Global ETD Search

501	Investigating TRPV4 Signaling in Choroid Plexus Culture Models Louise Susannah Hulme (12456711) 12 July 2022 (has links) <p>Hydrocephalus is a neurological disorder characterised by the pathological accumulation of cerebrospinal fluid (CSF) within the brain ventricles. Surgical interventions, including shunt placement, remain the gold standard treatment option for this life-threatening condition, despite these often requiring further revision surgeries. Unfortunately, there is currently no effective, pharmaceutical therapeutic agent available for the treatment of hydrocephalus. CSF is primarily produced by the choroid plexus (CP), a specialized, branched structure found in the ventricles of the brain. The CP comprises a high resistance epithelial monolayer surrounding a fenestrated capillary network, forming the blood-CSF barrier (BCSFB). The choroid plexus epithelium (CPe) critically modulates CSF production by regulating ion and water transport from the blood into the intraventricular space. This process is thought to be controlled by a host of intracellular mediators, as well as transporter proteins present on either the apical or basolateral membrane of the CPe. Though many of these proteins have been identified in the native tissue, exactly how they interact and modulate signal cascades to mediate CSF secretion remains less clear.</p> <p><br></p> <p>Transient potential receptor vanilloid 4 (TRPV4) is a non-selective cation channel that can be activated by a range of stimuli and is expressed in the CP. TRPV4 has been implicated in the regulation of CSF production through stimulating ion flux across the CPe. In a continuous CP cell line, activation of TRPV4, through the addition of a TRPV4 specific agonist GSK1016790A, stimulated a change in net transepithelial ion flux and increase in conductance. In order to develop a pharmaceutical therapeutic for the treatment of hydrocephalus, we must first understand the mechanism of CSF secretion in health and disease. Therefore, a representative <em>in vitro</em> model is critical to elucidate the signaling pathways orchestrating CSF production in the CP.</p> <p><br></p> <p>This research aims to characterize an <em>in vitro</em> culture model that can be utilized to study both the BCSFB and CSF production, to investigate and identify additional transporters, ion channels and intracellular mediators involved in TRPV4-mediated signaling in the CPe, primarily through a technique called Ussing-style electrophysiology which considers electrogenic ion flux across a monolayer. These studies implicated several potential modulators, specifically phospholipase C (PLC), phosphoinositide 3-kinase (PI3K), protein kinase C (PKC), intermediate conductance K+ channel (IK), transmembrane member 16A (TMEM16A), cystic fibrosis transmembrane conductance regulator (CFTR) and protein kinase A (PKA), in TRPV4-mediated ion flux.</p> Signal transduction Plant cell and molecular biology Animal cell and molecular biology Animal physiology - biophysics Animal physiology - cell Animal physiology - systems Neurosciences not elsewhere classified Choroid Plexus Epithelium In vitro model Continuous cell line Ussing-style electrophysiology Cell signaling Cell Biology Molecular Biology Neuroscience Pharmacology Physiology Signal Transduction
502	The implication of the microRNA Let-7f in the degeneration and dysfunction of retinal pigment epithelial cells Ortiz, Christina 02 1900 (has links) L'épithélium pigmentaire rétinien (EPR) est une monocouche formée de cellules hautement spécialisées et uniques dont les nombreuses fonctions servent à maintenir une vision adéquate. En revanche, ces fonctions spécifiques rendent les cellules de l’EPR particulièrement vulnérables au stress oxydant. Avec le vieillissement, les cellules de l’EPR peuvent dégénérer et devenir non-fonctionnelles, donnant lieu à plusieurs maladies telles que la dégénérescence maculaire liée à l'âge (DMLA). Dans les pays occidentaux, la DMLA est la principale cause de cécité et de déficience visuelle chez les personnes âgées. En fait, environ 90 % des patients atteints de la DMLA souffrent de la forme sèche, pour laquelle il n'existe aucun traitement. Des années de recherche ont établi que le stress oxydant est un contributeur majeur à la pathogenèse de la DMLA sèche. De nombreuses études ont montré que le stress oxydant induit les cellules de l’EPR à libérer des vésicules extracellulaires (VEs). Nos propres travaux ont démontré que les VEs peuvent induire le stress oxydant et la sénescence chez les cellules de l’EPR. Comme d'autres, nous avons constaté que les VEs étaient enrichis en microARNs. Grâce au séquençage d’ARN, nous avons identifié le let-7f comme étant l'un des microARNs les plus abondants contenus dans ces vésicules. L'objectif de ce mémoire a été l’exploration entre la relation let-7f et la dégénérescence des cellules de l’EPR. Nos résultats ont démontré une régulation et une augmentation de l’expression du let-7f dans les cellules de l’EPR sous stress oxydant, in vitro et in vivo. De plus, la surexpression du let-7f a généré un stress oxydant, le dysfonctionnement et la sénescence des cellules humaines de l’EPR (ARPE-19). De plus, l’inhibition du let-7f à protéger ces cellules contre les conséquences néfastes induites par l’iodate de sodium. En somme, les résultats de ce travail suggèrent fortement que le let-7f est impliqué dans la dégénérescence des cellules de l’EPR et pourraient aider à la découverte de nouveaux processus pertinents dans la pathogenèse de la DMLA sèche. / The retinal pigment epithelium (RPE) is a highly specialized and unique monolayer of cells whose many functions are vital for maintaining proper vision. In turn, these specific functions render RPE cells particularly vulnerable to oxidative injury. With age, RPE cells can degenerate and become dysfunctional, giving rise to various disorders such as age-related macular degeneration (AMD). In western countries, AMD is the primary cause of blindness and visual impairments in the elderly. In fact, approximately 90% of all AMD patients suffer from the dry form of the disease, for which there exist no approved treatment. Decades of research have established that chronic oxidative stress is a major contributor to the pathogenesis of dry AMD. Numerous studies have shown that oxidative stress induces RPE cells to release extracellular vesicles (EVs) which participate in cell-to-cell communication. Our recent work has demonstrated that EVs alone were sufficient in inducing oxidative stress and senescence in RPE cells. Consistent with others, we found that EVs released by RPE cells were enriched in microRNAs. RNA-sequencing identified let-7f as one of the most abundant miRNAs contained in these vesicles. Despite being one of the first miRNAs to be discovered, the role of let-7f in RPE cells has remained essentially unexplored. The aim of this dissertation was to investigate the relationship between let-7f and RPE cells in regards to their degeneration and dysfunction. Our results revealed that the expression of let-7f increased and was regulated by oxidative stress in RPE cells, in vitro and in vivo. In addition, let-7f overexpression promoted oxidative stress, cellular dysfunction and senescence in human RPE (ARPE-19) cells. Finally, inhibition of let-7f exhibited protective effects against sodium iodate-induced oxidative injury. Overall, the findings in this work provide strong evidence that let-7f is implicated in the degeneration of RPE cells and further mechanistic investigation may help to uncover novel insights into the genesis of dry AMD. Épithélium pigmentaire rétinien MicroARN let-7f Stress oxydant Sénescence Dysfonctionnement Iodate de sodium Vésicules extracellulaires Retinal pigment epithelium Dry age-related macular degeneration MicroRNA let-7f Oxidative stress Senescence Dysfunction Sodium iodate Extracellular vesicles
503	Development of Bimodal Gene Expression Analysis and Allele-Specific Competitive PCR for Investigation of Complex Genetic Traits, Lung Cancer Risk Blomquist, Thomas M. 04 August 2010 (has links) No description available. Bioinformatics Biology Biomedical Research Biostatistics Cellular Biology Computer Science Epidemiology Experiments Genetics Health Health Care Molecular Biology Oncology Pathology Pharmacology Philosophy Public Health allele-specific expression lung cancer complex genetic traits dispersion bimodal kurtosis variance
504	Étude de stratégies thérapeutiques complémentaires visant à favoriser la résolution des paramètres du syndrome de détresse respiratoire aiguë dans des modèles in vivo Aubin Vega, Mélissa 04 1900 (has links) Le syndrome de détresse respiratoire aiguë (SDRA) est une forme de défaillance respiratoire sévère, cause majeure de mortalité (~30-45%) chez les adultes et enfants dans les unités de soins intensifs. En dépit des progrès dans la prise en charge du patient, il n’existe à ce jour aucun traitement curatif pharmacologique efficace. Le SDRA peut se développer à la suite d’une atteinte pulmonaire directe (ex. pneumonie) ou indirecte (ex. septicémie) dont les principales caractéristiques sont des lésions épithéliales alvéolaires et endothéliales vasculaires, le développement d’un oedème pulmonaire et une réponse inflammatoire exacerbée durant la phase aiguë exsudative. La résolution de ces paramètres est critique afin d’éviter l’établissement irréversible de fibrose, entraînant une défaillance respiratoire. Le caractère hétérogène du SDRA et l’implication d’une multitude de mécanismes lésionnels rendent le développement de nouvelles thérapies plus difficile. Nous avons posé l’hypothèse que la restauration de l’intégrité épithéliale, en parallèle de la résolution de l’inflammation et la résorption de l’oedème, est critique pour la résolution de la phase exsudative du SDRA. Nous avons donc postulé que des stratégies combinant des effets bénéfiques sur la clairance liquidienne et proréparatrice constitueraient une voie intéressante pour la restauration de l’intégrité fonctionnelle de l’épithélium alvéolaire. L’objectif général de mon projet de doctorat était donc d’évaluer différentes stratégies, ciblant 1) l’inflammation, 2) le canal sodique ENaC impliqué dans la clairance liquidienne et 3) les canaux potassiques ayant un rôle pro-réparateur, avec des modèles complémentaires in vivo de lésions aiguës induites, mimant des paramètres de SDRA. Nous pensons que cette étude aura apporté de nouvelles connaissances sur la physiopathologie du SDRA et les mécanismes de résolution des paramètres caractéristiques de ce syndrome. Mon projet met particulièrement en lumière que de cibler une seule composante telle que l’inflammation ou la clairance liquidienne n’est pas suffisante et que des composés permettant de restaurer l’intégrité fonctionnelle alvéolaire sont nécessaires. / Acute respiratory distress syndrome (ARDS) is a severe form of respiratory failure, a leading cause of death (~30-45%) among adults and children in intensive care units. Despite advances in the management and care of ARDS patients, there is currently no effective curative pharmacological treatment. The ARDS can develop following a direct (e.g. pneumonia) or indirect (e.g. sepsis) lung injury, the main features of which are alveolar epithelial and endothelial vascular injury, the development of pulmonary edema, and an exacerbated inflammatory response during the exsudative acute phase. The resolution of these parameters is critical to avoid the irreversible establishment of fibrosis leading to respiratory failure. The heterogeneous nature of ARDS and the involvement of various lesional mechanisms complicate the development of new therapeutic strategies. We hypothesized that the epithelial restoration, in parallel with inflammatory resolution and edema resorption, is critical for the resolution of the acute exsudative phase of ARDS. Therefore, we postulated that strategies combining beneficial effects on fluid clearance and pro repair may be an interesting way to restore the functional integrity of the alveolar epithelium. The general objective of my PhD project was to evaluate different strategies targeting 1) the inflammation, 2) the sodium channel ENaC involved in fluid clearance, and 3) potassium channels playing pro repair role, using complementary in vivo models of acute lung injury mimicking ARDS parameters. We believe that these studies have provided new insight on the pathophysiology of ARDS and the mechanisms of resolution of the characteristic parameters of this syndrome. In particular, my project highlights that focusing on a single component such as inflammation or fluid clearance is not sufficient and that compounds will restore functional alveolar integrity are needed. canaux potassiques épithélium alvéolaire inflammation pulmonaire dommages pulmonaires réparation épithéliale oedème pulmonaire transport ionique membranaire potassium channels alveolar epithelium pulmonary inflammation lung damage epithelial repair pulmonary edema acute respiratory distress syndrome ion membrane transport Physiology / Physiologie (UMI : 0719)
505	The Effects of Lactate Receptor G Protein-Coupled Receptor 81 (GPR81) on the Integrity of the Choroidal Vasculature Yang, Xiaojuan 02 1900 (has links) No description available. G protein-coupled receptor 81 (GPR81) Hydroxycarboxylic acid receptor 1 (HCA1) Lactate Néovascularisation choroïdienne (NVC) Choroidal neovascularization (CNV) Aged-related macular degeneration (AMD) Épaississement choroïdien Choroidal thickening Amincissement choroïdien Choroidal thinning Stress oxydatif Oxidative stress Stress ER ER stress Réponse au stress intégrée (ISR) Integrated stress response (ISR) Dérèglement métabolique Metabolism dysregulation Rétine externe Outer retina Épithélium pigmentaire rétinien (EPR) Retinal pigment epithelium (RPE)
506	Development of Sensitive In Vitro Assays to Assess the Ocular Toxicity Potential of Chemicals and Ophthalmic Products McCanna, David January 2009 (has links) The utilization of in vitro tests with a tiered testing strategy for detection of mild ocular irritants can reduce the use of animals for testing, provide mechanistic data on toxic effects, and reduce the uncertainty associated with dose selection for clinical trials. The first section of this thesis describes how in vitro methods can be used to improve the prediction of the toxicity of chemicals and ophthalmic products. The proper utilization of in vitro methods can accurately predict toxic threshold levels and reduce animal use in product development. Sections two, three and four describe the development of new sensitive in vitro methods for predicting ocular toxicity. Maintaining the barrier function of the cornea is critical for the prevention of the penetration of infections microorganisms and irritating chemicals into the eye. Chapter 2 describes the development of a method for assessing the effects of chemicals on tight junctions using a human corneal epithelial and canine kidney epithelial cell line. In Chapter 3 a method that uses a primary organ culture for assessing single instillation and multiple instillation toxic effects is described. The ScanTox system was shown to be an ideal system to monitor the toxic effects over time as multiple readings can be taken of treated bovine lenses using the nondestructive method of assessing for the lens optical quality. Confirmations of toxic effects were made with the utilization of the viability dye alamarBlue. Chapter 4 describes the development of sensitive in vitro assays for detecting ocular toxicity by measuring the effects of chemicals on the mitochondrial integrity of bovine cornea, bovine lens epithelium and corneal epithelial cells, using fluorescent dyes. The goal of this research was to develop an in vitro test battery that can be used to accurately predict the ocular toxicity of new chemicals and ophthalmic formulations. By comparing the toxicity seen in vivo animals and humans with the toxicity response in these new in vitro methods, it was demonstrated that these in vitro methods can be utilized in a tiered testing strategy in the development of new chemicals and ophthalmic formulations. in vitro Alternative Methods alamarBlue rhodamine tight junctions bovine lens viability toxicity mitochondria mitochondria integrity benzalkonium chloride sodium dodecyl sulphate sodium lauryl sulfate scanning electron microscopy Draize Draize maximal average scores zonula occludens cosmetic directive PETA humane society animal league ICCVAM ECVAM JaCVAM BCOP bovine cornea ocular irritants ocular irritation ocular toxicity human corneal epithelial cells vitro confocal confocal microscopy metabolic activity optical quality reactive oxygen species contact lens contact lens care solutions barrier function microbial keratitis mulitipurpose solutions tight junction cell damage claudin ZO-1 occludin MDCK Madin Madin-Darby canine kidney cells cornea human cornea human corneal epithelium epithelial cell line human corneal epithelial cell line sodium fluorescein sodium fluorescein permeability fluorescein permeability ocular surface cell monolayer Araki-Sasaki cell physiology risk assessment safety assessment ScanTox scanning laser lens epithelium corneal cells in vitro model ophthalmic formulations multiple instillation back vertex animal testing rabbit testing alternatives to animal testing cultured bovine lens toxicity of chemicals irritation irritants laser scanner organ culture delayed toxicity toxins hydrogen peroxide cornea toxicity cornea toxicity models prediction of human toxicity no observable adverse effect level lowest observable adverse effect level NOAEL LOAEL toxicity thresholds safety factors cornea uncertainty factors preservatives disinfectants ophthalmic products preclinical preclinical testing epithelial barrier drug penetration clinical confocal microscopy animal rights rabbit cornea human cornea human clinical effects toxic animal rights activist sensitive measures toxic effect toxicity threshold agar overlay agar diffusion agar overlay method agar diffusion method cytochrome cytochrome c apoptosis necrotic apoptotic necrosis caspase rhodamine 123 resazuran resorufin cell death cell viability metabolic dye microsomal microsomal enzymes cytotoxicity cytotoxic cytotoxic effect MTT XTT WST-1 plasma membrane mitochondrial mitochondrial morphology ocular toxicity potential ocular toxicity human corneal epithelial cell line confocal analysis corneal epithelium cell fluorescence alamarBlue assay rhodamine dye animal welfare toxic injury degraded mitochondria epithelial monolayer disinfectants membrane integrity eye toxicity eye viability dye toxicity in humans human toxicity effects on the mitochondria mitochondrial toxicity in vitro battery in vitro test battery ophthalmic eye drop direct contact product safety cytotoxicity potential molecular molecular biology refine reduce replace sensitivity and relevance sensitivity relevance rabbit ocular irritation test product development cytotoxicity models cytotoxicity alternative methods replacements for animal testing three r's beagle tiered testing tiered testing strategy replacements animal testing mechanistic toxicity cornea mitochondria dose response threshold Vision Science and Biology
507	Development of Sensitive In Vitro Assays to Assess the Ocular Toxicity Potential of Chemicals and Ophthalmic Products McCanna, David January 2009 (has links) The utilization of in vitro tests with a tiered testing strategy for detection of mild ocular irritants can reduce the use of animals for testing, provide mechanistic data on toxic effects, and reduce the uncertainty associated with dose selection for clinical trials. The first section of this thesis describes how in vitro methods can be used to improve the prediction of the toxicity of chemicals and ophthalmic products. The proper utilization of in vitro methods can accurately predict toxic threshold levels and reduce animal use in product development. Sections two, three and four describe the development of new sensitive in vitro methods for predicting ocular toxicity. Maintaining the barrier function of the cornea is critical for the prevention of the penetration of infections microorganisms and irritating chemicals into the eye. Chapter 2 describes the development of a method for assessing the effects of chemicals on tight junctions using a human corneal epithelial and canine kidney epithelial cell line. In Chapter 3 a method that uses a primary organ culture for assessing single instillation and multiple instillation toxic effects is described. The ScanTox system was shown to be an ideal system to monitor the toxic effects over time as multiple readings can be taken of treated bovine lenses using the nondestructive method of assessing for the lens optical quality. Confirmations of toxic effects were made with the utilization of the viability dye alamarBlue. Chapter 4 describes the development of sensitive in vitro assays for detecting ocular toxicity by measuring the effects of chemicals on the mitochondrial integrity of bovine cornea, bovine lens epithelium and corneal epithelial cells, using fluorescent dyes. The goal of this research was to develop an in vitro test battery that can be used to accurately predict the ocular toxicity of new chemicals and ophthalmic formulations. By comparing the toxicity seen in vivo animals and humans with the toxicity response in these new in vitro methods, it was demonstrated that these in vitro methods can be utilized in a tiered testing strategy in the development of new chemicals and ophthalmic formulations. in vitro Alternative Methods alamarBlue rhodamine tight junctions bovine lens viability toxicity mitochondria mitochondria integrity benzalkonium chloride sodium dodecyl sulphate sodium lauryl sulfate scanning electron microscopy Draize Draize maximal average scores zonula occludens cosmetic directive PETA humane society animal league ICCVAM ECVAM JaCVAM BCOP bovine cornea ocular irritants ocular irritation ocular toxicity human corneal epithelial cells vitro confocal confocal microscopy metabolic activity optical quality reactive oxygen species contact lens contact lens care solutions barrier function microbial keratitis mulitipurpose solutions tight junction cell damage claudin ZO-1 occludin MDCK Madin Madin-Darby canine kidney cells cornea human cornea human corneal epithelium epithelial cell line human corneal epithelial cell line sodium fluorescein sodium fluorescein permeability fluorescein permeability ocular surface cell monolayer Araki-Sasaki cell physiology risk assessment safety assessment ScanTox scanning laser lens epithelium corneal cells in vitro model ophthalmic formulations multiple instillation back vertex animal testing rabbit testing alternatives to animal testing cultured bovine lens toxicity of chemicals irritation irritants laser scanner organ culture delayed toxicity toxins hydrogen peroxide cornea toxicity cornea toxicity models prediction of human toxicity no observable adverse effect level lowest observable adverse effect level NOAEL LOAEL toxicity thresholds safety factors cornea uncertainty factors preservatives disinfectants ophthalmic products preclinical preclinical testing epithelial barrier drug penetration clinical confocal microscopy animal rights rabbit cornea human cornea human clinical effects toxic animal rights activist sensitive measures toxic effect toxicity threshold agar overlay agar diffusion agar overlay method agar diffusion method cytochrome cytochrome c apoptosis necrotic apoptotic necrosis caspase rhodamine 123 resazuran resorufin cell death cell viability metabolic dye microsomal microsomal enzymes cytotoxicity cytotoxic cytotoxic effect MTT XTT WST-1 plasma membrane mitochondrial mitochondrial morphology ocular toxicity potential ocular toxicity human corneal epithelial cell line confocal analysis corneal epithelium cell fluorescence alamarBlue assay rhodamine dye animal welfare toxic injury degraded mitochondria epithelial monolayer disinfectants membrane integrity eye toxicity eye viability dye toxicity in humans human toxicity effects on the mitochondria mitochondrial toxicity in vitro battery in vitro test battery ophthalmic eye drop direct contact product safety cytotoxicity potential molecular molecular biology refine reduce replace sensitivity and relevance sensitivity relevance rabbit ocular irritation test product development cytotoxicity models cytotoxicity alternative methods replacements for animal testing three r's beagle tiered testing tiered testing strategy replacements animal testing mechanistic toxicity cornea mitochondria dose response threshold Vision Science and Biology

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