Series: Discussion Papers SFB International Tax Coordination

Research investigating the regulation of muscle fibre type has traditionally been conducted in vivo, analyzing global changes at a whole muscle level. Broadly, this thesis aimed to explore more “molecular” approaches, utilizing molecular and cell biology to understand the expression and regulation of myosin heavy chain (MyHC) isoforms as an indicator of muscle fibre composition. The mRNA expression profile of six MyHC isoform genes during C2C12 myogenesis was elucidated to reveal that the C2C12 cell line mimics developing fast-twitch muscle fibres. Additional characterization of the C2C12 cell line revealed a dramatic restructuring of metabolic gene expression during the switch from proliferating to fully differentiated C2C12 muscle cells. Post-mitotic muscle cells exhibited increased glycolytic gene expression and reduced oxidative gene expression and an increase in gene expression of enzymes involved in redirecting glucose carbons into ATP generating pathways and away from macromolecule biosynthesis (p<0.01 for all genes). The dynamic plasticity of MyHC isoform gene expression was compared between C2C12 muscle cells and fully differentiated adult muscle. Exposure of adult muscle to the beta-adrenergic agonist, Ractopamine, induced dynamic transitions in MyHC isoform expression, from the IIA/IIX isoforms to increased IIB isoform expression (p<0.05 for all genes). An acute exposure of C2C12 muscle cells to Ractopamine was capable of inducing an exclusive and rapid induction of the MyHC IIB isoform gene expression during myogenesis (p<0.001). The C2C12 cell line was utilized as a host environment for a molecular-based approach to understand the role of the promoter sequence in regulating the species-differential induction of the MyHC IIB gene during myogenesis. A 3bp miss-match in the CArG-Ebox region (at -74bp, -68bp and -48bp) of the proximal MyHC IIB promoter was identified that dictates the differential expression of MyHC IIB in pigs and humans.

No description available.

Phosphoinositides are lipid signals that control cellular processes and are particularly closely associated with the control of membrane trafficking. PtdIns(3,5) \(\char{cmmi10}{0x50}\)\(_2\) is the most recently identified phosphoinositide and was previously recognised as controlling events in the late endocytic system between the late endosome and the vacuole/lysosome. Primarily associated with retrograde trafficking from the vacuole/lysosome to the late endosome/MVB, PtdIns(3,5) \(\char{cmmi10}{0x50}\)\(_2\) is generated by the kinase Fab1p (PIKfyve in animals). In mammalian cells, PtdIns(3,5) \(\char{cmmi10}{0x50}\)\(_2\) has also been implicated in control of ill-defined trafficking pathways close to the Golgi; for example, the recycling of mannose-6-phosphate receptor (M6R) back to the Golgi and also the trafficking of some types of ion and nutrient channels from the Golgi to the cell surface. This thesis describes attempts to study putative PtdIns(3,5) \(\char{cmmi10}{0x50}\)\(_2\) dependent trafficking in the early endocytic system of \(\char{cmmi10}{0x53}\). \(\char{cmmi10}{0x63}\)\(\char{cmmi10}{0x65}\)\(\char{cmmi10}{0x72}\)\(\char{cmmi10}{0x65}\)\(\char{cmmi10}{0x76}\)\(\char{cmmi10}{0x69}\)\(\char{cmmi10}{0x73}\)\(\char{cmmi10}{0x69}\)\(\char{cmmi10}{0x61}\)\(\char{cmmi10}{0x65}\) using two model proteins; the recycling of Vps10p from late endosome to Golgi and of Chs3p from recycling endosome to Golgi.

The introduction of electron capture dissociation mass spectrometry in 1998 has provided a unique technique for the analysis of peptides and proteins, especially for the identification and localisation of posttranslational modifications. Despite many successes debate continues on the radical-based mechanism of ECD. This thesis explores ECD behaviour in a wide range of PTMs with the intention of furthering our knowledge of the radical-based mechanism. Studies were undertaken on the effect of 3-nitrotyrosine, which is an electron withdrawing group, on ECD. The presence of nitration dramatically decreases peptide backbone sequence coverage but results in the presence of abundant small neutral losses. The key finding is the insight provided into the hierarchy of the various proposed ECD mechanisms. ECD of cysteine bound modifications is shown to result in the fragmentation of the sulfur-modification bond and backbone sequence coverage is highly diminished when analysing S-nitrosopeptides. ECD behaviour of hydrogen-deficient radical peptides is highly dependent on gas-phase peptide structure, with electron capture typically resulting in an increase in charge-reduced precursor intensity. Comparisons of the intermolecular phospho-guanidinium bond strengths between phospho-serine, -threonine and -tyrosine were undertaken. ECD of these complexes results in the retention of the noncovalent bond allowing backbone sequence coverage.

The principal aim of this project is to examine posture and muscle activity when using an ergonomically designed saddle seat compared with a conventional seat during common dental procedures with the dental students and practising dentists. The study was conducted with practising dentists across the West Midlands and the dental students in the School of Dentistry – University of Birmingham. The study is mainly divided into a questionnaire survey of practising dentists, a questionnaire survey of dental student posture in the dental schools across the U.K, postural analysis, and a daily symptom survey of practising dentists and dental students, and finally the EMG analysis of practising dentists and dental students working posture. This thesis has established the relationship between posture, seating and muscle activity and indicates that use of an ergonomic aid (dental operator stool) may improve posture, decrease pain and muscle activity and may decrease the development of musculoskeletal disorders among dental students and dentists.

Chemotaxis underlies many physiological processes and is also hijacked by metastatic cancer cells to enter blood or lymphatic vessels enabling travel around the body. Using a novel migration assay we sought to determine the molecular mechanisms by which vesicle trafficking regulates chemotactic invasion. We show that dynamin is necessary for EGF-dependent migration and proceeding RNAi studies found clathrin-mediated endocytosis, but not caveolar endocytosis, to be necessary for migration in MDA-MB-231 cells. Using TIRF microscopy to investigate a role for endocytosis of integrins during chemotactic invasion we found no significant colocalisation of focal adhesion markers with endocytic markers. Inhibiting endocytosis also had no effect on the number or localisation of focal adhesions. Studies investigating EGFR showed that during EGF-directed chemotactic invasion of human breast cancer cells, EGFR endocytosis is polarised to the front of migrating cells and occurs via clathrin-mediated endocytosis. Analysis of exocytic trafficking of EGFR found this to be polarised towards the front of cells during chemotactic invasion. Finally we used FLIM-FRET microscopy to show that cells migrating in an EGF-dependent manner have increased signalling of Scr and Akt Biosensors. These experiments begin to investigate the link between endocytosis and signalling, an area not yet very well studied.

Musculoskeletal disorders are among the most important health problem affecting the quality of life and contributing to a high burden on healthcare systems worldwide. Understanding the molecular mechanisms underlying these disorders is crucial for the development of efficient treatments. In this thesis, musculoskeletal disorders including muscle wasting, bone loss and cartilage deformation have been studied using systems biology approaches. Muscle wasting occurring as a systemic effect in COPD patients has been investigated with an integrative network inference approach. This work has lead to a model describing the relationship between muscle molecular and physiological response to training and systemic inflammatory mediators. This model has shown for the first time that oxygen dependent changes in the expression of epigenetic modifiers and not chronic inflammation may be causally linked to muscle dysfunction. Bone and cartilage deformation observed in ageing, arthritis and multiple myeloma (MM) patients have also been investigated by using a novel modularization approach developed within this thesis. This methodology allows integration of multi-level dataset with large interaction networks. It aims to identify sub-networks with genes differentially expressed between experimental conditions that are co-regulated across samples in different biological systems. This study has identified several potential key players such as Myc, DUSP6 and components of Notch that could enhance osteogenic differentiation in MM patients. In conclusion, this thesis present the effectiveness of systems biology approaches in understanding complex diseases and these approaches could be applied for studying other systems and datasets.

A third of over 65s have at least one fall per year whilst a quarter of over 45s endure foot pain. Footwear is associated with both fall risk and foot pain hence its investigation is of great importance. This thesis explores the potential benefits of minimalist footwear for the older adult population. Chapter 2 ascertained the kinematic and kinetic differences between walking barefoot versus in footwear whilst highlighting the limited research on minimalist footwear, older adults and muscle activity differences. Accordingly, Chapter 3 outlined that minimalist footwear is kinematically more similar to barefoot, irrespective of age, thus offering a viable alternative. Similarly, Chapter 4 showed walking in minimalist footwear and walking unshod exhibit similar lower leg muscle activation patterns whilst differences exist to conventional footwear. Chapter 6 demonstrated how increasing intrinsic foot strength improved functional and static balance whilst Chapter 7 showed promise for minimalist footwear improving foot strength, functional balance, balance confidence as well as reducing foot and joint pain in a sample of older adults. In conclusion, this thesis highlights the need for future work to continue to investigate minimalist footwear in both older adults and other age groups for benefits to stability, foot health and joint pain.

Symptoms of gastro-oesophageal reflux disease are equally common in both sexes and at all ages. However, complications of gastro-oesophageal reflux disease such as reflux oesophagitis, Barrett's oesophagus and oesophageal adenocarcinoma, although more common in men, increase sharply in older women, suggestive of a protective effect of female sex hormones in menstruating women. Oestrogen has anti-inflammatory properties, improves healing in oral and skin wounds and may therefore reduce the severity of reflux-induced oesophageal mucosal injury, consequently protecting women from developing severe reflux oesophagitis. Long-term oestrogen treatment with hormone replacement therapy seems to be additionally associated with a reduction in the risk of oesophageal cancer. Moreover, there are gender-specific genotypic differences in the response of oesophageal mucosa to chronic acid reflux suggestive of multiple factors that may play a role in explaining the male predominance of oesophageal adenocarcinoma. Finally, oestrogen has no association with the severity of acid reflux once adjustment is made for the influence of increasing body mass index in women undergoing oesophageal pH monitoring. The gender difference in the prevalence of gastro-oesophageal reflux disease and its complications may thus be related to the effect of female sex hormones, particularly oestrogen and its 'protective' effect in pre-menopausal women.