This thesis advances the understanding of mitochondrial dynamics in adipocytes. Chapter 1 outlines mitochondrial dynamics, health, function and quality control in the context of obesity and T2DM. Chapters 2 and 3 are concerned with characterising mitochondrial dynamics in human abdominal subcutaneous adipose tissue and how the balance of this process may be augmented by adiposity and bariatric surgery. As an extension of this, Chapter 4 explores the potential regulatory role of p38 within adipogenesis in the 3T3-L1 cell line model together with the interplay between p38 and mitochondrial dynamics throughout differentiation. As part of this study, the impact of modulating p38 by chemical means or via the introduction of an overexpression vector was used to investigate the functional consequences of this gene on mitochondrial bioenergetics. The subtle balance of mitochondrial fusion and fusion markers is critical, not only for controlling the abundance and bioenergetic health of mitochondria but also, for overall cellular health. Finally, Chapter 5 extends analysis of the regulatory role of p38 on mitochondrial dynamics through computational modelling of the p38β. From the entire AstraZeneca compound collection, a series of novel and putative p38β inhibitors were selected for further functional assays to assess the effect of these compounds on mitochondrial bioenergetics in 3T3-L1.

Dematin is an actin-binding and -bundling protein that was first identified in human erythrocytes where it is important for providing mechanical stability. Two splice variants exist in vivo of 48 KDa and 52 KDa, each of which comprises a disordered C-terminal core domain and a short, highly-ordered, N-terminal headpiece (HP). Expression of dematin has now been demonstrated in a wide range of tissues and functional roles are emerging for dematin as a tumour suppressor, and regulator of wound healing, via negative regulation of RhoA. Despite these advances, the in vivo regulation of dematin remains poorly understood. This thesis therefore aimed to characterise the regulation of dematin by two known cytoskeletal coordinators; the p38 mitogen-activated protein-kinase (MAPK), and the adaptor protein 14-3-3. Dematin was confirmed as a substrate of p38, and multiple phosphorylation sites were identified in both the core and HP regions of the protein using mass spectrometry and in vitro kinase assays. Moreover pulse-chase degradation analysis identified dematin as a short-lived protein, with a calculated half-life of 5.49 +/- 0.44 hours. Further experiments confirmed that this propensity for degradation is at least in part due to the presence of a PEST motif, with the half-life of the dematin_ΔPEST construct significantly increased compared to wild-type dematin at 7.64 +/- 0.72 hours. Despite identifying p38 phosphorylation sites within and flanking the PEST motif, hyper-activation or inhibition of p38 had no significant effect on protein stability. Finally, it was shown that although p38 phosphorylated the dematin HP at Ser383, this was not sufficient to disrupt the association of the HP and core regions which is known to inhibit actin bundling. Additionally dematin was identified as a novel substrate of the 14-3-3 family of adaptor proteins. The 14-3-3β isoform was shown to bind to 52 KDa dematin at two phosphorylated motifs; RKTRS269LP and RGNS333LP. It was not possible to identify the kinases that regulate these motifs, but Akt, PKA, or AMPK were ruled out as potential candidates. Immunofluorescence staining and confocal imaging confirmed that 14-3-3 regulates dematin by altering its subcellular distribution, which is likely to be via masking of an actin-binding motif. Wild-type dematin was distributed throughout the cytoplasm in C2C12 skeletal muscle cells, whereas the dematin_S269/333A binding mutant localised strongly to the F-actin cytoskeleton. This localisation result was confirmed in quantitative sedimentation experiments, which showed a statistically significant decrease in both the actin-binding (42.8%) and -bundling (30.1%) ability of dematin upon 14-3-3 association. In this thesis I have characterised both the p38 MAPK and the adaptor protein 14-3-3 as novel regulators of dematin, adding considerably to the understanding of dematin regulation in vivo. This provides a basis for further investigation into the ability of dematin to coordinate the actin cytoskeleton, which has implications for a wide array of dematin functions due to its ubiquitous tissue expression profile.

Deep somatic pain originating from synovial joints is a major clinical problem as it is the primary reason for loss of joint mobility and function in musculoskeletal disorders. Musculoskeletal disorders, including osteoarthritis (OA), are the most prevalent cause of disability worldwide with an estimated 1 in 3 adults affected. Current therapies for the treatment of joint pain have limited effectiveness and certain drugs produce unwanted side effects, preventing their long-term use. Targeting pain at the level of the joint may have the potential to maximise treatment efficacy, whilst reducing possible non- specific side effects associated with systemic drug treatment. Identification of novel analgesic targets that inhibit peripheral mechanical sensitization during joint pain states will be critical to the development of improved analgesics for these conditions. Based on recent preclinical findings, the orphan G protein- coupled receptor GPR55 has controversially been suggested to be the novel third cannabinoid receptor and has been identified as a potential novel target for the treatment of pain. Very few studies have investigated the effects of GPR55 activation on nociceptive processing in vivo and only one at the level of the joint during acute inflammatory arthritis. The aim of this thesis was to investigate the role of GPR55 in the modulation of joint afferent mechanosensitivity in vivo, and whether this role is altered in an experimental model of OA during established pain. Electrophysiological recordings of joint afferent nociceptors, taken from the saphenous nerve, which innervates the knee joint via the medial articular nerve, were carried out in anaesthetised rats under non-pathological conditions (naïve rats) and in a model of OA following the development of pain behaviour, 14 days following knee joint injection of monosodium iodoacetate (MIA) and compared to saline control rats. Effects of peripheral administration of the putative endogenous GPR55 agonist L-α-lysophosphatidylinositol (LPI) on the mechanically-evoked responses of joint nociceptors were studied in naïve rats and in MIA and saline rats. An involvement of GPR55 in the effects of LPI was investigated using pre-administration of the GPR55 antagonist cannabidiol. Further, the role of GPR55 in endogenously modulating joint afferent mechanosensitivity was investigated following the peripheral administration of cannabidiol alone. GPR55 expression in knee innervating L3- L5 rat DRGs was studied by immunohistochemistry. Joint nociceptors in MIA rats were mechanically sensitized compared to the saline rats at 14 days post-injection confirming the development of peripheral sensitization during established pain behaviour. LPI (150, 250μM) inhibited joint nociceptor mechanically-evoked responses in naïve and saline rats and inhibited peripheral sensitization in MIA rats. Cannabidiol blocked the LPI- induced inhibition of joint nociceptor mechanosensitivity in all groups of rats confirming an involvement of GPR55 in these effects. Cannabidiol alone had no effect on mechanically-evoked responses of joint nociceptors in naïve, saline and MIA rats indicating that any endogenous GPR55 tone does not modulate joint afferent mechanosensitivity. GPR55 expression was detected in small, medium and large neurones (and possibly satellite glial cells) of L3-L5 DRG. The findings of this thesis provide compelling evidence that activation of GPR55 in vivo modulates the mechanosensitivity of joint afferent nociceptors and this inhibitory effect is maintained during established peripheral sensitization and pain behaviour following OA development. GPR55-mediated control of joint afferent mechanosensitivity during established OA pain and expression of GPR55 in sensory neurones at the level innervating the joint highlights GPR55 as a potential new peripheral target for the modulation of joint pain including during OA. The findings of this thesis support further studies aimed at investigating the clinical utility of GPR55 agonists for the treatment of OA pain.

The overall aim of this thesis was to determine the effects of administration of recombinant human erythropoietin) r-HuEpo on endurance performance and to explore the psychosocial effects of taking this drug. In addition, the effects of injecting a Placebo, believed to be an erythropoietin-like substance was assessed, quantitatively and qualitatively, to determine what role the placebo effect may play in mediating the effects of r-HuEpo on endurance performance. All tests were also carried out in the field to ensure that studies had high ecological validity. The first study, presented in this thesis (Chapter 3), aimed to assess the validity of the Cosmed K4b2 portable metabolic analyser (K4b2) in measuring VO2 during submaximal and maximal running velocities in an outdoor environment. Nineteen trained male volunteers (age: 22.9 ± 1.0 years; weight: 74.1 ± 1.8 kg; height: 179.2 ± 1.4 cm; mean ±SD; VO2max 59.37 ± 0.30; mean ± SEM) completed maximal continuous incremental running tests which involved 3 minute exercise stages at running speeds between 8 to 16 km·h-1 in 2 km·h-1 increments. Measured gas exchange variables included fractional content of expired oxygen (FEO2) and fractional content of expired carbon dioxide (FECO2), oxygen uptake (VO2), carbon dioxide output (VCO2), ventilation (VE) and respiratory exchange ratio (RER). The Douglas bag method was used for comparison purposes. The typical error in VO2 measurement for the K4b2 outdoors compared to the Douglas bag method was 6.5 ml.kg-1.min-1. This degree of error for the measurement of was considered unacceptable and it was therefore decided that the K4b2 was not accurate enough to justify its use in subsequent studies. The aim of Chapter 4 was to assess the effect of r-HuEpo administration on haematological, psychological and performance measures. Seven well-trained males (age: 25.7 ± 2.2 y; BMI 22.5 ± 0.7 kg.m-2; VO2max: 57.7 ± 2.9 ml.kg-1.min-1) participated in a 10 week protocol divided into three phases; baseline (2 weeks), r-HuEpo administration (4 weeks) and post administration (4 weeks). Fifty IU.kg-1 subcutaneous injections were administered every two days during the r-HuEpo administration phase. Blood measures included the determination of hemoglobin concentration ([Hb]), haematocrit (Hct), reticulocytes (ret) and total haemoglobin mass (tHb). Cognitive State Anxiety Inventory CSAI-2) and Profile of Mood States (POMS) were utilised for psychological measures. Maximal oxygen uptake was measured and 3 km time trial performance was assessed at baseline, immediately post (i.e. at 4 weeks) and 4 weeks post r-HuEpo administration. Cognitive State Anxiety questionnaires were obtained immediately prior VO2max and 3 km time trials. Profile of Moods States questionnaires were obtained at baseline, immediately and 4 weeks post. At immediately the post r-HuEpo administration time-point, [Hb], Hct, ret and tHb significantly increased compared to baseline (P < 0.05) by 21.2%, 21.3%, 75.0% and 11.7%, respectively. At 4 weeks post r-HuEpo administration, [Hb], Hct and tHb decreased compared to the immediately post time-point, but remained significantly elevated compared to baseline by 7.9%, 9.3% and 6.9%, respectively (P < 0.05). Ret decreased to below baseline levels (by 41.7%) 4 weeks post r-HuEpo administration (P <0.05). VO2max did not significantly change (P = 0.07) over the course of the protocol, but was numerically 5.2% higher immediately post r-HuEpo administration when compared to baseline. Running speed at which the LT occurred was significantly higher by 6.4% (P < 0.05) immediately post r-HuEpo administration when compared to baseline. Running speed at OBLA was significantly higher immediately post r-HuEpo administration when compared to baseline by 5.9% (P < 0.05) and this remained elevated 4 weeks post, by 5.3%, when compared to baseline (P < 0.05). The %VO2max at LT was significantly higher immediately post r-HuEpo administration when compared to baseline by 6.6% (P < 0.05). Lactate turn-point values were also significantly higher (P < 0.05) immediately post r- HuEpo administration when compared to baseline and 4 weeks post by 9.6 and 7.6%,respectively. The CSAI-2 cognitive subscale decreased significantly (18.3%; P<0.05)immediately post. The POMS tension subscale decreased significantly immediately and 4 weeks post baseline (61% and 50% respectively; P<0.05). Three km time trial performance significantly improved immediately post r-HuEpo administration by 4.9% (P < 0.05) when compared to baseline. Faster times were sustained 4 weeks post r-HuEpo administration, when compared to baseline, by 3.9% (P < 0.05). Changes in [Hb]significantly correlated with changes in VO2max (r = 0.985; P = 0.016) with a tendency for changes in Hct to correlate with changes in VO2max (r = 0.807; P = 0.053). A significant negative relationship was found between changes in %VO2max at LT with changes in [Hb](r = -0.882; P = 0.048) and Hct (r = -0.926; P = 0.024) immediately post r-HuEpo administration. This study confirmed r-HuEpo increases [Hb] and Hct, and these were associated with improvements in VO2max. Three km time trial performance improved immediately post r-HuEpo administration by 4.9%. However, no clear association was found between [Hb], Hct or VO2max and 3 km performance, suggesting that factors other than changes in [Hb], Hct and VO2max are likely to have influenced running performance. Positive changes were also observed in psychological components which warrant further investigation. Performance benefits persisted 4 weeks post r-HuEpo administration. Chapter 5 aimed to assess the effect of r-HuEpo use on psychosocial factors associated with the practice. The use of substance abuse in sport, with the aim of improving performance, has been prevalent in sport for decades. Recent studies, have attempted to measure athlete viewpoints on doping in sport. However, few studies have been able report viewpoints related to illegal substance use, whilst participants are actually being administered with a banned substance. Six well-trained males (age: 26 ± 2 y; BMI: 22 ± 1 kg.m-2; VO2max: 58 ± 3 ml.kg-1.min-1) individually participated in semi-structured interviews after completing the trial involving the administration of r-HuEpo by subcutaneous injection for 4 weeks. Participants individually participated in semistructured interviews which were carried out within 2 weeks of completing the study. Varied responses were given in relation to psychosocial impacts and experiences. Five participants reported feeling a substantial performance enhancing effect. One individual expected a greater performance effect. Four individuals stood by their initial stance of ‘drugs should be banned in sport’. One individual entertained the idea that drugs should be allowed, but in a ‘controlled’ manner. One participant suggested that his views had changed, since being involved in the trial, sighting being geographically disadvantaged in the UK in comparison competitors with access to higher altitude. However, the emotions of guilt, shame and/or embarrassment were prominent themes which may be considered as a significant deterrent to doping. Chapter 6 aimed to determine whether an injectable placebo, claiming to be a legalsubstance with similar effects to r-HuEpo, would improve endurance running performance in simulated race conditions. Fifteen endurance-trained male volunteers (age: 27.5 ± 6.8 years, height: 1.79 ± 0.05 m, body mass: 73.4 ± 7.6 kg: BMI 22.9 ± 2.0 kg.m-2) successfully completed the randomised cross-over study design consisting of 3 km competition races before and after a 7-day ‘control’ phase and ‘placebo’ phase. During the placebo phase, participants self-administered daily subcutaneous saline injections in the belief that they were receiving a performance enhancing drug called OxyRBX.

The majority of musculoskeletal patients attend with Lumbo-Pelvic and Hip dysfunction complaining of difficulty during walking and running. Clinicians commonly use weight bearing tests to examine the components of walking and running but these tests use postures and movements that are different to those found during gait. Few studies have examined the relationship between these tests and gait. The aim of this study was to investigate the validity of the Trendelenburg Test, Single Leg Squat and Corkscrew Test as measures of dynamic stability of the Lumbo-Pelvic and Hip region in healthy participants and professional football players during gait. This was a laboratory based study using an experimental, repeated measures design. 18 full time professional football players and 14 healthy participants were recruited. Movement data was captured using a ten camera system using the CAST technique. This study found that for walking there should be observable movement of all the regions and in all planes except at the: lumbar spine; thoracic spine ; trunk in the sagittal plane and lumbar spine; pelvis in the coronal plane. For running there should be observable movement of all the regions and planes. Recommendations are made for changes in the interpretation of the Trendelenburg Test and Single Leg Squat. New values for the interpretation of the Corkscrew Test were also established. Professional football players exhibited differences in their movement patterns at the hip, pelvis and trunk when compared to the healthy participants. Using the Corkscrew and Single Leg Squat Tests in combination allows clinicians to comprehensively examine the sagittal and coronal plane range of movement of the lumbar and thoracic spine relevant to walking. Similar movements occurred during the tests and both walking and running, but the similarities occurred only in specific regions and planes. Hence the tests were found to be task, region and plane specific. A greater understanding of the clinical tests and their relationship to gait may help clinicians to implement evidence based examination, sub-classify and treat Lumbo-Pelvic and Hip dysfunction. This will be of greatest use when examining and treating populations who have been found to have Lumbo-Pelvic and Hip dysfunction such as young males and professional football players.

Methylglyoxal is a physiological dicarbonyl metabolite and potent argininedirected glycating agent. It often modifies proteins at functional sites producing loss of positive charge, structural distortion and inactivation. Plasma methylglyoxal is increased in hyperglycaemia associated with diabetes and is linked to the development of vascular complications of diabetes – particularly nephropathy, retinopathy and neuropathy. The effects of dicarbonyl glycation on beta cells and involvement in early stage dysfunction and development of type 2 diabetes mellitus are not known. The aim of this project was to investigate the effect of dicarbonyl protein glycation on beta cell function and related involvement in the development of diabetes. Studies were performed in an in vitro model of beta cell dysfunction - MIN6 insulinoma cells incubated under low and high glucose concentrations, and in a pre-clinical in vivo model of decline of glucose tolerance preceding development of type 2 diabetes - high fat diet-induced insulin resistant mice. Dicarbonyl metabolism and protein damage by glycation and oxidation were studied by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry. Localisation of methylglyoxal glycation adducts within the pancreas were visualised by immunostaining. Interactions between the extracellular matrix protein, collagen IV, and MIN6 cells in vitro were investigated and impairments in adhesion were assessed following glycation with methylglyoxal. Impairments in adhesion of MIN6 cells to methylglyoxal-glycated collagen IV were assessed using atomic force microscopy force spectroscopy. The results show that MIN6 cells were resistant to accumulation of methylglyoxal when incubated in high glucose concentration although the flux of methylglyoxal was increased 41%. Glycation of collagen IV by methylglyoxal impairs binding to MIN6 cells in vitro resulting in a 91% decrease in the energy necessary to detach cells from the extracellular matrix protein. In high fat diet fed mice the concentration of methylglyoxal in the pancreas was increased. Visualisation of MG-H1 adduct residues in the pancreas showed they were predominantly on the extracellular matrix. In conclusion, protein glycation by methylglyoxal occurs in MIN6 cells in vitro and in the mouse pancreas in vivo. Although the methylglyoxal concentration in the pancreas of high fat diet fed, insulin resistant mice was increased, the lack of a concurrent increase in methylglyoxal protein glycation adducts suggests there may be increased turnover of methylglyoxal-modified proteins. Impairment of beta cell attachment to the extracellular matrix protein, collagen IV, by methylglyoxal and increased protein turnover stimulated by an increased rate of methylglyoxal glycation may impair beta cell function in pre-diabetes in vivo. Glycation by methylglyoxal may contribute to beta cell glucotoxicity and dysfunction with progression to type 2 diabetes mellitus.

A critical deficit in many studies using iPS cell-derived neurons is the electrophysiological properties of these cells. Using short differentiation protocols this deficit is more significant, likely due to a lack of differentiated astrocytes in these cultures. As a strategy for combatting this deficit, this study utilises astrocyte secreted factors, in the form of astrocyte conditioned medium (ACM). ACM generated cultures of neurons producing spontaneous activity more reminiscent of neurons in vivo after only 3 weeks of differentiation. Further, this study finds that voltage activated Ca2+ currents are enhanced at a very early time point using ACM and these channels, along with GABAA receptors, are vital to neuronal functional maturation. This demonstrates an endogenous mechanism present in the early stages of functional development, where neurons exhibit excitatory responses to GABA which drive the activation of voltage activated Ca2+ channels. In addition to finding that astrocyte secreted factors evoke a gain of this endogenous, activity led mechanism for functional maturation, this study also investigates methods of enhance functional maturation by manipulating Ca2+ influx. However, in the absence of ACM, direct manipulation of this endogenous mechanism appeared limited by GABA becoming an inhibitory neurotransmitter as the cells functional matured. These strategies for enhance functional maturation are then assessed using iPS cell-lines generated for a Huntington disease study, finding that GABAA and increased Ca2+ concentration in the medium evoked both increased and more consistent functional properties in theses neurons at week 2. In addition to developing enhanced protocols for neuronal differentiation, a novel protocol for producing cultures of iPS cell-derived astrocytes in the absence neurons. These cultures could provide an invaluable tool, alongside iPS cell-derived neurons, for modelling neurodegenerative disease mechanisms in both cell types.

Synaptic plasticity is the ability of neuronal synapses to strength and weaken from a set threshold determined by previous 'experience' thus representing a cellular basis for learning and memory. There are different forms of synaptic plasticity including Hebbian, homeostatic synaptic scaling as well as experience-dependent plasticity such as when animals are exposed to environmental enrichment (EE). Small protrusions which receive excitatory inputs known as dendritic spines are able to change morphology which has also been linked to trafficking of glutamate AMPA receptors (AMPAR) in response to neuronal activity. Brain-derived neurotrophic factor (BDNF) is a neutrophin involved in regulating both transcription and translation during synaptic plasticity. A plasticity-related gene, activity-regulated cytoskeleton-associated protein (Arc) controls the endocytosis of AMPAR nonetheless downstream signalling pathways are still largely unknown. The nuclear kinase, mitogen-and stress-activated protein kinase 1 (MSKI) regulates gene transcription in a BDNF-dependent manner. Thus we wanted to investigate whether MSKI plays a role in regulating Arc expression in response to BDNF-induced synaptic plasticity. In order to test this hypothesis we used MSKI kinase dead (MSK1 KD) transgenic mouse which had an inactivating knock-in mutation in the MSK1 N-terminus kinase domain. Dissociated hippocampal neurones were cultured from MSK1 KD and wild-type (WT) mice and stimulated with BDNF to monitor Arc protein expression. MSKI KD neurones showed a delayed response in BDNF-induced Arc upregulation. Interestingly in the presence of tetrodotoxin (TTX) which reduces BDNF levels, MSKI KD mice failed to show homeostatic scaling of synaptic transmission, Arc expression and spine morphological changes in vitro. After measuring the same parameters, EE-induced plasticity was unaffected in MSK1 KD adult mice but this may be a reflection of the experimental protocol used highlighted in the literature. The link between Arc and the cytoskeleton is unfamiliar and thus our finding of a novel interacting partner associated with clathrin-mediated endocytosis will carve out innovative mechanisms involved in synaptic plasticity. MSK1 acts an important homeostat during TTX-induced up-scaling of Arc protein and may influence structural effects on spine morphology. However MSKI may more of a local homeostat rather than generalised as its role in BDNF or experience-dependent stimulation was not as clear. Arc is an important synaptic mediator and thus linking it to the cytoskeleton could bridge the gap in understanding the mechanisms underlying learning and memory.

The growing knowledge of cell biology and evidence for the role of β-catenin signalling network in homeostasis and carcinogenesis encourages further investigation into the regulatory network of nuclear β-catenin signalling complex. While the role of canonical Wnt signalling in the development of both normal tissue and malignant tumours is well documented, the molecular basis of these functionally distinct nuclear transcriptional programs is poorly understood. Many proteins are associated with cytoplasmic β-catenin for regulation of Wnt/β-catenin pathway activities. However, in the nucleus, the LEF/TCF family of transcription factors, which have DNA binding properties, remains the sole focus as unambiguous partners of β-catenin. In addition to LEF/TCFs, interaction of β-catenin with several other transcriptional co-activators and/or co-repressors is required for gene regulation. This regulation may also be influenced by alterations of β-catenin protein such phosphorylation of β-catenin which dramatically alters its trafficking and function. Delineation and functional description of nuclear cofactors that interact with unphosphorylated (i.e. nuclear) β-catenin will further unravel the mechanisms of β-catenin-mediated nuclear transcription, and may also identify whether distinct patterns of transcriptional cofactors are engaged in normal development versus tumour progression. Human FLYWCH1, a conserved member of the mammalian C2H2 zinc finger proteins, was identified as one of the phosphorylation-independent Catenin- Interacting-Proteins (CIPs) in a recent screening performed in Dr Nateri's laboratory using a modified yeast-2-hybrid RRS. FLYWCH1 is a previously uncharacterized protein with no known function in mammals. Herein, we have shown that; i) in human cells, FLYWCH1 physically interacts with β-catenin and represses its transcriptional activity, ii) it regulates the expression of some if not all downstream target genes, iii) in the intestine, Flywch1 marks the crypt-based columnar-cells (CBCs), which function as stem cells, but does not mark any of the differentiated cells in normal villi, iv) FLYWCH1 expression is strongly down-regulated in CRC cell lines but its expression is up-regulated and restricted to a subpopulation of tumour cells in both human and ApcMin/+ mouse. Our data also showed that v) FLYWCH1 controls CRC cell morphology and inhibits cell migration through up-regulation of E-cadherin which may not be related to ZEB2-mediated EMT. Collectively, our data suggest that FLYWCH1 is a novel nuclear β-catenin interacting protein that inhibits cell motility by antagonizing the activity of Wnt/β-catenin signalling pathway. As changes in cell motility is a key step toward invasion and metastasis, FLYWCH1, therefore, may function as a metastasis-suppressing factor which could potentially be of use in the therapeutic field of colon cancer to control cancer spread.

Ion channels are transmembrane proteins which allow small ions to flow across the membrane downhill along the electrochemical gradient, with high effciency and selectivity over the different ion species, and which play crucial roles in a wide range of vital physiological functions. Research on the channels selective for potassium ions have attracted a great deal of attention over the past decade. This is because of the availability of three dimensional microscopical structures and because they provide a paradigm for the study of the complex superposition of the permeation of the ions and structural rearrangements which is responsible for the regulation of the current in ion channels. In the present work a thorough study of the strong coupling between permeation and the dynamics of the protein in the potassium ion channel KcsA is presented, based on Molecular Dynamics and Metadynamics calculations, which reveals the clear links between the function and the structure of the protein. The molecular determinants for the conformational changes of the pore region have been identified and described in details. The relationship between these rearrangements and the gating process known as "C-type inactivation", found in a variety of potassium ion channels, have been investigated and a mechanism has been proposed for the process. The knowledge acquired from these investigations is finally applied to unveil the driving forces and energetics associated with the permeation and selectivity properties of KcsA channel.