As relatives share genes that are identical by descent, organisms can gain additional fitness benefits by improving the reproductive success of known kin. There are a number of costs associated with close inbreeding, including an increased likelihood of the expression of recessive deleterious alleles and reduced survivorship. The ability to recognise kin therefore enables individuals to improve their inclusive fitness and avoid problems associated with close inbreeding. Female house mice (Mus musculus domesticus) will nest and nurse offspring communally. Choosing an appropriate nest partner is therefore important and competition between nesting females can result in reproductive inhibition and infanticide. Kin selection theory suggests that females could gain inclusive fitness benefits from nesting with relatives. This thesis explores the mechanisms of kin recognition in female house mice in the contexts of social partner choice and inbreeding avoidance. Female house mice recognised unfamiliar relatives, suggesting a phenotype matching mechanism for kin recognition. Females were presented with maternal and paternal half-siblings to investigate recognition template formation. Females nested with maternal but not paternal half-sisters, suggesting that female house mice may use a recognition template learnt from their mother for social partner choice decisions. However females avoided both maternal and paternal half-brothers suggesting that females may use a match-to-self mechanism for inbreeding avoidance. Female house mice were able to identify relatives from urine, suggesting that genetic markers are present in urine. To investigate the molecular markers of kin recognition mice were bred to control for the major histocompatibility complex (MHC) and major urinary proteins (MUPs). Females nested with females that matched for MHC or MUP type, suggesting that both gene families may be used for kin recognition between females. A non-significant trend was observed for females to avoid males that matched themselves for MUP type, but females showed no avoidance of males that matched for MHC type. A small pilot experiment investigated the physiological effects of female social environment. Housing with unfamiliar females resulted in short-term decreases in female body mass and urinary protein concentration but an increase in urinary creatinine concentration. Competition between unfamiliar females may have resulted in a decreased water uptake and an increase in scent marking which could explain the physiological changes observed. Together these results suggest that female house mice may use two mechanisms of kin recognition. For social partner choice females may use a match-to-maternal MHC and MUP type mechanism, whilst for inbreeding avoidance females may use a match-to-own MUP type mechanism. The possibility of a single species using two separate mechanisms suggests that kin recognition may be considerably more complex than previously thought.

Several inherited diseases such as Huntington disease and myotonic dystrophy type 1 are associated with the expansion of repeats. A high level of age-dependent instability has been observed in the expanded alleles present in the germline and soma. The present study covers the investigation of other non-disease-associated expanded microsatellites in order to explore their variability, which in turn could be used to predict age in the general population. First, using Tandem Repeats Finder, 23 pure loci with alleles >50 repeats in at least one of two reference genomes were identified. Several loci among those show high levels of variation in the general population with a considerable proportion of large expanded alleles. SP-PCR analysis revealed that these loci showed a relatively low level of somatic instability and mostly only small length changes. The SP-PCR approach is laborious and time consuming; these disadvantages could potentially be overcome by the use of new technologies based on high throughput analysis. In this study a target enrichment sequencing approach using custom bait and Illumina Paired-End Sequencing using Illumina GA IIx platform to capture and sequence sequences in question was used. 25,539 pure (100% match) mono, di, and trinucleotides with unique flanking sequences and copy number ≥5 were investigated. Similarly, telomeric regions, DNA repair genes, the DMPK region and SNPs were captured using custom designed baits. The experiment generated millions of reads for each sample, ranging from 5.5 to 9.5 million reads. Subsequently, CLC Genomics Workbench and Bowtie 2 were used to map reads to the HG19 reference genome and to count the number of reads aligned to each DNA sequence. The effect of age at sampling on the generated reads was also investigated. The data showed a significant correlation between unique sequence reads and age at sampling. Microsatellites, including mono, di and tri nucleotides, were successfully captured using custom baits based on unique flanking sequences. The microsatellite total reads were found to be correlated with age at sampling. Genotyping was found to be successful for di, tri and tetra nucleotide loci however PCR slippage was commonly found in the dinucleotide repeats. By contrast due to the extensive PCR slippage observed in mononucleotides, reliable genotyping was difficult to achieve. Unlike microsatellites, no significant correlation was observed between DNA sequences of repair genes or the autosome gene read counts with age at sampling. Thereby, this feature could be exploited to normalise the read counts such as of telomeres. Thousands of human SNPs were successfully captured using baits having a single mismatch. Baits were designed to ensure an equal capture for both alleles that could exist at a single SNP. The overall coverage of SNPs was noticeably good and used to generate reliable genotype. Age-dependent telomere shortening was estimated. The relative telomere length showed a significant correlation with age at sampling. The data show four major variants of telomeric repeats, TTAGGG, TTGGGG, TGAGGG and TAAGGG with a shortening rate of 60 bp/year. In summary, the primary aim of this project was not achieved due to the low level somatic instability of microsatellite and low-resolution power of traditional SP-PCR. However, the success with the NGS observed in this study, indicate a reasonable potential of using microsatellites to estimate ageing in humans.

Mutation introduces change at the sequence level. There is a critical mutation rate above which changes occur too frequently for natural selection to maintain the population's genetic makeup. This thesis examines the relationship between this critical mutation rate and the number of individuals in the adapting population. It presents an algorithmic method capable of providing widely applicable results in haploid and diploid populations, and varies this method against analytical models for the error threshold. Use of the method led to the discovery of an exponential relationship between the critical mutation rate and population size, particularly strong in small populations with 100 individuals or less, contradicting the existing idea that critical mutation rate and population size are independent. The critical mutation rate (and error threshold) were found to be lower in diploids due to differences in recombination. Analysis of the survival-of-the-fittest to survival-of-the-flattest transition enabled improvement of existing definitions of the critical mutation rate. Development of a faster algorithm capable of running experiments with parameter values within the range found in nature began the process of bridging the gap between artificial and biological evolution. A link was established between the exponential model and natural mutation rates. Increasing the gene length by a factor of 10 was found to decrease both the critical mutation rate and error threshold by an order of magnitude. Natural mutation rates lie below these values, although further work is required to establish any trend. A potential link has been established between the critical mutation rate, error threshold, and optimal mutation rate control theory. Future work may develop the algorithmic method to include more complex features of biological populations, and go on to determine the effect the exponential model can have on population extinction, recovery, and conservation.

No description available.

No description available.

The establishment of left-right (LR) asymmetry in animal development remains an unanswered, fundamental question in biology. Many mechanisms of symmetry-breaking have been proposed and supported, although as yet no universal mechanism has been verified across bilaterian animals. Snails provide an invaluable study organism for understanding LR asymmetry, due to the prevalence of chirally variable species. In the pond snail Lymnaea stagnalis LR asymmetry and resulting shell-coiling direction is a well described genetically tractable trait, inherited through a maternal effect. However, the ‘chirality gene’ is still unknown. In L. stagnalis, clockwise (dextral) coiling is the dominant genotype, therefore snails with homozygote genotype ‘DD’ or heterozygote ‘Dd’ both produce dextral offspring, whereas those with the homozygote recessive genotype ‘dd’ have anticlockwise (sinistral) coiling offspring. To further the Davison research group’s ongoing characterisation of the chirality gene in L. stagnalis, this project focussed on gene expression patterns exhibited between chiral genotypes. Differential gene expression was explored via a candidate gene approach, performing quantitative real-time PCR (qPCR) experiments on specific genes of interest, and also a transcriptomic sweep, utilising next generation sequencing. To enable accurate quantification of gene expression by relative qPCR, first, stable endogenous control genes had to be established. In light of general failings of the previously published control genes to meet the criteria for appropriate use of qPCR, five genes were verified for use as stable endogenous controls in L. stagnalis embryo, ovotestis and foot tissue, for the accurate comparison of gene expression between and within chiral genotypes. These endogenous control genes will enable other researchers of L. stagnalis to rapidly identify stable controls for relative qPCR experiments. qPCR experiments were performed to compare gene expression of 13 candidate genes between chiral genotypes in the single-cell embryo, ovotestis and foot tissues. Significant differential expression was observed between chiral genotypes only in the diaphanous related formin gene, Ldia2, and two actin-related protein genes, Larp2/3 1a and Larp2/3 3. A frameshift mutation in the sinistral copy of Ldia2, discovered by the Davison research group, has identified Ldia2 as the primary candidate for the causal gene in LR asymmetry determination in L. stagnalis. In support of this, Ldia2 mRNA was found to be dramatically underrepresented in the sinistral one cell embryo and significantly reduced in the sinistral ovotestis tissue, yet not in the somatic foot tissue. Ldia2 was also the only gene found to be overrepresented in the embryo tissue relative to the ovotestis and foot tissue, providing further support for the functional importance the gene in early development. The expression level of Ldia2 in the heterozygote genotype groups was calculated to be halfway between that of the homozygote groups, indicating equal expression dominance of the alleles at the chirality locus. The expression pattern observed in the actin-related proteins was less clear and will require further analysis to infer any true biological meaning. However due to the close interaction of actin-related proteins and formins the differential expression observed in the embryo tissue provides functional support for the role of Ldia2 in chiral dimorphism. Next generation transcriptome sequencing methods were employed to gain a transcriptome-wide scan of patterns of gene expression in the ovotestis tissue of snails of differing chiral genotype. A comparative analysis was initiated trialling a novel reduced-representation sequencing method, expression RAD sequencing (eRAD) and traditional RNA Seq. eRAD applies the method of restriction-site associated DNA Sequencing (RADSeq) to the transcriptome by utilising double-stranded complementary DNA (cDNA) in place of genomic DNA. Due to delays in sequencing, the RNA Seq data was not received in sufficient time to perform the comparative assessment within this thesis. Consequently, only the eRAD data is presented here. The eRAD data failed to identify reliable differences in gene expression between chiral genotypes, although did provide a transcriptomic resource of de novo assembled contigs, which has been verified through further analyses. Overall the lack of differential expression identified between chiral genotypes in both the qPCR and eRAD analyses has indicated that the sinistral morph of L. stagnalis does not exhibit a large-scale loss of gene function and pleiotropic effects on gene expression. Therefore, the negative consequences of chiral reversal in L. stagnalis, such as the low hatch rate observed in sinistral broods, may all result from the single chirality gene polymorphism.

No description available.

The insect Malpighian tubule is involved in osmoregulation, detoxification and immune function, physiological processes which are essential for insect development and survival. As the Malpighian tubules contain many ion channels and transporters, they could be an effective tissue for targeting with novel pesticides to control populations of Diptera. Many of the insecticide compounds used to control insect pest species are no longer suited to their task, and so new means of control must be found. The malarial mosquito, Anopheles gambiae, spreads the Plasmodium parasite which is responsible for over one million deaths each year, and is one of the species on which many current insecticides are no longer effective. Anopheles is notoriously difficult to study due to a lack of natural mutation stocks and transgenic capabilities, as well as the difficulties involved with maintaining a colony. The fruit-fly Drosophila melanogaster is a useful model organism for Anopheles, and previous studies suggest that the mechanisms of Malpighian tubule function are well conserved between the two species. Following microarray investigations to identify genes which were highly enriched in both the Anopheles and Drosophila Malpighian tubules, four homologous genepairs were selected, AGAP097752 and CG15406, AGAP012251 and Picot, AGAP009005 and ZnT35C, and AGAP002587 and CG8028. Analysis of the Anopheles Malpighian tubule microarray data-set showed ion channels and transporters to be highly expressed in the tubules, although similarly to Drosophila, very few of the renal up-regulated genes have been characterised. The gene-pairs chosen were all novel, but putatively predicted to be involved in sugar transport, phosphate transport, zinc transport and monocarboxylate transport respectively. These are functions which are likely to be essential, but so far remain unstudied in the insect renal system. The gene-pairs were chosen with two main purposes; to determine how closely expression of the genes was conserved between Anopheles and Drosophila, and also to determine which of the genes were essential, and could therefore be effective insecticide targets. The homologous gene-pair AGAP007752 and CG15406 have well-conserved expression in the Malpighian tubules, suggesting that they are functionally important genes. This was shown in Drosophila, where knockdown of CG15406 4 expression was lethal to the fly. A direct role in tubule fluid secretion was not found, and experiments to determine the sugars transported by CG15406 were inconclusive, possibly due to an abundance of highly-expressed sugar transporters in the tubules. The inorganic phosphate co-transporters AGAP012251 and Picot also show conservation of expression in the Malpighian tubules, and are likely to be involved in the transport of inorganic phosphate into the tubules for incorporation into metallo-organic concretions. In the Anopheles tubules the concretions are found in the main segment, in the Drosophila tubules they are located in the distal initial and transitional segments, where AGAP012251 and Picot are expressed. Picot is essential for Drosophila development through to adulthood, and for survival as an adult, although the transporter does not appear to be directly involved in fluid secretion. Expression of neither AGAP012251 nor Picot is confined to the tubules. The putative zinc transporters AGAP009005 and ZnT35C show a highly conserved expression pattern, and appear to be involved in the secretion of excess zinc from the Malpighian tubules. ZnT35C is essential early-on in Drosophila development, and for survival in the adult fly. Similarly to Picot and CG15406, there is no direct role for ZnT35C in fluid secretion from the tubules under normal zinc conditions. The putative monocarboxylate transporters AGAP002587 and CG8028 are not as well conserved, as AGAP002587 is highly upregulated in the tubules of female mosquitoes both before and after a bloodfeed, whereas CG8028 has no sex-specific up-regulation. CG8028 is not essential for Drosophila development or survival, and plays no discernable role in fluid secretion. The data collected during this investigation suggests that in general there is a high level of conservation of expression between homologous transport genes in the Anopheles and Drosophila Malpighian tubules. The three gene-pairs which show the greatest conservation of expression are also essential for development and survival in Drosophila. This suggests that cross-species studies are an effective way of finding essential and important genes. The data collected also suggests that Drosophila is a reliable model for Anopheles, and could be used as a high-throughput system of finding genes which could be effective insecticide targets in Diptera.

Haemopoietic stem cells (HSC) maintain lifelong haemopoiesis through the monitoring and production of cells from multiple haemopoietic cell lineages. A key property of HSC is their ability to maintain quiescence. Quiescence refers to a state of inactivity in which the cell is not dividing and remains dormant. It is this property of the HSC that is thought to maintain genomic integrity and to allow the HSC to sustain haemopoiesis over the period of a lifetime. However, the regulation of quiescence in this context is not well understood. Numerous studies have aimed to understand the molecular mechanisms underlying HSC quiescence using high-throughput approaches. A previous microarray study by our group aimed to understand the transcriptional differences between quiescent and proliferating human HSC. Data from this microarray showed that the most up regulated group of genes in quiescent compared to proliferating human HSC were chemokine ligands, specifically within the CXC family. Although this was a novel finding at the time, the biological function of these chemokine genes was not studied until the current work presented here. In this thesis, we aimed to extend foregoing research and importantly, investigate the role of CXC chemokines in HSC properties, using both human and mouse systems. First, we validated the results from the microarray study using gene expression analyses to show that chemokine ligands CXCL1 and CXCL2 were significantly up regulated in quiescent HSC (CD34+CD38-) in comparison to more proliferative progenitors (CD34+CD38+). Focusing on CXCL1, we showed positive expression of the ligand protein in human stem/progenitor cells using immunofluorescence and western blotting on human primary CD34+ cells. In addition, we identified positive expression of receptor CXCR2 by gene and protein analyses on CD34+ cells, indicating the presence of an autocrine chemokine signalling loop. To determine the biological function of CXCL1/CXCR2 signalling in human HSC, we used shRNA to reduce CXCL1 expression and a commercially available inhibitor (SB-225002) to block CXCR2 receptor signalling. Experiments on cell lines expressing CXCL1 and CXCR2 (HT 1080) showed that reduction of CXCL1 and over-expression reduced or increased cell viability and proliferation respectively. Experiments on human primary CD34+ cells revealed that reduction of CXCL1 induced apoptosis and reduced colony formation. Similarly, inhibition of CXCR2 signalling in CD34+ cells using SB-225002 induced apoptosis and reduced colony formation in a dose dependent manner. However, due to human sample availability and technical challenges, experiments need repeated in order for a valid conclusion to be made and statistical analysis could not be carried out for some primary experiments. In addition, further experimental work is required to conclusively prove that human stem/progenitors express CXCL1 and CXCR2 as different techniques showed varying results. In summary, we provide some evidence that CXCL1 and CXCR2 is expressed by human HSC and may be an important survival pathway in normal human HSC which requires further experimental data to provide valid conclusions. In order to gain a deeper understanding of the biological function of chemokine signalling in HSC biology, we used an in vivo murine system. First, we examined mRNA transcripts of CXC chemokines in mouse HSC populations. We screened a small selected group of CXC chemokines using primitive mouse HSC and single cell quantitative PCR using the Fluidigm™ platform. Gene expression analyses identified that Cxcr2 and Cxcl4 mRNA transcripts were detected including in the most rare, primitive HSC fraction. To elucidate the mechanism of action, we used a transgenic reporter and knock out mouse models for both genes of interest. Analysis of a Cxcr2 null mice model (Cxcr2-/-) validated previous research in which animals lacking Cxcr2 show disrupted haemopoiesis with an expansion of myeloid cells in the haemopoietic organs. Interestingly, within the current work, analysis of steady state haemopoiesis revealed an expansion of the most primitive HSC in the BM of animals lacking Cxcr2 and enhanced mobilisation demonstrated by an increase in the stem/progenitor activity in the spleen and PB. HSC functional analyses using BM reconstitution assays with wildtype (WT) or Cxcr2-/- HSC showed that there was a trend towards a reduction in engraftment in animals transplanted with HSC lacking Cxcr2. However, this result was not statistically significant due to high sample variability and due to time constraints and the length of this assay, this was not repeated. The data suggests that Cxcr2 expressing HSC may be important for stem cell maintenance via a cell autonomous mechanism however experiments are required to be repeated to draw valid conclusions. Cxcl4-Cre transgenic mice containing a RFP construct under the control of the Rosa26 promoter (Cxcl4-Cre) showed RFP expression in HSC and progeny. RFP expression in HSC populations was in accordance with Cxcl4 mRNA transcripts therefore suggesting RFP expression was correlated with endogenous Cxcl4 expression. Interestingly, flow cytometry analysis identified that not all (~50%) HSC showed positive expression for RFP. Flow cytometry sorting of positive and negative populations revealed that cells with enhanced colony formation potential reside within the RFP (Cxcl4) positive fraction. To extend this data, we aimed to knock out and reduce Cxcl4 expression and examine the phenotype. Targeted deletion of Cxcl4 in vitro using a Cxcl4 shRNA vector demonstrated that Cxcl4 reduction in vitro diminished colony formation in primary and secondary replating assays. Since data for human CXCL4 mRNA were not conclusive from the original microarray, we reassessed the relevance of CXCL4 in the human system. Gene expression analyses showed that CXCL4 transcripts were indeed detected and furthermore, up regulated in primitive HSC (CD34+CD38-CD90+) compared with proliferative progenitors (CD34+CD38+). Collectively, the data indicates that CXCL4 may play an important role in mouse and human HSC biology, however further experimental work is required to address this. In summary, the data presented in this thesis demonstrate that several chemokines including CXCL1, CXCL4 and receptor CXCR2 may have key roles in HSC survival and maintenance, both in the mouse and human systems. However, increased biological replicates and further experiments are required to draw valid conclusions. Enhanced understanding of the regulation of stem cell properties is critical for improving our ability to manipulate normal stem cells in vitro and in vivo. Furthermore, understanding normal stem cell regulation is fundamental for the research of diseases such as leukaemia in which leukaemic stem cells are less sensitive to drug treatment.

Atherosclerosis, a chronic inflammatory disorder of the vasculature, is one of the major causes of cardiovascular disease and is responsible for most deaths in western societies. The disease is characterised by a number of steps that occur during the lifespan of an individual, including fatty streak formation, development of complex lesions containing a fibrous cap, and thinning and rupture of such plaques leading to thrombosis and clinical complications of this disease. Macrophages play key roles during all stages of this disease such as foam cell formation, amplification of the inflammatory response and control of plaque stability. The actions of macrophages during this disease are regulated by cytokines present in atherosclerotic lesions such as interferon-γ (IFN-γ), tumour necrosis factor-like protein 1A (TL1A) and interleukin-17 (IL-17). Statins are widely used for the primary and secondary prevention of atherosclerosis and its complications because of their ability to inhibit cholesterol biosynthesis and, thereby, plasma levels of pro-atherogenic low density lipoprotein. However, statins have actions beyond lowering cholesterol levels, the so-called pleiotropic effects, and includes acting in an anti-inflammatory manner. Unfortunately, the anti-inflammatory effects of statins are not fully understood and therefore formed the focus of studies in this thesis using a combination of human macrophage THP-1 cell line, primary cultures of human monocyte-derived macrophages and mouse RAW264.7 macrophage cell line. Simvastatin generally acted in an anti-inflammatory manner in macrophages in relation to the expression of several pro-atherogenic genes, such as monocyte chemoattractant proein-1 (MCP-1) and intercellular adhesion molecule-1 (ICAM-1),regulated by IFN-γ, TL1A or IL-17. Such an anti-inflammatory action also extended to another statin, Atorvastatin. The inhibitory action of Simvastatin on IFN-γ induced expression of MCP-1 and ICAM-1 was reversed, at least in part, by intermediates of the 3-hydroxy-3-methyl-glutaryl coenzyme A pathway such as farnesyl pyrophosphate and geranylgeranyl pyrophosphate. This suggested a potential role for monomeric G-proteins that require such intermediates for activation. In addition, Simvastatin inhibited the phosphorylation-mediated activation of signal transducer and activator of transcription-1 (STAT1), a key transcription factor in IFN-γ signalling, on tyrosine 701 and serine 727 in response to the cytokine. The effect of Simvastatin on MAP Kinase (MAPK) pathways in macrophages was also analysed. The statin attenuated the IFN-γ induced activation of p38 MAPK and extracellular signal activated kinase (ERK)-1/2. Simvastatin also affected the constituve expression of many components of the MAPK pathways (e.g. ERK-1/2) along with downstream genes involved in atherosclerosis (e.g. ATP-binding cassette transporters-A1 and-G1). The effect of Simvastatin on lipid profile of THP-1 and RAW264.7 macrophages was also investigated. Simvastatin does not affect total polar lipids and triacylglycerol. The statin also had no significant effect on fatty acid distribution into polar lipids and triacylglycerol. The studies presented in this thesis provide insights into the actions, and potential mechanisms, underlying the anti-inflammatory effects of statins on human macrophages along with their effects on lipid profiles. Such studies are essential given the widespread use of statins and a need to gain a deeper understanding of their actions.