ITC/USA 2011 Conference Proceedings / The Forty-Seventh Annual International Telemetering Conference and Technical Exhibition / October 24-27, 2011 / Bally's Las Vegas, Las Vegas, Nevada / The integrated Network Enhanced Telemetry (iNET) program has developed standards for network-based telemetry systems, which implementers and range users of Telemetry Network System (TmNS) equipment can use to promote interoperability between components. While standards promote interoperability, only implementation of the standards can ensure it. This paper discusses the tools that are being developed by the iNET program which implement the technologies and protocols specified in the iNET standards in order to ensure interoperability between TmNS components and provide a general framework for device development. Capabilities provided by the tools include system management, TmNS message processing, metadata processing, and time synchronization.

Biogenesis of the multivesicular body (MVB) organelle is an important process for regulation of signalling in the cell. Signal receptors embedded within the outer MVB membrane can be sorted into intralumenal vesicles which bud away from the cytosol to within the MVB preventing further signalling. Sorting of receptors, invagination of the membrane and release of vesicles into the MVB lumen are mediated by the endosomal sorting complexes required for transport (ESCRT) along with a range of accessory proteins including histidine domain protein tyrosine phosphatase (HD-PTP). HD-PTP is a multidomain protein which makes several interactions with ESCRT partners, including ESCRT-0, ESCRT-I and ESCRT-III. This thesis focusses specifically on the interaction between HD-PTP CC domain and Ubap1 (ESCRT-I), and the two interactions of HD-PTP Bro and PRR domains with STAM2 (ESCRT-0) SH3 and Core domains. To address the structure of HD-PTP, multiple techniques were used: X-ray crystallography, which gives high resolution structural information; small angle X-ray scattering (SAXS), which gives low resolution data for large non-crystallisable units in their solution state; and double electron-electron resonance (DEER) spectroscopy, which gives high resolution nanometre-range distance constraints between cysteines labelled with methanethiosulfonate spin label (MTSL). It was shown by X-ray crystallography that HD-PTP has an elongated CC domain, in stark contrast to its homologues ALIX and Bro1 which both have V-shaped CC domains. The CC domain showed limited flexibility both by SAXS and DEER. Further investigation showed that there was no significant conformational change upon binding its ESCRT-I partner Ubap1. The multidomain structure of HD-PTP Bro1-CC-PRR was described by SAXS, showing that these domains form an extended arrangement in solution. In addition, SAXS was also used to analyse the structure of these domains in complex with STAM2 (ESCRT-0), which showed that STAM2 is simultaneously tethered by the Bro1 domain and PRR. The Bro-CC-PRR portion of HD-PTP, has 9 cysteines, so with the aim of measuring local structural information in the CC domain alone, alternative spin labelling methods were investigated. Use of a bromoacrylaldehyde spin label (BASL), instead of MTSL, allowed more selective labelling of surface exposed cysteines, and avoided labelling most of the cysteines in the Bro1 domain. This novel method allowed the shape of the CC domain to be monitored during STAM2 binding and showed that there is no induced conformational change.

La mitochondrie joue un rôle majeur dans la mort cellulaire par nécrose et apoptose. Un des phénomènes hautement régulés conduisant à cette mort cellulaire est la transition de perméabilité mitochondriale qui est médiée par l'ouverture du pore de transition de perméabilité (PTP) localisé dans la membrane interne. Dans les mitochondries de foie de rat, il a été constaté que les analogues de l'ubiquinone ont trois types d'action sur l'ouverture du PTP : l'induction, l'inhibition ou l'interaction sans effet régulateur. Bien que l'inhibition du PTP soit habituellement protectrice de la mort cellulaire, il a été rapporté que des quinones inhibitrices provoquent une toxicité sur certaines lignées cellulaires.<br />Dans notre étude, nous nous sommes attachés à comprendre d'où peuvent provenir ces divergences, en travaillant sur des lignées hépatocytaires immortalisées et cancéreuses. Nous avons observé que la régulation du PTP par les analogues de l'ubiquinone peut être bouleversée par l'immortalisation et/ou la cancérisation, alors que l'effet de la ciclosporine A demeure inchangé. Tant en culture qu'en co-culture, cette caractéristique unique permet une toxicité ciblée, même entre deux lignées cellulaires très proches. Dans certaines lignées, certaines ubiquinones inhibitrices entraînent malgré tout une mort cellulaire. Nous avons pu montrer que cette toxicité est due à l'augmentation de la production radicalaire. Ce travail ouvre de nouvelles perspectives dans l'utilisation du PTP comme cible moléculaire de thérapie anticancéreuse sélective.

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<P>To examine the effects of purported fiber-type transformation following spinal cord injury (SCI), twitch contractile properties of the soleus, tibialis anterior (TA) and thenar muscles were examined in individuals with chronic (> 4 years) SCI. Furthermore, the force-frequency relationship, fatigue and posttetanic potentiation (PTP) of the paralyzed TA muscle were also evaluated. Nine adults with SCI (22-59 yrs; lesion level C3-T2) and 9 age-and gender-matched able-bodied controls (AB) participated in this study.</P><P>On the first visit to the laboratory, the maximum twitch response for all three muscles was determined by delivering a series of single stimuli with gradually increasing intensity. For evaluation of PTP, tetanic stimulation (100Hz) was applied to the TA for 5 seconds followed by single twitches delivered at 5 seconds after tetanus and then at 30-second intervals for 4 minutes posttetanus. On a second visit, the force-frequency relationship (FFR) and 15Hz fatigue of the TA was evaluated. One second bursts ranging from 1-1OOHz were delivered randomly with 2 minutes of rest in between each frequency for assessment of FFR. Following a 1 0-minute rest period, the first fatigue protocol was given, consisting of 1-minute of tetanic stimulation at 15Hz. At the third session, the 30Hz fatigue of the TA was performed, consisting of 1-minute of tetanic stimulation at 30Hz.</P><P>In the soleus muscle, the AB had a higher peak twitch torque (PT) and M-wave amplitude compared to.the SCI group (14.2 ± 3.9Nm vs. 8.9 ± 6.1Nm; p = 0.058, and 13.5 ± 5.3mV and 5.5 ± 4.0mV; p < 0.05, respectively). Contractile speed was not significantly different between groups. Time to peak torque (TPT) was longer in AB (111.5 ± 15.4ms) compared to the SCI (76.7 ± 25.0ms; p<0.05) due to the larger twitches; however, the rates of torque development (RTD) were similar between groups. In the TA muscle, AB and SCI had similar PT (2.8 ± 0.5Nm and 3.2 ± 1.2Nm, respectively). TA contractile properties were faster in SCI, as seen by significantly shorter TPT and faster RTD (p<0.05). M-wave amplitude of AB was significantly greater than the SCI group, 8.3 ± 2.6mV versus 4.2 ± 1.7mV, respectively (p<0.05). Finally, in the thenar muscle, PT appeared to be smaller in AB compared to SCI, 1.7 ± 0.8Nm versus 2.9 ± 1.3Nm, respectively (p = 0.094). The RTD was faster in the SCI group compared to AB (p<0.05).</P><P>Evaluation of FFR revealed that the curve of the SCI was shifted to the left of that of AB. The F50 (frequency required to elicit 50% of maximum peak torque) was significantly lower in the SCI compared to AB, 6.7 ± 3.4Hz and 16.7 ± 4.1Hz, respectively (p<0.05). Following the fatigue protocols, SCI group tended to fatigue more rapidly and to a greater extent than AB at both frequencies, however this was only significant at 15Hz. The M-wave declined with fatigue (30Hz) in both groups, but this decline tended to be more rapid in SCI.</P><P>For the assessment of PTP, both groups started off with similar baseline twitches in their TA muscle (2.7 ± 0.3Nm and 2.9 ± 0.8Nm, respectively). At 5 seconds following tetanus, PT was significantly greater in both groups, but the amount of potentiation was greater in SCI versus AB (p = 0.058). Over the 4-min recovery period, PT declined in both groups until it was no longer significantly greater than baseline by 3 minutes 30 seconds. The potentiated twitch of both groups was faster than at baseline. RTD increased significantly by an average of 56% in the AB group and 91% in the SCI group and was significantly greater in SCI compared with AB at 30-150 seconds post-tetanus (p<0.05). At 5 seconds post-tetanus, RTR was significantly faster in both groups and had increased by 77% and 53% in the AB and SCI groups, respectively. The recovery ofRTD and R TR over the 4 minutes occurred more rapidly in AB versus SCI.</P><P>In conclusion, changes in contractile properties following SCI differ between muscle groups; faster contractile properties indicative of fiber type transformation are more evident in TA and thenar muscle groups, compared with the soleus. The smaller M-waves seen in the lower extremities support the significant muscle atrophy following SCI. Furthermore, the predicted transformation towards a higher proportion of fast-twitch fibers following paralysis was supported by a trend for decreased fatigue resistance and significantly greater PTP in the SCI group. The FFR data, however, did not support this predicted fiber type transformation, shifting to the left instead of the right. This leftward shift of FFR has been reported in other paralyzed human muscle presenting with faster contractile speeds; the mechanisms behind this warrant further investigation.</P> / Thesis / Master of Science (MSc)

La mitochondrie est un carrefour d’informations au centre du fonctionnement cellulaire puisque son rôle physiologique consiste à récupérer l’énergie fournie par la dégradation des produits issus de notre alimentation pour produire de l’ATP, par le processus d’oxydation phosphorylante. Cependant, des altérations du fonctionnement de la mitochondrie peuvent être responsables de nombreuses pathologies. Parmi les stress métaboliques pouvant entraîner un dysfonctionnement mitochondrial, l’ischémie-reperfusion est un phénomène présent également dans de nombreuses situations pathologiques. L’objectif de ce travail consiste à développer une approche méthodologique basée sur la microscopie confocale et l’analyse d’images afin de décortiquer les conséquences cellulaires des stress métaboliques induits lors d’épisodes de privation de substrats associée ou non à une privation partielle ou totale d’oxygène. Après avoir mis au point le programme d’analyse d’images basée sur la méthode du « tophat », deux approches ont été développées pour visualiser et quantifier la fonction mitochondriale. La première, qui combine le marquage du TMRM et l’autofluorescence du NADH, a permis de mettre en évidence des différences de réponses au stress d’ischémie-reperfusion au niveau de la chaîne respiratoire ou de l’ouverture du PTP pour les quatre types cellulaires testés : HMEC-1, INS1, RT112 ou hépatocytes primaires. La seconde approche a consisté à tester l’utilisation de biosenseurs permettant de suivre les variations de concentration d’ATP (Ateam) ou d’activation de l’AMPK (AMPKAR). Les conditions expérimentales réalisées dans ce travail n’ont pas permis de valider leur utilisation. / Mitochondria form an information hub at the center of the cellular metabolism because of its physiological role consisting in the porduction of ATP from the degradation of porducts stemming from our food through the OXPHOS process. However, changes in the functionnig of the mitochondria can be responsible for numerous diseases. Among the different foms of metabolic stress leading to mitchondrial dysfunctions, ischemia-reperfusion can be found in numerous pathological situations. This work aims at developing a methodological approach based on confocal microscopy and image analysis to dissect –at cell level- the consequences of metabolic stress induced by episodes of deprivation in substrata associated or not with hypoxia or anoxia. Having developed the program of image analysis based on the « tophat » method, two approaches were designed to vizualize and quantify the mitochondrial function. The first one, combining TMRM labelling with NADH fluorescence made it possible to highlight some differences in the response to the stress caused by ischemia-reperfusion at the level of the respiratory chain or concerning the PTP opening in the four cellular types that were tested : HMEC-1, INS1, RT112 or pirmary heaptocyes. The second approach consisted in testing the use of biosensors designed to follow the variations of ATP concentration (ATeam) or the activation of AMPK (AMPKAR). The experimental conditions established in this work did not allow us to validate their use.

L’adrénomédulline (AM) est un des principaux facteurs de croissance impliqués dans la formations de nouveaux vaisseaux. L’AM est responsable de la formation de jonctions adhérentes stables entre cellules endothéliales vasculaires via le maintien d’un état déphosphorylé du complexe d’adhésion VE-cadhérine/caténines. La phosphorylation de tyrosines est un évènement régulé par un équilibre entre protéine tyrosine kinases et protéine tyrosine phosphatases (PTP). Peu de choses sont encore connues sur le rôle des PTPs dans les voies de signalisation de l’AM au niveau des cellules endothéliales. La SHP2 a été décrite comme étant capable de déphosphoryler le complexe d’adhésion. Son association avec la β-caténine lui permet de contrôler le niveau de phosphorylation du complexe et de maintenir l’association entre VE-cadhérine et caténines. Nous avons ainsi émis l’hypothèse selon laquelle l’AM puisse agir sur la SHP2 permettant ainsi le contrôle de la formation du complexe d’adhésion VE-cadhérine-β-caténine. Nos travaux ont mis en évidence une augmentation de l’activation de la SHP2 induite par l’AM dans les cellules endothéliales entrainant sa localisation au niveau de la membrane et la stabilisation de l'adhésion cellulaire induite par la VE-cadhérine en réduisant le niveau de phosphorylation de cette dernière. Le blocage de la SHP2 entraine des effets opposés avec une inhibition de la déphosphorylation induite par l’AM de la VE-cadhérine sur les tyrosines 731 et 658. En résumé, l’AM régule l’activité de la SHP2 via sa phosphorylation sur la tyrosine 542 ce qui entraine une stabilisation des contacts cellules-cellules via une diminution de la phosphorylation de la VE-cadhérine. / Adrenomedullin (AM) is one of the main factors in the formation of tumor neo-vessels. It's responsible for stable adherent junction formation between vascular endothelial (VE) cells by maintaining VE-cadherin/catenins adhesion complex in a dephosphorylated status. Indeed, AM blockade induces phosphorylation of VE-cadherin in tyrosine 731, which is followed by disruption of VE-cadherin-mediated cell-cell contacts of endothelial cells (ECs), thereby leading to EC adhesion loss and tumor vessels disruption. Tyrosine phosphorylation events are controlled by the balance of activation of protein tyrosine kinases and protein tyrosine phosphatases (PTPs). Little is known about the role of endogenous PTPs in AM signaling in ECs. SHP2 is capable of dephosphorylating the complex. Its association with β-catenin allows it to control the dephosphorylated steady state of the complex and to maintain the VE-cadherin/β-catenin association. To study the mechanism of AM on the inter-endothelial junction stabilization, we hypothesized that AM may act on SHP2 allowing a control upon formation of VE-cadherin-β-catenin complex. In this study, we found that SHP2 activity is markedly increased by AM. In ECs, AM-induced phospho-SHP2 Y542 activity to localize at the human umbilical vein endothelial cell membrane and stabilizes VE-cadherin-mediated cell-cell adhesions by reducing VE-cadherin tyrosine phosphorylation. SHP2 inhibition causes opposite effects with inhibiting AM-induced dephosphorylation of VE-cadherin at Y731 and Y658. In summary, AM regulates SHP2 activity through phosphorylation of Y542, which stabilizes cell-cell adhesions through reducing tyrosine phosphorylation of VE-cadherin.

Made available in DSpace on 2016-03-15T19:37:54Z (GMT). No. of bitstreams: 1 Fernando Biazi Nascimento.pdf: 3885211 bytes, checksum: 52d266afc3ffddce2e0abc915e2471a1 (MD5) Previous issue date: 2014-10-23 / Fundo Mackenzie de Pesquisa / The present work consists of an implementation of time distribution protocol based on PTP disclosed in the IEC 61588:2009 / IEEE 1588-2008 standard to be used in low-power wireless devices. The distribution of time is important to determine the order of occurrence of events marked in distinct counts that can then be related. And the problems of lack of synchronicity are evident in circumstances ranging from the study of historical facts up to the investigation of intruders in modern equipments connected to the internet. The work included development of a completely new software for the microcontroller MSP430F2274TM using the CC2480TMnetwork controller. The implementation of the protocol considers one of the mechanisms described by the standard and remains very close to it, not fully conformant mainly because of lack of resources on the used device, but the expected behavior was kept. The devices synchronize the time between them and sintonize their time counting, in a way to reduce, as much as possible, the adjustments of further synchronizations. / O presente trabalho consiste em uma implementação de protocolo de distribuição de tempo baseado no PTP, definido na norma IEC 61588:2009/IEEE 1588-2008 a ser utilizado em dispositivos sem fio de baixo consumo de energia. A distribuição de tempo é importante para determinar a ordem de ocorrência de eventos marcados em contagens distintas que podem então ser relacionadas. E os problemas de falta de sincronia são evidentes em circunstâncias que vão de estudo de fatos históricos até a verificação de intrusos em equipamentos modernos conectados à internet. O trabalho contou com desenvolvimento de um software completamente novo para o micro-controlador MSP430F2274TM utilizando o controlador de rede CC2480TM. A implementação do protocolo considera um dos mecanismos apresentados pela norma e ficou muito próxima, não atendendo-a plenamente principalmente por falta de recursos no dispositivo utilizado, mas manteve o comportamento previsto. Os dispositivos sincronizam os tempos entre eles e sintonizam suas contagens de tempo, de forma a reduzir, tanto quanto possível, o ajuste de futuras sincronizações.