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Immunosenescence is characterized by deterioration of the immune system caused by aging which induces changes to innate and adaptive immunity. Immunosenescence affects function and phenotype of immune cells, such as expression and function of receptors for immune cells which contributes to loss of immune function (chemotaxis, intracellular killing). Moreover, these alterations decrease the response to pathogens, which leads to several age-related diseases including cardiovascular disease, Alzheimer's disease, and diabetes in older individuals. Furthermore, increased risk of autoimmune disease and chronic infection is increased with an aging immune system, which is characterized by a pro-inflammatory environment, ultimately leading to accelerated biological aging. During the last century, sedentarism rose dramatically, with a concomitant increase in certain type of cancers (such as breast cancer, colon, or prostate cancer), and autoimmune disease. Numerous studies on physical activity and immunity, with focus on special populations (i.e., people with diabetes, HIV patients) demonstrate that chronic exercise enhances immunity. However, the majority of previous work has focused on either a pathological population or healthy young adults whilst research in elderly populations is scarce. Research conducted to date has primarily focused on aerobic and resistance exercise training and its effect on immunity. This review focuses on the potential for exercise training to affect the aging immune system. The concept is that some lifestyle strategies such as high-intensity exercise training may prevent disease through the attenuation of immunosenescence. In this context, we take a top-down approach and review the effect of exercise and training on immunological parameters in elderly at rest and during exercise in humans, and how they respond to different modes of training. We highlight the impact of these different exercise modes on immunological parameters, such as cytokine and lymphocyte concentration in elderly individuals.

Cancer progression in skeletal muscle (SkM) is very rare, and mechanisms remain unclear. This study assessed the potential of SkM (myocyte)-derived EVs (C2C12-EVs) as anti-cancer agents. Using murine in vitro models, we showed that following treatment with C2C12-EVs, lung carcinoma cells failed to colonise SkM cells, and that C2C12-EVs selectively exerted apoptosis on cancer cells. Uptake of C2C12-EVs by carcinoma cells caused changes in lysosomal function and mitochondrial membrane properties inducing cell death with elevated caspase 3 and 9. The C2C12-EVs also inhibited cell proliferation, affecting cell cycle arrest at S phase and inhibited cell migration. Proteomic analysis of C2C12-EV cargoes highlighted functional enrichment pathways involved in lysozyme function, HIF-1 and PI3K-Akt signalling, regulation of actin cytoskeleton, pyruvate metabolism, platelet activation, and protein processing in ER. Decorin, a muscle cell-specific cytokine released from myocytes in response to stress, was significantly enriched in C2C12-EVs and may contribute to C2C12-EVs’ inhibitory activity on cancer cells. C2C12-EVs may suppress cancer and potentially be used as therapeutic agents for cancer metastasis.

During cell invasion, large Extracellular Vesicle (lEV) release from host cells was dose‐dependently triggered by Trypanosoma cruzi metacyclic trypomastigotes (Mtr). This lEV release was inhibited when IP3‐mediated Ca2+ exit from the ER and further Ca2+ entry from plasma membrane channels was blocked, but whilst any store‐independent Ca2+ entry (SICE) could continue unabated. That lEV release was equally inhibited if all entry from external sources was blocked by chelation of external Ca2+ points to the major contributor to Mtr‐triggered host cell lEV release being IP3/store‐mediated Ca2+ release, SICE playing a minor role. Host cell lEVs were released through Mtr interaction with host cell lipid raft domains, integrins, and mechanosensitive ion channels, whereupon [Ca2+]cyt increased (50 to 750 nM) within 15 s. lEV release and cell entry of T. cruzi, which increased up to 30 and 60 mpi, respectively, as well as raised actin depolymerization at 60 mpi, were all reduced by TRPC inhibitor, GsMTx‐4. Vesicle release and infection was also reduced with RGD peptide, methyl‐β‐cyclodextrin, knockdown of calpain and with the calpain inhibitor, calpeptin. Restoration of lEV levels, whether with lEVs from infected or uninfected epithelial cells, did not restore invasion, but supplementation with lEVs from infected monocytes, did. We provide evidence of THP‐1 monocyte‐derived lEV interaction with Mtr (lipid mixing by R18‐dequenching; flow cytometry showing transfer to Mtr of R18 from R18‐lEVs and of LAP(TGF‐β1). Active, mature TGF‐β1 (at 175 pg/×105 in THP‐1 lEVs) was detected in concentrated lEV‐/cell‐free supernatant by western blotting, only after THP‐1 lEVs had interacted with Mtr. The TGF‐β1 receptor (TβRI) inhibitor, SB‐431542, reduced the enhanced cellular invasion due to monocyte‐lEVs.

Microvesicles shed from cells carry constituents of the cell cytoplasm, including, of importance in multidrug resistance to cancer chemotherapy, drugs that the tumor cell attempts to efflux. To see whether such drugs could be used at lower concentrations with the same efficacy, it was first shown that microvesiculation of prostate cancer (PCa) cells, PC3, could be inhibited pharmacologically with calpeptin (calpain inhibitor) and by siRNA ( CAPNS1 ). In cells treated with docetaxel (DTX), this inhibition resulted in a third-fold increase in intracellular concentrations of DTX. As a result, 20-fold lower concentrations of DTX (5 nM) could be used, in the presence of calpeptin (20 μM) inducing the same degree of apoptosis after 48 h in PC3 cells, as 100 nM of DTX alone. Inhibition of microvesiculation similarly improved combination chemotherapy (DTX and methotrexate). In a mouse xenograft model of PCa, DTX (0.1 mg/kg) together with calpeptin (10 mg/kg), administered i.p., significantly reduced tumor volumes compared to DTX alone (0.1 mg/kg) and brought about the same reductions in tumor growth as 10 mg/kg of DTX alone. As well as further reducing vascularization, it also increased apoptosis and reduced proliferation of PC3 cells in tumor xenografts.

The main aim of this study was to investigate a possible functional connection between sigma-1 receptors and voltage-gated sodium channels (VGSCs) in human breast cancer cells. The hypothesis was that sigma-1 drugs could alter the metastatic properties of breast cancer cells via the VGSC. Evidence was found for expression of sigma-1 receptor and neonatal Na v 1.5 (nNa v 1.5) expression in both MDA-MB-231 and MDA-MB-468 cells. Sigma-1 drugs (SKF10047 and dimethyltryptamine) did not affect cell proliferation or migration but significantly reduced adhesion to the substrate. Silencing sigma-1 receptor expression by siRNA similarly reduced the adhesion. Blocking nNa v 1.5 activity with a polyclonal antibody (NESOpAb) targeting an extracellular region of nNa v 1.5 also reduced the adhesion in both cell lines. Importantly, the results of combined treatments with NESOpAb and a sigma-1 drug or sigma-1 siRNA suggested that both treatments targeted the same mechanism. The possibility was tested, therefore, that the sigma-1 receptor and the nNa v 1.5 channel formed a physical, functional complex. This suggestion was supported by the results of co-immunoprecipitation experiments. Furthermore, application of sigma-1 drugs to the cells reduced the surface expression of nNa v 1.5 protein, which could explain how sigma-1 receptor activation could alter the metastatic behaviour of breast cancer cells. Overall, these results are consistent with the idea of a sigma-1 protein behaving like either a “chaperone” or a regulatory subunit associated with nNa v 1.5.

The main aim of this study was to investigate a possible functional connection between sigma-1 receptors and voltage-gated sodium channels (VGSCs) in human breast cancer cells. The hypothesis was that sigma-1 drugs could alter the metastatic properties of breast cancer cells via the VGSC. Evidence was found for expression of sigma-1 receptor and neonatal Na sub(v)1.5 (nNa sub(v)1.5) expression in both MDA-MB-231 and MDA-MB-468 cells. Sigma-1 drugs (SKF10047 and dimethyltryptamine) did not affect cell proliferation or migration but significantly reduced adhesion to the substrate. Silencing sigma-1 receptor expression by siRNA similarly reduced the adhesion. Blocking nNa sub(v)1.5 activity with a polyclonal antibody (NESOpAb) targeting an extracellular region of nNa sub(v)1.5 also reduced the adhesion in both cell lines. Importantly, the results of combined treatments with NESOpAb and a sigma-1 drug or sigma-1 siRNA suggested that both treatments targeted the same mechanism. The possibility was tested, therefore, that the sigma-1 receptor and the nNa sub(v)1.5 channel formed a physical, functional complex. This suggestion was supported by the results of co-immunoprecipitation experiments. Furthermore, application of sigma-1 drugs to the cells reduced the surface expression of nNa sub(v)1.5 protein, which could explain how sigma-1 receptor activation could alter the metastatic behaviour of breast cancer cells. Overall, these results are consistent with the idea of a sigma-1 protein behaving like either a \"chaperone\" or a regulatory subunit associated with nNa sub(v)1.5.

The main aim of this study was to investigate a possible functional connection between sigma-1 receptors and voltage-gated sodium channels (VGSCs) in human breast cancer cells. The hypothesis was that sigma-1 drugs could alter the metastatic properties of breast cancer cells via the VGSC. Evidence was found for expression of sigma-1 receptor and neonatal Nav1.5 (nNav1.5) expression in both MDA-MB-231 and MDA-MB-468 cells. Sigma-1 drugs (SKF10047 and dimethyltryptamine) did not affect cell proliferation or migration but significantly reduced adhesion to the substrate. Silencing sigma-1 receptor expression by siRNA similarly reduced the adhesion. Blocking nNav1.5 activity with a polyclonal antibody (NESOpAb) targeting an extracellular region of nNav1.5 also reduced the adhesion in both cell lines. Importantly, the results of combined treatments with NESOpAb and a sigma-1 drug or sigma-1 siRNA suggested that both treatments targeted the same mechanism. The possibility was tested, therefore, that the sigma-1 receptor and the nNav1.5 channel formed a physical, functional complex. This suggestion was supported by the results of co-immunoprecipitation experiments. Furthermore, application of sigma-1 drugs to the cells reduced the surface expression of nNav1.5 protein, which could explain how sigma-1 receptor activation could alter the metastatic behaviour of breast cancer cells. Overall, these results are consistent with the idea of a sigma-1 protein behaving like either a \"chaperone\" or a regulatory subunit associated with nNav1.5.

Microvesicles (or MVs) are plasma membrane-derived vesicles released from most eukaryotic cells constitutively during early apoptosis or at higher levels after chemical or physical stress conditions. This review looks at some of the functions of MVs in terms of intercellular communication and ensuant signal transduction, including the transport of proteins (unconventional protein export) as well as of mRNA and microRNA. MVs also have roles in membrane repair, the removal of misfolded proteins, and in the control of apoptosis. We also discuss the role MVs have been shown to have in invasive growth and metastasis as well as in hypoxia in tumours and cerebral ischaemia. The association of MVs in infectious and autoimmune disease is also summarised together with their possible use as therapeutic agents.

The main aim of this study was to investigate a possible functional connection between sigma-1 receptors and voltage-gated sodium channels (VGSCs) in human breast cancer cells. The hypothesis was that sigma-1 drugs could alter the metastatic properties of breast cancer cells via the VGSC. Evidence was found for expression of sigma-1 receptor and neonatal Na 1.5 (nNa 1.5) expression in both MDA-MB-231 and MDA-MB-468 cells. Sigma-1 drugs (SKF10047 and dimethyltryptamine) did not affect cell proliferation or migration but significantly reduced adhesion to the substrate. Silencing sigma-1 receptor expression by siRNA similarly reduced the adhesion. Blocking nNa 1.5 activity with a polyclonal antibody (NESOpAb) targeting an extracellular region of nNa 1.5 also reduced the adhesion in both cell lines. Importantly, the results of combined treatments with NESOpAb and a sigma-1 drug or sigma-1 siRNA suggested that both treatments targeted the same mechanism. The possibility was tested, therefore, that the sigma-1 receptor and the nNa 1.5 channel formed a physical, functional complex. This suggestion was supported by the results of co-immunoprecipitation experiments. Furthermore, application of sigma-1 drugs to the cells reduced the surface expression of nNa 1.5 protein, which could explain how sigma-1 receptor activation could alter the metastatic behaviour of breast cancer cells. Overall, these results are consistent with the idea of a sigma-1 protein behaving like either a \"chaperone\" or a regulatory subunit associated with nNa 1.5.

The research presented in this thesis encompasses a detailed biophysical and biochemical analysis of extracellular vesicles: in particular microvesicles (MVs). The characterisation utilised novel applications of existing protocols, demonstrated for the first time within this thesis, resulting in an up-to-date and greatly improved series of techniques for the analysis of microvesicles.Application of these techniques revealed two distinct subtypes of MVs that can be collected from the same parent cell types in culture, originating via different pathways of biogenesis. cMVs are constitutively released and sMVs stimulated for release. They express distinct biochemistries leading to the potential to perform different physiological functions. These subtypes exhibit distinct FACs morphologies, distinct protein, non-specific carbohydrate and nucleic acid profiles. The techniques used demonstrated in this thesis also show that the MVs have remarkable differences in size as shown for the first time by TEM, flow cytometry, DLS and Nanosight particle tracking.FT-IR spectroscopy was used for the first time to profile similarities and differences between cell lines by analysis of the fingerprint region [1800cm1 to 900cm1). The MV subtypes derived from parent cells were analysed using FT-IR to demonstrate similarity in non-disclosed protein expression. BCA protein analysis, modified schiffs test, spectroscopic and quartz crystal microbalance techniques demonstrated cMVs to be smaller, denser and more slowly released from cells than the sMVs are larger, lighter and released sMVs rapidly in response to a stress stimuli.Analysis of calcium homeostasis has revealed that MVs contain high calcium levels, that are deliverable to cells via MVs as cargo. Furthermore, the MV subtypes express different arrays of deliverable proteins and receptors that were transiently expressible upon the recipient cell.Novel research into the role of ultra low-frequency magnetic fields (ULMFs) on cellular viability was explored. Results suggested that 0.3pT 6V A/C 10Hz ULMF exposure to cells increased intracellular Ca2+ levels via transiently induced plasma membrane pores. Furthermore, ULMFs stimulated MV release from cells and negated apoptosis by export of pro-apoptotic agents, increasing the health of the cells. ULMFs can be used to significantly enhance the uptake promoting cellular sensitivity to anti-cancer drugs, such that 10% of the therapeutic dose used with ULMFs was able to achieve current levels of therapeutic response. ULMFs significantly reduce HeLa migration during wound healing although they increased cell proliferation, possibly by releasing MVs.The exciting potential of this research may lead to new possibilities in cancer therapy. Using ULMFs in conjunction with chemotherapy may increase the success rates of existing therapies and reduce unwanted side effects. The data presented in this thesis will allow researchers to better understand subtle differences in MV biogenesis, how this reflects in their composition and morphology. It is the hoped that this research will lead to more laboratories engaging MV research using the techniques pioneered in this thesis to engage within many new areas of biology and biochemistry. This work offers a significant contribution to the field of MV research, but represents the tip of the iceberg.