With increasing globalization, new infectious diseases are being discovered and spread quickly around the world. The traditional ways for the detection and identification of infectious pathogens are time-consuming and usually require specific facilities. This may delay effective treatment and lead to the spread of infectious disease, especially in the early stages of an epidemic. Therefore, accurate and efficient methods for identification of causative pathogens are very important. Here, we take advantages of new synthetic tools and nucleic acid technologies to develop novel infectious disease diagnostic methods that are user-friendly and have high sensitivity and specificity. These include: 1). The development of a rapid ultrasonic DNA isothermal amplification method with multiplexed melting analysis; 2). The development of an origami device based nucleic acid multiplexed detection method; 3). The development of a novel branched Hybridization Chain Reaction (HCR) assay. 1. Rapid ultrasonic isothermal amplification of DNA with multiplexed melting analysis-applications in the clinical diagnosis of sexually transmitted diseases (1) In this project, surface acoustic wave (SAW) signals are generated by interdigitated transducer (IDT) on LiNbO3 and propagated into disposable silicon superstrates on which a droplet of Loop-mediated isothermal amplification (LAMP) reaction mixture has been placed. As SAW interacts with the LAMP mixture, its energy is transferred into the LAMP mixture and causes the temperature of the mixture to increase. By controlling the SAW generation voltage, the temperature of the LAMP mixture could be maintained within a certain range as necessary for the LAMP reaction. During the process of LAMP amplification, a lot of double-strand DNA (dsDNA) is produced; this can incorporate specific fluorescent dyes and result in an exponential increase in fluorescence signal intensity. Also, by gradually increasing the SAW generation voltage, a SAW actuation-based DNA melting method could be used for multiplex detection. Ten-fold serially diluted targets from 105 copies/reaction to 10 copies/reaction were used to quantify the analytical sensitivity of the SAW-LAMP system and measurable signals were found down to 10 copies/reaction. Compared to a Peltier-LAMP system, SAW actuation enables the amplification to be performed more rapidly, about 18.23 % +/- 2.5 faster. Six clinical samples were used to demonstrate the clinical validation of SAW-LAMP by comparing with results from qPCR. The use of a SAW actuation-based DNA melting method distinguished the difference between melting temperatures of C. trachomatis amplicon (79.65 +/- 0.14 °C), and N. gonorrhoea (82.55 +/- 0.53 °C). 2. Development of paper origami device based nucleic acid multiplex detection for infectious diseases diagnostics In this project, a paper-folding origami device to manipulate malaria-infected blood samples was described. Through the simple process of paper folding, the nucleic acid of parasites in the blood sample could be extracted, purified and eluted. The extracted nucleic acid was then amplified with a multiplexed colorimetric LAMP assay in a plastic plate. Finally, the amplification products of multiplexed colorimetric LAMP assay were detected within an array with a low cost hand-held torch by naked eye. The multiplexed colorimetric LAMP assays for Plasmodium pan, Plasmodium falciparum, Plasmodium vivax with an internal control (IC) were investigated. The analytical sensitivity of colorimetric LAMP assays was tested by WHO International Standard DNA, with the limit of detection down to 105 IU/ml. Serially diluted quantified hCMV genomic DNA was used to demonstrate the DNA recovery of our origami device, which was between 60-70%. Serial dilution of a known infected blood samples (from 100 parasites/μl to 1 parasites/μl) were used to study the analytical sensitivity and obtain an LOD of the origami device to 5 parasites/μl. 80 fully characterised fresh malaria infected blood samples were used to assess the clinical validation effect of our origami device through a double blind, randomized controlled, clinical trial. All samples were also tested with commercially available LAMP kit and benchmark real-time PCR assay. The coincidence of our method and benchmark PCR were 88.75% (71/80) for Plasmodium pan, 90% (72/80) for P. falciparum and 93.75% (75/80) for P. vivax. Similarly, the coincidence between our method and the LAMP kit for Plasmodium pan and P. falciparum were 90% (72/80) and 92.50% (74/80) respectively. Using benchmark PCR as a gold standard for the detection of Plasmodium pan, P. ovale, P. falciparum and P. vivax, the sensitivity for our tests was of 85.5%, 92.9%, 61.1% and 81.0%, respectively. While the specificity are 100%, 94.2%, 98.5% and 98.30% respectively. We also established our origami device can diagnose species type from stored samples (either frozen, fixed, or dried). 3. Development of a novel branched Hybridization chain reaction (HCR) By increasing the dimensionality of an HCR system, a novel branched HCR product with complex branched structures instead of linear constructs has been developed. To validate the principle of a transition from a 1D chain to a higher dimension, we adapted a 3-arm branching construct to enable it to form a chain reaction by incorporating hybridization tails onto its sequences. The novel branched HCR reaction can form three-arm junction units with the introduction of a specific initiator. The three-armed units formed not only freed initiators to start of another cycle of HCR, but also bonded to each other to form complex and branched products. We also show that the highly branched polymers produced allow label-free acoustic mass sensing. The product of branched HCR was detected by Love Wave (LW) biosensor with the limit of detection at 2 nM, meaning 0.1% - 0.2% working frequency shift. Based on the branched HCR, we also design a new multiplexed HCR mechanism, where a single reaction is able to detect the presence of different initiators. It is based on designing primers that carry additional hairpin structures, which cross-react specifically upon initiation, yielding branching, thus opening up new applications for this enzyme-free, label-free DNA detection system.

The role of outdoor adventure programmes as a recognised approach for the effective treatment of psychological issues has, in recent years, reflected the growing interest in the development of adventure therapy. Although there has been an increased awareness of the possibilities of such a therapeutic approach there is limited practice, and thus very little instruction for how to implement such approaches, especially from a UK perspective. The aim of this study was to develop adventure therapy practice in the UK by specifically developing an intervention for women with eating disorders. Given that this specific approach for working with eating disorders didn't exist at the time of this study, this thesis is based on the principles of action research -a key aspect of the research process was the development of adventure therapy practice itself. Given the gender considerations of working in an outdoor adventure setting and the fact that eating disorders are largely a female phenomenon this study also took a feminist approach to ensure that disordered eating was in fact not reinforced by any adventure therapy approach developed. The thesis itself describes in detail the processes of developing the adventure therapy intervention and the associated experience of the six women who were recruited and took part in the intervention. The dilemmas and decisions made with regard to a number of issues in implementing an adventure therapy approach are examined, for example, facilitating therapeutic processes in an outdoor setting, identifying issues related to eating disorders that might arise in an outdoor adventure context, and examining feminist principles in action (e. g. reflexivity). As well as the six women's experiences of the different aspects of the adventure therapy intervention, the overall impact of the intervention for each woman is also examined. Data collected from a range of tools completed by the women, including personal information sheets, the Eating Disorders Inventory (EDI), personal journals, individual interviews and a final focus group indicate changes in most, but not all of the women. The results suggests that for the women with less chronic eating disorder symptoms positive change across a range of clinical symptoms were evident, including reduced troubled eating behaviours, improved body image, and motivation for change, albeit to different degrees for each woman. And for the one woman with the most chronic symptoms, although the intervention was a positive experience there was no evidence to suggest the intervention had any sustained impact. Although, the results from this study are not representative of a large clinical population of women, there is an indication that the intervention did initiate therapeutic change for some of the women and thus suggests that adventure therapy has the potential to be an effective therapeutic treatment for eating disorders and is, therefore, worthy of further investigation. Inevitably, in continuing to develop work in this area many questions and issues are raised as result of the action research process and the thesis concludes with a consideration of some of the needs of developing future adventure therapy research and practice in the UK.

This complex practice as research project was designed to interrogate the potential of 'Traumaturgy', an emergent dramaturgical methodology, in addressing the many challenges of writing, staging, and performing, autobiographical trauma narratives and to understand the impact of this process on the psychic, and somatic memories, of the autobiographical performer. The methodology was designed to motivate complex reflections on personal and cultural traumata as critical provocations for the re-writing and performing of the memory-scripts associated with the autobiographical traumatic life events such as adoption, which are explored through the traumaturgical performance process. Rather than distracting the psyche from the autobiographical traumatic experience, the traumaturgy model functions by seeking to establish new internal cognitive networks: positive associations that might facilitate an empowering, liberating transition, initiated through the act of traumaturgically framed narrative performance. Models of trauma intervention locate narrative reconstructions of the traumatic experience as a central focus for the process of recovery (Eagle., 2000; Herman, 1992; Schwartz & Prout, 1991) etc, however, unlike expressive therapies (see Glading, 1991; Moreno, 1975) which exist within the relative safety of the applied theatre space, key to this methodology is the achievement of strategic closure, by returning the performance to the traditional theatre environment and inviting an audience to play the role of witness. This creative synthesis between trauma theory and dramaturgical responses to the staging, and performing of post-traumatic memory based materials, forms the axis of this methodological approach. The research-sharing event In Search of Duende, which represented the performative articulation of this thesis, culminated in the performance of the play Dancing For Franco, which sought to re-write, and re process the researcher’s autobiographical trauma-based memory scripts through its witnessed performance. The play takes the somatic language of flamenco intertwoven with the adoption narratives of the researcher, and individuals affected by the Francoist system of illegal baby theft which are collectively known as the Niños Robados (Spain’s Stolen Children), and the fictional narratives of created characters, to understand how the traumaturgy model might instigate transformational processes within the autobiographical performer.

Orthopaedic infections can be polymicrobial existing as a microbiome. Infections often incorporate staphylococcal species, including Staphylococcus aureus. Such infections can lead to life threatening illness and implant failure. Furthermore, biofilm formation on the implant surface can occur, increasing pathogenicity, exacerbating antibiotic resistance and altering antimicrobial mechanism of action. Bacteria change dramatically during the transition to a biofilm growth state: phenotypically; transcriptionally; and metabolically, highlighting the need for research into molecular mechanisms involved in biofilm formation. Metabolomics can provide a tool to analyse metabolic changes which are directly related to the expressed phenotype. Here, we aimed to provide greater understanding of orthopaedic infection caused by S. aureus and biofilm formation on the implant surface. Through metagenome analysis by employing: implant material extraction; DNA extraction; microbial enrichment; and whole genome sequencing, we present a microbiome study of the infected prosthesis to resolve the causative species of orthopaedic hip infection. Results highlight the presence of S. aureus as a primary cause of orthopaedic infection along with Enterococcus faecium and the presence of secondary pathogen Clostridium difficile. Although results were hindered by the presence of host contaminating DNA even after microbial enrichment, conclusions could be made over the potential increased pathogenicity caused by the presence of a secondary pathogen and highlight method and sample preparation considerations when undertaking such a study. Following this finding, studies were focused on an orthopaedic clinical isolate of S. aureus and a metabolome extraction method for staphylococcal biofilms was developed using cell lysis through bead beating and solvent metabolome extraction. The method was found to be reproducible when coupled with liquid chromatography-mass spectrometry (LC-MS) and bioinformatics, allowing for the detection of significant changes in metabolism between planktonic and biofilm cultures to be identified and drug mechanism of actions (MOA) to be studied. Metabolomics results highlight significant changes in a number of metabolic pathways including arginine biosynthesis and purine metabolism between the two cell populations, evidence of S. aureus responding to their changing environment, including oxygen availability and a decrease in pH. Focused investigations on purine metabolism looking for biofilm modulation effects were carried out. Modulation of the S. aureus biofilm phenotype was observed through the addition of exogenous metabolites. Inosine increased biofilm biomass while formycin B, an inosine analogue, showed a dispersal effect and a potential synergistic effect in biofilm dispersal when coupled with gentamycin. Changes in metabolism between planktonic cells and biofilms highlight the requirement for antimicrobial testing to be carried out against planktonic cells and biofilms. Untargeted metabolomics was used to study the MOA of triclosan in S. aureus. The triclosan target and MOA in bacteria has already been characterised, however, questions remain over its effects in bacteria. Although the use of triclosan has come under increasing speculation, its full effects are still largely unknown. Results show that triclosan can induce a cascade of detrimental events in the cell metabolism including significant changes in amino acid metabolism, affecting planktonic cells and biofilms. Results and conclusions provide greater understanding of orthopaedic infections and specifically focus on the S. aureus biofilm, confirming S. aureus as a primary cause of orthopaedic infection and using metabolomic analysis to look at the changing state of metabolism between the different growth states. Metabolomics is a valuable tool for biofilm and drug MOA studies, helping understand orthopaedic infection and implant failure, providing crucial insight into the biochemistry of bacteria for the potential for inferences to be gained, such as the MOA of antimicrobials and the identification of novel metabolic drug targets.

It has been proposed that polyphenol-rich foods have a role in disease prevention and are associated with health benefits due to their antioxidant, anti-inflammatory, prebiotic, and antibacterial properties. However, associated health benefits depend on their intake, metabolism, and bioavailability. The metabolism and the bioavailability of polyphenols have been studied in young adults and show substantial variability. As the majority of polyphenols are metabolised in the colon, this may result in different bioactive microbial metabolites in the large intestine where they may have an impact on the risk of colorectal cancer (CRC). This variability could be due to: 1) dietary habits including intake polyphenol-rich foods; 2) ethnic-specific colonic microbiota; and 3) ageing and its effect on colonic physiology. Little is known about the impact of ethnicity, ageing, and the risk of CRC on polyphenol metabolism. Therefore, this thesis aimed to investigate the effect of the factors that could have an impact on the colonic metabolism of dietary polyphenols in a human feeding study measuring the biomarkers of polyphenol metabolism, colonic fermentation, and gut health; and an in-vitro faecal fermentation study measuring the colonic metabolites of quercetin-3-O-rutinoside (rutin). The first aim of this thesis (Chapter 3) was to examine the effect of ethnicity (Europeans versus Indians) on polyphenol metabolism. The findings of this study suggest that ethnicity could have a role on the colonic metabolism of polyphenols which could be due to the differences in disease incidence between countries such as the lowest risk of CRC in India among the world. The Indian group excreted less urinary phenolic acid after the high-polyphenol diet compared to the Europeans; however, Indians were more capable and faster in metabolizing rutin in the in-vitro model. This could be due to the differences in: 1. Genetics and its effect on gastrointestinal tract absorption. 2. Gut microbiota, as Indians have a significantly higher level of Bifidobacterium. 3. Gut environment, in particular the colonic pH and SCFA could have an influence as the colonic pH was lower in the Indian group. 4. Cultural daily diet between groups, as Indians significantly consumed a high amount of onions, tomatoes, chillies, spices, curry-based products, and yoghurt. These food types are high in polyphenols, fibre, and probiotics. The second study of this thesis aimed to investigate the effect of ageing on polyphenol metabolism. The results suggest another factor, ageing, which could influence the colonic metabolism of polyphenols. The older group excreted less urinary phenolic acid and some of the acid was not detected in certain of the participants’ urine compared to the younger group. However, the sum of the phenolic acid that formed after the faecal fermentation of rutin was not significantly different between the groups. This could suggest different reasons behind these variations. First, the lack of absorption of some phenolic acids by the older group as ageing was shown to decrease the colonic absorption. Secondly, the effect of ageing on gut microbiota composition and function. Thirdly, changes in dietary habits and physical activity may be influenced by ageing. Thus, this may suggest that older people can have fewer benefits of polyphenol metabolites which could be associated with an increase in risk for age-related diseases including CRC. As the risk of CRC is different between countries and increases with age, the supportive findings of the first and second study suggest that ethnicity and ageing could have a role on the metabolism of polyphenols so this raises the questions whether a low intake of polyphenols can be one of the factors that may lead to CRC, or whether polyphenols can reduce the risk of CRC due to their colonic health benefits. Therefore, the last study examined the metabolism of polyphenols on patients who are at risk of CRC (history of polyps). No significant differences were observed between the healthy control and polypectomy groups in terms of the sum urinary phenolic acid excretion and phenolic acid formation in the faecal fluids. However, some phenolic acids were not detected in all of the urine samples of the polypectomy group as well as one acid in the faecal fermentation fluids, while some of the acids were not detected in few participants in the healthy group. No hard conclusion can be made from this study due to the small sample size. However, this study gives us an idea that there could be differences if a larger sample size were used. Therefore, more studies are needed to determine the effect of CRC risk as being one of the factors that can influence the metabolism of polyphenols. In conclusion, the work of this thesis showed that ethnicity, ageing, and gut health are likely some of the key factors that could contribute to the variations in polyphenol metabolism which were observed previously by many in-vivo and in-vitro studies. These variations could result in bioavailability variation and consequential differences in the biological activity of polyphenol metabolites leading to differences in health and optimal health among individuals.

Introduction: Over the past decades, several labelling techniques have been used in an attempt to track stem cells using magnetic resonance imaging (MRI). However, very few of these were able to definitely determine the precise location of stem cells within a living organism and monitor throughout a long term period, without loss or diffusion of the signal. A novel MRI cell tracking method described in 2005 proposed that reporter genes that could effectively increase the iron content of a target cell would allow a stronger contrast when imaged via MR. Being a fundamental part of the iron metabolism, transferrin receptor 1 (TfR 1) and ferritin heavy chain 1 (Fth 1) were naturally suggested to have the potential to increase the iron load of cells when overexpressed. More recently, there has been some interest in the reporter gene MagA, which is a known iron transporter found in magnetotactic bacteria. Aim: To evaluate the suitability of using TfR 1, Fth 1 and MagA as potential magnetic reporter genes for MRI-based cell tracking. Methods: Several cell and stem cell lines were transduced with a 2nd generation HIV-based lentiviral system containing one or more magnetic reporters. Viral transduction resulted in genome incorporation of bicistronic construct(s) with TfR 1 gene alongside a gene encoding a green fluorescent reporter (GFP) and/or Fth 1 and MagA gene alongside a red fluorescent reporter (RFP). This allowed for identification and monitoring of positive cells with complementing imaging modalities: MRI and fluorescence based methods. Transgenes were evaluated for integration stability over passages and their influence on iron homeostasis was assessed; also, integration and/or overexpression were confirmed at the mRNA and protein level. Finally, the influence of magnetic reporters on intracellular iron retention and MRI contrast capacity was tested both in vitro and in a model organism, the chick embryo. Results: After analysing all three potential magnetic reporters, TfR 1 was found to be the most promising, as its overexpression induced an adjustment of iron homeostasis in Chinese hamster ovary K1 cells, leading to higher intracellular iron accumulation relative to controls. The same adjustment was found in mouse mesenchymal stem cells (mMSC), but only when TfR 1 was overexpressed in conjunction with Fth 1, also leading to an increase in iron retention capacity. However, a limitation was found when overexpressing Fth 1 in mMSC, as permanent iron supplementation was needed in order to keep these cells viable. In contrast with previous studies, MagA gene integration posed some restrictions in certain cell lines studied. The results presented here show that while some cell types are able to stably maintain MagA expression over several passages, others fail to survive and die shortly after transduction, suggesting that a potential toxic effect may be originating from MagA gene integration. From the surviving cells, two were compared side by side and contradictory results were obtained, demonstrating that MagA would only be a suitable magnetic reporter for some cell types. Conclusion: The results obtained with this project are of relevance for reporter gene-based MRI cell tracking as they show that no single magnetic reporter is capable of generating detectable MRI contrast for a global cohort of cell lines. On the contrary, overexpression of endogenous genes or integration of foreign genes should be performed with caution and analysed on a case by case basis. Finally, for some cell type and magnetic reporter gene combinations, this study suggests that MRI could be a promising method for the longitudinal monitoring of engrafted cells, especially when the cells have been cultured in media supplemented with low concentrations of iron.

Hospital-acquired infections, a large proportion of which are derived from contact transmission, represent a massive global challenge. It has been proved that surface modification of biomaterials with Ag or Cu has evolved as a potentially effective method for preventing bacterial proliferation on the devices surfaces. However, thin antimicrobial coatings on materials such as austenitic stainless steels can be easily worn and removed in relative motion with other surfaces. The purpose of this study is to develop multi-functional stainless steel surfaces which combine greatly improved wear resistance, at least maintain corrosion resistance and provide long-lasting, high efficacy, antimicrobial effects. In this thesis a series of surface engineering technologies, including active screen co-alloying, active screen plasma duplex alloying and double glow plus active screen duplex plasma alloying, were developed for surface alloying stainless steel with Ag or Cu and N; the phase constitution, microstructure, composition, and surface roughness of the alloyed surfaces were fully characterized, and the surface hardness, wear resistance, bonding strength, antimicrobial efficiency and corrosion behaviour of the treated surfaces were evaluated. In addition, further inspection of the wear mechanisms and corrosion mechanisms were conducted on post-exposure surfaces. It was found that the adhesive wear mechanism of austenite can be reduced by this alloying combination and the wear resistance was improved by up to 1000 times, and the Ag/Cu alloyed surface was bactericidal and growth-inhibitive for many pathogens including E. coli NCTC 10418 and S. epidermidis NCTC 11047 effectively up to 99%/6h. The mechanism of bactericidal efficiency of Ag/Cu is found dependent on the structure of the bacterial membrane and a higher efficiency of antibacterial agents is found associated with the higher elemental concentration of copper and silver. With regard to corrosion, it is affected largely by the configuration of surface structure and several corrosion mechanisms were evolved. One principal conclusion was that it is feasible to generate long lasting antimicrobial stainless steel surface to fulfil growing demands from industry for practically robust multifunctional medical device surfaces.

Aims: This thesis investigated the health and supportive care needs of surgical lung cancer patients to address gaps in the evidence base and inform future service developments. Additionally, the prognostic significance of smoking behaviour was investigated. Methods: Semi-structured interviews were conducted with 29 surgical (VATS and thoracotomy) lung cancer patients to explore health, functioning, smoking, satisfaction with recovery and preferences for a tailored rehabilitation programme. Interviews were analysed using framework approach. Systematic literature searches were conducted to review evidence of the association between smoking history or continued smoking after diagnosis and prognosis. Survival estimates were combined where possible using a random effect inverse variance model. Results: Most participants experienced difficulty during recovery. Breathlessness and pain emerged as dominant health challenges. Participants were open to being offered smoking cessation support. From 78 and 10 studies, preliminary evidence was found that both smoking history and continued smoking are associated with prognosis, respectively. Analyses indicated that smoking-associated increased risk may be mediated through cancer-related pathways. Conclusions: Many surgical lung cancer patients’ supportive care needs are not being met. Well-developed treatments and services for management of breathlessness, pain and smoking cessation may improve quality of life and health outcomes after lung cancer surgery, and require further testing.

Hepatitis C Virus (HCV) is a global health problem, with over 170 million infected individuals worldwide. 70-80% of infected individuals develop progressive disease, and approximately 2% of these acquire hepatocellular carcinoma (HCC). HCV entry is dependent on tetraspanin CD81, scavenger receptor BI, and tight junction proteins claudin-1 and occludin. Tetraspanins are involved in multiple biological functions including cell-ECM adhesion and motility. An actin polymerization-dependent cell spread was observed upon ligation of CD81 on hepatoma cells. Importantly, HCV infection perturbed CD81-dependent cell spread, suggesting HCV infection may modulate CD81 function in hepatoma cells. Functional assays demonstrated that CD81 expression and HCV infection promote hepatoma cell motility. These findings allude to a link between HCV infection and associated HCC development. Establishment of a chronic infection demonstrates that HCV can escape from the host adaptive immune responses. We developed an in vitro cell culture system to monitor viral transmission in the presence of neutralizing antibodies (nAb). Separation of producer and target cells ablated nAb resistant transmission, suggesting that cell-cell contact was essential. Furthermore nAb resistant transmission was dependent upon all four co-receptors. These observations confirm HCV immune evasion by cell-to-cell transfer and have major implications for anti-glycoprotein targeted therapies.

CD4+ T cells play a key role in orchestrating adaptive immunity. Their activation requires antigen presentation via MHC II proteins on antigen presenting cells (APC). Exosomes are membrane vesicles released by various cell types including APCs. APC-derived exosomes are MHC class II-positive and can induce CD4+ T cell responses. MHC II delivery to the cell surface and/or exosomes might be influenced by tetraspanins, a family of transmembrane proteins. We have prepared exosomes derived from Epstein-Barr virus (EBV)-infected human B lymphoblastoid cell lines (LCLs) and shown by Western blotting and immunoelectron microscopy that they contain MHC class II and tetraspanins including CD63, CD81 and CD82. Such LCLs as well as LCL-derived exosomes can mediate immunologically specific recognition by MHC class II matched EBV antigen-specific CD4+ T cell clones when directly added to the T cells. Using shRNA, we have decreased CD63 expression in LCLs and had been studying the effect of such downregulation on LCL as well as LCL-derived exosome mediated antigen presentation. Despite an unaltered level of MHC II, CD63low LCLs showed to be hyperstimulatory. In spite of a similar depletion of CD63 in exosomes derived from CD63low LCLs, the CD4+ T cell stimulation by these exosomes was unaltered. In search for the mechanism of this phenomenon we found a higher level of exosome secretion by CD63low LCLs. We speculate that CD63 may influence T cell stimulation by exosome trafficking as well as exosome release.