In the past decade, limited progress has been made in identifying genetic associations with performance and health-related fitness phenotypes due to the use primarily of the traditional candidate-gene approach involving small sample sizes and few coordinated research efforts. Much of the genetic data relating to human performance has been generated while exploring the aetiology of lifestyle-related disorders such as obesity and type 2 diabetes mellitus (T2DM). As of 2008, over 200 autosomal gene entries and quantitative trait loci have been reported to be significantly associated with performance and health-related fitness. However, most genetic findings to date have been inconclusive due to studies employing relatively small sample sizes and predominantly single-gene approaches which are especially prone to type I errors. It is widely accepted that there will be many genes involved in sporting performance and health-related fitness phenotypes, and hence it is timely that genetic research has moved to the genomics era with the use of a genome-wide approach (e.g. genotyping a large number of variants simultaneously across the entire human genome) in a well-phenotyped, large cohort. This thesis summarizes the recent findings of genetic predisposition to elite human performance by using the conventional candidate-gene approach as well as the unbiased genome-wide approach (i.e. genome-wide association studies, GWASs). The current candidate gene study focused on investigating whether polymorphisms in the angiotensin-converting enzyme (ACE) and α-actinin-3 (ACTN3) genes are associated with elite swimmer status (stratified by swimming distance) in Caucasians and East Asians. ACE I/D and ACTN3 p.R577X polymorphisms were genotyped for 200 elite Caucasian swimmers (short and middle distance, ≤ 400 m, n = 130; long distance, > 400 m, n = 70) and 326 elite Japanese and Taiwanese swimmers (short distance, ≤ 100 m, n = 166; middle distance, 200–400 m, n = 160). Logistic regression and multiple-testing adjustment were applied to test for these genetic associations. ACE I/D was found to be associated with swimmer status in Caucasians, with the D allele being overrepresented in short-and-middle-distance swimmers with the largest effect being observed for the I-allele-dominant model (odds ratio = 1.90; logistic regression p = 0.001; permutation test p = 0.0005). In East Asians, however, the I allele was overrepresented in the short-distance swimmer group under the D-allele-dominant model (odds ratio = 1.52; logistic regression p = 0.012; permutation test p = 0.0098). The ACE I/D association findings in the elite swimmer cohorts showed that different risk alleles responsible for the associations were observed in swimmers of different ethnicities. ACTN3 p.R577X was not statistically significantly associated with swimmer status in either Caucasian or East Asian population. The lack of associations between the functional ACTN3 p.R577X polymorphism and elite swimmer status in both cohorts were in contrast to many associations with power-/sprint- performance in other sports previously reported. Since current sample size is relatively modest, larger studies will be required to further confirm these results, which, however, have highlighted that it is probable that the genes studied here are not the resulting variants responsible for the phenotypes of interest, despite the associations reported by previous candidate-gene studies in other sports. The present GWAS were conducted in an attempt to identify common polymorphisms associated with elite sprint and endurance status in Jamaicans, African-Americans and Japanese, respectively. These unique athlete cohorts comprised of athletes of the highest standard including world record holders, world champions, Olympians and winners of other international events. Following exclusion of individuals and markers failing the quality control filters, 609,801 autosomal SNPs in 88 Jamaican sprint athletes and 87 Jamaican controls, 637,991 autosomal SNPs in 79 African-American sprint athletes and 391 African-American controls, and 541,179 autosomal SNPs in 114 Japanese athletes (including 60 endurance and 54 sprint athletes) and 116 Japanese controls, were available for association analyses. 17, 7, 36 and 21 SNPs were associated with elite athlete status at a p < 0.00005 threshold of significance in elite Jamaican sprint, African-American sprint, Japanese sprint and Japanese endurance GWAS sets, respectively. Meta-analyses were performed for SNPs with unadjusted association p < 0.00005 across the sprint GWAS sample sets (i.e. Jamaican sprint, African-American sprint, Japanese sprint GWAS cohorts), using the fixed-effects model. The top 17 SNPs (unadjusted p < 0.00005) from the Jamaican sprint cohort were extracted from the association results of African-American sprint, Japanese sprint cohorts, respectively, for the combined effects to be calculated using a meta-analysis method. The same procedure was also applied to the top hits in African-American and Japanese cohorts. The combined odds ratio for the top meta-analysis hit was 2.61 (p = 0.000000466) with the allele G associated with elite sprint status in Jamaicans, African-Americans and Japanese. Although meta-analysis has increased the sample size and power to detect associations in the current GWAS, independent replication of these associations followed by functional studies of replicated SNPs are required. The results of the association studies presented here are the very first positive findings from GWAS involving world-class athletes and these encouraging findings provide further evidence of the importance of genetic predisposition to elite human performance. GWAS of athletes of the highest performance caliber as well as the application of meta-analysis across several initial GWASs seemed to help to circumvent the need for very large cohort of elite athletes and increase the study power. Nevertheless, future GWAS involving large well-funded collaborations using larger cohorts of elite athletes will be necessary in order to explore further the genetic architecture underlying elite human performance. Such initiatives may also allow gene x gene and gene x environment interactions to be explored to some extent, as well as the predictive utility of this genomic research.

In the first part of the thesis we focus on a hierarchical analysis on multiple, single cell, Nrf2 reporter levels in nucleus and cytoplasm, observed in human endothelial HMEC-1 in vitro cells (Xue et al., 2015a). Nrf2 is a transcription factor that regulates the expression of several defensive genes protecting against various cellular stresses and forms of oxidation. This analysis aims to gain an insight into this essential cellular protective mechanism. We propose a reaction network based on five reactions, including a distributed delay and a non-linear term, for longitudinal measurements of the amount of Nrf2 in nucleus and cytoplasm. The diffusion approximation (DA) is used to approximate this Markov jump process with a stochastic delay differential equation (SDDE). Since this continuous process is only observed at discrete time points, a second approximation, the Euler-Maruyama approximation (EMA) of the DA, is needed to obtain an approximate likelihood for this bivariate process. Furthermore, to make use of multiple single cell data, we embed the model in a Bayesian hierarchical framework. Moreover, a measurement equation, which involves a proportionality constant and a bivariate normal error, for the nuclear and cytoplasmic measurements, is necessary to relate the original unobserved population levels, X, to the observations, Y. This introduces a hidden Markov process for X and a Bayesian analysis is performed, via a data augmentation procedure, to explore the high dimensional posterior space which includes a bivariate latent process X for every cell. We show results obtained on simulation studies, proving the validity of the methodology, and on a real data application, composed of 35 single cell fluorescent xvi levels under the basal condition, and of 36 under the induction by a stimulant, both observed every two minutes for 1.5-7 hours. In the second part of the thesis we describe the analysis of a switch gene model for mRNA populations. We consider a gene that switches, with exponential waiting times, between a more active ON state and a less active OFF state, where the gene transcribes mRNA at a higher and a lower rate, respectively. We observe, via a measurement equation, the mRNA level in each cell, which is assumed to have reached a steady state. We analytically derive the stationary distribution of such a model and infer its parameters from experimental data, again via hierarchical Bayesian inference. The mRNA populations are only observed up to a proportionality constant and with a second source of white noise attributed to the measurement process. As in the previous case, we use a data augmentation procedure to explore the posterior space of the latent data. The analysis is repeated for different levels of induction by tetracycline, a stimulant, which results in increased gene expression. We particularly focus on studying how the stimulation affects the system.

Small RNA are short, 18-25 nt molecules that regulate gene expression in plants and animals. Two main types, microRNA (miRNA) and short interfering RNA (siRNA) perform this regulation by transcript silencing, translation inhibition, DNA methylation and chromatin remodeling. This thesis is an investigation into small RNA activity in archaeological plant material, specifically barley grain from Qasr Ibrim, a multi-period archaeological site in southern Egypt. It is of particular interest due to its unusual phenotype, suggestive of stunted development that is unexpected in a staple, domesticated cultivar, and the unusual level of DNA and RNA preservation attributable to the extremely arid climate at the site. The research presented here is a comparative analysis of small RNA profiles and epigenetic states of Qasr Ibrim barley and modern, unstressed counterparts. It concludes that differential microRNA and epigenetic profiles are the result of stress response, adaptation, dormancy and / or viral infection unique to the archaeological grain. The primary method of investigation was generation of small RNA sequence data using the Illumina GAIIx platform. This was followed by extensive bioinformatic analysis (RNA diagenesis patterns, miRNA prediction, siRNA target prediction and small genome in silico reconstruction) the results of which were in turn validated experimentally (genomic methylation states, locus-specific methylation analysis and direct miRNA detection). The research represents a twofold contribution to knowledge: first, proof-of-principle that biologically meaningful archaeological RNA can be extracted despite its relative instability to DNA, and second that a unique miRNA profile and epigenetic state is detectable in this particular cultivar of archaeological barley.

DNA associates with proteins to form chromatin which is essential for the compaction of the DNA into the cell nucleus and is highly dynamic in order to allow the different biological processes of the DNA to occur. Chromatin compaction is achieved at different hierarchical levels: the 10nm fibre (DNA associates to nucleosomes formed by different histones), the Higher Order Chromatin fibre and the 300 nm chromosome structures. This study has shown that both H1 and H4 histones play a crucial role in preserving meiotic as well as mitotic chromosome structure and functional genome integrity in Arabidopsis. The role of the different linker histone H1 isoforms as well as the core histone H4 in Arabidopsis thaliana was investigated using T-DNA and RNAi mutant lines which showed different meiotic defects. Chromosomal breaks as well as non-homologous connections in the h4RNAi were linked to 45S/5S rDNA disorganisation, suggesting that H4 preserves chromosome integrity at these rDNA regions. Ath1.1 mutant presented univalents and reduced chiasma frequency at metaphase I, linked to a severe defect in ASY1 localisation on the meiotic chromosome axes. Thus, indicating that histone H1.1 is vital for proper chromatin axis organization that permit normal loading of recombination machinery proteins in Arabidopsis.

There is no information available in the scientific literature that documents a specific assessment protocol for analysing a soccer player’s maximal power capabilities. As soccer-associated muscular power has not previously been investigated, it is not known how important power is in elite soccer and, if it is, the physiological and genetic determinants of soccer-associated power remain unknown. Such information could be used to optimise soccer-specific talent identification and development strategies. With this in mind, the overriding aim of our thesis was to investigate the physiological and genetic factors underpinning powerful actions in elite youth soccer. When devising an assessment of soccer-associated muscular power, there needs be a detailed analysis of the specific actions performed during elite competitive match-play that can be described as powerful. The aims of our first experimental study (Chapter Three) were to compare the frequency and durations of powerful actions during competitive English Premier League [under 18 (U18) and under 21 (U21)] elite youth soccer matches using a novel soccer specific powerful action (SSPA) notational analysis coding system. We found that while elite soccer match-play requires players to perform powerful actions in multiple directions [68 horizontal accelerations (in the horizontal-forward or mediolateral directions), eight sprints, and six vertical jumps (three bilateral and three unilateral)], horizontal accelerations of short duration (< 1.5 s) from different starting speeds were the most dominant type of explosive action. This activity profile provides a strong rationale for devising a muscular power assessment protocol that evaluates the ability to produce maximal power in multiple directions, from a unilateral stance. Our data also suggests that such a protocol could provide a specific lower body power profile in elite soccer players (ESP). The aim of our second study (Chapter Four) was therefore, to determine whether countermovement jumps (CMJs) in different directions [CMJs: bilateral vertical (BV), unilateral vertical (UV), unilateral horizontal-forward (UH) and unilateral medial (UM)] assessed independent lower-limb power qualities, and if CMJ performance differed between ESP (representing an English Premier League Academy regularly at U18 and under U21 levels) and non-elite soccer players (NSP). We found that unilateral CMJs in different directions assessed independent peak vertical power (V-power) and resultant take-off velocity capabilities, and the UH CMJ required significantly greater bicep femoris electromyographic (EMG) activation in comparison all other CMJs. Moreover, in comparison to NSP, ESP achieved greater V-power during all CMJs (p≤0.032) except for BV (p=0.197), and also achieved greater UH CMJ projectile range (51.6 ± 15.4 vs. 40.4 ± 10.4 cm, p=0.009). Our results suggest that unilateral CMJs in different directions, but not the commonly used BV CMJ, are determinants of U18 and U21 elite soccer playing status and can be used by applied practitioners as independent assessments of soccer-associated muscular power. As the physiological determinants of performance are of use to the applied practitioner for informing talent identification criteria, and prescribing detailed training intervention strategies, the primary aims of the third and fourth studies were to investigate the neuromuscular (Chapter Five) and tendon (Chapter Six) determinants of unilateral CMJs oriented in different directions. Our data suggests that unilateral CMJ performance is associated with direction-specific neuromuscular and tendon properties in U18 and U21 ESP. While UV CMJ performance was related to the size (quadriceps femoris muscle volume and physiological cross sectional area), architecture (vastus lateralis pennation angle) and ability to activate (vastus lateralis EMG activation level) the knee extensor muscles, UH CMJ performance was related to the elongation and compliance properties of the patellar tendon, and was inversely correlated with vastus lateralis fascicle pennation angle. Our findings highlight the importance of targeting specific neuromuscular and tendon properties when assessing and developing muscular power performance in U18 and U21 ESP. Many physiological changes occur during puberty (Viru et al., 1999) and our findings in Chapters Four, Five and Six may only be applicable in U18 and U21 ESP. Therefore, in our fifth study (Chapter Seven) we aimed to investigate the importance of acceleration, sprint, horizontal-forward CMJ and vertical CMJ capabilities at different stages of maturation in elite youth soccer. Elite soccer players and CON were grouped using years from/to predicted peak height velocity (PHV, a measure of growth velocity and an indirect measure of pubertal phase) to determine maturation status (ESP: pre-PHV, n=100; mid-PHV, n=25; post-PHV, n=88; CON: pre-PHV, n=44; mid-PHV, n=15; post-PHV, n=54). By comparing performance of ESP and control participants (CON) matched for maturation status, we found that acceleration and sprint performance were associated with elite youth soccer at all stages of maturation, but maximal power (horizontal-forward and vertical jumping) capabilities may only be important for elite youth soccer at mid- and post-peak height velocity. Our data could imply that assessments of acceleration and sprint capabilities should be included in soccer talent identification protocols at all stages of maturation, but maximal power should only be included at mid- and post-PHV. The purpose of our sixth experimental study (Chapter 8) was to investigate if specific gene single nucleotide polymorphisms [SNPs: ACTN3 R577X (rs1815739), BDNF G > A (rs6265), COL5A1 C > T (rs12722), and COL2A1 C > T (rs2070739)] played a role in determining elite youth soccer player status, and speed and power capabilities, in ESP and CON at different stages of maturation. We found that ACTN3 R- and BDNF G-allele frequencies were more frequent in post-PHV compared to pre-PHV ESP. Moreover, while the COL2A1 CC genotype was associated with greater horizontal power and faster 20 m sprint performance, BDNF GG genotype appears to positively influence 20 m sprint performance during the pre-PHV period only. Overall, our findings illustrate that elite soccer may require different genetic profiles before and after maturation, and genetic screening could be included in talent identification criteria to help predict maximal power and sprint potential in ESP. In summary, we devised a muscular power assessment battery that measured independent power qualities and could discriminate between U18 and U21 ESP and NSP. Our subsequent analysis showed that the physiological factors underpinning unilateral CMJ performance were direction-specific, and UV and UH CMJ capabilities were underpinned by separate neuromuscular and tendon properties, and should be assessed and developed, independently in U18 and U21 ESP. We then recruited a larger cohort of ESP and CON, at different stages of maturation, and demonstrated that muscular power was important for elite soccer performance at mid and post-PHV, but not pre-PHV. Finally, we showed that genetic profiles of ESP differed between pre- and post-PHV, and that certain gene variants [COL2A1 C > T (rs2070739), BDNF G > A (rs6265)] were associated with specific power and speed capabilities in ESP. Overall, our studies provide novel information that could have significant implications on soccer-associated power related talent identification and training intervention strategies in elite youth soccer academies.