Essential hypertension (EH) is considered one of the major contributors to the present pandemic of cardiovascular disease (CVD). EH has a largely obscure aetiology, which lies upon both environmental risk factors and underlying genetic traits. The stroke-prone spontaneously hypertensive rat (SHRSP) is an excellent model of human EH and exhibits salt sensitivity. Two quantitative trait loci (QTL) for blood pressure (BP) regulation have been identified on rat chromosome 2 (chr.2). On this basis, previous work in our laboratory focused on construction of chr.2 congenic strains, on both the SHRSP and Wistar-Kyoto (WKY) genetic backgrounds. In combination with microarray gene expression profiling in kidney from salt-loaded rats, two positional candidate genes for salt-sensitive hypertension were identified. Sphingosine-1-phosphate receptor 1 (S1pr1) and vascular adhesion molecule (Vcam1) lie on the chr.2 congenic interval implicated in salt-sensitivity. Additionally, studies on vascular smooth muscle cells (VSMC) demonstrated enhanced S1PR1-mediated sphingosine signalling in SHRSP compared to WKY. Finally, glutathione S-transferase mu 1 (Gstm1) was identified as another chr.2 candidate gene for BP regulation, lying outside the region implicated in salt-sensitivity. This project attempts to comprehensively investigate the potential role of altered S1PR1 signalling in BP regulation and salt-sensitivity, through comparative proteomic and metabolomic profiling in WKY, SHRSP and chromosome 2 congenic and transgenic stains (WKY.SPGla2a, SP.WKYGla2a, SP.WKYGla2k and Gstm1-transgenic). Characterisation of S1PR1 expression in renal and vascular tissue from 21 week-old salt-loaded rats, demonstrated below detection protein levels across parental and congenic strains. To further investigate the effect of the congenic interval and Gstm1 on salt-sensitivity and BP regulation and identify putative biomarkers, high-throughput metabolomic screening of urine and plasma was conducted in parental, SP.WKYGla2k congenic and Gstm1-transgenic strains, on a normal-salt and high-salt diet. In both urine and plasma, salt-loading affected processes implicated in CVD, including inflammatory response, free radical scavenging and lipid metabolism. In urine, oleic acid, implicated in regulation of renin levels, was increased in the SHRSP and transgenic salt-sensitive strains compared to the WKY and 2k congenic salt-resistant strains, upon salt-loading. In plasma, known biomarkers of CVD were altered in SHRSP compared to the other three strains, at normal-salt, including L-proline and linoleic acid. Upon salt-loading, glutathione disulfide and sphingosine-1-phosphate (S1P) were identified in high levels in the salt-sensitive strains. However, at normal-salt S1P was decreased in SHRSP compared to WKY and 2k congenic strains. Therefore, characterisation of the impact of S1P/S1PR1 signalling in the vasculature across the different strains was further investigated. Initially, structure, mechanical properties and vascular reactivity of mesenteric resistance arteries (MRA) were studied in 16 week-old parental and reciprocal 2a congenic strains (WKY.SPGla2a and SP.WKYGla2a). There was no significant remodelling observed across the strains. However, SHRSP vessels were stiffer and this phenotype was under the control of the congenic segment. SHRSP exhibited hypercontractility, which was mediated by RhoA/Rock signalling pathway and was corrected by the transfer of the congenic interval in SP.WKYGla2a. SHRSP also displayed endothelial dysfunction, which was related to reduced nitric oxide (NO) bioavailability and was not improved by the congenic interval. The predominant regulatory mechanisms of contraction and relaxation in MRAs from WKY and WKY.SPGla2a were demonstrated to be different compared to SHRSP. Subsequently, representation of these physiological differences in MRAs, at the molecular level, was investigated along with the effect of S1P-signalling in HTN. Comprehensive, high-throughput proteome profiling of S1P-stimulated primary mesenteric VSMCs from parental and 2a-reciprocal congenic strains, was achieved through triple stable isotope labelling (SILAC), LC-MS/MS analysis and MaxQuant quantification. Detection of few abundant phosphorylated proteins was attributed to lack of enrichment for phosphoproteome. Therefore, focus was placed on proteins whose differential expression between SHRSP and WKY was genetically regulated. These proteins mapped to pathways implicated in BP-regulation, including oxidative stress, vascular tone regulation and vascular remodelling. Glutathione S-transferase mu 1 (GSTM1) was upregulated in SHRSP, as opposed to down-regulated NAD(P)H oxidase quinone 1 (NQO1) and heme oxygenase 1 (HMOX1), suggesting different antioxidant mechanisms in health and disease. Natriuretic peptide receptor C (NPR3) which is implicated in vascular relaxation was increased in SHRSP, along with activators of RhoA contractile mechanism, such as caveolin1 (CAV1). Furthermore, RhoA/Rock signalling pathway was highly altered in SHRSP. Finally, differentially expressed proteins were related to sphingosine signalling, including superoxide dismutase 2 (SOD2) and collagen type III, alpha 1 (COL3A1). To further investigate the metabolic effect of sphingosine signalling across the strains, and assess the contribution of the congenic interval, metabolomic profiling of primary mesenteric VSMCs from parental and SP.WKYGla2a congenic strains, was performed at basal conditions and upon S1P-stimulation. A labelling-free, untargeted approach was employed, using HILIC-MS analysis and data processing through IDEOM. The effect of the congenic interval on the metabolic profile of SP.WKYGla2a was more profound under basal conditions. S1P-stimulation induced greater responses in SHRSP than WKY, indicating altered signalling. Furthermore the responses were different in each strain, suggesting a combined effect of the genetic background and the congenic interval on S1P signalling regulation. Inosine, which is implicated in purine metabolism, was significantly decreased in SHRSP compared to SP.WKYGla2a, at basal conditions, but was increased upon-S1P stimulation, implying that this S1P effect depends on the congenic interval. Moreover, tyramine, which has vasodilatory properties, was increased in stimulated SHRSP compared to basal conditions, indicating potential relation of sphingosine signalling with BP-regulation. This study has combined high-throughput proteomic and metabolomic screenings with congenic and transgenic strains to capture a clearer picture of the pathophysiological processes that underlie HTN in SHRSP. Individual metabolites and proteins or pathways and processes identified to be altered in HTN, through this work, can be used for generation of new testable hypothesis towards the development of new therapeutic approaches against HTN.

It is now accepted that there is a link between obesity and several diseases such as cardiovascular disease (CVD), diabetes, rheumatoid arthritis (RA), and atherosclerosis with the common initiating factor in pathogenesis being a state of low grade, chronic inflammation. This state, characterised by elevated levels of pro-inflammatory cytokines such as interleukin (IL) 6, leads to sustained activation of inflammatory signalling pathways such as the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway and subsequently pathogenesis. Suppressor of cytokine signalling (SOCS) 3 is inducible by several stimuli including IL6 and 3'-5'-cyclic adenosine monophosphate (cAMP), and via these routes has been demonstrated to terminate IL6 signalling thus quenching JAK/STAT signalling and an inflammatory response. While SOCS3 was primarily characterised as a competitive inhibitor of intracellular signalling, it also functions as specificity factor for an elongin-cullin-SOCS (ECS)-type E3 ubiquitin ligase. In this role, it has been demonstrated to direct ubiquitin-mediated proteasomal degradation of several substrates and lysosomal routing. However, the full spectrum of SOCS3-dependently ubiquitinated substrates is unknown. Given that JAK/STAT signalling is critical in the development of chronic inflammatory disorders, delineating the role of SOCS3 as an E3 ligase might be therapeutically beneficial. However, given the broad range of SOCS3 stimuli, the availability of certain SOCS3 substrates might be conditional on the route of SOCS3 induction. Using a global proteomics approach, this study aimed to identify SOCS3-dependently ubiquitinated substrates in response to cAMP and thus elaborate on the already well-established role of cAMP in inflammation. Differentially stable isotope labelling of amino acids in cell culture (SILAC)-labelled, tandem affinity purified ubiquitinomes of wild type (WT) murine embryonic fibroblasts (MEFs) and SOCS3-/- MEFs, each expressing epitope-tagged forms of ubiquitin, were compared using mass spectrometry (MS) following cAMP-mediated SOCS3 induction. Using this approach, proteins modified by SOCS3 with the epitope-tagged form of ubiquitin should be enriched in WT MEFs but not SOCS3-/- MEFs. MaxQuant analysis of raw mass spectromeric data identified several candidate SOCS3 substrates. Of these, SOCS3 was found to interact with PTRF/cavin-1, a regulator of caveolae formation and stability. Other substrates were tested but with limited success. Co-immunoprecipitation studies showed that SOCS3 could precipitate cavin-1 however the interaction was reduced following the inhibition of protein tyrosine phosphatases (PTPs) using sodium orthovanadate and hydrogen peroxide. This was surprising since all known SOCS3 substrates are tyrosine-phosphorylated prior to interacting with SOCS3 via its Src-homology (SH) 2 domain. Consistent with this finding, SOCS3 did not interact with known cavin-1 tyrosine-phosphorylated peptides spotted on a peptide array. However, a full-length cavin-1 peptide array spotted with non-tyrosine-phosphorylated peptides showed specific interactions at multiple sites. It is proposed that this interaction might influence the localisation and stability of either protein. While SOCS3 was demonstrated to impact cavin-1 ubiquitination, the mechanism by which it does so or the functional consequence is still not clear. Immunoprecipitation of cavin-1 following the introduction of SOCS3 was accompanied by a shift in the polyubiquitin signal from a high molecular weight, seen with cavin-1 alone, to a low molecular weight. Furthermore, an enhanced K48-polyubiquitin signal was detectable in this low molecular weight fraction, which was focused around the molecular weight of cavin-1. It is not known if this ubiquitin signal is SOCS3-dependent. In conclusion, the project has identified and validated a novel substrate of SOCS3. However, the mechanism by which SOCS3 regulates cavin-1 ubiquitination or the biological function of the interaction is currently unknown.

The structure and properties of a nascent pectin-xyloglucan complex in etiolated pea epicotyls were investigated. Membrane pellets were prepared and incubated with UDP-[14C] galactose, and were extracted with six different reagents: Tris-HC1 buffer (pH 7.4); 50mM EDTA/50mM PO4 (pH6.8) at 100°C; 50mM EDTA/59mM PO4 (pH6.8) at 25°C; phospholipase C with 100 ml of 0.1M Tris-HC1 pH7.4 at 25°C; trypsin at 25°C and 0.1% Triton X-100 at 25°C. The best extractant used to solubilise the pectin-complex from the pellets was 50nM EDTA/50mM PO4 pH 6.8 at 100°C. Aqueous solutions of pectic polyuronides tend to associate covalently into multichain aggregates. Because of the tendency of pectins to aggregate in solution, the effect of a number of eluents on the behaviour of the complex on gel filtration was studied. 10mM EDTA/10nM PO4/1M NaC1 (pH6.8) was chosen as the best eluent to minimise this aggregate formation. On gel filtration using Sepharose CL-2B with this eluent, the complex eluted with a Kav of around 0.8, corresponding to a molecular size approximately 200 kDa, as judged by dextran standards. In other solvents tested, aggregation appeared to occur. On Sepharose CL-4B and CL-6B columns using the same EDTA/Pi/NaC1 solvent, the apparent molecular size was significantly reduced by a xyloglucan-specific endoglucanase, confirming the presence of a xyloglucan rather that a glucan in the complex. Polygalacturonase caused a greater decrease in apparent molecular size, to 10-20 kDa while endo-1 4-b-galactanase converted the radioactivity to [14C] galactobiose and [14C] galactose. When the [14C] galactose-labelled complex was incubated in solution with 3MM paper, the radioactivity was almost completely absorbed onto the paper over a period of about 6 hours. This confirmed the presence of xyloglucan attached to pectin. A new model for the structure of the nascent pectin-xyloglucan complex is proposed.

To date, investigation and policy development relating to antimicrobial resistance (AMR) have focused largely on describing patterns of resistance to individual antimicrobials, often from restricted data sources. What this approach fails to accommodate is the complexity of AMR, such as genetic linkage of resistance determinants, which could potentially lead to inaccurate inference. Novel approaches are therefore required to bring new perspectives on the epidemiology of AMR. These studies take an ecological perspective of AMR, examining several key issues: The relative contribution to AMR from animal and human populations; the potential differences between passive and active surveillance of AMR; the associations of age, antimicrobial treatment and a shared environment with AMR diversity; the relationships between AMR phenotype and genotype; and exploring the additional information provided by DNA sequencing data. Novel ecological and epidemiological approaches were developed to examine long-term passive surveillance data of AMR in Salmonella Typhimurium DT104 (DT104) isolates from concurrently sampled and sympatric human and animal populations in Scotland. By examining the diversity and the phenotypic and temporal relatedness of the resistance profiles, the most likely source of resistance was assessed. The conclusions were that whilst ecologically connected, animals and humans have distinguishable DT104 communities, differing in prevalence, linkage, and diversity. Furthermore, the sympatric animal population is unlikely to be the major source of resistance for human DT104. As robust data are critical to any analysis, the potential differences between data collected by different surveillance types were examined. These systems are not generally designed to detect emerging resistances. The diversity of phenotypic AMR from passive and active surveillance data of poultry Salmonella Heidelberg and swine Salmonella Typhimurium var. 5- isolates derived from animals and foods-of-animal origin were contrasted to assess their suitability for detecting emerging resistance patterns. Results indicated that active and passive surveillance approaches were potentially sampling from distinguishable microbiological communities and are therefore complementary, and that passive surveillance is better at detecting rare profiles. The ecological and epidemiological approach was also applied to a different organism, in a different ecosystem. In order to assess the association of age and antimicrobial exposure with AMR diversity, E. coli from cows and calves on seven dairy farms were examined. There were distinguishable populations of resistance phenotypes on the farms, associated with both age and treatment. Multivariable models were developed to examine simultaneously the association of age, treatment, time, and farm with AMR diversity. These results indicated that there may be particular animal husbandry or farm management practices which influence AMR diversity, and which appear to be different for co-habiting young and adult dairy cattle. The majority of AMR data collected by surveillance systems are phenotypic in nature. However, it is often the underlying genotype that is of interest, which until now could only be achieved with the application of molecular methods. A novel latent class Bayesian model was developed to infer the prevalence of various AMR determinants in a population given a sample of phenotypes, which was applied to animal and human DT104 data. Differences were demonstrated in the estimated prevalences of a number of AMR determinants between the two populations, further supporting the previous observations that the epidemiologies of the organism, the resistance determinants, or both, are distinguishable. To obtain a greater resolution with which to compare AMR in different populations, a second-generation sequencing platform was used to obtain DNA sequencing data from select animal and human DT104 isolates. The objectives were to determine the diversity of the bacteria and of the resistance determinants. Whilst analysis of the resistance determinants is ongoing, preliminary results have suggested that the subtypes of DT104 infecting animals and humans are indeed similar. Overall, these studies comprise application of novel methods and frameworks for the analysis of AMR. The implication of these studies is that greater and more explicit thought is needed regarding the design, collection, analysis, and interpretation of AMR data properly to inform policy.

The thesis focuses on characterisation of two different protein families, kinesins and ubiquitin ligases, that are involved in different biological processes. Kinesins constitute a superfamily of microtubule-based motor proteins, fulfilling important cellular functions, ranging from intracellular transport to cell division. They also play a role in primary cilia and Hedgehog signalling. Ubiquitin ligases are enzymes that catalyse the transfer of ubiquitin to the lysine residues of the substrate. Marking protein substrates with ubiquitin alters their functions and fates. With nearly 700 different ubiquitin ligases in humans, they control a vast array of cellular processes. The first part of this thesis summarises results relating to kinesins. Protocols for creation of a panel of kinesin motor domains, a useful tool for anti-mitotic inhibitors specificity testing are presented. Furthermore, biochemical, kinetic and structural features of a poorly described kinesin motor protein - Kif15 are reported. Kif15's motor domain structure is described and compared to Eg5's catalytic core. Moreover, the influence of Eg5 inhibitors on Kif15 ATPase activity is investigated. Scouting for small molecules targeting Kif15 is also performed. Kif15 interaction with microtubules in various nucleotide-bound states is characterised. The possibility of a secondary microtubule binding site in the tail of Kif15 is examined. The binding of Kif15 with partner proteins is also investigated. Additionally the high-resolution structure of the human Kif7 motor domain is presented, providing the first step towards structural characterisation of this Kinesin-4 family member. The second part of this thesis concerns a ubiquitin ligase, Trim28. Trim28 was first reported as a transcription corepressor, working by recruiting proteins that drive the heterochromatin state, whilst its mechanism of action as a ubiquitin ligase remains elusive. Attempts to crystallise and determine the three-dimensional structure of Trim28 are described. Additionally, Trim28 functioning as an E3 ubiquitin ligase, and its interactions with E2s, KRAB domains of various zinc finger proteins and with the Mage-C2 protein are investigated. The results provide the foundation for future studies on Trim28.

Background Night Eating Syndrome (NES) was characterised in 2003 as comprising; morning anorexia, evening hyperphagia, night-time awakenings, consumption of high calorie snacks during awakenings and an absence of other eating disorders (ED). Method An identification study was conducted in a hospital–based UK obesity clinic with 81 individuals undertaking a diagnostic interview and completing a proposed screening tool. Full NES (n=7) and partial NES (n=24) individuals were combined into one Night Eating Behaviour (NEB) group (n=31) and compared to all other participants (n=50). NEB characteristics were identified through qualitative thematic analysis of interview data, based on techniques used by Grounded Theorists. NEB individuals (n=28) were compared with matched controls. In a separate prevalence study, night-eating, sleep quality and suspected Obstructive Sleep Apnoea (OSA) were estimated in 103 participants using the Pittsburgh Sleep Quality Index, Epworth Sleepiness Scale and a validated Night Eating Questionnaire. Correlational analysis was also performed. Results In the identification study, full NES was rare (9%). Comparison of the NEB and non-NEB group showed significant differences in mood (p=0.001), work status (p= 0.03), perceived lack of control over eating (p= 0.03) and variability in sleep duration (p<0.01). The study tool successfully identified NEB, but not other ED. Interview analysis identified the compulsive nature of night-eating and chaotic eating patterns. A variety of physical factors affected night-time awakenings. The matched control comparison showed no difference in levels of significant life events, childhood-onset obesity and poor sleep quality. Prevalence study results showed; suspected NES 14.6% (n=15), based on a lower cut score of >25, 3.9% (n=4), based on a higher cut score of >30, poor sleep quality 74% (n= 76) and suspected OSA 32% (n= 33). A strong relationship (r = 0.55, p = < 0.001) between night-eating and poor sleep quality was found, with day-time sleepiness having no influence over this relationship. Conclusion Comparison of the study findings with new NES criteria (2010) shows poor differentiation between NES and morbid obesity. In obese populations, a shift of focus is proposed to an alternative ‘Impaired Sleep Syndrome’ of which night-eaters may be a sub-group, often with severe depression. Night-eaters also exhibit too many features of other ED for this relationship to be ignored.

Diabetes (DM) increases mortality after myocardial infarction and deteriorates post-ischaemic cardiac remodelling. This study investigated possible implications of thyroid hormone (TH) signalling in either reducing or preventing this response. TH signalling has a regulatory role in metabolism, cardiac function, growth and ischaemic stress. Acute myocardial infarction (AMI) was induced in age–match healthy control rats (AMI-C) and in streptozotocin (STZ)-induced type I diabetic (DM) animals (DM+AMI) using 35 mg/kg body weight while sham operated animals served as controls (SHAM). The results show that AMI in tissue hypothyroidism caused significant down-regulation of TH receptors, TRα1 and TRβ1, in the diabetic myocardium without changes in T3, T4 levels in plasma. This response was associated with increased expression of β-MHC and distinct changes in cardiac function and geometry. Ejection fractions (EF%) was decreased in DM-AMI as compared to DM+AMI animals. Systolic and diastolic chamber dimensions were increased without concomitant increase in wall thickness and thus, WTI (the ratio of LVIDd/2*Posterior Wall thickness), an index of wall stress, was significantly elevated. The absence of wall thickening in DM+AMI hearts was associated with changes in stretch-induced kinase hypertrophic signalling p38 MAPK. In contrast, ERK, p-ERK and p-p38 MAPK levels were not changed in DM+AMI as compared to non-infarcted hearts (DM+SHAM). TH administration after AMI prevented hypothyroidism and resulted in decreased β-MHC expression, increased wall thickening and normalized wall stress, while stretch-induced p38 MAPK activation was restored. The results show that diabetes can exacerbates post-ischaemic cardiac remodelling and tissue hypothyroidism and TH treatment can prevent this response and improve cardiac haemodynamics.

The aims of this thesis were to characterise further lesion-induced impairments in unilateral rodent models of Parkinson’s disease (PD) on a more cognitive level and to investigate the effects of cell replacement therapies on these tests. Chapter 3.1 deals with the effects of dopamine depletion on a lateralised choice reaction time task in the Skinner box as this apparatus is more widely available than the 9-hole boxes on which initial studies have been based. Unilateral near complete lesions of the nigro-striatal pathway induced a stable side bias that was comparable to the lesion-induced deficits that have been reported in the 9-hole box apparatus. Chapter 3.2 reports on the effects of similar lesions on a more spatial reaction time task and the effects of engraftment of dopamine rich tissue in the denervated striatum. The lesions induced a spatial bias that was only marginally improved by the cell transplantation, clearly showing the limitations of ectopic graft placement. Nevertheless, small but significant improvements on that task could be shown as grafted animals performed with higher accuracy and had reduced movement times compared to the lesion only counterparts. Chapter 3.3 explores the lesion-induced deficit in more detail by implementing an error correction rule on the operant task to enforce a change in the animals’ response strategy. The results of this chapter confirmed earlier findings, that the dopamine depletion produced by the lesion gives rise to a strong near hole bias on the contralateral side which did not recover, even with extensive post lesion testing, i.e. the lesion-induced deficit is most likely to be caused by a misrepresentation of response space, rather than caused by a shift in response strategy. The second strand of this thesis focuses on the development of mouse models of similar dopamine-depleting lesions that are typically used in rat models of PD. In Chapter 3.4 the three most common lesion models are compared to each other on an extensive battery of simple motor tests. The aim was both to characterise the behavioural impact of dopamine depletion in different sites, as well as to identify appropriate hand tests, capable of distinguishing lesions greater than 70% depletion. The differences and similarities between lesions were evaluated and correlations between behavioural performance and nigral cell loss were observed. In Chapter 3.5 I developed parameters which allowed application of the lateral choice reaction time task to mouse models of dopamine depletion. Here I demonstrate the effects of two lesions, either to the medial forebrain bundle or the substantia nigra, on the same task conducted in mice. Lesioned mice of the former group displayed a stronger deficit largely because of the larger dopamine depletion. Subsequently, in Chapter 3.6 I characterise the effects of primary fetal tissue grafts on the previously established model and task. Primary fetal tissue was able to ameliorate some of the lesion-induced deficits on an operant choice reaction time task and a series of simple motor screens. The results of both strands of research in the present thesis have implications for the understanding of the cognitive and motor deficits that are induced by the most commonly used lesion model of PD and for the parameters that can be recovered by cell replacement therapies. The primary fetal tissue will serve as a baseline, against which future stem cell based therapies can be measured

Currently, UK forensic service providers (FSPs) tend to employ three geographically-broad databases when estimating profile frequencies based on a standard SGM Plus® DNA profile. These estimations will typically include correction factors to take into account issues such as substructuring of populations and sampling inefficiencies. It has been shown previously that regional genetic variation within the UK ‘Caucasian’ population is negligible but consideration has to be made for profiles which may originate from an individual of a more genetically isolated population. Samples were collected from Indian, Pakistani and UK (white British) donors; as well as Kalash individuals, a small population from the Khyber Pakhtunkhwa region in the North West of Pakistan. These were profiled using the SGM Plus® and Identifiler® kits and databases for each population were compiled. The greatest pairwise FST was seen between the Kalash and Indian population at 2.9 %. Allele frequency data were collected for each population and each sample’s profile frequency was estimated against all other databases to see whether samples reported a more conservative profile frequency (higher match probability) in their cognate database or in that of another population. A combined database comprising the Indian, Pakistani and previously published Bangladeshi data was also formed and used to calculate the level of correction required to make all samples of a population report a more conservative profile frequency in this combined database as opposed to their cognates. At the standard FST correction of 3 % – the minimum correction used by some FSPs, 94 % of the UK samples reported a more conservative profile frequency in the South Asian database; the lowest proportion that did so from all four populations. The Kalash dataset required the highest correction factor at FST = 12 % to make 100 % of samples report more conservative match probabilities when measured against the combined database. It was established that the current levels of correction applied to profile frequency calculations were more than sufficient; with random match probabilities remaining in the order of less than one in one billion for all samples in all databases with a correction of FST = 5 %. Although significant pairwise FST differences were observed as well as significant differentiation between populations across all SGM Plus® loci, no evidence of substructuring was detected using a program which employs a Bayesian probabilistic clustering approach, STRUCTURE, likely due to an insufficient number of samples and number of loci tested. Marked differences were seen in allele frequencies of the Kalash population, which also exhibited the highest affiliation to their cognate database, at least 80 %, with or without correction. AMOVA analysis also confirmed the greatest variance between groups was seen when the Kalash were kept as a separate entity from the other South Asian populations. Although current UK practice for applying FST correction prior to estimating STR match probabilities seems generous, there will be occasions when an estimation may appear less conservative when based on a broad database. Conversely, in this study, the one in one billion match probability ceiling threshold was not exceeded for any sample being compared to all databases. Therefore, although consideration should be given to a suspect’s reference population prior to frequency estimation, the current correction factors applied should be sufficient in the vast majority of cases. In instances where partial profiles are obtained, this caused little effect on the estimation of geographic origin, compared to full profiles, with the populations used in this study.

It has been established in a number of research publications that a careful study of general handwriting features based on class characteristics could indicate either the place or the country where the writer was first taught to write. Using these studies as the basis, this research was carried out explicitly to understand the characteristics of Arabic handwriting. The aim of this study was to determine the presence of any particular features or characteristics that may be common to individuals of a given region or nationality. This was done by obtaining samples of handwriting collected from individuals of four countries including; Jordan, Morocco, Oman and Tunisia, where Jordon and Oman are considered to be Eastern Arab world and Morocco and Tunisia in the Western Arab world. An attempt was made to establish whether it was possible to determine either the region or nationality of the writer of an Arabic passage of text, based on the formation and the style of the handwriting using specific Arabic characters. Different steps were taken towards the identification of the class characteristics of Arabic handwriting in this study starting with the collection of 600 handwriting samples from the participants in four Arabic countries employing; 150 handwriting samples from each. Ten different characters and one word were selected for examination, with more than one form of each character in different positions being identified and the handwriting samples classified accordingly. In total, 221 class characteristics were identified from the samples based on different criteria including the shape, number of strokes, pen movement and starting point. Tests of association using chi-squared on individual characters showed that the p-value is less than 0.001 in every case. Correspondence analysis was used to produce a plot of relative similarities where the different countries appear as discernible, but overlapping groups. ANOSIM showed these groups to be statistically different (R = 0.321 p = 0.0002, 1000 permutations). Tree analysis was used to create a classification system and blind tests were conducted to test the accuracy of the classification system. On the basis of the statistics used, significant differences were found in character forms used by the individuals from the four Arabic countries, suggesting that either region or nationality of the writer may potentially be predicted with a useful degree of accuracy. Though the samples were obtained from only four countries out of a total of 22 Arab countries and only ten characters and one word out of 28 characters were chosen in this study, the results obtained are valuable and useful, particularly to Forensic Document Examiners (FDEs). In turn this could be implemented in practice in a situation where a questioned document containing Arabic text is presented and the suspected author could have come from one of the four considered countries.