Phonation is a complex process requiring the controlled exhalation of air through the larynx. Within the larynx there is a specialist tissue structure known as the vocal folds, which under muscular control captures energy within the airflow and transfers it to a dynamic phenomena, analogous to a static fluid wave, known as the mucosal wave. This mucosal wave causes the vocal folds to open and close rhythmically, thus modulating the airflow, which can then be manipulated in the vocal tract to create the sounds that we know as speech. The purpose of the research detailed in this thesis is the quantification of the biomechanical properties of the vocal folds. There is a major gap in knowledge relating to the elastic properties of the vocal fold as the only reliable apparatus available to determine these properties rely on dissecting the tissue out of anatomical context. The author's research is dedicated to developing methods to measure these properties from intact larynges, and from patients in vivo. This is to enable a better understanding of how this complex tissue structure works; to assist with the derivation of mathematical models of phonation; and to provide methods to assess objectively the effectiveness of tissue engineering therapies used to repair scarred vocal folds. The author devised a new and novel apparatus to obtain data from excised tissue and in vivo. A key principle of these devices is that they directly measure the mechanical properties of intact larynges, which contrasts to methods reported by the majority of other researchers. The author also managed a number of research grant funded projects, in his capacity as PI, which deployed the devices. The author developed most of the software and the mathematical techniques used to analyse the data. Details of the apparatus devised to obtain data from both excised larynges and in vivo are given, which required the derivation of devices capable of measuring micrograms of force and displacement resolutions at micron level. Also given are the mathematical models used to transform the raw data into the fundamental material property known as shear modulus. The results include measurements of the shear modulus of a group of 20 excised vocal folds, of varying ages and both sexes. Also given are the results of similar data obtained from eight volunteer patients in vivo. The anisotropic nature of vocal fold tissue is quantified and iso-contour maps presented showing the variation of elasticity with respect to anatomical position. Early results are given that quantify the change in vocal fold tension with respect to electrical stimulation of the recurrent and superior laryngeal nerves in a canine model.A lso given are the resultso f a study that demonstratedth at hyaluronica cid tissue augmentation could restore vocal fold pliability in a rabbit model.

Osteoarthritis (OA) is a degenerative joint condition that affects roughly one third of adults over the age of 60. In the UK, this amounts to over 8 million people with the total cost of the disease to the economy estimated at £12 billion. Symptoms can include joint pain, stiffness, effusion (swelling of the affected joint) and reduced mobility. Whilst the symptoms and diagnosis of the disease have been clearly defined in medical research, the underlying causes are not yet fully understood. It is thought that biomechanical factor’s, and walking kinematics in particular, play a key role in OA aetiology. Furthering the understanding of these factors could lead to better treatments and help reduce prevalence through preventative measures. A gait analysis protocol suitable for a clinical environment was developed to analyse the Newcastle Thousand Families Study birth cohort. This presented a unique opportunity to study an existing cohort of adults who are representative of the overall population. Gait analysis was performed on every able cohort member who attended for clinical assessment over a period of 16 months. Females showed more significant differences in their gait than males. Of the differences found in males, most were found to be associated with altered cadence. Some variables among female participants were found to be associated with altered cadence, as well as body mass index (BMI), pain and stiffness. It was concluded that female gait is more susceptible to kinematic changes but that these changes are adaptations that slow disease progression. Males do not make these adaptations and show higher prevalence at later OA grades. Differences in cadence were thought to account for most differences in gait kinematics with BMI, pain and stiffness also contributing. Overall, none of the variables measured seem likely to have caused the initiation of OA, however there is potential that the variables showing significant associations between grade 0 and 1 (particularly cadence) could be used for the prediction of OA incidence from gait and could be used as a supporting measure for other diagnostic tools.

Ca2+ -activated Cl- channels (CaCCs) are activated by an increase in intracellular [Ca2+] and as vascular smooth muscle cells (VSMCs) activity accumulate Cl- ions, opening of these channels leads to Cl- ion efflux and membrane depolarisation. There is very little known on how these channels are regulated, the aim of this project was to investigate the regulation of CaCCs in VSMCs. Initial experiments were carried out to characterise CaCC currents (lClca) in isolated rat pulmonary artery smooth muscle cells (PASMCs). Native CaCCs are proposed to be encoded by TMEM16A. The presence of this gene in PASMCs shown using quantitative PCR and protein expression with Western Blot. Furthermore novel modulators of currents produced by overexpression of TMEM16A were shown to modulate native lClca. The majority of the work, investigated the novel finding that CaCCs are regulated by the membrane phospholipid phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2). Using whole cell electrophysiology, a reduction in PI(4,5)P2 levels via either phospholipase C activation, PI4K inhibition, PIP2 scavenging or absorption, increased lC/ca. Conversely intracellular enrichment of PI(4,5)P2 inhibited lClca. Furthermore, PI(4,5)P2and other phospholipids were shown to directly bind to TMEM16A isolated from rat pulmonary artery and TMEM16A-eGFP expressed in HEK293 cells. This data suggest that PI(4,5)P2 plays a negative role on CaCC activation. Finally the interaction of CaCCs and the cystic fibrosis transmembrane conductance regulator (CFTR) channel in mesenteric artery smooth muscle (MASMCs) was studied. TMEM16A and CFTR proteins were shown to locate <40 nm of each other with proximity ligation assay. In isometric tension recordings TMEM16A (T16inh- AOl) and CFTR (oxo-CFTR-172) inhibitors caused potent relaxation on preconstricted vessels, with oxo-CFTR-172 showing paradoxical effect. Increasing CFTR activity either by directly or indirectly decreased the potency of T16Ainh-AOl, whereas decreasing CFTR activity increased the potency of T16Ainh-AOl. This suggests that CFTR negatively regulates TMEM16A.

The subjects of these studies are the major components of the mammalian muscle spindle, which is an encapsulated proprioceptor serving to monitor skeletal muscle length and length change. Those components are: specialized intrafusal muscle fibres; sensory nerve endings that form intimate contacts with the intrafusal fibres; and motor nerve fibres by means of which the central nervous system can exercise control over the sensitivity of the spindle. My first important contribution was to establish the number of types of intrafusal fibre (1-8, 11). Their different mechanical properties help to shape the responses of the sensory endings in characteristic ways (papers 15 and 42). Detailed reconstructions of sensory endings revealed recognizable features of the primary ending that were consistently associated with the different intrafusal fibres (10, 13, 18, 20, 33). The sites of nerve impulse generation and coding are being studied in relation to the branching pattern of the sensory nerve fibres (45, 50, 55). Analysis of the innervation of individual spindles has revealed the interplay of random and deterministic factors in spindle construction (20, 36, 37, 40, 41, 44, 48, 52, 53). As yet it is unknown how the differences that exist between muscles in this respect are related to their specific roles in motor control or kinaesthesia. However, reflex activity appears to be grossly disturbed in muscles that have been reinnervated following nerve section, since functional endings may be formed in inappropriate locations (22, 25, 28- 31,34, 38, 39, 43, 46). The motor innervation of the spindle was for long controversial, especially concerning the distribution of the different functional categories of axon. I have pursued histophysiological and probabilistic approaches to this problem, about which there now appears to be a large measure of agreement in favour of my conclusions (9, 12, 19, 21, 23, 26, 27, 35, 41, 42, 44, 48, 49). Papers 1-6 in the following list are based on work that originally formed part of a thesis presented in candidature for the degree of Ph. D. in the Faculty of Medicine, University of Sheffield. For each full paper of which 1 am a co-author an estimate of my contribution to the overall effort is given as a percentage in the list.

NBRl (Neighbour of BRCAJ) is a ubiquitously expressed scaffold protein that mediates intracellular functions through protein-protein interactions. The C-terminal UBA domain of Nbrl binds mono- and polyubiquitinated chains whilst the N-terminal PB1 domain interacts with the structurally similar protein p62. The aims of this thesis were to further characterise the biological functions of Nbrl by identifying novel protein interactors. A yeast-2-hybrid screen revealed a direct interaction between Nbrl and the autophagic effector protein LC3. This suggested a role for Nbrl in the autophagic degradation of polyubiquitinated proteins. A further yeast-2-hybrid screen identified a direct interaction between Nbrl and the microtubule associated protein MAP IB. It is hypothesised that via its interaction with MAP IB and LC3, Nbrl targets ubiquitinated proteins to the microtubule network where they can be transported to sites of autophagosomal formation. Further putative interactors were identified for Nbrl including the ubiquitin conjugating enzyme, UBE20 and the eukaryotic translation elongation factor eEFl A. These suggested a role for Nbrl as a scaffold to facilitate protein ubiquitination and degradation by the proteasome as eEFl A is involved in the cotranslational degradation of polyubiquitinated proteins. -- In bone, Nbrl was previously identified as a key regulator of osteoblast differentiation and activity. The work described in this thesis strengthened these observations and highlighted that the interaction between Nbrl and p62 is also important for the maintenance of bone. Mice with the NbrlD50R point mutation which inhibits the interaction between Nbrl and p62 displayed an early osteoporotic phenotype due to altered osteoblast function and osteoclast size and nucleation that was subsequently resolved with age.

Regular participation in intense physical activity induces characteristic physiological ECG and echocardiographic changes. These are well described in Caucasian athletes (WA). Male black athletes (MBAs) demonstrate a higher prevalence of repolarisation anomalies and a greater maximal left ventricular wall thickness (mLVWT). The longitudinal significance of this is unclear. There is no published data on female black athletes (FBAs). Methods Between 1996 and 2010, 1144 elite BAs (21% female) underwent a standard pre-participation screen, including a 12-lead EGG and 2-D trans-thoracic echocardiogram. These were compared with 2059 similar WAs (12% female), 259 sedentary black controls (BCs; 54% female), and 52 black patients with hypertrophic cardiomyopathy (HCM). Any healthy subject exhibiting a mLVWT of >11mm (females) or >13mm (males), underwent comprehensive examination to look for phenotypic features of HCM. Male athletes were followed up for 69.7±29.6 months. Results BAs demonstrated a greater mLVWT (Females 9.2±1.2mm vs. 8.6±1.2mm, P<0.001: Males 10.6 ±1.6mm vs. 10.0±1.2, P<0.001) than WA and BC subjects. Eight FBA (3%) exhibited a mLVWT >11mm (12 to 13mm) compared with none of the FWA. T wave inversions (TWI) were present in 82.7% of HCM patients, 22.8% MBAs, 14% FBAs, 10.1% BCs, 3.7% MWA and 2% FWA (P<0.001). The major determinant of TWI in healthy subjects was black ethnicity. In BAs, TWI were confined predominately to the anterior leads (V1-4). Anterior TWI were infrequent amongst BCs (4.2%) and HCM patients (3.8%). TWI in the lateral leads were frequently seen amongst HCM patients(79.6%), but rare in all healthy individuals. During follow-up, one MBA survived a cardiac arrest, and two athletes (one MBA and one MWA) were diagnosed with HCM. Conclusion: Systematic physical exercise in BAs is associated with greater LV hypertrophy and higher prevalence of TWI than in similar WAs. However, a mLVWT >13mm (females) or >16mm (males) or deep TWI in the inferior/lateral leads are rare and warrant investigation.

Hepcidin, the iron regulatory peptide, has emerged as the master regulator of systemic iron homeostasis. Under normal circumstances, hepcidin expression is upregulated by excess iron and inflammation; and downregulated by iron deficiency, anaemia and hypoxia. Insights gained into the pathogenesis of iron-storage disorders such as hereditary haemochromatosis (HH) and hypotransferrinaemia (HPX), have contributed to the identification of the molecular mechanisms governing hepcidin expression. In these disorders, hepcidin expression is inappropriately low, causing increased absorption of iron by the gut thus leading to iron overload. The precise mechanism by which hepcidin is suppressed in these disorders is unclear. The regulation of hepcidin is principally transcriptional, where the Bone Morphogenetic Protein (BMP) pathway has been shown to be a major network governing hepcidin expression. The current study utilised the HPX mouse model to identify potential regulators of hepcidin that are produced locally in liver with a focus on the regulation of hepcidin by the BMP signalling pathway. A known regulator of the BMP pathway, bone morphogenetic protein [BMP]-binding endothelial cell precursor-derived regulator (BMPER), was found to be overexpressed in the HPX mouse liver. Soluble BMPER peptide in excess strongly inhibited BMP-dependent hepcidin promoter activity in both HepG2 and Huh7, cells abolishing the effects of BMP2 and attenuating the effects of BMP6. These effects correlated with reduced cellular pSMAD levels. Addition of recombinant BMPER peptide to primary human hepatocytes strongly downregulated hepcidin mRNA levels and abolished the effects of BMP2. These effects were reflected in vivo where the injection of mice with recombinant BMPER peptide significantly reduced hepcidin mRNA expression which correlated with increased serum iron levels. Thus, the protein may play an important role in suppressing hepcidin production to increase iron availability under conditions of chronic anaemia. Using the same HPX model, several other genes related to the BMP pathway also demonstrated differential gene expression. Atonal Homologue 8 (ATOH8), a basic helix-loop helix (bHLH) transcription factor, was previously shown to be regulated by iron loading however its precise role in iron metabolism was unknown. The studies presented in this thesis identified hepatic ATOH8 mRNA and protein expression to be robustly downregulated in various mouse models of altered iron metabolism where increased erythropoietic activity was shown to suppress hepcidin. Further investigations demonstrated that ATOH8 expression in HEK-293 cells directly regulated the hepcidin promoter and increased cellular pSMAD levels, thereby establishing a hitherto missing link between the regulation of hepcidin, erythropoietic activity and the BMP/SMAD pathway. Finally, the little known BMP, BMP8b, and the clotting factor, von Willebrand Factor which were also found to be significantly increased in the liver of the HPX mouse, were investigated as potential regulators of hepcidin. BMPSb has the potential to form heterodimeric complexes with other BMP members and therefore could be important in modulating BMP signalling and thus in turn hepcidin promoter activity. Whereas von Willebrand factor contains BMP binding sites and therefore may sequester BMPs, thus inhibiting BMP signalling. However the present study was unable to show consistent effects of either molecule on hepcidin promoter activity and so the roles of BMPSb and von Willebrand Factor in iron metabolism, if any, remains unclear. In conclusion, the analyses presented in this thesis demonstrate the novel molecular interactions governing hepcidin regulation by the BMP pathway. The new knowledge generated will be useful in the development of therapeutic strategies to diagnose, prevent, or mitigate disorders of iron homeostasis.

The stretch-shortening cycle (SSC) is an intricate muscle action, which is fundamental to successful plyometric performance. However, there currently exists minimal research examining the effects of age, maturation and trainability on the development of SSC in male youths. Study one of this thesis examined the reliability and validity of a series of SSC-type activities in young adolescents, as assessed by a mobile contact mat in comparison with force plate analysis. Squat jump height was the most reliable measure (CV = 8.64%), while reactive strength index (RSI) during maximal hopping and leg stiffness during sub-maximal hopping produced moderate reliability (CV = 10.17% and 13.98% respectively). Measures of leg stiffness were in agreement (TEE = 6.5-7.5%) with force plate data during sub-maximal hopping, but not during maximal hopping (TEE = 41.9%). Test reliability was moderate to good for the range of performance variables, and leg stiffness measures during sub-maximal hopping were valid. Subsequently, it was concluded that the contact mat served as a replicable assessment tool to assess SSC function in youths. Previous research has identified that a number of physical abilities undergo nonlinear development throughout childhood, however research for SSC development is lacking. Study two of the thesis examined the effects of age and maturation on the natural development of SSC function in boys aged 7-17 years. Peak height velocity (PHV) was used as a central reference mark to assess maturational status, and significant differences were apparent three years either side of (PHV) for SJ, CMJ, RSI and leg stiffness development (P <0.05). Results suggested the possible existence of periods of accelerated adaptation, however it remained unclear as to whether exposure to the correct training stimulus during these timeframes would promote adaptation above and beyond that of natural growth alone. The third study examined the neural regulation of SSC function in youths during both sub-maximal and maximal hopping. Fifteen-year olds produced greater levels of absolute and relative leg stiffness than both twelve- and nine-year olds during sub-maximal hopping. During the sub-maximal hopping condition, the youngest children spent longer in contact with ground, less time in the air, and utilised less muscle activity contribution during the short-latency stretch-reflex phase. When performing maximal hopping, both 15- and 12- year olds recorded greater RSI values than nine-year olds. When expressed as a percentage of total feedforward activity, preactivation of the soleus muscle in the 100 ms prior to ground contact was significantly greater in 15-year olds compared to the younger children when hopping maximally. Therefore, results would suggest a greater reliance on feedforward and immediate feedback mechanisms with advancing age, which help to increase absolute and relative stiffness. The final study examined changes in SSC performance following a 4-week plyometric training programme. Exposure to the plyometric training resulted in 12- and 15- year olds making significant improvements in both absolute and relative leg stiffness, and 12-year olds achieving significant training adaptations in RSI. Control groups revealed no performance changes, and in some cases a significant decline in performance. This study demonstrated that a 4-week training programme can have a positive effect on SSC function, and that trainability may be age or maturation dependent for various measures associated with leg stiffness. The research has enhanced our understanding of the impact of age and maturity on the natural development of SSC function in male youths. It has highlighted that natural development alone can result in performance changes and that, amongst other variables, neural regulation appears to be a confounding factor. Additionally, it was identified that the SSC is sensitive to change when exposed to the correct training stimulus, suggesting the possible existence of trainability.

Dental Age Assessment (DAA) is used to estimate age when date of birth is uncertain. This thesis utilised radiographs archived at the Eastman Dental Hospital and King‟s College Dental Hospital. All teeth developing on the left side were assessed using an eight stage system (Demirjian 1973) and a twelve stage system (Haavikko 1970). The ages of attainment for each Tooth Development Stage (TDS) provided the Reference Data Set (RDS). Dental Age (DA) was calculated using weighted averages. DA estimates using the eight and twelve stage systems were compared, as were the effects of gender and ethnicity. The relative distal root canal widths (RCW) of the 1st, 2nd and 3rd permanent molars were assessed using a five category system designed by the investigator. In addition ossification of the sternoclavicular joint (SCJ) was also assessed using a five stage system (Schmeling 2004). The stages and the categories were related to age. The improvement obtained by combining DA, RCW and SCJ data was explored. A total of 2,622 subjects comprised the RDS with 45% male and 55% female, and an age range of 3 - 35 years. The main ethnic group was White (70%) followed by Black (13%), Mixed (5%) Asian (3%) and „not recorded‟ for 9% of subjects. The mean difference between DA & CA was -0.15 years (SD 1.3) for males and -0.14 years (SD 1.4) for females respectively using 12 stages, and -0.14 years for both genders using 8 stages. The greater ease of use of the 8 stage system makes it preferable for DAA. The combination of DA, SCJ & RCW showed that DA was the best predictor of an individual with teeth still developing. For subjects with no teeth still developing, SCJ can be used to estimate the age of an individual.

Phonation is a complex process requiring the controlled exhalation of air through the larynx. Within the larynx there is a specialist tissue structure known as the vocal folds, which under muscular control captures energy within the airflow and transfers it to a dynamic phenomena, analogous to a static fluid wave, known as the mucosal wave. This mucosal wave causes the vocal folds to open and close rhythmically, thus modulating the airflow, which can then be manipulated in the vocal tract to create the sounds that we know as speech. The purpose of the research detailed in this thesis is the quantification of the biomechanical properties of the vocal folds. There is a major gap in knowledge relating to the elastic properties of the vocal fold as the only reliable apparatus available to determine these properties rely on dissecting the tissue out of anatomical context. The author's research is dedicated to developing methods to measure these properties from intact larynges, and from patients in vivo. This is to enable a better understanding of how this complex tissue structure works; to assist with the derivation of mathematical models of phonation; and to provide methods to assess objectively the effectiveness of tissue engineering therapies used to repair scarred vocal folds.