Abstract Neurofibromatosis type 1 syndrome (NF1) is caused by mutations of the NF1 gene. The NF1 protein (neurofibromin) contains a domain which is related to the GTPase-activating protein (GAP) and accelerates the switch of active Ras-GTP to inactive Ras-GDP. The NF1 protein has been referred to as a tumor suppressor, since the cells of malignant schwannomas of NF1 patients may display a loss of heterozygosity of the NF1 gene. Psoriasis is characterized by hyperproliferation of the epidermis and by down-regulated levels of NF1 mRNA and protein. Ca2+ is an universal signal transduction element modulating cell growth and differentiation. Many cell types coordinate their activities by transmitting waves of elevated intracellular calcium levels from cell to cell. The propagation of calcium waves had not been studied previously in human keratinocytes. Thus, the aim of the present study was to find out which pathways may play a role in Ca2+ signaling at different extracellular calcium concentrations in NF1 and and psoriatic keratinocytes versus normal control keratinocytes. The results demonstrated that NF1 and psoriatic keratinocytes have a tendency to form cultures characterized by altered Ca2+-mediated cell signaling compared to normal keratinocytes. Specifically, the main route of calcium-mediated signaling was gap-junctional in normal keratinocytes. In contrast, ATP-mediated calcium signaling predominated and capacitative calcium influx was defective in NF1 and psoriatic keratinocytes. The results of the present study suggest that mutations of the NF1 tumor suppressor gene or lowered levels of NF1 protein/mRNA may eventually lead to altered intercellular communication.

Abstract Respiratory syncytial virus (RSV) infection may be associated with the development of asthma and atopy. The aim of the present study was to investigate this association and the related immunological mechanisms. Seventy-six children admitted to Oulu University Hospital in 1991–1994 for an RSV infection at an age of less than 12 months and healthy controls were called for a visit at the age of 6–10 years. Twenty subjects (26%) had asthma compared with 12 controls (16%) (difference 11%, 95% confidence interval (CI) –3% to 24%). Asthma had been diagnosed significantly earlier in the subjects. Eight per cent of the subjects had at least one positive skin prick test as compared with 43% of the controls (difference –35%, 95% CI –50% to –19%). Serum concentrations of interferon-γ and soluble intercellular adhesion molecule -1 were significantly higher among the subjects than among the controls and among the subjects with asthma or current wheezing than among the corresponding controls. All children born in Finland in 1986–1995 were arranged in birth cohorts by month and year of birth and grouped by exposure to an RSV epidemic at age 0–6 months, resulting in 97 exposed and 23 unexposed cohorts. The proportions of children taking asthma medication or receiving special reimbursement for asthma medication in 1995–2002 were similar in the unexposed and exposed cohorts. Altogether 47 children born between August and November 2001 with a cord blood sample taken were admitted to hospital (n = 26) or seen in an outpatient department (n = 21) for RSV infection before the age of six months. Twenty-eight children had some other respiratory viral infection and 84 children formed a group of healthy controls. High scores on a factor combining the cord blood interleukin-6 and interleukin-8 responses (as derived by factor analysis) were shown in logistic regression analysis to predict hospitalization for RSV infection by comparison with the healthy controls (odds ratio 2.29, 95% CI 1.21 to 4.33). We suggest that RSV does not induce asthma but inborn features of immunity affect the severity of RSV infection and the postinfectious development of asthma.

The contractile state of microcirculatory vessels is a major determinant of the blood pressure of the whole systemic circulation. Continuous bi-directional communication exists between the endothelial cells (ECs) and smooth muscle cells (SMCs) that regulates calcium (Ca2+) dynamics in these cells. This study presents theoretical approaches to understand some of the important and currently unresolved microcirculatory phenomena. Agonist induced events at local sites have been shown to spread long distances in the microcirculation. We have developed a multicellular computational model by integrating detailed single EC and SMC models with gap junction and nitric oxide (NO) coupling to understand the mechanisms behind this effect. Simulations suggest that spreading vasodilation mainly occurs through Ca2+ independent passive conduction of hyperpolarization in RMAs. Model predicts a superior role for intercellular diffusion of inositol (1,4,5)-trisphosphate (IP3) than Ca2+ in modulating the spreading response. Endothelial derived signals are initiated even during vasoconstriction of stimulated SMCs by the movement of Ca2+ and/or IP3 into the EC which provide hyperpolarizing feedback to SMCs to counter the ongoing constriction. Myoendothelial projections (MPs) present in the ECs have been recently proposed to play a role in myoendothelial feedback. We have developed two models using compartmental and 2D finite element methods to examine the role of these MPs by adding a sub compartment in the EC to simulate MP with localization of intermediate conductance calcium activated potassium channels (IKCa) and IP3 receptors (IP3R). Both models predicted IP3 mediated high Ca2+ gradients in the MP after SMC stimulation with limited global spread. This Ca2+ transient generated a hyperpolarizing feedback of ~ 2-3mV. Endothelium derived hyperpolarizing factor (EDHF) is the dominant form of endothelial control of SMC constriction in the microcirculation. A number of factors have been proposed for the role of EDHF but no single pathway is agreed upon. We have examined the potential of myoendothelial gap junctions (MEGJs) and potassium (K+) accumulation as EDHF using two models (compartmental and 2D finite element). An extra compartment is added in SMC to simulate micro domains (MD) which have NaKα2 isoform sodium potassium pumps. Simulations predict that MEGJ coupling is much stronger in producing EDHF than alone K+ accumulation. On the contrary, K+ accumulation can alter other important parameters (EC Vm, IKCa current) and inhibit its own release as well as EDHF conduction via MEGJs. The models developed in this study are essential building blocks for future models and provide important insights to the current understanding of myoendothelial feedback and EDHF.

[EN] Heart failure (HF) constitutes a major public health problem worldwide. Operationally it is defined as a clinical syndrome characterized by the marked and progressive inability of the ventricles to fill and generate adequate cardiac output to meet the demands of cellular metabolism that may have significant variability in its etiology and it is the final common pathway of various cardiac pathologies. Much attention has been paid to the understanding of the arrhythmogenic mechanisms induced by the structural, electrical, and metabolic remodeling of the failing heart. Due to the complexity of the electrophysiological changes that may occur during heart failure, the scientific literature is complex and sometimes equivocal. Nevertheless, a number of common features of failing hearts have been documented. At the cellular level, prolongation of the action potential (AP) involving ion channel remodeling and alterations in calcium handling have been established as the hallmark characteristics of myocytes isolated from failing hearts. At the tissue level, intercellular uncoupling and fibrosis are identified as major arrhythmogenic factors. In this Thesis a computational model for cellular heart failure was proposed using a modified version of Grandi et al. model for human ventricular action potential that incorporates the formulation of the late sodium current (INaL) in order to study the arrhythmogenic processes due to failing phenotype. Experimental data from several sources were used to validate the model. Due to extensive literature in the subject a sensitivity analysis was performed to assess the influence of main ionic currents and parameters upon most related biomarkers. In addition, multiscale simulations were carried out to characterize this pathology (transmural cardiac fibres and tissues). The proposed model for the human INaL and the electrophysiological remodeling of myocytes from failing hearts accurately reproduce experimental observations. An enhanced INaL appears to be an important contributor to the electrophysiological phenotype and to the dysregulation of calcium homeostasis of failing myocytes. Our strand simulation results illustrate how the presence of M cells and heterogeneous electrophysiological remodeling in the human failing ventricle modulate the dispersion of action potential duration (APD) and repolarization time (RT). Conduction velocity (CV) and the safety factor for conduction (SF) were also reduced by the progressive structural remodeling during heart failure. In our transmural ventricular tissue simulations, no reentry was observed in normal conditions or in the presence of HF ionic remodeling. However, defined amount of fibrosis and/or cellular uncoupling were sufficient to elicit reentrant activity. Under conditions where reentry was generated, HF electrophysiological remodeling did not alter the width of the vulnerable window (VW). However, intermediate fibrosis and cellular uncoupling significantly widened the VW. In conclusion, enhanced fibrosis in failing hearts, as well as reduced intercellular coupling, combine to increase electrophysiological gradients and reduce electrical propagation. In that sense, structural remodeling is a key factor in the genesis of vulnerability to reentry, mainly at intermediates levels of fibrosis and intercellular uncoupling. / [ES] La insuficiencia cardíaca (IC) constituye un importante problema de salud pública en todo el mundo. Operacionalmente se define como un síndrome clínico caracterizado por la incapacidad marcada y progresiva de los ventrículos para llenar y generar gasto cardíaco adecuado para satisfacer las demandas del metabolismo celular, que puede tener una variabilidad significativa en su etiología y es la vía final común de varias patologías cardíacas. Se ha prestado mucha atención a la comprensión de los mecanismos arritmogénicos inducidos por la remodelación estructural, eléctrica, y metabólica del corazón afectado de IC. Debido a la complejidad de los cambios electrofisiológicos que pueden ocurrir durante la IC, la literatura científica es compleja y, a veces equívoca. Sin embargo, se han documentado una serie de características comunes en corazones afectados de IC. A nivel celular, se han establecido como las características distintivas de los miocitos aislados de corazones afectados de IC la prolongación del potencial de acción (PA), que implica la remodelación de los canales iónicos y las alteraciones en la dinámica del calcio. A nivel de los tejidos, el desacoplamiento intercelular y la fibrosis se identifican como los principales factores arritmogénicos. En esta tesis se propuso un modelo celular computacional para la insuficiencia cardíaca utilizando una versión modificada del modelo de potencial de acción ventricular humano de Grandi y colaboradores que incorpora la formulación de la corriente tardía de sodio (INaL) con el fin de estudiar los procesos arritmogénicas debido al fenotipo de la IC. Los datos experimentales de varias fuentes se utilizaron para validar el modelo. Debido a la extensa literatura en la temática se realizó un análisis de sensibilidad para evaluar la influencia de las principales corrientes iónicas y los parámetros sobre los biomarcadores relacionados. Además, se llevaron a cabo simulaciones multiescala para caracterizar esta patología (en fibras y tejidos transmurales). El modelo propuesto para la corriente tardía de sodio y la remodelación electrofisiológica de los miocitos de corazones afectados de IC reprodujeron con precisión las observaciones experimentales. Una INaL incrementada parece ser un importante contribuyente al fenotipo electrofisiológico y la desregulación de la homeostasis del calcio de los miocitos afectados de IC. Nuestros resultados de la simulaciones en fibra ilustran cómo la presencia de células M y el remodelado electrofisiológico heterogéneo en el ventrículo humano afectado de IC modulan la dispersión de la duración potencial de acción (DPA) y el tiempo de repolarización (TR). La velocidad de conducción (VC) y el factor de seguridad para la conducción (FS) también se redujeron en la remodelación estructural progresiva durante la insuficiencia cardíaca. En nuestras simulaciones transmurales de tejido ventricular, no se observó reentrada en condiciones normales o en presencia de la remodelación iónica de la IC. Sin embargo, determinadas cantidades de fibrosis y / o desacoplamiento celular eran suficientes para provocar la actividad reentrante. En condiciones donde se había generado la reentrada, el remodelado electrofisiológico de la IC no alteró la anchura de la ventana vulnerable (VV). Sin embargo, niveles intermedios de fibrosis y el desacoplamiento celular ampliaron significativamente la VV. En conclusión, niveles elevados de fibrosis en corazones afectados de IC, así como la reducción de acoplamiento intercelular, se combinan para aumentar los gradientes electrofisiológicos y reducir la propagación eléctrica. En ese sentido, la remodelación estructural es un factor clave en la génesis de la vulnerabilidad a las reentradas, principalmente en niveles intermedios de fibrosis y desacoplamiento intercelular. El remodelado electrofisiológico promueve la arritmogénesis y puede ser alterado dependi / [CAT] La insuficiència cardíaca (IC) constitueix un important problema de salut pública arreu del món. A efectes pràctics, es defineix com una síndrome clínica caracteritzada per la incapacitat marcada i progressiva dels ventricles per omplir i generar el cabal cardíac adequat, per tal de satisfer les demandes del metabolisme cel·lular, el qual pot tenir una variabilitat significativa en la seua etiologia i és la via final comuna de diverses patologies cardíaques. S'ha prestat molta atenció a la comprensió dels mecanismes aritmogènics induïts per la remodelació estructural, elèctrica, i metabòlica del cor afectat d'IC. A causa de la complexitat dels canvis electrofisiològics que poden ocórrer durant la IC, trobem que la literatura científica és complexa i, de vegades, equívoca. No obstant això, s'han documentat una sèrie de característiques comunes en cors afectats d'IC. A nivell cel·lular, com característiques distintives dels miòcits aïllats de cors afectats d'IC, s'han establert la prolongació del potencial d'acció (PA), que implica la remodelació dels canals iònics, i les alteracions en la dinàmica del calci. A nivell dels teixits, el desacoblament intercel·lular i la fibrosi s'identifiquen com els principals factors aritmogènics. Per tal d'estudiar els processos aritmogènics a causa del fenotip de la IC, es va proposar un model cel·lular computacional d'IC utilitzant una versió modificada del model de potencial d'acció ventricular humà de Grandi i els seus col·laboradors, el qual incorpora la formulació del corrent de sodi tardà (INaL). Amb l'objectiu de validar el model es van utilitzar dades experimentals de diverses fonts. A causa de l'extensa literatura en la temàtica, es va realitzar una anàlisi de sensibilitat per tal d'avaluar la influència de les principals corrents iòniques i els paràmetres sobre els biomarcadors relacionats. A més, es van dur a terme simulacions multiescala per a la caracterització d'aquesta patología (fibres i teixits transmurals). El model proposat per al corrent de sodi tardà i la remodelació electrofisiològica dels miòcits de cors afectats d'IC van reproduir amb precisió les observacions experimentals. Una INaL incrementada sembla contribuir de manera important al fenotip electrofisiològic i a la desregulació de l'homeòstasi del calci dels miòcits afectats d'IC. Els resultats de les nostres simulacions en fibra indiquen que la presència de cèl·lules M i el remodelat electrofisiològic heterogeni en el ventricle humà afectat d'IC modulen la dispersió de la durada del potencial d'acció (DPA) i el temps de repolarització (TR). La velocitat de conducció (VC) i el factor de seguretat per a la conducció (FS) també es van reduir en la remodelació estructural progressiva durant la IC. A les nostres simulacions transmurals de teixit ventricular, no s'observà cap reentrada ni en condicions normals ni en presència de la remodelació iònica de la IC. No obstant això, amb determinades quantitats de fibrosi i/o desacoblament cel·lular sí que es provocà l'activitat reentrant. I amb les condicions que produïren la reentrada, el remodelat electrofisiològic de la IC no va alterar l'amplada de la finestra vulnerable (FV). Tanmateix, nivells intermedis de fibrosi i el desacoblament cel·lular sí que ampliaren significativament la FV. En conclusió, nivells elevats de fibrosi en cors afectats d'IC, així com la reducció d'acoblament intercel·lular, es combinen per augmentar els gradients electrofisiològics i reduir la propagació elèctrica. Per tant, la remodelació estructural és un factor clau en la gènesi de la vulnerabilitat a les reentrades, principalment en nivells intermedis de fibrosi i desacoblament intercel·lular. / Gómez García, JF. (2015). Multiscale Modeling and Simulation of Human Heart Failure [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/52389 / TESIS

This thesis intends to utilize biomechanics to study the endothelial biomechanical response in a static hyperglycemic microenvironment. Hyperglycemia is a diabetic condition with abnormally high levels of glucose in the bloodstream. The effects of hyperglycemia over time lead to vascular complications resulting in patients being more prone to cardiovascular diseases. Current studies have focused on the molecular mechanisms affected by hyperglycemia; however, the mechanical mechanisms by which hyperglycemia causes vascular structural and functional changes are understudied. Therefore, to study the effects of hyperglycemia in the endothelium, Human Umbilical Vein Endothelial Cells (HUVEC) were cultured under three glucose conditions: normal glucose (4 mmol/l D-glucose), high glucose (30 mmol/l D-glucose), and an osmotic control (4 mmol/l D-glucose + 26 mmol/l D-mannitol). To evaluate the biomechanical response, we used traction force microscopy and monolayer stress microscopy to measure the cell-substrate tractions and cell-cell intercellular stresses. For the RMS tractions, HUVEC monolayers exposed to high glucose decreased by 10%, while the osmotic control decreased by 17% compared to the normal glucose. HUVEC monolayers exposed to high glucose produced average normal stresses that were 53% lower than monolayers exposed to normal glucose, while the osmotic control decreased by 51% compared to the normal glucose. For the maximum shear stresses, HUVEC monolayers exposed to high glucose decreased by 20%, while the osmotic control decreased by 14% compared to the normal glucose. To conclude this study, we report that hyperglycemia lowers the biomechanical response in the endothelium compared to normal glucose conditions. These results will contribute to understanding the specific role hyperglycemia has on endothelial mechanics and its role in the progression and development of cardiovascular diseases in diabetic patients.

No description available.

No description available.

We report the assembly of three-dimensional (3D) liver sinusoidal mimics comprised of primary rat hepatocytes, human or rat liver sinusoidal endothelial cells denoted as hLSECs and rLSECs respectively, and an intermediate chitosan-hyaluronic acid (HA) polyelectrolyte multilayer (PEM). The height of the PEMs ranged from 30-55nm and exhibited a shear modulus of ~ 100kPa. Primary rat hepatocytes coated with 5 and 15 PE layers exhibited stable urea and albumin production over a seven day period and these values were either comparable or superior to that in a collagen sandwich (CS). Hepatocyte-PEM-hLSEC liver mimics exhibited stable urea production and increasing albumin secretion over the culture period in comparison to hepatocyte-LSEC samples. In the 3D liver mimics, hLSEC phenotype was maintained and verified by the uptake of acetylated low-density lipoprotein (AcLDL). A sixteen-fold increase in CYP1A1/2 activity was observed for hepatocyte-PEM-10,000 hLSEC samples, thereby, suggesting that interactions between hepatocytes and hLSECs play a key role in enhancing hepatic phenotypes in in vitro cultures. As the first step towards elucidating key signaling pathways involved in cell-cell communications, global genome-wide transcriptional profiles of primary hepatocytes cultured in CS and hepatocyte monolayers (HMs) were performed over an eight-day period using DNA microarray measurements and Gene Set Enrichment Analysis (GSEA) in order to derive biologically meaningful information at the level of gene sets. The gene expression in CS cultures steadily diverged from that in HMs. Gene sets up-regulated in CS are those linked to liver metabolic and synthetic functions, such as lipid, fatty acid, alcohol and carbohydrate metabolism, urea production, and synthesis of bile acids. Monooxygenases such as CYP enzymes were significantly up-regulated starting on day 3 in CS cultures. These results serve as a baseline for further investigation into the systems biology of engineered liver tissues. 3D hepatic constructs were also assembled with primary rat hepatocytes and rLSECs, and a chitosan-HA PEM. In these hepatic models, the phenotype of hepatocytes and rLSECs were maintained. rLSEC phenotype was verified over a twelve-day period through immunostaining with the sinusoidal endothelial-1 (SE-1) antibody. In contrast, rLSECs cultured as monolayers lost their phenotype within 3 days. A two-fold increase in albumin production was observed only in the 3D liver models. rLSEC-PEM-hepatocyte cultures exhibited three- to six-fold increased CYP1A1/2 and CYP3A enzymatic activity. Well-defined bile canaliculi were observed in only 3D hepatic constructs. In summary, these results indicate that the layered rLSEC-PEM-hepatocyte constructs can be used as liver models for future studies. / Ph. D.

La Protéine Cellulaire du Prion (PrPc), isoforme non pathogène de la Protéine Scrapie, est une protéine ubiquitaire qui a été impliquée dans de nombreux processus cellulaires tels que la prolifération, la migration, l’adhésion, la différenciation et l’apoptose, par des mécanismes qui restent en grande partie à élucider. L’épithélium intestinal est en constant renouvellement et son homéostasie repose sur une régulation fine et coordonnée de l’ensemble de ces processus. Notre équipe s’est intéressée au rôle de la PrPc dans l’épithélium intestinal et a mis en évidence son expression dans le type cellulaire majoritaire de cet épithélium, les entérocytes, et sa double localisation selon leur état de différenciation. En effet, dans les cellules différenciées, la PrPc est majoritairement présente au niveau des desmosomes, alors que dans les cellules prolifératives, elle est principalement nucléaire. Nous mettons en évidence que la PrPc desmosomale est impliquée dans le maintien et l’intégrité de l’ensemble des jonctions intercellulaires (jonctions serrées, adhérentes et desmosomes) et contribue à la fonction de barrière de l’épithélium intestinal. La PrPc nucléaire, quant à elle, interagit avec plusieurs effecteurs de la voie de signalisation Wnt : la -caténine, la -caténine et le facteur de transcription TCF7L2. Dans ce contexte, nous révélons la capacité de la PrPc nucléaire à moduler l’expression de gènes cibles de la voie Wnt canonique. L’ensemble de ces travaux permet de révéler la PrPc comme un nouvel élément clé de l’homéostasie de l’épithélium intestinal – du maintien de la fonction de barrière jusqu’à la régulation de l’expression de gènes – et de définir la PrPc comme un nouveau membre de la famille des protéines NACos. / The cellular Prion Protein (PrPc), the normal conformer of the Scrapie protein, is a ubiquitous protein, which has been involved in several cellular processes such as proliferation, migration, adhesion, differentiation and apoptosis, through mechanisms that are not fully characterized. Intestinal epithelium is renewing constantly and its homeostasis requires a fine and coordinated regulation of all these processes. Our team has focused on PrPc functions in this tissue and has demonstrated that it is expressed in enterocytes, the major cell type in the intestinal epithelium, with a dual localization depending on the differentiation state of the cells. Indeed, in differentiated cells PrPc is localized in desmosomes while being mostly in the nucleus in proliferative cells. We demonstrated the involvement of desmosomal PrPc in the maintenance and integrity of all the intercellular junctions (tight, adherens junctions and desmosomes) and its requirement for the intestinal barrier function. PrPc in the nucleus interacts with key effectors of the Wnt pathway: -catenin, -catenin and the transcription factor TCF4/TCF7L2. In this context, we revealed the ability of nuclear PrPc to modulate the expression of a subset of Wnt target genes. Altogether, this work highlights the role of PrPc as a new key element of the intestinal epithelial homeostasis – from the barrier function to gene regulation – and allows considering PrPc as a new member of the NACos family proteins (proteins associated with the Nucleus and Adhesion Complexes).

No description available.