Fosfolipases D (FLD) do veneno das aranhas do gênero Loxosceles são capazes de causar entre outros efeitos, uma forte agregação plaquetária cujo mecanismo ainda não foi elucidado. Portanto, para estudar o papel das FLDs nesta atividade, uma FLD recombinante de L. gaucho (LgRec1) foi fusionada com a proteína fluorescente verde (EGFP) e utilizada como uma sonda para detectar a interação de LgRec1 com plaquetas. Essa quimera, denominada EGFP-LgRec1, manteve as principais características da LgRec1. A microscopia confocal das plaquetas mostrou que LgRec1 não requer componentes plasmáticos para se ligar às plaquetas, embora estes sejam necessários para que a LgRec1 induza agregação. Além disso, foi observado que a ação da LgRec1 leva à exposição de fosfatidilserina. Contudo, esta exposição não está relacionada à morte celular. Portanto, este trabalho mostrou que uma FLD de Loxosceles se liga a plaquetas, promovendo a exposição de fosfatidilserina, possibilitando a ligação de fatores de coagulação e resultando na agregação plaquetária. / Phospholipases D (PLD) from spider venom of the genus Loxosceles are capable of causing, among other effects, a strong aggregation of platelets and its mechanism has not yet been elucidated. Therefore, to study the role of PLDs in this activity, a recombinant L. gaucho PLD (LgRec1) was fused with a green fluorescent protein (EGFP) and used as a probe to detect the interaction of LgRec1 with platelets. This chimera, named EGFP-LgRec1, remained the main activities of LgRec1. Platelet confocal microscopy has shown that LgRec1 does not require plasma components to bind to platelets, although these are required for LgRec1 to induce aggregation. In addition, it has been observed that the action of LgRec1 leads to exposures of phosphatidylserine. However, this exposure is not related to cell death. Therefore, this work showed that a Loxosceles PLD binds to platelets, promoting an exposure of phosphatidylserine, that may act as a scaffold for coagulation factors, resulting in platelet aggregation.

Mathematical models of signal transduction pathways are characterized by a large number of proteins and uncertain parameters, yet only a limited amount of quantitative data is available. The dissertation addresses this problem using two different approaches: the first approach deals with a model simplification procedure for signaling pathways that reduces the model size but retains the physical interpretation of the remaining states, while the second approach deals with creating rich data sets by computing transcription factor profiles from fluorescent images of green-fluorescent-protein (GFP) reporter cells. For the first approach a model simplification procedure for signaling pathway models is presented. The technique makes use of sensitivity and observability analysis to select the retained proteins for the simplified model. The presented technique is applied to an IL-6 signaling pathway model. It is found that the model size can be significantly reduced and the simplified model is able to adequately predict the dynamics of key proteins of the signaling pathway. An approach for quantitatively determining transcription factor profiles from GFP reporter data is developed as the second major contribution of this work. The procedure analyzes fluorescent images to determine fluorescence intensity profiles using principal component analysis and K-means clustering, and then computes the transcription factor concentration from the fluorescence intensity profiles by solving an inverse problem involving a model describing transcription, translation, and activation of green fluorescent proteins. Activation profiles of the transcription factors NF-κB, nuclear STAT3, and C/EBPβ are obtained using the presented approach. The data for NF-κB is used to develop a model for TNF-α signal transduction while the data for nuclear STAT3 and C/EBPβ is used to verify the simplified IL-6 model. Finally, an approach is developed to compute the distribution of transcription factor profiles among a population of cells. This approach consists of an algorithm for identifying individual fluorescent cells from fluorescent images, and an algorithm to compute the distribution of transcription factor profiles from the fluorescence intensity distribution by solving an inverse problem. The technique is applied to experimental data to derive the distribution of NF-κB concentrations from fluorescent images of a NF-κB GFP reporter system.

Extended periods of oxygen deprivation cause brain death in mammals but the western painted turtle overwinters in anoxic mud for months without damage. Neural protection is achieved through decreases in the whole cell currents of N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (NMDAR and AMPAR) that are dependent on a mild increase in intracellular calcium from the mitochondria. The goal of this research was to determine if natural anoxic decreases in reactive oxidative species (ROS) serve as the signal to bring about these changes. Reductions in cellular ROS levels were demonstrated to have no effect on AMPAR currents or intracellular calcium and produced massive increases in NMDAR currents, indicating that ROS depression does not directly mediate anoxic alterations. Interestingly, mammalian neural tissue also experiences a similar increase in NMDAR whole cell current in response to reducing agents suggesting a possible conserved mechanism for normoxic receptor control.

L’adhésine impliquée dans l’adhérence diffuse (AIDA-I) est une adhésine bactérienne présente chez certaines souches d’Escherichia coli qui, associée aux toxines Stx2e ou STb, contribue à l’apparition de la maladie de l’œdème ou de la diarrhée post-sevrage chez les porcelets. AIDA-I est un autotransporteur qui confère des capacités d’autoaggrégation, de formation de biofilms et d’adhésion. L’objectif principal du projet de recherche consistait en la recherche de récepteur(s) potentiel(s) d’AIDA-I. Les bactéries pathogènes adhèrent aux cellules-cibles soit en liant directement des molécules à la surface cellulaire ou en utilisant des molécules intermédiaires qui permettent de diminuer la distance séparant la bactérie de la cellule-cible. Puisque le sérum est un fluide qui contient de nombreuses molécules, celui-ci a été utilisé comme matériel de départ pour l’isolement de récepteur(s) potentiels. Nous avons isolé un récepteur potentiel à partir du sérum porcin : l’apolipoprotéine A-I. L’interaction entre l’apolipoprotéine A-I et AIDA-I a été confirmée par ELISA et microscopie à fluorescence. La capacité à envahir les cellules épithéliales offre aux pathogènes la possibilité d’établir une niche intracellulaire qui les protègent contre les attaques du milieu extérieur. La présente étude a démontré que la présence d’AIDA-I en tant que seul facteur de virulence chez une souche de laboratoire permet de conférer la capacité d’envahir les cellules sans promouvoir la survie intracellulaire. L’étude de la souche sauvage 2787, exprimant AIDA-I en association avec d’autres facteurs de virulence, a démontré une différence significative pour les phénotypes d’invasion et de survie intracellulaire face à la souche de laboratoire exprimant AIDA-I. / The adhesin involved in diffuse adherence (AIDA-I) is a bacterial adhesin associated with some Escherichia coli strains that might, when associated with toxin Stx2e or STb, contribute to the development of edema disease or post-weaning diarrhea in piglets. AIDA-I is an autotransporter that mediates various phenotypes such as adhesion, autoaggregation and biofilm formation. The main aim of our project was to find potential receptor(s) for AIDA-I. Pathogens can either bind cell directly by targeting exposed cell surface molecules or use an intermediate molecule as a bridge to lessen the space separating them from their target cell. Serum is known to contain a wide range of molecules so it has been used as raw material for the isolation of a putative receptor for AIDA-I. We isolated a putative receptor for AIDA-I: the apolipoprotein A-I. The interaction between the apolipoprotein A-I and AIDA-I was confirmed by ELISA and fluorescent microscopy. The capacity to invade epithelial cell enables pathogens to create an intracellular niche that protects them against attacks from the extracellular environment. The present report has shown that the presence of AIDA-I as the sole virulence factor in a laboratory strain, enable bacteria to invade cultured cells but does not promote intracellular survival. Studies conducted on wild-type strain 2787, which express AIDA-I in association with other virulence factors, has shown a significant difference in invasion and intracellular survival phenotypes compared to the laboratory strain expressing AIDA-I.

Extended periods of oxygen deprivation cause brain death in mammals but the western painted turtle overwinters in anoxic mud for months without damage. Neural protection is achieved through decreases in the whole cell currents of N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (NMDAR and AMPAR) that are dependent on a mild increase in intracellular calcium from the mitochondria. The goal of this research was to determine if natural anoxic decreases in reactive oxidative species (ROS) serve as the signal to bring about these changes. Reductions in cellular ROS levels were demonstrated to have no effect on AMPAR currents or intracellular calcium and produced massive increases in NMDAR currents, indicating that ROS depression does not directly mediate anoxic alterations. Interestingly, mammalian neural tissue also experiences a similar increase in NMDAR whole cell current in response to reducing agents suggesting a possible conserved mechanism for normoxic receptor control.

Fosfolipases D (FLD) do veneno das aranhas do gênero Loxosceles são capazes de causar entre outros efeitos, uma forte agregação plaquetária cujo mecanismo ainda não foi elucidado. Portanto, para estudar o papel das FLDs nesta atividade, uma FLD recombinante de L. gaucho (LgRec1) foi fusionada com a proteína fluorescente verde (EGFP) e utilizada como uma sonda para detectar a interação de LgRec1 com plaquetas. Essa quimera, denominada EGFP-LgRec1, manteve as principais características da LgRec1. A microscopia confocal das plaquetas mostrou que LgRec1 não requer componentes plasmáticos para se ligar às plaquetas, embora estes sejam necessários para que a LgRec1 induza agregação. Além disso, foi observado que a ação da LgRec1 leva à exposição de fosfatidilserina. Contudo, esta exposição não está relacionada à morte celular. Portanto, este trabalho mostrou que uma FLD de Loxosceles se liga a plaquetas, promovendo a exposição de fosfatidilserina, possibilitando a ligação de fatores de coagulação e resultando na agregação plaquetária. / Phospholipases D (PLD) from spider venom of the genus Loxosceles are capable of causing, among other effects, a strong aggregation of platelets and its mechanism has not yet been elucidated. Therefore, to study the role of PLDs in this activity, a recombinant L. gaucho PLD (LgRec1) was fused with a green fluorescent protein (EGFP) and used as a probe to detect the interaction of LgRec1 with platelets. This chimera, named EGFP-LgRec1, remained the main activities of LgRec1. Platelet confocal microscopy has shown that LgRec1 does not require plasma components to bind to platelets, although these are required for LgRec1 to induce aggregation. In addition, it has been observed that the action of LgRec1 leads to exposures of phosphatidylserine. However, this exposure is not related to cell death. Therefore, this work showed that a Loxosceles PLD binds to platelets, promoting an exposure of phosphatidylserine, that may act as a scaffold for coagulation factors, resulting in platelet aggregation.

No description available.

<p dir="ltr">DNA is organized into chromatin – a complex polymeric structure which stores information and controls gene expressions. Advancements in microscopy have enabled us to see chromatin in motion – which was previously thought to be static, and these motions contribute to various cellular functions. In my thesis I will demonstrate the molecular tools and biophysical approaches our lab has developed to uncover the mysteries of chromatin dynamics and structures at the single nucleosome resolution; I will also discuss how these new discoveries in chromatin enable us to explore its role in cell functions. This dissertation will first describe the technology advancement of live-cell image analysis; particularly, I will discuss the utilization of AI to improve the spatial and temporal resolution of chromatin imaging. Then I will show complex nature of chromatin where depending on the temporal scale of observation we see a different behavior and how computer simulations can see these differences. Following that, I will introduce our investigation on the role of chromatin motion in DNA damage and repair. Afterwards, I will discuss how the cell regulates its chromatin dynamics in response to the metabolism indicators AMPK (AMP-activated protein kinase). I will also show how chromatin motion and structure behave without the presence of key proteins such as RAD51 that aid in DNA damage. Finally I will go over future directions and improvements we can do to our current techniques to improve our understanding of chromatin’s role is various biological functions. We expect that the exploration of the spatiotemporal dynamics in live cells will facilitate the diagnosis, treatment, and prevention of cancers.</p>

Indiana University-Purdue University Indianapolis (IUPUI) / Advancements in human genomics have simultaneously enhanced our basic understanding of the human body and ability to combat debilitating diseases. Historically, research has shown that there have been many hindrances to realizing this medicinal revolution. One hindrance, with particular regard to the kidney, has been our inability to effectively and routinely delivery genes to various loci, without inducing significant injury. However, we have recently developed a method using hydrodynamic fluid delivery that has shown substantial promise in addressing aforesaid issues. We optimized our approach and designed a method that utilizes retrograde renal vein injections to facilitate widespread and persistent plasmid and adenoviral based transgene expression in rat kidneys. Exogenous gene expression extended throughout the cortex and medulla, lasting over 1 month within comparable expression profiles, in various renal cell types without considerably impacting normal organ function. As a proof of its utility we by attempted to prevent ischemic acute kidney injury (AKI), which is a leading cause of morbidity and mortality across among global populations, by altering the mitochondrial proteome. Specifically, our hydrodynamic delivery process facilitated an upregulated expression of mitochondrial enzymes that have been suggested to provide mediation from renal ischemic injury. Remarkably, this protein upregulation significantly enhanced mitochondrial membrane potential activity, comparable to that observed from ischemic preconditioning, and provided protection against moderate ischemia-reperfusion injury, based on serum creatinine and histology analyses. Strikingly, we also determined that hydrodynamic delivery of isotonic fluid alone, given as long as 24 hours after AKI is induced, is similarly capable of blunting the extent of injury. Altogether, these results indicate the development of novel and exciting platform for the future study and management of renal injury.