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Pharmacological regulators of T cell calcium storesPeters, Alister Michael 01 January 2004 (has links) (PDF)
Calcium is a crucial intracellular messenger controlling a plethora of important intracellular events. Elevated [Ca2+]i levels regulate numerous processes due to the versatility of the Ca2+ signaling in terms of speed, amplitude and spatia-temporal patterning. Depletion of intracellular calcium stores functions as a primary trigger for a message that is translated to the plasma membrane, resulting in the slow activation of plasma membrane Ca2+ influx channels, which allow entry of external calcium. Since these channels depend upon the state of filling of the intracellular Ca2+stores, these influx channels are called store-operated channels (SOCs). It is unclear how empty intracellular stores signal activation of plasma membrane capacitative calcium entry (CCE). In order to bridge the gaps in our understanding of calcium's role in T cell regulation, this project was designed to look at the effects of various pharmacological regulators of T cell calcium stores. At the start, the effort was directed at the regulation of the microsomal calcium A TPases, considering these are perhaps the most essential mediators of intracellular calcium storage. Thapsigargin (TG) and cyclopiazonic acid (CPA) were shown to be the most potent inhibitors of the Ca2+ -ATPase, also a new Ca2+ regulator aaptamine was shown to exert more modest inhibition of the Ca2+ pump. We also characterized a novel compound, gingerol, findings its actions are to stimulate Ca2+ pumps. Cell growth assays revealed an important role for ryanodine receptors (RyRs) in regulating T cell growth. RyR activators CMC and PCB95 dramatically altered T cell growth patterns leading to significantly reduced cell viability. In contrast RyR antagonist dantrolene appeared to induce growth arrest in that cell proliferation was curtailed, yet cells remained viable.
Cell viability studies revealed that the Ca2+ pump regulators TG and aaptamine were also observed to reduce cell growth rates, presumably as a result of their ability to deplete Ca2+ stores (100 nM TG was able to decrease cell viability by 90% within 24 hrs of exposure). PCB95 was able to decrease cell viability by 50% within 24 of hrs exposure and CMC decreased cell viability by 75% within 24 hrs and further over a 48 hr period. 2-APB and aaptamine were cytotoxic at higher doses. The inositol 1 ,4,5 -trisphosphate receptor (IP3R) pathway was also found to be critically linked to T cell growth control. We observed that the IP3R modulator, 2- aminoethoxydiphenyl borate (2-APB) induced antigen-like Ca2+ spike that correlates with suppression of T cell growth rates.
In this study we have identified two novel T cell Ca2+ store regulators, aaptamine and gingerol. We also find that Ca2+ stores are indeed sensitively linked to T cell growth regulation. Depletion of Ca2+ stores with SERCA inhibitors as well as both RyR and IP3R activators profoundly suppress T cell proliferation most likely via activation of apoptosis.
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Mechanisms by which hypoxia augments Leydig cell viability and differentiated cell function in vitroKukucka, Mark A. 06 June 2008 (has links)
The 1980's heralded the discovery and identification of extra-pituitary sources of the neurohypophysial hormone oxytocin in non-neural tissues of several animal species. The presence, location and biosynthesis of significant amounts of oxytocin in the ovarian corpus luteum was followed by the immunocytochemical demonstration of an oxytocin-like peptide in the testicular interstitial cells. Leydig cells, which comprise up to 80% of the testicular intertubular cell population, are known to synthesize testosterone in situ. Indirect evidence indicated that an oxytocin-like peptide was also present in Leydig cells.
The question arose whether this peptide was synthesized de novo by Leydig cells or was taken up and stored by the cells following biosynthesis at some other intra- and/or extra-gonadal source(s). Since luteinizing hormone (LH) and ascorbate are known to augment the production of oxytocin in ovarian granulosa cells, varying concentrations of these two stimulants were used to monitor the biosynthesis of oxytocin from isolated Leydig cells in culture. / Ph. D.
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Structural complexity of the co-chaperone SGTA: a conserved C-terminal region is implicated in dimerization and substrate quality controlMartínez-Lumbreras, S., Krysztofinska, E.M., Thapaliya, A., Spilotros, A., Matak-Vinkovic, D., Salvadori, E., Roboti, P., Nyathi, Yvonne, Muench, J.H., Roessler, M.M., Svergun, D.I., High, S., Isaacson, R.L. 08 June 2020 (has links)
Yes / Protein quality control mechanisms are essential for cell health and involve delivery of proteins to
specific cellular compartments for recycling or degradation. In particular, stray hydrophobic proteins are captured in
the aqueous cytosol by a co-chaperone, the small glutamine-rich, tetratricopeptide repeat-containing protein alpha
(SGTA), which facilitates the correct targeting of tail-anchored membrane proteins, as well as the sorting of membrane
and secretory proteins that mislocalize to the cytosol and endoplasmic reticulum-associated degradation. Full-length
SGTA has an unusual elongated dimeric structure that has, until now, evaded detailed structural analysis. The Cterminal region of SGTA plays a key role in binding a broad range of hydrophobic substrates, yet in contrast to the
well-characterized N-terminal and TPR domains, there is a lack of structural information on the C-terminal domain. In
this study, we present new insights into the conformation and organization of distinct domains of SGTA and show that
the C-terminal domain possesses a conserved region essential for substrate processing in vivo.
We show that the C-terminal domain region is characterized by α-helical propensity and an intrinsic ability to
dimerize independently of the N-terminal domain. Based on the properties of different regions of SGTA that
are revealed using cell biology, NMR, SAXS, Native MS, and EPR, we observe that its C-terminal domain can
dimerize in the full-length protein and propose that this reflects a closed conformation of the substrate-binding domain.
Our results provide novel insights into the structural complexity of SGTA and provide a new basis
for mechanistic studies of substrate binding and release at the C-terminal region. / MRC New Investigator Research Grant: G0900936; BBSRC grants: BB/L006952/1 and BB/L006510/1; BBSRC grant: BB/N006267/1; Wellcome Trust Investigator Award in Science: 204957/Z/16/Z; BBSRC grant: BB/J014567/1
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Autocrine mechanisms of action of insulin-like growth factor-I (IGF-I) and hormonal regulation of expression of IGF-finding proteins in mammary epithelial cellsRomagnolo, Donato 06 June 2008 (has links)
Limited information is available concerning the molecular and cellular mechanisms that regulate expression of insulin-like growth factor-I (IGF-I) and IGF-binding proteins (IGFBPs) genes in mammary epithelial cells. To test the hypothesis that IGF-I affects growth of bovine mammary epithelial cells through an autocrine and/or paracrine pathway, several cell lines were developed expressing an ovine exon-2 containing IGF-I cDNA under the control of the mouse mammary tumor virus-long terminal repeat (pMMTV-IGF-I), early simian virus (pSV40-IGF-I), and herpes simplex thymidine kinase (pTK-IGF-I) promoters. Stably transfected clones were generated by cotransfection of clonal MAC-T cells with the IGF-I expression vectors and a plasmid conferring resistance to hygromycin-B (HYG-B), using a calcium phosphate precipitation procedure. Induction of the MMTV-LTR with the glucocorticoid dexamethasone (DEX) was required for enhanced expression of IGF-I in MD-IGF-I (MD=Mammary Derived) cells, whereas SV40-IGF-I cells constitutively expressed the highest levels of IGF-I, followed by TK-IGF-I cells. Activity of the MMTV promoter in MD-IGF-I cells was coordinately regulated by lactogenic hormones and extracellular matrix. Acute secretion of DEX-induced recombinant IGF-I by MD-IGF-I cells stimulated cell proliferation through an autocrine/paracrine pathway and triggered the expression of IGFBP-3. Neither acute nor constitutive expression of IGF-I affected expression of type 1 IGF receptor mRNAs, but down-regulated cell surface receptor levels, in the order SV40-> TK- > MD-IGF-I. Secretion of IGF-I-induced IGFBP-3 potentiated the mitogenic actions of IGF-I as evidenced by enhancement of [³H]thymidine uptake into DNA of parental MAC-T cells. This study provides evidence that local production of IGF-I can stimulate cell proliferation of bovine mammary epithelial cells through an autocrine/paracrine mode of action. We suggest that secretion of IGF-I-induced IGFBP-3 by bovine mammary epithelial cells enhances cell responsiveness to IGF-I, but does not prevent down-regulation of the IGF-I receptor in cells constitutively expressing IGF-I. / Ph. D.
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A comparative analysis of the G1/S transition control in kinetic models of the eukaryotic cell cycleConradie, Riaan 12 1900 (has links)
Thesis (PhD (Biochemistry))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT:
The multiplication of cells proceeds through consecutive phases of growth and division
(G1, S, G2 and M phases), in a process known as the cell cycle. The transition between
these phases is regulated by so-called checkpoints, which are important to ensure proper
functioning of the cell cycle. For instance, mutations leading to faulty regulation of the
G1/S transition point are seen as one of the main causes of cancer.
Traditionally, models for biological systems that show rich dynamic behavior, such
as the cell cycle, are studied using dynamical systems analysis. However, using this
analysis method one cannot quantify the extent of control of an individual process in
the system. To understand system properties at the process level, one needs to employ
methods such as metabolic control analysis (MCA). MCA was, however, developed
for steady-state systems, and is thus limited to the analysis of such systems, unless the
necessary extensions would be made to the framework. The central question of this thesis focuses on quantifying the control in mathematical
models of the G1/S transition by the individual cell cycle processes. Since MCA was
never applied to the cell cycle, several new methods needed to be added to the framework.
The most important extension made it possible to follow and quantify, during a
single cell cycle, the control properties of the individual system processes.
Subsequently, these newly developed methods were used to determine the control
by the individual processes of an important checkpoint in mammalian cells, the restriction
point. The positioning of the restriction point in the cell cycle was distributed over
numerous system processes, but the following processes carried most of the control:
reactions involved in the interplay between retinoblastoma protein (Rb) and E2F transcription
factor, reactions responsible for the synthesis of Delayed Response Genes and
Cyclin D/Cdk4 in response to growth signals, the E2F dependent Cyclin E/Cdk2 synthesis
reaction, as well as the reactions involved in p27 formation. In addition it was
shown that these reactions exhibited their control on the restriction point via the Cyclin
E/Cdk2/p27 complex. Any perturbation of the system leading to a change in the
restriction point could be explained via its e ect on the Cyclin E/Cdk2/p27 complex,
showing a causal relation between restriction point positioning and the concentration of
the Cyclin E/Cdk2/p27 complex.
Finally, we applied the new methods, with a modular approach, to compare a number
of cell cycle models for Saccharomyces cerevisiae (budding yeast) and mammalian cells
with respect to the existence of a mass checkpoint. Such a checkpoint ensures that cells
would have a critical mass at the G1/S transition point. Indeed, in budding yeast, a
correction mechanism was observed in the G1 phase, which stabilizes the size of cells
at the G1/S transition point, irrespective of changes in the specific growth rate. This in
contrast to the mammalian cell cycle models in which no such mass checkpoint could
be observed in the G1 phase.
In this thesis it is shown that by casting specific questions on the regulation and
control of cell cycle transition points in the here extended framework of MCA, it is
possible to derive consensus answers for subsets of mathematical models. / AFRIKAANSE OPSOMMING:
Die selsiklus bestaan uit agtereenvolgende groei- en delingsiklusse wat tot selvermeerdering
lei. Die siklus word gekenmerk deur onderskeie fases (G1, S, G2 en M) wat
deur sogenaamde beheerpunte gereguleer word. Hierdie beheerpunte verseker dat selvermeerdering
nie ongekontroleerd kan plaasvind nie en mutasies wat lei tot foutiewe regulering
van die G1/S transisiepunt word as een van die hoofoorsake van kanker beskou.
Die hoofdoel van hierdie studie was om die beheer wat selsiklusprosesse op die G1/S
transisie uitoefen met behulp van wiskundige modelle te kwantifiseer. Omdat biologiese
sisteme soos die selsiklus ryk dinamiese gedrag vertoon, word hulle tradisioneeldeur
middel van dinamiese sisteemanalise bestudeer. Die analisemetode beskik egter nie oor
die vermoë om die hoeveelheid beheer wat afsonderlike sisteemprosesse op 0n sisteemeienskap
uitoefen te kwantifiseer nie. Om sisteemeienskappe op prosesvlak te verstaan
moet metodes soos metaboliese kontrole analise (MKA) ingespan word. MKA was egter
ontwikkel om sisteme in 0n bestendige toestand te analiseer en aangesien MKA nog nooit vantevore vir selsiklus analises gebruik was nie, moes nuwe MKA tegnieke gedurende
die studie ontwikkel word. Die belangrikste van die metodes maak dit moontlik
om beheer (soos uitgeoefen deur die onderskeie sisteemprosesse) oor 0n enkele selsiklus
na te volg en te kwantifiseer. Die nuut-ontwikkelde metodes was vervolgens gebruik
om te bepaal hoe een so 0n beheerpunt in soogdierselle - die restriksiepunt - deur die
onderskeie sisteemprosesse beheer word.
Die studie het aangedui dat die posisie van die restriksiepunt tydens die selsiklus
deur ’n verskeidenheid sisteemprosesse beheer word. Die bevinding was dat vier prosesse
beduidend meer beheer op die posisie van die restriksiepunt uitoefen: Reaksies
wat betrekking het op die wisselwerking tussen retinoblastoma proteïen (Rb) en E2F
transkripsiefaktor; reaksies verantwoordelik vir die sintese van vertraagde responsgene
en Siklien D/Cdk4 in respons tot groeiseine; die E2F afhanklike Siklien E/Cdk2 sintesereaksie;
sowel as die reaksies betrokke in p27 vorming. Daar was ook aangetoon
dat hierdie reaksies hul beheer op die posisie van die restriksiepunt deur die Siklien
E/Cdk2/p27 kompleks uitoefen, siende enige sisteemversteuringe (wat tot veranderinge
in die restriksiepuntposisie aanleiding gee) deur veranderinge in die kompleks verklaar
kon word - 0n observasie wat aandui dat daar 0n kousale verhouding is tussen die posisie
van die restriksiepunt en die Siklien E/Cdk2/p27 kompleks.
Die nuut-ontwikkelde metodes was verder gebruik om 0n verskeidenheid selsiklusmodelle
van Saccharomyces cerevisiae (bakkersgis) en soogdierselle met 0n modulêre
aanpak te vergelyk om te bepaal of daar 0n massa beheerpunt in beide soogdier- en bakkersgisselle
bestaan. Daar word gepostuleer dat hierdie beheerpunt verseker dat selle
0n kritiese massa by die G1/S transisiepunt bereik. Die resultate van die studie dui
daarop dat bakkersgis, anders as soogdierselle, oor so 0n korreksiemeganisme beskik.
Die meganisme stabiliseer die grootte van selle in die G1 fase ondanks veranderinge in
die groeitempo van die selle, sodat massa homeostaties by die G1/S transisiepunt gehandhaaf
word. Die studie het getoon dat moeilike vrae met betrekking tot die selsiklus
beantwoord kan word deur van wiskundige modelle gebruik te maak en die probleme in
die nuut-ontwikkelde metaboliese kontrole analise raamwerk te giet.
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Komparace kontrolních mechanismů mezinárodněprávní ochrany lidských práv / Comparison of control mechanisms in the international legal protection of human rightsŘíhová, Petra January 2015 (has links)
Comparison of the Control Mechanisms of the International Protection of Human Rights This Diploma Thesis deals with the Control Mechanisms in the field of International Protection of Human Rights. In this Diploma Thesis are introduced universal and regional control mechanisms and they are analysed with respect to their functioning. The aim of this Thesis is the comparison of the control mechanisms in the several aspects, especially in the terms of their efficiency, and their evaluation based on the undertaken comparison. Another purpose is to answer the question whether or not we have in the contemporary International Law functional control mechanisms of the protection of human rights and which of them can be seen as the most effective. The comparison in this Thesis emphasis on the possibility of the individual complaints. The comparison includes the formal aspects of monitoring bodies, their regulation in the relevant treaties and analysis of their functions and their efficiency. In Thesis are included and evaluated also available statistical data concerning e.g. the number of ratifications or the number of individual complaints which were lodged before the monitoring body. With respect to the topic, the Thesis is divided into the three chapters. The first chapter deals with the brief introduction...
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Over-Expression of BDNF Does Not Rescue Sensory Deprivation-Induced Death of Adult-Born Olfactory Granule CellsUnknown Date (has links)
It is of interest to understand how new neurons incorporate themselves into the
existing circuitry of certain neuronal populations. One such population of neurons is that
which are born in the subventricular zone (SVZ) and migrate to the olfactory bulb where
they differentiate into granule cells. Another area of interest is the role of brain-derived
neurotrophic factor (BDNF) on the survival and overall health of these neurons. This
study aimed to test whether or not BDNF is a survival factor for adult-born granule cells.
Here were utilized a transgenic mouse model over-expressing BDNF under the α-
calcium/calmodulin-dependent protein kinase II (CAMKIIα) promoter, and tested its
effect on olfactory granule cells under sensory deprived conditions. Results from this
experiment indicated that there was no significant difference in cell death or cell survival when comparing transgenic and wild type animals. We concluded that BDNF is not a
survival factor for adult-born granule cells. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2016. / FAU Electronic Theses and Dissertations Collection
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Of Mice, Men and Memories: The Role of the Rodent Hippocampus in Object RecognitionUnknown Date (has links)
Establishing appropriate animal models for the study of human memory is
paramount to the development of memory disorder treatments. Damage to the
hippocampus, a medial temporal lobe brain structure, has been implicated in the memory
loss associated with Alzheimer’s disease and other dementias. In humans, the role of the
hippocampus is largely defined; yet, its role in rodents is much less clear due to
conflicting findings. To investigate these discrepancies, an extensive review of the rodent
literature was conducted, with a focus on studies that used the Novel Object Recognition
(NOR) paradigm for testing. The total amount of time the objects were explored during
training and the delay imposed between training and testing seemed to determine
hippocampal recruitment in rodents. Male C57BL/6J mice were implanted with bilateral
dorsal CA1 guide cannulae to allow for the inactivation of the hippocampus at discrete
time points in the task. The results suggest that the rodent hippocampus is crucial to the
encoding, consolidation and retrieval of object memory. Next, it was determined that there is a delay-dependent involvement of the hippocampus in object memory, implying
that other structures may be supporting the memory prior to the recruitment of
hippocampus. In addition, when the context memory and object memory could be further
dissociated, by altering the task design, the results imply a necessary role for the
hippocampus in the object memory, irrespective of context. Also, making the task more
perceptually demanding, by requiring the mice to perform a two-dimensional to three-dimensional
association between stimuli, engaged the hippocampus. Then, in the
traditional NOR task, long and short training exploration times were imposed to
determine brain region activity for weak and strong object memory. The inactivation and
immunohistochemistry findings imply weak object memory is perirhinal cortex
dependent, while strong object memory is hippocampal-dependent. Taken together, the
findings suggest that mice, like humans, process object memory on a continuum from
weak to strong, recruiting the hippocampus conditionally for strong familiarity.
Confirming this functional similarity between the rodent and human object memory
systems could be beneficial for future studies investigating memory disorders. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2016. / FAU Electronic Theses and Dissertations Collection
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Presynaptic Determinants of Synaptic Strength and Energy Efficiency at Drosophila Neuromuscular JunctionsUnknown Date (has links)
Changes in synaptic strength underlie synaptic plasticity, the cellular substrate for learning and memory. Disruptions in the mechanisms that regulate synaptic strength closely link to many developmental, neurodegenerative and neurological disorders. Release site probability (PAZ) and active zone number (N) are two important presynaptic determinants of synaptic strength; yet, little is known about the processes that establish the balance between N and PAZ at any synapse. Furthermore, it is not known how PAZ and N are rebalanced during synaptic homeostasis to accomplish circuit stability. To address this knowledge gap, we adapted a neurophysiological experimental system consisting of two functionally differentiated glutamatergic motor neurons (MNs) innervating the same target. Average PAZ varied between nerve terminals, motivating us to explore benefits for high and low PAZ, respectively. We speculated that high PAZ confers high-energy efficiency. To test the hypothesis, electrophysiological and ultrastructural measurements were made. The terminal with the highest PAZ released more neurotransmitter but it did so with the least total energetic cost. An analytical model was built to further explore functional and structural aspects in optimizing energy efficiency. The model supported that energy efficiency optimization requires high PAZ. However, terminals with low PAZ were better able to sustain neurotransmitter release. We suggest that tension between energy efficiency and stamina sets PAZ and thus determines synaptic strength. To test the hypothesis that nerve terminals regulate PAZ rather than N to maintain synaptic strength, we induced sustained synaptic homeostasis at the nerve terminals. Ca2+ imaging revealed that terminals of the MN innervating only one muscle fiber utilized greater Ca2+ influx to achieve compensatory neurotransmitter release. In contrast, morphological measurements revealed that terminals of the MN inner vating multiple postsynaptic targets utilized an increase in N to achieve compensatory neurotransmitter release, but this only occurred at the terminal of the affected postsynaptic target. In conclusion, this dissertation provides several novel insights into a prominent question in neuroscience: how is synaptic strength established and maintained. The work indicates that tension exists between energy efficiency and stamina in neurotransmitter release likely influences PAZ. Furthermore, PAZ and N are rebalanced differently between terminals during synaptic homeostasis. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2015. / FAU Electronic Theses and Dissertations Collection
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Perfil diário e os mecanismos de produção de melatonina pela glândula pineal de ratos diabéticos por estreptozotocina. / Pineal melatonin production in Streptozotocin-diabetic rats: mechanisms and microdialysis daily profile.Amaral, Fernanda Gaspar do 27 October 2009 (has links)
A pineal participa da sincronização do organismo pela síntese de melatonina. Diabetes é um distúrbio metabólico caracterizado por hiperglicemia. Diante da controvérsia sobre a síntese de melatonina em animais diabéticos, esse trabalho objetivou avaliar as alterações da glândula pineal mediante o diabetes induzido por STZ (60mg/kg, i.p.). Ratos wistar (250-280g, 12h/12h claro/escuro) foram utilizados em todos os procedimentos que envolveram técnicas de FACS, microdiálise, HPLC, radiometria da atividade enzimática e qPCR. Os resultados mostraram que o diabetes causa diminuição (50%) e perda do perfil mono/bifásico da síntese pineal de melatonina, que não é causada por necrose ou apoptose dos pinealócitos e reflete um desarranjo no metabolismo pineal, evidenciado pela diminuição na atividade da AANAT (55%). Observou-se também um desbalanço rítmico de fatores determinantes como a expressão do receptor ß1 e a atividade e expressão das enzimas TPH1 e HIOMT. A menor concentração de melatonina circulante pode ser um fator contribuinte para o desenvolvimento da doença. / The gland is involved in the organism synchronization via its hormone melatonin. Diabetes involves hyperglicemia and insulin synthesis/signaling impairment. The aim of this work was to evaluate the pineal melatonin synthesis in STZ-diabetic rats (60mg/kg, i.p.). Male wistar rats (250-280g, 12h/12h light/dark) were used as the animal model and FACS, microdialysis, HPLC, enzyme activity assay and qPCR were the techniques used to evaluate the pineal phisiology. The results show a decrease in pineal melatonin (50%) and a circadian profile impairment that were not due to necrosis or apoptosis. This finding reflects an important impairment in the pineal metabolism that was related to a decrease in AANAT activity (55%). An alteration in the rhthmicity of important factors, such as the ß1-adrenergic receptor expression and the TPH1 and HIOMT activity and expression, was also observed. This decrease in circulating melatonin may be of fundamental importance to the establishment and maintenance of diabetes.
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