Molecular Simulations of Protein-Induced Membrane Remodeling

Membranes organize much of the cell and host a great deal of molecular machinery required to integrate signals from the outside, regulate the surrounding matrix, change shape, move, and grow. Understanding how a dense forest of proteins, sugars, and biomarkers modulates the shape of the cell is necessary to produce more detailed, accurate predictions of cell behavior, particularly in the studies of cell signaling processes that lead to oncogenesis. In this dissertation, I will present a series of molecular models which, when combined with continuum models and both in vitro and in vivo experiments, describe the molecular basis for membrane morphology changes. In particular, we investigate the mechanisms by which proteins assemble on a bilayer undergoing thermal fluctuations. This work serves to quantify and explain a series of biophysical experiments in molecular detail, and contributes to the development of multiscale models for predicting cell fate

A Rewriting-based, Parameterized Exploration Scheme for the Dynamic Analysis of Complex Software Systems

Los sistemas software actuales son artefactos complejos cuyo comportamiento es a menudo extremadamente difícil de entender. Este hecho ha llevado al desarrollo de metodologías formales muy sofisticadas para el análisis, comprensión y depuración de programas. El análisis de trazas de ejecución consiste en la búsqueda dinámica de contenidos específicos dentro de las trazas de ejecución de un cierto programa. La búsqueda puede llevarse a cabo hacia adelante o hacia atrás. Si bien el análisis hacia adelante se traduce en una forma de análisis de impacto que identifica el alcance y las posibles consecuencias de los cambios en la entrada del programa, el análisis hacia atrás permite llevar a cabo un rastreo de la procedencia; es decir, muestra como (partes de) la salida del programa depende de (partes de) su entrada y ayuda a estimar qué dato de la entrada es necesario modificar para llevar a cabo un cambio en el resultado. En esta tesis se investiga una serie de metodologías de análisis de trazas que son especialmente adecuadas para el análisis de trazas de ejecución largas y complejas en la lógica de reescritura, que es un marco lógico y semántico especialmente adecuado para la formalización de sistemas altamente concurrentes. La primera parte de la tesis se centra en desarrollar una técnica de análisis de trazas hacia atrás que alcanza enormes reducciones en el tamaño de la traza. Esta metodología se basa en la fragmentación incremental y favorece un mejor análisis y depuración ya que la mayoría de las inspecciones, tediosas e irrelevantes, que se realizan rutinariamente en el diagnostico y la localización de errores se pueden eliminar de forma automática. Esta técnica se ilustra por medio de varios ejemplos que ejecutamos mediante el sistema iJulienne, una herramienta interactiva de fragmentación que hemos desarrollado y que implementa la técnica de análisis de trazas hacia atrás. En la segunda parte de la tesis se formaliza un sistema paramétrico, flexible y dinámico, para la exploración de computaciones en la lógica de reescritura. El esquema implementa un algoritmo de animación gen érico que permite la ejecución indeterminista de una teoría de reescritura condicional dada y que puede ser objeto de seguimiento mediante el uso de diferentes modalidades, incluyendo una ejecución gradual paso a paso y una fragmentación automática hacia adelante y/o hacia atrás, lo que reduce drásticamente el tamaño y la complejidad de las trazas bajo inspección y permite a los usuarios evaluar de forma aislada los efectos de una declaración o instrucción dada, el seguimiento de los efectos del cambio de la entrada, y obtener información sobre el comportamiento del programa (o mala conducta del mismo). Por otra parte, la fragmentación de la traza de ejecución puede identificar nuevas oportunidades de optimización del programa. Con esta metodología, un analista puede navegar, fragmentar, filtrar o buscar en la traza durante la ejecución del programa. El marco de análisis de trazas gen érico se ha implementado en el sistema Anima y describimos una profunda evaluación experimental de este que demuestra la utilidad del enfoque propuesto.Frechina Navarro, F. (2014). A Rewriting-based, Parameterized Exploration Scheme for the Dynamic Analysis of Complex Software Systems [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/44234TESI

Gene Expression in Dwarf Mistletoe Related to Explosive Seed Dispersal with special Attention to Aquaporins

Worldwide, mistletoe plants are important forest pests, found in all continents aside from Antarctica. In North America, control of conifer parasitism by dwarf mistletoes, specifically Arceuthobium americanum, is challenging. Dwarf mistletoes are dioecious parasitic flowering plants with a unique and effective explosive seed dispersal mechanism. Due to the large accumulation of water within the fruit, which increases both the fruit size and its internal hydrostatic pressure, the transport of water into the fruit and the possible involvement of plasma membrane intrinsic proteins (PIPs), particularly aquaporins (AQPs), were the focus of the investigation. Notably, previous research also demonstrated that cuticle thickening, declining stomata number, and thermogenesis are potential “players” in forcible discharge. As such, molecular work was performed here to reveal genes involved in physiological processes potentially associated with explosive seed dispersal and to examine the expression of these genes. Therefore, the overarching goal of this project was to obtain gene sequencing and expression data for Arceuthobium spp. to gain insights into the involvement of AQPs and other proteins likely responsible for generating high hydrostatic pressure and discharge. Many methods were employed to extract the RNA from the fruit, including both traditional and commercially available methods, and following successful extraction, a cDNA library was constructed. A non-standard heterologous microarray hybridization, unconventionally used for transcription analysis of non-model species, employing both the MicroCASTer handheld system and an Affymetrix Arabidopsis Gene 1.0 ST microarray was used to determine gene sequence and expression. Five genes sequences were obtained from the handheld system: AQP2, ABC transporter, Ribulose Bisphosphate Carboxylase-Oxygenase, Alkaline/neutral invertase, and Sterol-14 demethylase. To confirm that AQP2 codes for plasma membrane intrinsic protein 2 (PIP2), gateway cloning followed by a physiological study with stopped-flow spectroscopy was undertaken. Despite sequence divergence between the two species, 24.2% of Arabidopsis Gene 1.0 ST array was hybridized to dwarf mistletoe RNA; this hybridization identified approximately 1,000 genes that are highly differentially expressed in dwarf mistletoe during fruit development. Further investigation of the Affymetrix results revealed several additional mistletoe genes, Stomatal cytokinesis defective2, Movement protein binding protein, Eceriferum1, and SHINE2, that are seasonally up and down regulated (May vs. September) and are likely involved in the explosive seed dispersal process. With these results in hand, research can advance towards molecular insights into not only explosive seed dispersal in dwarf mistletoe, but also into other processes such as development, reproduction, photosynthesis light response, and parasitism-associated gene expression within dwarf mistletoe and related plants. Ideally, this work will provide new opportunities for investigating novel avenues of plant-plant pest control

Verification of temporal-epistemic properties of access control systems

Verification of access control systems against vulnerabilities has always been a challenging problem in the world of computer security. The complication of security policies in large- scale multi-agent systems increases the possible existence of vulnerabilities as a result of mistakes in policy definition. This thesis explores automated methods in order to verify temporal and epistemic properties of access control systems. While temporal property verification can reveal a considerable number of security holes, verification of epistemic properties in multi-agent systems enable us to infer about agents' knowledge in the system and hence, to detect unauthorized information flow. This thesis first presents a framework for knowledge-based verification of dynamic access control policies. This framework models a coalition-based system, which evaluates if a property or a goal can be achieved by a coalition of agents restricted by a set of permissions defined in the policy. Knowledge is restricted to the information that agents can acquire by reading system information in order to increase time and memory efficiency. The framework has its own model-checking method and is implemented in Java and released as an open source tool named \char{cmmi10}{0x50}\char{cmmi10}{0x6f}\char{cmmi10}{0x6c}\char{cmmi10}{0x69}\char{cmmi10}{0x56}\char{cmmi10}{0x65}\char{cmmi10}{0x72}. In order to detect information leakage as a result of reasoning, the second part of this thesis presents a complimentary technique that evaluates access control policies over temporal-epistemic properties where the knowledge is gained by reasoning. We will demonstrate several case studies for a subset of properties that deal with reasoning about knowledge. To increase the efficiency, we develop an automated abstraction refinement technique for evaluating temporal-epistemic properties. For the last part of the thesis, we develop a sound and complete algorithm in order to identify information leakage in Datalog-based trust management systems

A kinetic study of receptor activation of the G-protein gated K+ channel

The cloned G-protein gated inwardly rectifying K+ channel (a tetramer composed of Kir3.1-3.4 subunits) is activated by direct binding of Gβγ dimers, liberated by receptor activation of the Gi/o subfamily of heterotrimeric guanine nucleotide binding (G)-proteins. The interaction of these three membrane-associated components, G-protein coupled receptor (GPCR), heterotrimeric G-protein and channel, is rapid in native cells, with full channel activation via the GABA-B receptor occurring within a few hundred milliseconds (Sodickson & Bean, 1996 and 1998), and current deactivation occurring with a time constant of 1-2 seconds. Recent discovery of the Regulators of G-protein signalling (RGS) protein family has solved a major discrepancy between the slow deactivation of purified G-proteins and the fast deactivation of G-protein mediated signalling pathways. Their discovery has generated considerable interest in the kinetics of G-protein signalling and the organisation of these signalling components in the cell membrane. For these studies, the GIRK signalling system was reconstituted in mammalian HEK-293 cell lines, stably expressing the cloned neuronal channel subunits (Kir3.1 and Kir3.2A) plus a Gi/o-coupled GPCR (α2A adrenergic, A1 adenosine, D2 dopamine, M4 muscarinic and the heterodimeric GABA-B1b/2 receptors). Chapter 1 provides a general introduction to G-protein signalling and reviews our current understanding of the factors involved in the regulation of GERK channels. In Chapter 2, the methods and experimental protocols used in the study are described. In Chapter 3, I present a systematic analysis of the factors that contribute to the rapid activation of the channel complex, and in Chapter 4 the characteristic fast desensitisation of receptor-activated currents is examined. Factors influencing channel deactivation upon removal of agonist are explored in Chapter 5, and in Chapter 6 I describe the effects of the novel RGS protein family in these cell lines. Conclusions and future directions for this work are presented in Chapter 7

Rule-based Methodologies for the Specification and Analysis of Complex Computing Systems

Desde los orígenes del hardware y el software hasta la época actual, la complejidad de los sistemas de cálculo ha supuesto un problema al cual informáticos, ingenieros y programadores han tenido que enfrentarse. Como resultado de este esfuerzo han surgido y madurado importantes áreas de investigación. En esta disertación abordamos algunas de las líneas de investigación actuales relacionada con el análisis y la verificación de sistemas de computación complejos utilizando métodos formales y lenguajes de dominio específico. En esta tesis nos centramos en los sistemas distribuidos, con un especial interés por los sistemas Web y los sistemas biológicos. La primera parte de la tesis está dedicada a aspectos de seguridad y técnicas relacionadas, concretamente la certificación del software. En primer lugar estudiamos sistemas de control de acceso a recursos y proponemos un lenguaje para especificar políticas de control de acceso que están fuertemente asociadas a bases de conocimiento y que proporcionan una descripción sensible a la semántica de los recursos o elementos a los que se accede. También hemos desarrollado un marco novedoso de trabajo para la Code-Carrying Theory, una metodología para la certificación del software cuyo objetivo es asegurar el envío seguro de código en un entorno distribuido. Nuestro marco de trabajo está basado en un sistema de transformación de teorías de reescritura mediante operaciones de plegado/desplegado. La segunda parte de esta tesis se concentra en el análisis y la verificación de sistemas Web y sistemas biológicos. Proponemos un lenguaje para el filtrado de información que permite la recuperación de informaciones en grandes almacenes de datos. Dicho lenguaje utiliza información semántica obtenida a partir de ontologías remotas para re nar el proceso de filtrado. También estudiamos métodos de validación para comprobar la consistencia de contenidos web con respecto a propiedades sintácticas y semánticas. Otra de nuestras contribuciones es la propuesta de un lenguaje que permite definir y comprobar automáticamente restricciones semánticas y sintácticas en el contenido estático de un sistema Web. Finalmente, también consideramos los sistemas biológicos y nos centramos en un formalismo basado en lógica de reescritura para el modelado y el análisis de aspectos cuantitativos de los procesos biológicos. Para evaluar la efectividad de todas las metodologías propuestas, hemos prestado especial atención al desarrollo de prototipos que se han implementado utilizando lenguajes basados en reglas.Baggi ., M. (2010). Rule-based Methodologies for the Specification and Analysis of Complex Computing Systems [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/8964Palanci

Investigating Post-Translational Modification of the Net Protein Superfamily

The Net protein superfamily represents a group of actin binding proteins contain a novel actin binding domain at their N termini. The aim of this project was to investigate post-translational modification of these proteins in order to explore how the interaction of these proteins with actin is regulated. A bioinformatics-based approach was used to predict sites for multiple types of modification, with a focus on phosphorylation, that were conserved between members of the superfamily. It became clear that there was some level of conservation of predicted post-translational modification sites at the C-terminus in multiple Net families. Net4B was identified as having a high probability of being phosphorylated at the C-terminus and predicted to have its N- and C-termini proximal to one another in its tertiary structure, and following this mutant forms of the protein were created to investigate how its actin binding activity would be affected if one site, S509, was phosphorylated or dephosphorylated. The mutants were transiently expressed in Nicotania benthamiana and the appearance of the leaf cells assessed. Whilst phosphomimicry of S509 resulted in no appreciable change in the appearance of the GFP-tagged protein, mutation to a residue imitating a non-phosphorylatable serine resulted in the formation of punctae, in some cases much like the ‘beads-on-a-string’ seen in other members of the Net superfamily. This finding may have implications for the regulation of actin binding in other Net proteins and for other proteins outside of the superfamily. Two models are presented in both of these contexts. This project may also provide groundwork for future experiments concerning phosphorylation and acylation, and may illuminate the mechanism by which Net proteins interact with actin and with the membranes with which they are respectively localised

Definition of VPU sensitivity using a model VPU target and role of hydrophobicity of the membrane spanning domain in the viral envelope glycoprotein fusogenicity

Retroviral compatibility with diverse glycoproteins has been known and identified through the course of several studies. However, molecular mechanisms of glycoprotein acquisition are poorly defined. Glycoproteins are acquired by the virus as it buds out of the cell at the plasma membrane. Budding of retroviruses involves multiple interactions between viral and cellular proteins and a mature viral particle is the consummation of a regulated and a sequential process. Currently there are no drugs to target the assembly step of retrovirus. In the series of studies outlined here, we outline a physical factor, Vpu that contributes to glycoprotein exclusion from HIV particles. Using a model Vpu target, Gibbon ape Leukemia Virus (GaLV) Env, we have deduced the characteristics of a protein that is targeted by Vpu through its cytoplasmic tail domain (CTD). This unique observation of Vpu modulating the GaLV Env CTD allowed us to compare the two modes of Vpu mediated protein modulation- CTD mediated and membrane spanning domain (MSD) mediated. Subsequently, we studied the contribution of MSD hydrophobicity to Env recruitment to viral budding sites. Curiously, although hydrophobicity of MSD did not dictate Env recruitment, the helicity changes as a result of our mutations resulted in observation of the Env fusogenicity

Sands through the hourglass the structural and functional diversity of major intrinsic proteins

Major Intrinsic Proteins (MIPs) are an ancient family of integral membrane proteins that mediate the bidirectional flux of water and small solutes across cellular membranes. Genomic and phylogenetic analyses indicate that plants contain more MIP genes than their animal and microbial counterparts. An analysis of MIP structure also indicates that plant MIPs structurally diverse at the regions that control selectivity of these proteins. Homology modeling was performed using all 35 members of the MIP family from Arabidopsis thaliana. This analysis revealed that MIPs can be divided into 8 functional subgroups based on the amino acids in their selectivity determining ar/R regions. A broader phylogenetic analysis of all available MIP sequences indicates that 92 ar/R regions exist in this dataset, and that much of the diversity arises from plant sources.Homology modeling indicated that the Nodulin 26-like intrinsic protein (NIP) family of Arabidopsis could be divided into two subgroups based on ar/R classification: NIP subgroup I and II. Functional analysis indicates that these two subgroups are functionally distinct. NIP subgroup I forms aquaglyceroporin channels that are also permeable to ammonia, while NIP subgroup II channels are impermeable to water and capable of transporting larger solutes, such as urea. Site-directed mutagenesis studies were used to rationally interconvert the selectivity of these proteins by amino acid substitutions in the ar/R region. Finally, it was demonstrated that members of NIP subgroup II in Arabidopsis form physiologically relevant boric acid channels at the plasma membrane. Nodulin 26, the archetypal NIP, is phosphorylated by a calcium dependant protein kinase (CDPK) at Ser 262 in its C-terminus. This study demonstrates that nodulin 26 phosphorylation increases the water permeability of the channel.In addition, the C-terminus was found to constitute a protein interaction site for nodule cytosolic glutamine synthetase. Finally, the study was extended to characterize MIP channels from the common pea aphid (ApAQP1 and ApAQP2). The results indicate that ApAQP1 is a water-selective aquaporin that is involved in aphid gut osmoregulation, while ApAQP2 is a water channel that is permeable to an array of linear polyols. The potential physiological function of this channel is also discussed

The Role of Notch-1-Mediated Repression of PTEN on Growth and Cancer Stem Cell Survival in Trastuzumab Resistant, Her2+ Breast Cancer

Trastuzumab targets the ErbB2 (HER2) receptor on breast cancer cells to attenuate HER2 driven tumor formation. Trastuzumab reduces both downstream PI3K/Akt and MAPK pathway signaling as well as the breast cancer stem cell (BCSC) population. BCSCs are hypothesized to be responsible for tumor recurrence, metastasis, as well as drug resistance. Today, resistance to trastuzumab remains a major clinical problem for women diagnosed with HER2+ breast cancer. Attenuation of PI3K/Akt and MAPK pathways may occur through the tumor suppressor, PTEN. Women with HER2+ breast tumors expressing less PTEN and increased PI3K/Akt or MAPK activity have worse overall outcome. Previously we have shown that trastuzumab resistant cells have increased expression of Notch-1 which drives cell proliferation in vitro as well as tumor recurrence in vivo. Here, we show, to our knowledge for the first time, that Notch-1 directly represses PTEN transcript RNA expression to promote trastuzumab resistant HER2+ breast cancer cell proliferation at least in part through activation of ERK1/2. Furthermore, we demonstrate that Notch-1 mediated inhibition of PTEN promotes BCSC survival and self-renewal both in vitro and in vivo