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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

The role of microvesicles in the hyper-coagulation associated with prostate cancer

Al Saleh, Hassan Ali January 2017 (has links)
Patients with prostate cancer (PC) are at high risk of developing migratory thrombosis compared to healthy individuals. This is due to the haemostatic abnormality as a result of the presence of cancer, and is referred to as Trousseau’s syndrome. Trousseau's syndrome leads to increased mortality among cancer patients, and is considered the second cause of death after cancer itself. We investigated the role of microvesicles (MVs), which are circular membrane compartments shed from cancer as well as from healthy cells, in the development of Trousseau’s syndrome. We compared the pro-coagulant activities between MVs derived from PC cell lines with different oncogenic and metastatic characteristics, using chromogenic assays to determine their thrombin generation. Microvesicles from the more aggressive DU145vIII and more metastatic PC3-MLN4 show increased thrombin generation compared to MVs derived from DU145 and PC3. We also compared thrombin generation in MVs extracted from plasma of PC patients of various cancer stages. MVs from PC patients with a metastasized tumour had increased thrombin generation compared to patients with localized tumours. Finally, we transfected the CHO cell line with the human protease-activated receptor 1 (hPAR1), the principal receptor of thrombin. PC MVs led to the activation of PAR1 in CHO (hPAR1), indicating thrombin generation. Our in vitro studies suggest a potential role of PC MVs in the migratory thrombosis observed in Trousseau’s syndrome, due to their independent ability to generate active thrombin. We also demonstrated that thrombin generation of PC-derived MVs correlated with the oncogenic and metastatic characteristics of prostate cancer. / Thesis / Master of Science (MSc)
2

Neutrophil microvesicles restrict the phlogistic activation of macrophages

Rhys, Hefin Ioan January 2017 (has links)
Released in response to cellular activation, microvesicles are a major vector mechanism for the delivery of protein, nucleic acid and bioactive lipid payloads in local tissues and plasma. Large numbers of microvesicles (including those from neutrophils) are found within inflammatory sites, such as the rheumatoid synovium. Human neutrophil microvesicles promote tissue protection, and in some cases repair, by affecting function and phenotype of other inflammatory cells. Of these, tissue macrophages are central to the recovery of homeostasis after an inflammatory insult. The data herein indicate that microvesicles released by activated neutrophils impede lipopolysaccharide and interferon gamma-induced \M1-like" polarisation of macrophages via phosphatidylserine (PtdSer) exposure, and induce annexin A1-dependent release of transforming growth factor beta (TGFb). Macrophages treated with these vesicles stimulate the production of cartilage matrix from chondrocytes, and are unable to induce an inflammatory phenotype in fi broblasts. The efficacy of these vesicles is reproduced in two in vivo models of acute inflammation, zymosan-induced peritonits and K/BxN serum-transfer arthritis. Finally, the possibility of using both autologous, and cell-line-derived microvesicles as pharmacodynamic tools is explored. Microvesicles generated from neutrophils from patients with rheumatoid arthritis are found to be protective, and can outcompete the pro-inflammatory effects of both platelet microvesicles, and those isolated from synovial fluid of patients with rheumatoid arthritis. By building on the observation that anxA1 on microvesicles stimulates TGFb release in macrophages, a cell line was transfected to release anxA1+ microvesicles, and their e ects compared to those of their wild type counterparts.
3

THE PHYSICOCHEMICAL CHARACTERIZATION OF MICROVESICLES SECRETED BY SULFOLOBUS ACIDOCALDARIUS

Bonanno, Alexander P. January 2019 (has links)
Microvesicles secreted from the thermoacidophilic archaeon S. acidocaldarius (Sa-MVs) contain a membrane made exclusively of tetraether lipids and covered by crystalline surface layer proteins known as the S-layer. While tetraether lipids and S-layer proteins are known to be useful biomaterials, little has been done to exploit Sa-MVs for any scientific applications. In the present study, as the start point to explore this area, we isolated Sa-MVs and used dynamic light scattering, laser Doppler electrophoresis, and cryo-transmission electron microscopy (cryo-TEM) to characterize the particle size, size distribution, zeta potential, and morphology of Sa-MVs and tested their stabilities against temperature, pH, autoclaving, and the detergent Triton X-100. We found that, at the cell’s growth pH (~2.6) and growth temperature (75-80oC), Sa-MVs in the growth medium are ~180-183 nm in diameter with a polydispersity index (PDI) ≤ 0.15 and have a zeta potential of -0.5 mV. Sa-MVs in buffer exhibited long-term (at least 137 days) stability with no signs of vesicle disintegration or fusion. When the pH was decreased from 7.2 to 2.6, the average size of Sa-MVs was increased by ~40-45 nm, which probably came from conformational changes of S-layer proteins, in concomitant with vesicle aggregation, but not due to conformational changes in tetraether lipid headgroups. The isoelectric point (pI) for Sa-MVs in 1 mM KCl is 3.0 while that for the reconstituted liposomes (LUVMV) is estimated to be below 2.0. Sa-MVs dispersed in buffer at pH 2.6 change little in size over five autoclaving cycles, despite becoming slightly less spherical after autoclaving, while at this pH liposomes made of diester lipids cannot sustain multiple cycles of autoclaving. In addition, compared to diester and PLFE liposomes, Sa-MVs and LUVMV exhibit unusual resistance against the surfactant Triton X-100. Although some man-made liposomes such as PLFE liposomes are also stable against temperature, pH, and other environmental stressors, Sa-MVs are unique in that they are naturally occurring nanoparticles with a native membrane environment suitable for inserting additional lipids and membrane-bound proteins as needed. With their great stability presented here and the lack of cytotoxicity known in the literature, Sa-MVs hold great promise for technological applications. In addition to these biophysical techniques employed to characterize these microvesicles, a series of fluorescence experiments have also been conducted to gain further insight into how the membrane packing of these vesicles compares to tetraether as well as diester liposomes. Intrinsic protein fluorescence of native microvesicles was examined to characterize the dynamics of the S. acidocaldarius MVs. We have used the probe 6-lauroyl-2-dimethylaminonaphthalene (Laurdan) to monitor membrane packing and dynamics within the water-membrane interfacial region of the Sa-MVs. Specifically, we measured Laurdan’s generalized polarization (GP), which depends on the probe’s local polarity, probe location and nearby viscosity. We also measured Laurdan’s red edge excitation shift (REES), which depends on the dynamics of solvent relaxation around the fluorophore compared to the probe’s fluorescence lifetime. As temperature increased from 18 to 66.7 °C, GP decreased from 0.026 to -0.118. Comparing the GP values of reconstituted vesicles to that of the native Sa-MVs, it appears that the two curves are similar in both GP value and trend over increasing temperature range (values decrease from 0.112 to -0.215), which suggests that Laurdan in Sa-MVs resides in the lipid membrane, not in proteins and that Laurdan’s GP is not affected much by the presence of Sa-MV proteins. It is well known that, for liposomes made of diester lipids, the GP value of Laurdan fluorescence is high in the gel state and low in the liquid-crystalline state, with an abrupt change during the phase transition. However, Laurdan’s GP values obtained from liposomes comprised of tetraether lipids such as PLFE and Sa-MV lipids cannot be compared directly to those obtained from diester liposomes and cannot be interpreted simply based on membrane packing because probe location and chromophore orientation in tetraether liposomes could be distinctly different from those in diester liposomes. Our data show that the REES effect in PLFE LUVs is most pronounced among all the membranes examined showing a value of 10.58 nm at 24oC compared to 0.9 nm for DMPC LUVs at the same temperature. It is already known that membrane packing in PLFE liposomes is extraordinarily tight partly due to the strong hydrogen bond network in the polar head group regions where the phosphate and sugar moieties are abundant and partly due to the rigid and ordered dibiphytanyl hydrocarbon chains. Since the chromophore of Laurdan in PLFE liposomes is most likely located in the membrane-water interfacial region, it is not surprising that solvent tumbling around Laurdan in PLFE LUVs is much more restricted in PLFE LUVs than in DMPC LUVs, giving rise to a much higher REES value in PLFE LUVs than in DMPC LUVs. The REES values (10.3-14.1 nm) of Laurdan fluorescence in LUVs reconstituted from the extracted MV lipids are higher than those (9.3-10.6 nm) in PLFE LUVs, which suggests that the mobility of solvent molecules (including water and lipid polar groups) in the membrane-water interfacial regions of LUVMV is much less restricted than that in PLFE LUVs. Like PLFE, Sa-MV lipids are tetraethers. However, as mentioned earlier, PLFE lipids are different from Sa-MV lipids in their hydrophobic core composition. It is likely that their polar head groups are also different despite that the zeta potentials of LUVPLFE and LUVMV are virtually identical (-43 mV in 50 mM Tris buffer containing 10mM EDTA and 0.02% NaN3 at pH 7.2-7.6 and 25oC). The REES values (14.5-18.9 nm) of Laurdan fluorescence in Sa-MVs are higher still than those of both LUVPLFE and LUVMV, which is reasonable because tetraether lipids in Sa-MVs are covered with S-layer proteins. As a result, the mobility of solvent molecules around Laurdan’s chromophore in the lipid polar head group regions is more restricted than that in LUVPLFE or LUVMV. Free-standing planar membrane made of MV lipids built on a cellulose acetate partition and mounted onto a Teflon device sustained a nearly constant capacitance (~36-39 pF) for 8 h. Thereafter, the membrane collapsed as evidenced by a zero capacitance. In contrast, the planar membrane made of the diester lipid POPC had much lower stability, showing large fluctuations in capacitance before its collapse at 1.5 h, a very short lifetime typical for free-standing planar membranes made of diester lipids. The planar membrane made of the diester lipid DMPC also showed a short lifetime ~3h. In comparison, the planar membrane made of PLFE showed remarkable stability, exhibiting a constant capacitance for at least 11 days. Similar high stability of PLFE free-standing planar membranes over micro-pores on PDMS thin films in microchip platform was previously reported. Our data suggest that lipids extracted from S. acidocaldarius MVs are able to form fairly stable free-standing planar membranes across a pinhole on a solid support. However, even though both MV lipids and PLFE lipids are tetraethers, the planar membrane made of MV lipids is not as stable as that made of PLFE lipids. The molecular basis for the differential stability between planar membranes of MV lipids and PLFE lipids is not clearly understood at present, but the difference in stability is likely to originate from the chemical structure differences between PLFE lipids and MV lipids. As mentioned earlier, in terms of the hydrophobic cores, PLFE contains ~90% GDNT and ~10% GDGT, whereas MV lipids are mainly GDGT and GTGT, without any GDNT, and their headgroup structures are not known. We have also demonstrated the ability to observe channel activity in PLFE monolayers at a range of voltages from -200 to 200 mV. However, this was property was not replicated in lipids extracted from S. acidocaldarius microvesicles. In any case, our past and present data showed that archaeal tetraether lipids are excellent materials to make stable and yet biologically relevant free-standing planar membranes. / Biomedical Sciences
4

Measurement and characterisation of microvesicles and nanovesicles in pregnancy and pre-eclampsia

Dragovic, Rebecca January 2011 (has links)
Excessive release of syncytiotrophoblast vesicles (STBM) from the placenta into the maternal circulation may cause the inflammatory response, endothelial dysfunction and activation of the coagulation system characteristic of pre-eclampsia (PE). Consequently, other cell types including platelets, leukocytes, red blood cells (RBC) and endothelium may be activated to release cellular vesicles which exacerbate the disease. This thesis aimed to develop methodology for enumerating and phenotyping STBM and the other vesicle types to determine whether they could be used as biomarkers for PE. In vitro derived STBM and vesicles from the other cells of the vascular compartment were examined to select a suitable panel of antibodies to analyse these same vesicle types in plasma samples from non-pregnant (NonP), normal pregnant (NormP) and PE women. Our flow cytometer was shown to detect microvesicles ≥290nm, hence smaller nanovesicles and exosomes could not be detected by this method. Therefore, a novel technique for analysing both microvesicles and nanovesicles, Nanoparticle Tracking Analysis (NTA), was explored and was found to be able to detect vesicles as small as 70nm. The origins of the vesicles that change in pregnancy are not yet known. Flow cytometry and NTA were used in parallel to determine the size, number and phenotype of STBM and other cellular vesicles in NonP, NormP and PE women. Flow cytometry showed that majority of vesicles were derived from platelets, followed by RBC vesicles, leukocyte vesicles and STBM. NTA showed that the total number of vesicles in plasma was significantly elevated in NormP and late-onset PE women compared to NonP controls, and the vesicles were smaller in size. Similarly, flow cytometry showed differences in the composition of vesicles between pregnant and non-pregnant women, demonstrating that pregnancy affects vesicle release. However, no differences were found between NormP and PE women. This was probably due to the majority of samples studied being from late rather than early-onset PE. Thus, although this is the most comprehensive analysis of circulating vesicles in pregnancy to date, their use as biomarkers for PE remains an open question.
5

Apoptosis-driven microenvironmental conditioning by microvesicles in non-Hodgkin lymphoma

Patience, Lauren Alexandra January 2017 (has links)
Plasma membrane derived microvesicles (MV) are nanoscale particles released from cells both constitutively and in response to stimuli including stress, apoptosis and oncogenic transformation. Due to their mechanism of biogenesis, the majority of MV expose phosphatidylserine (PS) on their surface and as such can be identified by staining with annexin V (AxV). First observed nearly 40 years ago as coagulant ‘dust’ originating from activated platelets, MV were initially studied for their role in thrombosis. In more recent years it has become apparent that MV release is increased in several diseases including cancer; this, in conjunction with their ability to carry cargo such as proteins and nucleic acid species, strongly implicates them in disease pathology. Given their small size it is considered likely that MV are able to travel to distal sites within the body allowing the widespread dissemination of effects otherwise not achievable by their parent cells. In the context of malignancy, the contribution of MV is especially important in that MV have been demonstrated to have roles in oncogenic transformation, promotion of tumour growth and increasing metastatic potential. Although clearly important in pathogenesis, their small size makes qualitative and quantitative analysis extremely difficult. Furthermore, the study of MV has been greatly hampered by a lack of standardised protocols for their isolation and as such the majority of studies have been in vitro. In line with this, the relevance of observed effects to in vivo systems is often questioned; given the high quantities of MV used in in vitro systems, the question of whether these concentrations bear any relevance in vivo remain to be answered. We hypothesise that the high rates of apoptosis observed in many tumours, most notably in the high grade B cell malignancy, Non-Hodgkin’s lymphoma (NHL), provides an environment whereby MV are continually released into the surrounding milieu allowing for an amplification of effects. As apoptosis has been previously implicated in promoting tumourigenesis we propose that this is extended to include MV released from apoptotic tumour cells (aMV). Given the numerous technical challenges involved in MV research, initial studies involved identifying the limitations of the instruments available for MV analysis. Preliminary experiments identified considerable resolution issues with the older style EPICS XL flow cytometer (Beckman Coulter) and so a newer flow cytometer, The Attune™ (Thermo Fisher), capable of higher resolution was utilised for the remainder of the project. Despite this improvement, flow cytometry was demonstrated to be less effective at quantifying MV than nanoparticle tracking analysis (NTA). As the fluorescent capacity of NTA is still in its infancy, it was used in concert with flow cytometry in order to quantify and phenotype MV as accurately as possible. As there is currently no concensus on an optimal method of MV isolation subsequent studies focused on determining a method of MV isolation that was appropriate for our experimental system. To this end, centrifugation, filtration and antibody coated magnetic bead-based methods were all tested and their limitations identified. In terms of bead-based isolation strategies, the generation of AxV, protein S, gla domain and gas 6 fusion proteins was attempted with the intention to conjugate to magnetic beads and provide a novel means to isolate aMV. Unfortunately this aspect of the project was ultimately abandoned due to time constraints and although commerically available antibody coated beads were tested for their ability to isolate MV, later co-culture experiments demonstrated that the beads had off target effects that were deleterious to cells. As a result, centrifugation and filtration methods were next researched and validated extensively. TEM analysis of MV morphology identified damage likely induced by the high-speed centrifugation of a fragile apoptotic cell population. As such, a protocol combining low speed centrifugation and filtration was designed and validated by several methods including TEM and staining with AxV. The surface levels of parent cell markers (CD19 and CD20) and apoptosis associated proteins were compared in aMV and vMV (MV released from viable tumour cells) and results demonstrated that B cell surface markers were off loaded into MV to a greater extent following apoptosis. Additional phenotypic studies extended previous work from the group demonstrating the presence of apoptotic cell associated molecular patterns (ACAMPs) capable of binding a panel of antibodies to LPS. Flow cytometry results confirmed the presence of ACAMPs on aMV and results from co-culture experiments with CD14 positive and negative cells suggested that unlike recognition of LPS, binding via ACAMPs was not CD14 dependent. The protein and nucleic acid content of MV was also studied and interestingly, results demonstrated significantly increased quantities of DNA and RNA in aMV compared to vMV. Furthermore, aMV were also shown to contain the matrix metalloproteinases, MMP2 and MMP12 alluding to a role for aMV in angiogenesis. The final stage of the project was focused on determining the roles of aMV in the tumour microenvironment and effects relating to cell growth, cell cycle and angiogenesis were studied and compared to vMV. Results showed that both aMV conditioned supernatant and aMV concentrated by the centrifugation were able to significantly increase the growth of the parent cell population. Further studies using DAPI staining to determine the cell cycle status of cells co-cultured with aMV demonstrated an increase in DNA synthesis and cell division upon incubation with aMV. An in vitro angiogenesis assay was designed to determine any pro-angiogenic capabilities of aMV given the earlier results demonstrating the presence of MMPs. These results provided some of the most interesting findings of the project and showed that aMV were able to increase the angiogenic potential of human endothelial cells (HUVECs); an effect that was shown to be greatly reduced following storage at either 4 or - 80°C. These results demonstrated that aMV possess factors capable of manipulating the tumour microenvironment to favour disease progression and that previously described pro-tumour functions of MV are increased as a result of apoptosis. These findings have implications both in terms of extending the previously described hallmarks of cancer and also when designing a course of therapy whereby in some instances the generation of large amounts of apoptosis may in fact serve to promote regeneration of the tumour cell population.
6

Rôle des exosomes comme nouvelle voie de communication entre les neurones / Role of exosomes as a novel way of interneuronal communication

Javalet, Charlotte 30 September 2016 (has links)
Les exosomes sont des vésicules d’origine endosomale sécrétées par les cellules dans leur environnement après fusion à la membrane plasmique des endosomes multivésiculés. Les exosomes représentent un nouveau mode de communication entre les cellules en permettant un transfert direct de protéines, de lipides et d’ARN. L’objectif de ma thèse était d’étudier le rôle des exosomes dans la communication entre les neurones. Précédemment, le laboratoire a montré que les neurones sécrètent des exosomes de manière régulée par l’activité synaptique. Nous avons observé que les exosomes neuronaux ne sont endocytés que par les neurones. Après avoir montré qu’ils ne contiennent que des ARN courts, nous avons réalisé un séquençage complet de leurs microARN et observé que ces microARN étaient sélectivement exportés dans les exosomes. Nos observations suggèrent que les microARN contenus dans les exosomes peuvent modifier la physiologie des neurones receveurs. Nos résultats renforcent l’hypothèse du rôle des exosomes dans la communication entre les neurones via le transfert de microARN. / Exosomes are vesicles of endocytic origin released by cells into their environment following fusion of multivesicular endosomes with the plasma membrane. Exosomes represent a novel mechanism of cell communication allowing direct transfer of proteins, lipids and RNA. The goal of my PhD thesis was to study that exosomes represent a novel way of interneuronal communication. Our team has previously reported that neurons release exosomes in a way tightly regulated by synaptic activity. We observed that exosomes released by neurons are only endocytosed by neurons. We found that exosomes contain only small RNA and did a deep sequencing of all their microRNA. MicroRNA are selectively exported into exosomes. It seems that exosomal microRNA can modify the physiology of receiving neurons. Our results strengthen the hypothesis of the role of exosomes in the interneuronal communication by the way of microARN transfert.
7

Glycomic insights into microvesicle biogenesis

Batista, Bianca Stella 22 September 2011 (has links)
Cells can mediate intercellular communication by the secretion and uptake of microvesicles, nano-sized membranous particles that carry signaling molecules, antigens, lipids, mRNA and miRNA between cells. The biological function of these vesicles is dependent upon their composition and cellular origin which is regulated by mechanisms that are not well understood. Based on their molecular content, microvesicles may play a role in immune regulation, cancer progression, the spread of infectious agents and numerous other important normal and pathogenic processes. The proteomic content of microvesicles from diverse sources has been intensely studied. In contrast, little is known about their glycomic content. The glycosylation pattern of a protein or lipid plays a key role in determining its functional properties in several ways. Glycans can determine the trafficking of a protein to particular regions of the cell as well as the protein’s half life. In addition, the glycan-dervied oligomerization of glycolipids and glycoproteins is a known mechanism for the activation of receptors and recognition of ligands on the surface of the cell. Glycomic analysis may thus provide valuable insights into microvesicle function. I utilized lectin microarray technology to compare the glycosylation patterns of microvesicles derived from a variety of biological sources. When compared to cellular membranes, microvesicles were enriched in high mannose, polylactosamine, α2-6 sialic acid, and complex N-linked glycans but exclude terminal blood group A and B antigens. The polylactosamine signature in microvesicles from different cell lines derives from distinct glycoprotein cohorts. After treatment of Sk-Mel-5 cells with lactose to inhibit lectin-glycan interactions, secretion of microvesicle resident proteins was severely reduced. Taken together, this work provides evidence for a role of glycosylation in microvesicle-directed protein sorting. / text
8

Particle Balances in Therapeutic Extracellular Vesicle Development and in depth Characterization of Fluorescence Nanoparticle Tracking Analysis

Deighan, Clayton J. January 2015 (has links)
No description available.
9

Characterization and miRNA analysis of cancer cell-secreted microvesicles

Guzman, Nicole Denise 27 June 2012 (has links)
No description available.
10

BMI1 REDUCES ATM AND ATR ACTIVATION DURING DNA DAMAGE RESPONSE THROUGH BINDING TO NBS1 AND TOPBP1

LIN, XIAOZENG January 2017 (has links)
DNA damage response (DDR) maintains genome integrity through checkpoint activation and lesion repair. While ATM and ATR are essential in DDR, mechanisms regulating their activation remain unclear. BMI1 is a component of the polycomb repressive complex 1 (PRC1), and contributes to PRC1’s E3 ubiquitin (E3-Ub) ligase activity though binding the catalytic subunit RING2. BMI1 binds RING2 through its ring finger (RF) domain. The E3-Ub ligase activity contributes to BMI1-deirved facilitation of the homologous recombination-based repair of DNA double-stranded breaks (DSBs). My research demonstrates that BMI1 reduces ATM and ATR activation during DDR. DSBs and single-strand DNA (ssDNA) lesions respectively activate ATM and ATR. ATM subsequently phosphorylates CHK2 at threonine 68 (CHK2pT68) and induces G2/M arrest. ATR produces CHK1pS345 and S-phase arrest. Both kinases phosphorylate histone H2AX at serine 139 (γH2AX) to prepare for lesion repair. Hydroxyurea initiates DDR via producing ssDNA lesions, and increases ATR activation (phosphorylation of T1989/ATR pT1989), CHK1pS345, γH2AX, and S-phase arrest. These events were significantly reduced and enhanced following the respective BMI1 overexpression and BMI1 knockdown in MCF7 and DU145 cells. BMI1 also displays similar effects towards ATM during DDR induced by etoposide-caused DSBs. Activation of ATM and ATR requires the formation of the ATM-NBS1 and ATR-TOPBP1 complexes. We observed that BMI1 interacted with NBS1 or TOPBP1. Deletion of the RF domain from BMI1 did not affect the associations and also had no effects on BMI1’s activity in reducing ATM activation and ATR-mediated CHK1 pS345. Collectively, our research suggests that BMI1 attenuates ATM and ATR signaling independently of the E3-Ub ligase activity. Genotoxic treatments elicit DDR in cells that are directly exposed and also in cells that are not exposed, a phenomenon known as bystander effect (BE). However, it remains unclear what mediates the BE. Microvesicles are small membrane-enclosed sacks that are shed from donor cells and communicate specific messages to recipient cells. We demonstrated that microvesicles isolated from cells treated with etoposide and ultraviolet induced BE in recipient cells. Neutralization of microvesicles through annexin V reduced the microvesicles-associated BE. / Thesis / Doctor of Philosophy (PhD)

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