provides greater evidence that TbORC1/CDC6 provides an essential function in the parasite, since RNAi depletion of TbORC1/CDC6 in PCF cells has a less pronounced effect on growth. Nevertheless, attempts to generate TbORC1/CDC6 null mutants failed in PCF cells, consistent with an essential role in this life cycle stage also. To study the molecular interactors of TbORC1/CDC6, we performed immunoprecipitation analyses. From this, we have identified one protein (gene ID, Tb927.10.13380) that acts as a component of the T. brucei pre-replicative machinery, and suggest that this is a previously unidentified orthologue of Orc4. We also indentified a further protein (gene ID, Tb927.10.7980) that may also act in T. brucei DNA replication, but whose identity and function are unclear. TbORC1/CDC6 appears not to interact directly with the TbMCM helicase (for which orthologues of all subunits can be identified), consistent with previous observations from a number of eukaryotic organisms, and contrary to reports in some archaeal species. MCM subunits in T. brucei form at least one subcomplex (TbMCM2/4/6/7) homologous to that previously observed for human, yeast, Drosophila, Xenopus and mouse MCM proteins. Taken together, these data appears to refute the hypothesis that the DNA replication pre-RC machinery in T. brucei is analogous to archaea. Rather, we propose that TbORC contains at least two components, TbORC1/CDC6 and Tb927.10.13380, more analogous to the eukaryotic model, suggesting that origin designation is not carried out by a single protein. To identify potential replication origin sequences, we performed chromatin immunoprecipitation with functional, epitope-tagged TbORC1/CDC6 in PCF cells and, using a high-resolution tiling array (NimbleGen) for T. brucei, we have mapped TbORC1/CDC6 binding sites along all the megabase chromosomes in the genome. Analyses of chromosomes 1-10 showed that 278 binding sites are sparsely located within the core of chromosomes, of which 114 loci (40%) co-localise with probable RNA Polymerase II transcription start sites, perhaps consistent with an origin function. In addition, a further 330 binding sites are present as high density clusters in subtelomeric VSG arrays, and 81 binding sites are associated with sub-telomeric elements, perhaps consistent with a non-origin function. Consistent with these results, RNAi knockdown of TbORC1/CDC6 led to derepression of metacyclic Variant Surface Glycoprotein (VSG) genes, suggesting that TbORC1/CDC6 plays a role in the epigenetic silencing at VSG expression sites in PCF T. brucei. Similar analysis of VSG expression in BSF cells, and of BSF VSGs in PCF cells, was less conclusive, perhaps suggesting differential functions of TbORC1/CDC6 in different life cycle stages or at different VSG expression sites. These analyses shed new light on the architecture and potential function of TbORC1/CDC6 in T. brucei nuclear DNA replication in general, as well as a potential association between replication and antigenic variation in T. brucei.

Meiosis is a specialised form of cell division in which diploid cells divide to form four non-identical spores containing half the genetic complement of the parent. During this cell division program, much of the usual machinery regulating cell division is put to alternate use to allow the cells to undergo an extra round of division without an intervening phase of DNA synthesis. In particular, the end of the first division, meiosis I, must be regulated differently than the end of the mitotic division. We used the model organism Saccharomyces cerevisiae to determine some of these differences in regulation. The cell division program is driven by the sequential association of cyclins with the CDK (cyclin dependent kinase), leading to waves of kinase activity. Exit from mitosis requires the downregulation of CDK activity, and is coordinated by two signalling networks, the FEAR (Cdc14 Early Anaphase Release) network and the MEN (Mitotic Exit Network). Both networks initiate the release of the phosphatase Cdc14 from its inhibitor, Net1, to counter CDK activity. Exit from meiosis I similarly relies on Cdc14 activity, but is driven only by the FEAR network. Experimental results showed that the phosphorylation state and subcellular localisation of the meiotic cyclin, C1b1, are altered in meiosis I. We investigated this relationship and aimed to determine the kinase responsible. We used modelling techniques to explore several rationales for the specific regulation of C1b1. We examined the functional significance of C1b1 localisation, using localisation mutants, and made an investigation into Cdc14 release in meiosis I.

Trait-based approaches are emerging in various fields of ecology, and are here developed for coral reefs. ‘Traits’ means biological characteristics of each species; thus closely related species may have different traits and distantly related species may share common traits. This promotes understanding of a system better than using species alone. Chapter One summarizes existing methods, mostly from plant ecology, explores their utility for corals, and an extensive review then extracts candidate Scleractinian traits (Chapter Two). A dataset of 26 key traits from 231 species from Southwest Madagascar was then collected using 68 reefs of several typologies along several natural and ‘use’ gradients (Chapter Three). This used over 7,000 photo-quadrats on reefs spanning over 200 km (Chapter Four). Trait-based approaches require species-level identification. However, where species are difficult to distinguish, a species-replacement methodology facilitated translation of species to trait-combinations (Chapter Four). Inter-specific trait similarity between the 231 corals and their 26 traits is examined (Chapter Five). In total, 13 groups of corals with highly similar trait-combinations were identified, in which species are functionally equivalent and which therefore can be considered as functionally interchangeable parts in the ecosystem. However, because one quarter of species had unique trait combinations, a functional group approach to surveying reefs may not adequately describe existing trait diversity. Therefore a methodological alternative to using functional groups alone was developed. A trait-based similarity coefficient (Tsim) was developed to take into account both species and trait combination similarities between reefs (Chapter Six). A R-based package that calculates and visualizes Tsim is provided. Tsim’s characteristics were compared to species-based coefficients (Renkonen similarity). Tsim identifies functionally similar reefs missed using species identity alone (Chapters Six and Seven), and can be used to determine reefs that have highly similar trait combinations while being very dissimilar in terms of species.

The following thesis is a composite of two separate research projects which were undertaken by the author for the award of an MRes in Molecular and Cellular Biology within the School of Biosciences at the University of Birmingham. Part one of this thesis will detail the first research project which sought to characterise the contribution of the enzyme phospholipase B to the intercellular lifecycle employed by the pathogenic fungus Cryptococcus neoformans which has the ability to survive within the phagolysosome of immune cell macrophages. This project used cell culture and microscopy techniques as well as whole cell lipidomic analysis and found that many aspects of Cryptococcus neoformans parasitism of macrophages are modified by phospholipase B activity. Part two of this details the second project which examined the predatory bacteria Bdellovibrio bacteriovorous. This bacterium has a unique lifecycle which involves predation of other gram negative bacteria resulting in prey cell invasion and an interperiplasmic growth and replication phase. This project sought to purify and characterize two B. bacteriovorous C-di-GMP modifying enzymes – Bd2325 and Bd1971. Herein we report the first putative analyses of these proteins.

Population dynamics is the study of the abundance of a species at different life stages of a species, the interactions between these life stages and sometimes the interactions with other species. Stage-structured modelling is a popular approach for population dynamics studies. This approach examines populations based on their ecology and allows the incorporation of complex dynamic processes. Model outputs are sensitive to the parameter values. It then becomes crucial to accommodate and quantify parameter uncertainty. This is of particular importance when the population of interest is exploited and the risk of over-exploitaion or extinction needs to be assessed. When studying real world examples of populations exploited by fisheries, several additional problems often arise such as: multiple and heterogeneous sources of information (e.g. datasets collected at different spatial and temporal scales), missing observations, life stages of interest not directly observable. The Bayesian framework allows all of these issues to be handled within the general framework. Thus has proven its particular value in studying the dynamics of exploited populations. Indeed, unknown quantities have associated probability distributions reflecting their uncertainty. Dealing with variations in the interactions/processes between life stages or limited and indirect ecological data is also facilitated by Bayesian modelling. In this study, I examined a large Atlantic salmon population located in the Foyle catchment (Ireland). This population has been exploited for several centuries and particularly during the 20th century. This study focused on the period from 1959 to present for which most monitoring data is available from the Loughs Agency (formerly the Foyle Fisheries Comission). The Loughs Agency is responsible for the management of the salmon population. The aim of the agency is “to manage [the] fisheries towards maximum sustainable exploitation for commercial and recreational purposes”. In order to do so, it is important to understand the regulatory mechanisms occurring in the population in order to (i) estimate the number of fish returning to river, i.e. pre-fishery abundances (PFAs), and (ii) to derive standard reference points for assessing the population status with regards to its sustainable exploitation. To this end, a state-space model is implemented within a Bayesian framework. A life stage and spatially structured dynamic model describes the lifecycle of the Main components of Atlantic salmon in the Foyle catchment. Several empirical datasets related to the abundances of the stages at different scales of space and time, over a period of 50 years are brought together. Observations and process errors are taken into account ultimately allowing PFAs to be estimated. A retrospective analysis was also carried out providing insights on the historical status of the population and its exploitation. Geo unit specific abundances of the different states and their associated uncertainty are estimated. The main state of interest is the pre-fishery abundance (PFA), during the time-series considered (1959-2008) the salmon population reached its apex in the mid 1960’s. This was followed by a steep decrease until the mid 1970’s. From then to present, the population followed a slow declining trend with a slight recovery in the mid 1980’s. This decline is shown to be mostly due to a decline of the 0+ juvenile to returning adult survival which is accentuated some years by some overfishing.

An island system with corresponding mainland sites, was used to study woodland rodent dynamics and their parasite communities within a naturally fragmented landscape. The study site, hosts and parasite species investigated within this thesis allowed the investigation of how natural fragmentation affects demographic and population dynamics of rodents (chapter 3). Reduced habitat connectivity is known to affect nearly every process in biology. Low degrees of fragmentation and high connectivity between habitats have been shown to provide the most stable conditions for populations to persist, as movement of organisms is less restricted. It is shown that in contrast to previous studies on fragmented populations, the fragmented landscape of the islands had little effect on the demographic characteristics of rodent populations in comparison to those on the mainland. There were few difference found in the demographics of wood mice and bank voles when compared to mainland sites. The results from this study then allowed the broader question of how parasites dynamics are affected by the spatial structure of a host population to be addressed. Theory predicts that parasites are unable to persist in small, isolated host populations, due to small host population size as well as potential genetic factors increasing the risk of extinction. However parasites may become more prevalent in isolated populations as hosts may have a reduced ability to deal with infection. It is shown (chapter 4) that within this study system that despite some island populations being extremely small, there is no overall reduction in parasite species found within fragmented habitats. Furthermore, extinction of the parasites investigated within wood mice and bank voles is unlikely due to the direct life cycle of these parasites. Variation was seen in the prevalence of infection, however the majority of the parasite species on islands did not show a reduced prevalence of infection compared to mainland sites. Finally parasite co-infection and co-aggregation and their dependency on host characteristics in woodland rodents (chapter 5) were investigated. Parasite species infecting hosts are normally studied individually, however this is not what is seen within natural populations. Co-infection is an important concept within natural systems as there is a vast diversity of parasite species that create ample opportunity for concurrent infections. Therefore, it is proposed that studies should be focused on parasite interactions, as within host interactions can in turn affect the abundance and distribution at the level of the host population. This study focused on seven parasite taxa, and it was found that the maximum number of parasite species any individual was found to be infected with was five, with the mean number for both host species at around two. Parasites associations were also more common than expected within the same functional groups with co-occurrence being more common between parasite species associated with ectoparasites. Within this study, host aggregation was positively correlated with differing parasite taxa. Furthermore, looking at patterns of co-aggregation could aid in our understanding of parasite interactions within hosts. The nature of these interactions will determine whether aggregation is positively or negatively correlated across different parasite taxa. A small number of hosts maybe responsible for transmitting the majority of infections (20/80 rule). Identifying these individuals would be informative in helping to control disease spread. Host characteristics have been found to be informative in terms of single parasite species infections. Within this study it was found that juvenile bank voles were more likely to be co-infected than those within other age classes. No host characteristic explained patterns of co-infection in wood mice. In conclusion I found that natural fragmentation does not have an overall negative effect on rodent host dynamics nor does it reduce the number or prevalence of infection of parasite species able to infect hosts. This thesis has highlighted the importance for using natural wildlife systems in empirical studies, and the need to further address multiple parasite interactions within a host community.

Phosphoregulation is essential for the control of cell division. In yeasts and animals, premature entry into mitosis is prevented by the inhibitory phosphorylation of CDK by WEE1 kinase. WEE1 homologues have been identified in several species of higher plant, including Arabidopsis and tobacco. However, while WEE1 function has been confirmed in the DNA replication checkpoint in higher plants, a role for the protein in the G2/M transition during an unperturbed plant cell cycle is yet to be identified. To address this issue, the further characterisation of Arabidopsis WEE1 was completed, particularly focussing on the localisation and stability of the protein. A GFP-Arath;WEE1 construct under the 35S promoter was transformed into both Arabidopsis plants and the tobacco BY-2 cell line, and a nuclear localisation of the protein at interphase was confirmed. Additionally, the study of WEE1 subcellular localisation in different cell cycle phases revealed that the protein was absent during metaphase. Interestingly, levels of WEE1 degradation varied in different Arabidopsis root tissues, and the protein was absent in lateral root primordia. The proteasome inhibitor MG132 was used to demonstrate that Arath;WEE1 is degraded via the 26S proteasome pathway, as in yeasts and animals. Bimolecular fluorescence complementation confirmed an interaction between Arath;WEE1 and the F-box protein Arath;SKIP1 in vivo, which may target Arath;WEE1 for degradation. Tobacco BY-2 cells were stably co-transformed with BiFC constructs to facilitate the study of any changes in this interaction during the cell cycle. There was again no evidence of the interaction during metaphase, but a return of the signal during anaphase and telophase. The root phenotype of an Arath;SKIP1 knockdown line suggested that this F-box protein may target Arath;WEE1 for degradation early in development, but this requires confirmation. The work presented in this thesis describes, to my knowledge, the first investigation into the stability of Arath;WEE1 protein.

A novel toxicological tool, which consisted of a differentiated, 3-D, in vitro model of human respiratory epithelia, i.e. EpiAirway-100 cells (MatTek Corp., USA), was utilised to identify intelligent biomarkers in human respiratory epithelia following exposure to selected particle and vapour phase tobacco smoke components (TSC); thus providing a holistic approach towards the identification of biomarkers in the pulmonary epithelium. A range of TSC doses were used to elicit a classic dose response which was characterised by conventional toxicology techniques (transepithelial electrical resistance [TEER], cell viability [MTT] and protein assay). All four TSC appeared to induce similar stress responses (e.g. protective mechanisms) within the model. Histological (LM level) examination was utilised to investigate the morphological changes in cellular-tissue organisation in the ETM, following exposure challenges with the TSC. In conjunction with this, targeted immunohistochemistry assays were performed in order to better characterise the tissue response at the cellular level, i.e. basal, ciliated, intermediate and Goblet cells. Three discrete zones of tissue injury and repair were delineated: (1) the apical region, (2) the suprabasal region and (3) the basal zone (i.e. where basal cells acted as progenitors or stem cells). Toxicogenomics was carried out to identify early molecular markers for events in pulmonary injury. This study identified nine genes, with the potential of being genomic biomarkers, for specific disease mechanisms known to be associated with tobacco smoke. Another potential genomic biomarker was identified which was not previously related to tobacco smoke, PTGS1. Its induction could result in a disruption of tissue homeostasis that may lead to the onset of a number of human inflammatory diseases (e.g. chronic obstructive pulmonary disorder). The proteomic analysis identified two proteins (cystatin-A and ubiquitin) that were significantly induced for all TSC exposures and could be described as ‘defence proteins’. Consequently, they represented potential biomarkers of general lung injury. This study also identified three proteins (complement C3, calmodulin and CD9) that could be used as biomarkers for specific disease mechanisms (e.g. atherosclerosis).

Apicomplexan parasites of the genus Plasmodium are the causative agent of malaria, one of the most prevalent infectious diseases worldwide. Five different Plasmodium species can cause malaria in humans, leading to a total of approximately 500 million cases each year and of these, P. falciparum causes the most deadly form of the disease and is responsible for more than 1 million deaths annually. A major problem in the global fight against malaria is the widespread resistance of the parasites against the currently available drugs. It is of great importance to identify new drug target as well as to understand the mechanisms that lead to drug resistance in the first instance in order to potentially reverse the resistant phenotypes and to avoid the development of resistance in the future. The tripeptide glutathione (GSH) or γ-glutamylcysteinyl-glycine is the most abundant low molecular weight thiol in most eukaryotic organisms and serves a number of important functions as sulfhydryl-buffer, cofactor for enzymes and for the detoxification of xenobiotics and drugs. GSH is an important component of the antioxidant machinery and because malaria parasites live in an environment rich in iron and oxygen and thus increased oxidative stress, they depend on functional antioxidant systems. The biosynthesis pathway for GSH, consisting of γ-glutamylcysteine synthetase (γGCS) and glutathione synthetase (GS) is present in malaria parasites as well as in their host cells. Previous studies have shown that depletion of GSH has an antimalarial effect, but it remained unclear whether parasites were killed directly or died because their host cell could not survive the depletion of GSH. To address this question, the knockout of both genes encoding the enzymes of the GSH biosynthesis pathway in P. falciparum was attempted. While both gene loci were targeted by control constructs, the knockout of either pfγgcs or pfgs was impossible, indicating both genes are essential for parasite survival in the erythrocytic stages. To analyse the localization of γGCS and GS, GFP-tagged recombinant fusion proteins were expressed in the parasites and showed that GSH biosynthesis is cytosolic. Apart form its other functions GSH has previously been suggested to be involved in resistance to the antimalarial drug chloroquine (CQ). CQ was for a long time the first line antimalarial drug due to its high efficiency, low cost and low toxicity, but is now widely inefficient in the treatment of the disease. CQ resistance is associated with mutations in the CQ resistance transporter (PfCRT), a membrane protein of the digestive vacuole that allows the efflux of the drug form its site of action. However, PfCRT mutations alone cannot explain the full array of phenotypes found in resistant parasites. GSH is able to degrade heme, the target of CQ, in vitro and it has been suggested that elevated GSH levels contribute to CQ resistance. However, analyses of isogenic parasite lines bearing different forms of PfCRT in this study revealed lower GSH levels and higher susceptibility to inhibition of GSH biosynthesis in the CQ resistant lines. These changes did not correlate with changes in the expression of enzymes involved in the de novo biosynthesis or consumption of GSH. However, the cellular accumulation ratio for CQ indicated a decrease of free heme in the resistant parasites. Mutant forms of PfCRT expressed in oocytes of Xenopus laevis were able to transport GSH, while the sensitive wild-type form did not transport the tripeptide. The findings of this study suggest that in parasites bearing mutant PfCRT, GSH is transported into the digestive vacuole where it is able to contribute to resistance by degrading heme, before the tripeptide itself is degraded by peptidases inside the vacuole, consistent with the overall reduction of GSH levels in CQ resistant parasites.

Flavonoids and phenolic compounds (aka polyphenols) are phytochemicals, thought to participate in plant development and defence mechanisms. Polyphenols are ubiquitous plant secondary metabolites, and are usually found conjugated as glycosides or esters. These compounds have been of particular interest as part of the human diet, and have been the focus os many studies in nutrition research. Many epidemological studies have found a correlation between flavonoid intake and protection against certain chronic diseases such as cancer and cardiovascular events. The mechanisms underlying such benefits, however, remain to be unveiled, as judged by the number of in vitro and animal model intervention studies. The primary property attributed to polyphenolic and phenolic compounds relates to their antioxidant activities. Outcome from in vitro studies have established the ability of polyphenols to scavenge radical species and bind to metal ions, thus preventing the damage caused by oxidative stress. Recent progress in the field has broadened the knowledge of how polyphenols exert their beneficial effects, which appears to depend on more than simply antioxidant activity. Indeed, polyphenols are thought to actively participate in the modulatory effects involved in signal transduction in cells, responsible for the regulation of genes, apoptosis and cell proliferation.