Marine compound palytoxin induces apoptotic cell death via Mcl-1 and Bcl-2 down-regulation in human leukemia cells

학위논문 (석사)-- 서울대학교 대학원 : 약학과(의약생명과학전공), 2015. 8. Marc Diederich.Marine ecosystems contribute to a huge repository of pharmacologically active compounds. Palytoxin, one of the most toxic marine compounds, is known to be involved in the transformation of Na+/K+-ATPase into a cation channel inducing massive intracellular Na+ influx. Anti-cancer activity of palytoxin is an emerging area of research and especially palytoxin-induced cancer cell death mechanisms remain to be elucidated. Here we show that palytoxin induced cell death of various leukemia cell lines at low picomolar concentrations. Importantly, palytoxin did not affect viability of peripheral blood mononuclear cells (PBMC) cells from healthy donors and did not create systemic toxicity in zebrafish, thus demonstrating excellent differential toxicity. Cell death was characterized by nuclear condensation as demonstrated by Hoechst staining as well as by caspase activation demonstrated by western blot and luminescent caspase assays. Palytoxin triggers cleavage of initiator pro-caspases-8 and -9 as well as executioner pro-caspases-3 and -7 after 6 hours of treatment in a dose-dependent manner. As caspase activation is sensitive to pan-caspase inhibitor zVAD, we conclude that palytoxin induces apoptotic cell death. From a molecular point of view, palytoxin down-regulates anti-apoptotic Bcl-2 family proteins Mcl-1 and Bcl-xL in a dose-dependent manner. MG-132, a proteasome inhibitor, was able to prevent proteolysis of Mcl-1 whereas the three major proteasomal enzymatic activities were up-regulated by palytoxin. Palytoxin-induced dephosphorylation of Bcl-2 further exacerbates the pro-apoptotic effect of Mcl-1 and Bcl-xL degradation. As okadaic acid could rescue cell death triggered by palytoxin, we hypothesize involvement of protein phosphatase (PP)2A in Bcl-2 phosphorylation and induction of apoptosis by palytoxin. Altogether, we provide here first evidence of the role of palytoxin as a very potent and promising cancer-specific cytotoxic agent acting at low picomolar concentrations.ABSTRACT ..................................................................................................................................... 1 LIST OF ABBREVIAIONS ............................................................................................................ 3 INTRODUCTION ............................................................................................................................ 5 MATERIALS AND METHODS..................................................................................................... 7 1. Cells and medium ....................................................................................................................... 7 2. Compounds ............................................................................................................................... 7 3. Cell viability assessment ............................................................................................................ 8 4. Caspase 3/7 activity assay .......................................................................................................... 8 5. Proteasome activity assay........................................................................................................... 9 6. Cell lysate preparations and western blots ................................................................................. 9 7. Fluorescent microscopy analysis .............................................................................................. 12 8. Systemic toxicity in zebrafish .................................................................................................. 12 9. Differential toxicity effects on healthy peripheral blood mononuclear cells ........................... 13 10. Statistical Analysis ................................................................................................................. 13 RESULTS....................................................................................................................................... 14 1. Cytotoxic effect of palytoxin on human leukemia cells ....................................................... 14 Figure 1. Cytotoxic effect of Palytoxin on human leukemia cell lines ............................................ 15 2. Effect of palytoxin on healthy cells and organisms ............................................................. 16 Figure 2. Effects of Palytoxin on zebrafish and healthy cells .......................................................... 17 3. Palytoxin-induced cell death leads to caspase activation in U937 cells ............................. 18 Figure 3. Palytoxin-induced cell death leads to caspase activation in U937 cells ........................... 19 4. Palytoxin down-regulates expression of anti-apoptotic Bcl-2 family proteins .................. 20 Figure 4. Palytoxin down-regulates expression of anti-apoptotic Bcl-2 family proteins ................. 21 5. Mcl-1 is down-regulated by palytoxin in a proteasome dependent manner ..................... 21 Figure 5. Mcl-1 is ubiquitously down-regulated by palytoxin in a proteasome dependent manner 23 6. Bcl-2 serine 70 dephosphorylation induced by palytoxin is mediated through Protein Phosphatase 2A activation in U937 cells .................................................................................. 24 Figure 6. Bcl-2 serine70 dephosphorylation induced by palytoxin is mediated through protein phosphatase 2 activation in U937 cells ............................................................................................ 25 Figure 7. Schematic model of palytoxin-induced cell death ............................................................ 27 DISCUSSION ................................................................................................................................ 28 REFERENCES ............................................................................................................................... 31 요 약 ................................................................................................................................................ 34Maste

Finding Na,K-ATPase II - From fluxes to ion movements

After identification of the Na,K-ATPase as active ion transporter that maintains the Na+ and K+ concentration gradient across the membrane of virtually all animal cells, a long history of mechanistic studies began in which enzyme activity and ion-transport were intensively investigated. A basis for detailed understanding was laid in the so-called Post-Albers pump cycle. Developing new experimental techniques allowed the determination of different flux modes, the analysis of the kinetics of enzyme phosphorylation and dephosphorylation as well as of the transport of Na+ and K+ ions across the membrane. The accumulation of results from transport studies allowed the proposal of the gated channel concept that turned out to be a successful approach to explain the transport-related experimental findings. Eventually, it found its counterpart in the high-resolution structure of the ion pump. Recently it turned out that simple mutations of the Na,K-ATPase are the cause of several diseases

Substrate-dependent effects on the conformational equilibrium of the Na +, K +-ATPase monitored by VCF

The Na+,K+-ATPase was discovered more than 50 years ago, but even today the pumpcycle and its partial reactions are still not completely understood. In this thesis, Voltage Clamp Fluorometry was used to monitor the conformational changes that are associated with several electrogenic partial reactions of the Na+,K+-ATPase. The conformational dynamics of the ion pump were analyzed at different concentrations of internal Na+ or of external K+ and the influences on the conformational equilibrium were determined. To probe the effect of the internal Na+ concentration on the Na+ branch of the ion pump, oocytes were first depleted of internal Na+ and then loaded with Na+ using the epithelial sodium channel which can be blocked by amiloride. The conformational dynamics of the K+ branch were studied using different external K+ concentrations in the presence and in the absence of external Na+ to yield additional information on the apparent affinity of K+. The results of our Voltage Clamp Fluorometry experiments demonstrate that lowering the intracellular concentration of Na+ has a comparable effect on the conformational equilibrium as increasing the amount of K+ in the external solution. Both of these changes shift the equilibrium towards the E1/E1(P) conformation. Furthermore, it can be shown that the ratio between external Na+ and K+ ions is also a determinant for the position of the conformational equilibrium: in the absence of external Na+, the K+ dependent shift of the equilibrium towards E1 was observed at a much lower K+ concentration than in the presence of Na+. In addition, indications were found that both external K+ and internal Na+ bind within an ion well. Finally, the crucial role of negatively charged glutamate residues in the 2nd extracellular loop for the control of ion-access to the binding sites could be verified.In der vorliegenden Arbeit wurden die Methoden der ortsspezifischen Fluoreszenzmarkierung und der Voltage Clamp Fluorometrie (VCF) kombiniert um die Konformationsänderungen der Na+,K+-ATPase während verschiedener elektrogener Reaktionsschritte des Pumpzyklus zu untersuchen. Neben der extrazellulären Freisetzung von Na+ waren vor allem die interne Na+-Bindung und externe K+-Bindung von Interesse, da diese aufgrund ihrer geringen Elektrogenizität mit rein elektrophysiologischen Methoden nicht, oder nur ungenügend, erfasst werden können. Es sollten vor allem Informationen über die Dynamik der mit den Reaktionsschritten assoziierten Konformationsänderungen gewonnen werden und der Einfluss der Pumpsubstrate Na+ und K+ auf die Lage des Konformationsgleichgewichts geklärt werden. ..

Quaternary Organic Amines Inhibit Na,K Pump Current in a Voltage-dependent Manner: Direct Evidence of an Extracellular Access Channel in the Na,K-ATPase

The effects of organic quaternary amines, tetraethylammonium (TEA) chloride and benzyltriethylammonium (BTEA) chloride, on Na,K pump current were examined in rat cardiac myocytes superfused in extracellular Na+-free solutions and whole-cell voltage-clamped with patch electrodes containing a high Na+-salt solution. Extracellular application of these quaternary amines competitively inhibited extracellular K+ (K+o) activation of Na,K pump current; however, the concentration for half maximal inhibition of Na,K pump current at 0 mV (K0Q) by BTEA, 4.0 ± 0.3 mM, was much lower than the K0Q for TEA, 26.6 ± 0.7 mM. Even so, the fraction of the membrane electric field dissipated during K+o activation of Na,K pump current (λK), 39 ± 1%, was similar to λK determined in the presence of TEA (37 ± 2%) and BTEA (35 ± 2%), an indication that the membrane potential (VM) dependence for K+o activation of the Na,K pump current was unaffected by TEA and BTEA. TEA was found to inhibit the Na,K pump current in a VM-independent manner, i.e., inhibition of current dissipated 4 ± 2% of the membrane electric field. In contrast, BTEA dissipated 40 ± 5% of the membrane electric field during inhibition of Na,K pump current. Thus, BTEA inhibition of the Na,K-ATPase is VM-dependent. The competitive nature of inhibition as well as the similar fractions of the membrane electric field dissipated during K+o-dependent activation and BTEA-dependent inhibition of Na,K pump current suggest that BTEA inhibits the Na,K-ATPase at or very near the enzyme's K+o binding site(s) located in the membrane electric field. Given previous findings that organic quaternary amines are not occluded by the Na,K-ATPase, these data clearly demonstrate that an ion channel–like structure provides access to K+o binding sites in the enzyme

Toxicological effects of palytoxin after cutaneous exposure

2010/2011Palytoxin (PLTX) is a marine toxin identified in Palythoa zoanthid corals and Ostreopsis dinoflagellates, representing an increasing hazard for human health. Human poisonings attributed to PLTX exposure are usually associated to ingestion of contaminated seafood and to marine aerosol exposure during Ostreopsis blooms. However, also dermatological problems have been recently associated to PLTX cutaneous exposure during Ostreopsis blooms as well as after handling of Palythoa corals. Despite the increasing human cases of dermotoxicity attributed to PLTX, very few data about its dermal toxicity are presently available. Hence, the aim of this study is to investigate the cutaneous effects of PLTX characterizing its mechanism of action. Thus, this toxicological in vitro study has been carried out on spontaneously immortalized human keratinocytes (HaCaT cells), as a first-round screening of dermotoxicity. The entity of cytotoxicity induced by PLTX has been firstly investigated. A short time exposure (4 h) to PLTX reduces mitochondrial activity (MTT assay), cell mass (SRB assay) and plasma membrane integrity (LDH leakage) with different potencies (EC50 values of 6.1±1.3x10-11, 4.7±0.9x10-10 M and 1.8±0.1x10-8 M, respectively). All these effects are ouabain-sensitive corroborating the dependency of PLTX effects on the interaction with Na+/K+-ATPase. These results indicate that among the chain of intracellular events following the interaction of PLTX with the Na+/K+-ATPase the earliest is mitochondrial damage. This sustained cytotoxic effect can be explained by the high affinity of binding to HaCaT cells. Indeed, saturation experiment reveals a Kd affinity constant of 3.0±0.4x10-10 M after an exposure time as short as 10 minutes. A possible mechanism of mitochondrial dysfunction can be reactive oxygen species (ROS) overproduction. Among all, only superoxide anion (O2-) seems to be produced by the toxin after only 1 h, whereas neither nitric oxide nor peroxynitrite formation are detected. Hence, the mechanism of O2- production has been investigated. Real time PCR analysis together with western blot analysis suggest a possible involvement of NADPH oxidase (NOX) and inducible nitric oxide synthetase (iNOS) since an early increase of their gene and protein expression was observed after short (1 – 4 h) but not longer (24 h) exposure times. On the contrary, other enzymes involved in ROS production (i.e. COX-1, COX-2, XOD) seem to be not involved in PLTX effects. Moreover, using selective inhibitors of these enzymes, we found that only DPI, a nonspecific inhibitor of both NOX and NOS, is able to inhibit by 15%, 26% and 43% O2- production induced by 10-10, 10-9 and 10-8 M PLTX, respectively. However, NMMA, inhibitor of NOS, significantly reduces only O2- produced by high (10-8 M) but no by low (10-9 and 10-10 M) PLTX concentrations, whereas the selective inhibitor of NOX apocynin is totally ineffective. Moreover, since their co-administration does not reproduce DPI effect, a prominent role of these enzymes in causing PLTX-induced oxidative stress seems unlikely. Another feasible source of O2- is mitochondria itself and its production is regulated by H+ fluxes through mitochondrial membranes. Indeed, in presence of nigericin, an ionophore that reduces the H+ imbalance, PLTX-induced O2- is significantly reduced by 23% (10-9 M PLTX) and 24% (10-8 M PLTX). Furthermore, the co-administration with rotenone, a complex I inhibitor, that per se is ineffective, results in a further inhibition of O2- production (-32% and -43% in the presence of 10-9 and 10-8 M PLTX, respectively). Moreover, O2- production turned out to be ouabain-sensitive and Na+-dependent but Ca2+-independent. Thus, on the basis of these results it has been hypothesized that PLTX binding to Na+/K+-ATPase induces intracellular overload of Na+ followed by intracellular increase of H+ with a consequent ΔpH increase across H+-impermeable mitochondrial inner membrane and O2- overproduction by reverse electron transports through mitochondrial chain. Under oxidative stress conditions, mitochondrial dysfunction can be mediated by mitochondrial permeability transition pore (MPTP), which opening, indeed, is induced by PLTX already after only 5 minutes exposure. MPTP opening, which turned out to be cyclosporine A-independent, seems to be mainly induced by the sustained ionic imbalance, since in Na+-free, Ca2+-free medium and in presence of nigericin PLTX effect is strongly inhibited. The very rapid Na+-dependent opening of MPTP suggests that this is the peculiar mechanism of PLTX cytotoxicity and cell death primum movens. Cell death induced by the toxin seems to occur with necrotic-like features. PLTX, indeed, induces a concentration- and time-dependent as well as irreversible uptake of PI after only 1 h exposure and confocal images revealed dramatic morphological alterations such as plasma membrane ruptures and leakage of cytolpasmic content after 4 h. By contrast, caspasis 3/7, 8 and 9 are not activated by PLTX up to 24 h, neither under recovery conditions. Moreover, apoptotic bodies formation is not observed, discarding apoptosis occurrence. Finally, PLTX effects on some pro-inflammatory mediators such as cytokines (IL-1α, IL-6, IL-8 and TNF-α) and arachidonic acid metabolism products (PGE2 and LTB4) have been evaluated. The toxin (10-11 M) induces an early release of PGE2 that is time-dependent after 2 h exposure. On the contrary, even if an early gene expression (1–4 h) is observed, the toxin induces a delayed release of IL-6 and IL-8 (24 h), whereas no effects have been observed evaluating IL-1α and TNF-α. In conclusion, this study highlights the toxic in vitro properties of PLTX on human keratinocytes. The intracellular pathway of the sustained PLTX cytotoxicity leading to cell death has been characterized, as well as the inflammatory mediators involved in skin irritant properties of the toxin. These results can corroborate the use of non steroidal anti-inflammatory drugs in association with anti-inflammatory corticosteroids.La palitossina (PLTX) è una tossina marina identificata in coralli zoantidi appartenenti al genere Palythoa e dinoflagellati del genere Ostreopsis. Intossicazioni umane attribuite alla PLTX sono state solitamente associate all'ingestione di prodotti ittici contaminati, nonché da un'esposizione ad aerosol marino durante le fioriture di Ostreopsis. Tuttavia, anche problemi dermatologici sono stati recentemente associati alla PLTX in seguito ad esposizione cutanea durante fioriture di Ostreopsis o manipolando coralli Palythoa. Nonostante i crescenti casi di dermotossicità attribuiti alla PLTX, pochissimi dati sulla sua tossicità cutanea sono attualmente disponibili. Lo scopo di questo studio è stato, pertanto, indagare gli effetti cutanei della PLTX caratterizzando il suo meccanismo d'azione. E’ stato quindi effettuato uno studio tossicologico in vitro su cheratinociti umani spontaneamente immortalizzati (cellule HaCaT), considerate metodo predittivo per uno screening preliminare di dermotossicità. In primo luogo è stato caratterizzato il grado di citotossicità indotta dalla tossina. Un breve tempo d'esposizione (4 h) alla PLTX riduce l'attività mitocondriale (saggio MTT), la massa cellulare (saggio SRB) e l'integrità della membrana plasmatica (perdita LDH) con diversi valori di EC50 (6.1 ± 1.3x10-11, 4.7 ± 0.9x10-10 M e 1.8 ± 0.1x10-8 M, rispettivamente). Tutti questi effetti sono sensibili alla ouabaina, corroborando la dipendenza degli effetti della PLTX sull'interazione con la Na+/K+-ATPasi. Questi risultati indicano che fra la catena di eventi intracellulari dopo l'interazione con l’ATPasi il più sensibile è un danno mitocondriale. Questo effetto può essere spiegato dall’alta affinità di legame della tossina con le cellule HaCaT. Infatti, esperimenti di saturazione rivelano una costante di affinità (Kd) pari a 3,0 ± 0.4x10-10 M dopo un tempo di esposizione molto breve (10 minuti). Uno dei possibili meccanismi di disfunzione mitocondriale è una sovrapproduzione di specie reattive dell'ossigeno (ROS). Tra tutti, solo l’anione superossido (O2-) sembra essere prodotto dalla tossina dopo 1 h, mentre né ossido nitrico né formazione di perossinitrito sono stati rilevati. Quindi, il meccanismo di produzione di O2- è stato studiato. Analisi real time-PCR ed analisi western blot suggeriscono un possibile coinvolgimento della NADPH ossidasi (NOX) e della forma inducibile dell’ossido nitrico sintetasi (iNOS) poiché un aumento precoce della loro espressione genica e stata osservata dopo brevi (1 - 4 h) ma non lunghi (24 h) tempi di esposizione. Al contrario, altri enzimi coinvolti nella produzione di ROS (COX-1, COX-2, XOD) sembrano non essere coinvolti nel meccanismo di produzione di O2- da parte della tossina. Inoltre, tramite l'utilizzo di inibitori selettivi di questi enzimi, è emerso che solo il DPI, un inibitore non specifico sia di NOX che di NOS, è in grado di inibire del 15%, 26% e 43% la produzione di O2- indotta da 10-10, 10-9 e 10-8 M PLTX, rispettivamente. Tuttavia, l’NMMA, inibitore delle NOS, riduce in modo significativo solo O2- prodotto da alte (10-8 M), ma non basse (10-9 e 10-10 M) concentrazioni di PLTX, mentre l'inibitore selettivo delle NOX apocinina è totalmente inefficace. Inoltre, poiché la loro co-somministrazione non riproduce l’effetto inibitorio del DPI, un ruolo preminente di questi enzimi nel causare stress ossidativo sembra improbabile. Un'altra fonte possibile di O2- è il mitocondrio. La sua produzione è regolata dal flusso di H+ attraverso le membrane mitocondriali. Infatti, in presenza di nigericina, uno ionoforo che riduce lo squilibrio protonico, i livelli di O2- indotti dalla PLTX vengono significativamente ridotti del 23% (10-9 M PLTX) e 24% (10-8 M PLTX). Inoltre, la co-somministrazione con il rotenone, un inibitore del complesso I della catena mitocondriale di trasporto degli elettroni, che è di per sé inefficace, induce un’ulteriore inibizione di produzione di O2- (-32% e -43% in presenza di 10-9 e 10-8 M PLTX, rispettivamente). Inoltre, la produzione di O2- risulta essere ouabaina-sensibile e Na+-dipendente, ma Ca2+-indipendente. Pertanto, sulla base di questi risultati è stato ipotizzato che il legame della PLTX con la Na+/K+-ATPasi induce un aumento intracellulare di Na+ seguito da aumento intracellulare di H+ con un conseguente aumento di ΔpH attraverso la membrana mitocondriale interna con una sovrapproduzione di O2- indotta dal trasporto inverso degli elettroni attraverso la catena mitocondriale. In condizioni di stress ossidativo, la disfunzione mitocondriale può essere mediata dall’apertura dei pori di transizione mitocondriali (MPTP). La loro apertura, infatti, viene indotta dalla PLTX già dopo soli 5 minuti di esposizione. Tale apertura, che si è rivelata ciclosporinaA-indipendente, sembra principalmente indotta dallo squilibrio ionico indotto dalla tossina, poiché in terreni privo di Na+ e privo di Ca2+ e in terreno contenente nigericina, l’attività della tossina è fortemente inibita. La rapidissima apertura di MPTP suggerisce che questo è il peculiare meccanismo di citotossicità della tossina e il primum movens della cellule morte. La morte cellulare sembra verificarsi con un danno necrotico. La PLTX, infatti, induce un uptake di PI (marker di necrosi) in maniera concentrazione e tempo-dipendente. Tale uptake è inoltre irreversibile, dopo solo 1 h di esposizione e immagini ottenute al microscopio confocale rivelano drammatiche alterazioni morfologiche, quali rotture della membrana plasmatica e la perdita di contenuto citoplasmatico dopo 4 h. Al contrario, le caspasi 3/7, 8 e 9 non sono attivate dalla PLTX fino a 24 h, né sotto condizioni di recovery. Inoltre, la formazione di corpi apoptotici non è stata rilevata, scartando l’ipotesi di una morte di tipo apoptotico. Infine, gli effetti della PLTX su alcuni mediatori proinfiammatori quali citochine (IL-1α, IL-6, IL-8 e TNF-α) e metaboliti dell’acido arachidonico (PGE2 e LTB4) sono stati valutati. La tossina (10-11 M) induce una rapida produzione di PGE2 che è tempo-dipendente dopo 2 ore di esposizione. Al contrario, la tossina induce un rilascio ritardato di IL-6 e IL-8 (24 h), anche se alterazioni dell'espressione genica si sono osservate dopo breve tempo di contatto con la tossina (1-4 h). mentre non sono stati osservati effetti valutando IL-1α e TNF -α. In conclusione, questo studio mette in evidenza le proprietà tossiche in vitro della PLTX su cheratinociti umani. L’elevata citotossicità indotta dalla tossina conduce ad una morte cellulare di tipo necrotico mediata dai mitocondri. Infine, i mediatori infiammatori coinvolti nella proprietà irritanti della pelle della tossina sono stati caratterizzati, ponendo delle basi molecolari per spiegare l'utilizzo di farmaci anti-infiammatori non steroidei in associazione con corticosteroidi.XXIV Ciclo198

Modelado y análisis probabilístico de sistemas híbridos

Los sistemas híbridos, se han tornado de gran interés en la comunidad científica a partir del desafío que presenta el estudio de sus dinámicas, las continuas y las discretas, y el estudio y comprensión de sus interacciones. Estas puede tomar diversas formas, las más comunes ocurren cuando cambian de estado entre diferentes procesos continuos. Otras formas de interacción incluyen transiciones discretas que dependen de evoluciones continuas, u otras aparecen como resultados de una decisión, o por la ocurrencia de ciertos eventos. Un sistema híbrido probabilístico considera la distribución de probabilidad de ambas dinámicas, y se enfoca el análisis en lo referido a la alcanzabilidad probabilística El acercamiento numérico sufre del problema de explosión de estados y son computacionalmente muy exigentes. Un método alternativo de análisis es el realizado por medio de Model Checkers Probabilísticos. En nuestra línea de investigación proponemos el modelado y estudio de estos sistemas, como parte de la verificación y validación de sistemas desde un punto de vista de la ingeniería de software por medio de herramientas de model checking. En una primera etapa se centrará en modelos, específicamente de sistemas biológicos, con el objetivo de mejorar el poder predictivo de modelos formales existentes.Eje: Ingeniería de SoftwareRed de Universidades con Carreras en Informática (RedUNCI

Herramienta de modelado y análisis estocástico de sistemas biológicos

Los sistemas híbridos cuentan con la atención de gran parte de la comunidad científica por lo atractivo del estudio de sus dinámicas, las continuas y las discretas, y la comprensión de sus interacciones. Entre las más comunes se encuentran aquellas que cambian de estado entre diferentes procesos continuos. También se pueden encontrar formas de interacción que incluyen transiciones discretas supeditadas a evoluciones continuas, u otras como resultados de una decisión, o por la ocurrencia de determinados eventos. El estudio de la distribución probabilísticas de la dinámica discreta y la continua se lleva a cavo por medio del análisis del sistema híbrido estocástico (SHE) que lo modela. Debido a que el modelo numérico, de un SH, es afectado por el problema de la explosión de estados y de ser sumamente exigente en lo que a recursos se refiere, aparece como una opción aceptable la formulación del mismo sistema por medio de un SHP. Estos tipos de sistemas pueden ser analizados por medio de herramientas informáticas con solida base matemática como son los Model Checkers Probabilísticos. Los sistemas biológicos encuadran perfectamente en la clasificación de SHE. En el ámbito de estudio de SB no se cuenta con herramientas que permitan una traducción directa de un SHE, como por ejemplo un sistema de reacciones, a un modelo estocástico factible de ser analizado por herramientas informáticas disponibles en la actualidad como son los model checkers antes mencionados. En nuestra línea de investigación proponemos el estudio de factibilidad y de propuesta de desarrollo de una herramienta de análisis de SB basado en su formulación estocástica. Tenemos como hipótesis de trabajo que el desarrollo de este prototipo de herramienta que permite la obtención de un modelo estocástico a partir de su formulación por medio de reacciones que permitirá analizar el sistema e incrementar la productividad en el estudio de SBs habilitando a su verificación y validación con herramientas novedosas en el área biológica. En una primera etapa se centrará en la obtención de modelos estocásticos de un sistema de reacciones para luego habilitar el análisis basado en probabilidades y en simulaciones probabilísticas basadas en su semántica estocástica.Eje: Innovación en Sistemas de Software.Red de Universidades con Carreras en Informática (RedUNCI

Modelado y análisis probabilístico de sistemas híbridos

Los sistemas híbridos, se han tornado de gran interés en la comunidad científica a partir del desafío que presenta el estudio de sus dinámicas, las continuas y las discretas, y el estudio y comprensión de sus interacciones. Estas puede tomar diversas formas, las más comunes ocurren cuando cambian de estado entre diferentes procesos continuos. Otras formas de interacción incluyen transiciones discretas que dependen de evoluciones continuas, u otras aparecen como resultados de una decisión, o por la ocurrencia de ciertos eventos. Un sistema híbrido probabilístico considera la distribución de probabilidad de ambas dinámicas, y se enfoca el análisis en lo referido a la alcanzabilidad probabilística El acercamiento numérico sufre del problema de explosión de estados y son computacionalmente muy exigentes. Un método alternativo de análisis es el realizado por medio de Model Checkers Probabilísticos. En nuestra línea de investigación proponemos el modelado y estudio de estos sistemas, como parte de la verificación y validación de sistemas desde un punto de vista de la ingeniería de software por medio de herramientas de model checking. En una primera etapa se centrará en modelos, específicamente de sistemas biológicos, con el objetivo de mejorar el poder predictivo de modelos formales existentes.Eje: Ingeniería de SoftwareRed de Universidades con Carreras en Informática (RedUNCI

Modelado y análisis probabilístico de sistemas híbridos

Los sistemas híbridos, se han tornado de gran interés en la comunidad científica a partir del desafío que presenta el estudio de sus dinámicas, las continuas y las discretas, y el estudio y comprensión de sus interacciones. Estas puede tomar diversas formas, las más comunes ocurren cuando cambian de estado entre diferentes procesos continuos. Otras formas de interacción incluyen transiciones discretas que dependen de evoluciones continuas, u otras aparecen como resultados de una decisión, o por la ocurrencia de ciertos eventos. Un sistema híbrido probabilístico considera la distribución de probabilidad de ambas dinámicas, y se enfoca el análisis en lo referido a la alcanzabilidad probabilística El acercamiento numérico sufre del problema de explosión de estados y son computacionalmente muy exigentes. Un método alternativo de análisis es el realizado por medio de Model Checkers Probabilísticos. En nuestra línea de investigación proponemos el modelado y estudio de estos sistemas, como parte de la verificación y validación de sistemas desde un punto de vista de la ingeniería de software por medio de herramientas de model checking. En una primera etapa se centrará en modelos, específicamente de sistemas biológicos, con el objetivo de mejorar el poder predictivo de modelos formales existentes.Eje: Ingeniería de SoftwareRed de Universidades con Carreras en Informática (RedUNCI