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Mitochondrial Remodeling During Hyperosmotic StressZulys, Matthew 26 February 2009 (has links)
Hyperosmotic stress represents a major threat to cellular integrity and may lead to cell death via apoptosis. Accordingly, each cell reacts to hyperosmolarity with a set of functional and structural compensatory responses. Recently it has been shown that the mitochondria remodel during hyperosmotic stress. Although changes in mitochondrial dynamics could be crucial for both adaptation and apoptosis, hyperosmolarity-induced mitochondrial remodeling has not been characterized. We found that hyperosmotic stress translocates dynamin like protein 1 (DLP-1) to the mitochondria and induces DLP-1 mediated, F-actin-modulated, Rac-dependent fragmentation of these organelles in LLC-PK1 cells. Downregulation of DLP-1 mitigates the activation of the osmotic response element and increases the susceptibility of tubular cells to hyperosmotically-induced apoptosis, suggesting that DLP-1 (or mitochondrial fragmentation) may have a protective role during osmotic stress. The hyperosmolarity-triggered remodeling of the mitochondrion represents a hitherto unrecognized response to osmotic shock, which may have significant impact on adaptation and apoptosis.
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Mitochondrial Remodeling During Hyperosmotic StressZulys, Matthew 26 February 2009 (has links)
Hyperosmotic stress represents a major threat to cellular integrity and may lead to cell death via apoptosis. Accordingly, each cell reacts to hyperosmolarity with a set of functional and structural compensatory responses. Recently it has been shown that the mitochondria remodel during hyperosmotic stress. Although changes in mitochondrial dynamics could be crucial for both adaptation and apoptosis, hyperosmolarity-induced mitochondrial remodeling has not been characterized. We found that hyperosmotic stress translocates dynamin like protein 1 (DLP-1) to the mitochondria and induces DLP-1 mediated, F-actin-modulated, Rac-dependent fragmentation of these organelles in LLC-PK1 cells. Downregulation of DLP-1 mitigates the activation of the osmotic response element and increases the susceptibility of tubular cells to hyperosmotically-induced apoptosis, suggesting that DLP-1 (or mitochondrial fragmentation) may have a protective role during osmotic stress. The hyperosmolarity-triggered remodeling of the mitochondrion represents a hitherto unrecognized response to osmotic shock, which may have significant impact on adaptation and apoptosis.
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Akutní komplikace diabetu a jeho následky / Acute complications of diabetes mellitus and it's sequelFrank, Adrianna Natalie January 2010 (has links)
The basis of this thesis is intended to inform the reader about the general complications of acute diabetes mellitus and its consequences. It focuses on the general definitions of the diseases, etiology, morbidity, mortality, pathogenesis of the disease, clinical presentation, treatment, and future developments in hopes of treating the disease. The major focus highlights the differences between diabetic ketoacidosis and hyperglycemic hyperosmolar state, as well as understanding the complications of diabetic hypoglycemia. The most critical effects of these disorders are also emphasized; cerebral edema, vascular thrombosis, and hyperchloremic metabolic acidosis.
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Efficacy of hyper-osmotic agent (100% anhydrous glycerol) in tissue and light-activated micro-pattern drug delivery device in in vivo rabbit eyeZaman, Raiyan Tripti 13 July 2011 (has links)
My PhD research involves multi-disciplinary areas of study such as measuring perfusion of blood vessels in hamster dorsal skin using laser speckle imaging technique. In this study the changes were measured in blood flow velocity and diameters of micro vasculatures after the influence of glycerol application. The second study identifies the changes in morphology and optical properties of eye tissue after applying hyper-osmotic agent such as 100% anhydrous glycerol. Further investigation on the reversal process was performed without any application of 0.9% saline. The third study identified the variation in fluorescence in hamster dorsal skin tissue and enucleated porcine eyes with temperature. This study investigated the variation in fluorescence intensity with temperatures starting at 14°C and compared in vivo and in vitro results for consistency. The fourth study investigated an implantable drug delivery package that was fabricated using PMMA and implanted between the sub-conjunctival and super-scleral space and release the content of the device by either mechanical pressure or light-activated ophthalmic Nd:YAG laser after optically clearing the eye tissue by topical application of a hyper-osmotic agent, 100% anhydrous glycerol. A hyper-osmotic agent creates a transport region in the conjunctiva and sclera to get visual access of the compartments in the drug delivery package. This new technology would provide the option to the patient of one time implantation of the carrier system containing the drug. Each time the patient requires medication a ND-YAG or other laser beam will propagate through the cleared eye tissue to release the drug in measurable doses at the discretion of the doctor from the package directly in to the vitreous humor. In this study we have measured half-life of the dye in the vitreous humor or posterior chamber and biocompatibility. The last study had drawn distinction between the fluorescence signals based on the location (anterior or posterior chamber) of the 10% Na fluorescence dye in the in vivo rabbit and ex vivo pig eyes. / text
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Turgor regulation in species of Vaucheria (Xanthophyceae, Heterokontophyta) from habitats of contrasting salinitiesMuralidhar, Abishek January 2014 (has links)
Turgor regulation is the process by which walled organisms alter their internal osmotic potential to adapt to osmotic changes in the environment. Much of what we know regarding turgor regulation and osmotic adjustment in algae is limited to the green characean and chlorphytan algae. This thesis is an investigation of turgor regulation in two species of the yellow-green xanthophycean alga, Vaucheria.
The first part of this study involved the collection and identification of species of Vaucheria from contrasting habitats in New Zealand. Seven species of Vaucheria were identified based on the morphology of their reproductive structures. Two were described as new species (V. aestuarii and V. edaphica) and two others were reported for the first time from New Zealand (V. erythrospora and V. litorea). The genetic variation and phylogenetic position of these species were studied using phylogenetic analyses of rbcL sequences.
Two of the species from contrasting habitats were selected for a comparative study on turgor regulation. These were Vaucheria erythrospora, isolated from an estuarine habitat, and Vaucheria repens, isolated from a freshwater habitat. Using a single cell pressure probe to directly measure turgor after hyperosmotic shock, V. erythrospora was found to recover turgor after a larger shock than V. repens. Threshold shock values for this ability were > 0.5 MPa for V. erythrospora and < 0.5 MPa for V. repens. Recovery was more rapid in V. erythrospora than V. repens after comparable shocks. Growth studies showed that V. erythrospora was able to grow and maintain turgor over a wider range of NaCl concentrations. These responses are thought to underlie the ability of V. erythrospora to survive in an estuarine habitat and restrict V. repens to freshwater.
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The final part of this study investigated the mechanisms underlying turgor regulation in V. erythrospora. Different responses were observed depending on whether NaCl or sorbitol was used to elicit the shock. Membrane potential (Em) measurements showed a rapid depolarization of the plasma membrane in response to a NaCl-induced hyperosmotic shock, followed by a slower repolarization, and recovery almost back to the resting Em. MIFE recordings indicate a net K+ efflux, a response that has been reported in other systems. While recordings of Na+ fluxes were not possible due to the high external Na+, these may account for the depolarisation and recovery of turgor as turgor recovery was inhibited by the non-selective cation channels (NSCCs) inhibitor Gd3+ and was dependant on the external Na+ concentration. An equivalent sorbitol-induced hyperosmotic shock hyperpolarized the Em, followed by depolarization and recovery back to the resting Em. Net flux recordings showed that both K+ and Na+ were taken up in response to a sorbitol shock when there was a low external Na+ concentration (1mM). K+ was possibly taken up through inward rectifying K+ channels activated by membrane hyperpolarization. The ability of V. erythrospora to rapidly regulate turgor by taking up ions during hyperosmotic stress is the possible reason for its survival in an estuarine habitat.
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Role of ion channels in programmed cell death induced by hyperosmotic stresses in plant cells / Rôle des canaux ionique dans la mort cellulaire induit par stress osmotiqueMonetti, Emanuela 17 November 2014 (has links)
Le travaux présenté dans cette thèse concerne le rôle des canaux ioniques de la membrane plasmique en réponse à des stress salins et non salins ainsi qu’aux interactions possibles avec d’autres événements de signalisation conduisant à la mort cellulaire programmée (PCD). Nous avons montré que les réponses cellulaires précoces: tels que l`augmentation du calcium cytosolique et la production de ROS, classiquement impliqués lors de la PCD, ne semblaient pas être impliqué dans la mort cellulaire induite par les stress hyperosmotiques chez les cellules en culture de tabacco BY2 ou d’A. thaliana. Nous avons montré que, dans les cas de stress salin chez les cellules de BY2 un influx précoce de sodium à travers des canaux cationiques non spécifiques participe au développement de la PCD en entraînant un disfonctionement mitochondrial et la production de O2• - par des NADPH oxydases. Dans le cas de stress hyperosmotique non-ionique, nous avons observé une diminaution précoce de l’intensité des courants anioniques. Afin de poursuivre l’étude du rôle des canaux anioniques lors du stress hyperosmotique non salin, nous avons utilisé des cellules A.thaliana nous permettant de travailler avec le mutant de canal anionique SLAC1. Nous avons constaté que l’activation retardée des canaux SLAC1 participait au développement de la PCD induite par un stress hyperosmotique non salin. La réduction précoce de l'activité des canaux anioniques pourrait participer à la signalisation ou l'ajustement osmotique permettant l'adaptation et la survie cellulaire alors que des évènements retardés, à savoir la production d'anion superoxyde (O2• -) par les NADPH-oxydases et l'activation des canaux anioniques pourraient participer au développement de la PCD d'une partie de la population cellulaire. Nous avons aussi étudié le rôle potentiel des petits peptides appartenant à la famille des peptides FMRFamide décrite chez les métazoaires à l'osmorégulation chez des cellules d’A. thaliana. Des génes susceptibles de coder de tels peptides sont en effet présent dans le génome d’A. thaliana. En utilisant des peptides synthétiques, nous avons montré que ces FLPS putatifs pourraient participer aux réponses induites losr de stress hyperosmotique chez les plantes. Ce travail illustre la complexité et l'importance de la régulation des canaux ioniques dans les voies de signalisation et les processus conduisant à la PCD / The work presented in the present thesis relates to the role of ion channels in response to (ionic and non-ionic) hyperosmotic stresses and their interactions with signaling events leading to PCD in plant. Early cell responses such as cytosolic calcium increase and ROS production classically involved in PCD process, seems not to be involved in hyperosmotic-induced cell death in BY2 tobacco and A. thaliana cultured cells. When BY2 tobacco cells were subjected to hyperosmotic stress, an early influx of sodium through non-selective cation channels participates in the development of PCD through mitochondrial dysfunction and NADPH-oxidase-dependent O2•– generation. On the contrary, non-ionic hyperosmotic stress resulted in an early decrease in anion currents. To further investigate the role of anion channels in non-ionic hyperosmotic stress further experiments were conducted by using A.thaliana cells of the anion channel mutant SLAC1. Results showed that the delayed activation of SLAC1 channels was involved in the non-ionic hyperosmotic stress induced pathway leading to cell death. Interestingly, the early anion channel activity decrease could participate to signalisation or osmotic adjustment allowing cell adaptation and survival, when a second set of events, namely superoxide anion (O2•-) generation by NADPH-oxidase and anion channel activation could participate in PCD development of a part of the cell population. In addition, the potential role of small peptides belonging to the FMRFamide-like peptide (FLP) family described in metazoan in osmoregulation in A. thaliana was investigated. By using synthetic peptides, based on FLPs homolog genes existing in A. thaliana, it was possible to demonstrate that these putative FLPs are involved in hyperosmotic stress response. Overall, the present work shed light on the importance and the complexity of ion channels regulation in the signaling pathways and the processes leading to PCD
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Vias de sobrevivência e morte em queratinócitos submetidos ao estresse oxidativo e choque hiperosmótico / Survival and death signaling pathways in keratinocytes exposed to oxidative stress and hyperosmotic shockSilva, Rodrigo Augusto da 18 August 2018 (has links)
Orientador: Giselle Zenker Justo / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T14:42:19Z (GMT). No. of bitstreams: 1
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Previous issue date: 2011 / Resumo: A epiderme é constantemente confrontada por inúmeros agentes estressores. Variações na umidade ou exposição à radiação ultravioleta afetam o balanço osmótico e o estado redox celular alterando, assim, as características fisiológicas da pele. Em resposta aos diferentes estímulos os queratinócitos ativam vias distintas de sinalização. Portanto, o balanço entre as vias de sobrevivência e morte determina o destino celular. A fim de se determinar possíveis alvos moleculares associados a morte e sobrevivência de queratinócitos, vias de sinalização celular disparadas pela exposição ao choque hiperosmótico e estresse oxidativo foram investigadas em células HaCaT tratadas com sorbitol e peróxido de hidrogênio (H2O2) respectivamente. Os resultados obtidos neste estudo demonstraram que, em ambos os modelos, a redução da viabilidade celular dependeu da dose e do tempo de exposição ao agente extressor, apresentando valores de IC50 de aproximadamente 1 mol/L de sorbitol e 2 mmol/L de H2O2 após 2 e 4 h de exposição respectivamente. Os danos causados foram irreversíveis e estão associados à ativação da via intrínseca de morte celular apoptótica, acompanhada de perda da integridade da membrana lisossomal, extravasamento de catepsina B para o citosol e alterações morfológicas atípicas no citoesqueleto, principalmente no arranjo dos filamentos de actina. A investigação do status de funcionamento de proteínas quinases ativadas por mitógenos (MAPKs) e do estado redox celular indicou que esses eventos foram mediados por espécies reativas de oxigênio e pela ação da quinase c-Jun N-terminal (JNK). Adicionalmente, a exposição dos queratinócitos aos diferentes estímulos estressores foi acompanhada de ativação da proteína tirosina fosfatase de baixa massa molecular (LMWPTP), cuja relevância nos estudos de biologia celular aumentou nos últimos anos. A LMWPTP atua em importantes vias de sinalização que estão associadas à sobrevivência e morte celular. Cientificamente, este estudo é pioneiro ao demonstrar alterações no citoesqueleto e ação de proteínas quinases e fosfatases nos mecanismos que determinam o destino de queratinócitos expostos ao choque hiperosmótico e ao estresse oxidativo. De fato, o melhor conhecimento da relação entre as vias de sobrevivência e morte celular em queratinócitos é fundamental para promover o desenvolvimento de novas estratégias terapêuticas aplicadas às doenças dermatológicas. Desta maneira, o presente trabalho apresenta resultados inéditos, contribuindo no conhecimento da biologia dos queratinócitos e com sua aplicação no desenvolvimento da terapia dermatológica / Abstract: The epidermis is constantly confronted with multiple environmental stressors. Changes in humidity or exposition to UV radiation affect the redox state and osmotic balance, modifying the physiological characteristics of the skin. In response to different stresses, epidermal keratinocytes can activate distinct signaling pathways and the balance between death and life signals will determine the cell fate, leading to programmed cell death or cell survival. In order to determine the possible molecular targets associated to death and survival of keratinocytes, the signaling pathways activated by the exposition of HaCaT cells to sorbitol (hyperosmotic shock) and H2O2 (oxidative stress) were investigated. The results showed that in both models the reduction in cellular viability was time and dose-dependent, displaying IC50 values of 1 mol/L for sorbitol and 2 mmol/L for H2O2 after 2 and 4 h of exposition to the stressors, respectively. The damages caused by the stressors were irreversible and associated to the induction of the intrinsic apoptotic pathway, accompanied by the loss of lisosomal membrane integrity, release of cathepsin B to cytosol and atypical morphological alterations in cytoskeleton, particularly in the arrangement of actin filaments. Analysis of the functional status of mitogen-activated protein kinases (MAPKs) and the cellular redox state showed that such events were mediated by reactive oxygen species and occurred through c-Jun N-terminal kinase (JNK) activation. Additionally, exposure of keratinocytes to the different stress inducers was followed by low molecular weight tyrosine protein phosphatase (LMWPTP) activation, which is responsible for the regulation of important signaling pathways associated to cell survival and death. It is important to highlight the novelty of these results showing alterations in the cytoskeleton and the action of protein kinases and phosphatases during exposure of keratinocytes to hyperosmotic and oxidative stresses. In fact, the development of more efficacious therapies against skin diseases depends on the establishment of the relationships between the survival and death signaling pathways in keratinocytes. In this direction, this work contributes to a better understanding of the keratinocyte biology and the improvement of traditional dermatological therapies / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular
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A Model for Studying Vasogenic Brain EdemaShukla, Anshu 01 January 2006 (has links)
Convection-enhanced delivery (CED) is a proven method for targeted drug delivery to the brain that circumvents the blood-brain barrier (BBB). Little study has been conducted in understanding CED in pathological brain states. This is of importance when dealing with chemotherapeutic agent delivery to brain tumors, where vasogenic edema (VE) exists. The current study aims to characterize a model of VE suitable for studying CED.VE was produced in the right hemisphere of the rat brain using multiple infusions of hyperosmotic mannitol (0.25mL/kg/s over 30 seconds) delivered through the right internal carotid artery. Magnetic resonance imaging (MRI) revealed consistent edema formation and high water levels in the ipsilateral gray and white matter within an hour of the first infusion. Evan's Blue (EB) staining verified that VE has formed. However, apparent diffusion coefficient (ADC) and histological examination revealed also that some possible cytotoxic edema formed.This model provides a reproducible technique for generating a large area of edema for CED study. Further studies with lower doses of mannitol, while titrating to changes in ADC and values for fractional water content, may modify this model with a greater component of VE and less cerebral toxicity.
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The Role and Regulation of the Exchange Factor GEF-H1 in Tubular CellsWaheed, Faiza 01 September 2014 (has links)
The Rho family small GTPases are key regulators of the cytoskeleton, through which they impact and control many vital cellular functions, including growth, vesicle trafficking, intercellular junctions, transepithelial transport, migration, and gene transcription. Activation of Rho GTPases is induced by Guanine Nucleotide Exchange Factors (GEFs). We have previously shown that Tumour Necrosis Factor-α (TNF), plasma membrane depolarization, and immunosuppressive drugs activate RhoA through a specific exchange factor, GEF-H1. However, the question of whether other stimuli, such as hyperosmolarity, that activate RhoA, act through GEF-H1 and whether GEF-H1 activates other RhoGTPases was not known.
The overall objective of this research project has been to gain insights into the complex mechanism through which the Rho GTPases, Rac and RhoA, are regulated in tubular cells. Specifically, we wished to explore the role and pathway-specific regulation of GEF-H1 in hyperosmotic stress- and TNF-induced signalling in tubular cells.
In order to accomplish our goals, we optimized and used affinity precipitation assays to detect GEF-H1 activation (RhoA(G17A) and Rac(G15A)). We found that 1) GEF-H1 is activated by hyperosmotic stress and mediates the hyperosmolarity-induced RhoA activation, as well as nuclear translocation of the Myocardin-Related Transcription Factor (MRTF); 2) TNF induces activation of both Rac and RhoA through GEF-H1, but via different mechanisms. Epidermal Growth Factor Receptor (EGFR)- and Extracellular signal Regulated Kinase (ERK)-dependent phosphorylation at the Thr678 site of GEF-H1 is a prerequisite for RhoA activation only, while both Rac and RhoA activation require GEF-H1 phosphorylation on Ser885. Interestingly, Rac is required for TNF-induced RhoA activation.
Together these findings highlight a role for GEF-H1 as an osmosensitive molecule that regulates cellular reprogramming through MRTF. Importantly, we have also uncovered a novel mechanism explaining hierarchical activation of Rac and RhoA by TNF. Such a mechanism could be key in coordinating GEF function and fine-tuning Rac and RhoA activation.
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Studium odolnosti bakterií vůči vybraným stresovým faktorům / Study on resistance of bacteria to selected stress factorsMiléřová, Miluše January 2016 (has links)
The aim of the master thesis was to study the effect of the accumulation of polyhydroxyalkanoates (PHA) for bacterial resistance to selected stress factors. In the theoretical part the selected stress factors, polyhydroxyalkanoates and the involvement of polyhydroxyalkanoates into stress response of bacteria were reviewed. In the experimental part we used bacteria Cupriavidus necator H16 and its mutant strain Cupriavidus necator H16/PHB-4 unable of polyhydroxybutyrate (PHB) accumulation. The resistance of above-mentioned bacterial strains against thermal and osmotic stress was tested. According to the results of the experiment, when the bacteria were exposed to three different concentrations of NaCl (50, 100 and 200 g/l) PHB accumulating strain showed a higer resistance to hyperosmotic stress than the strain unable of PHB accumulation. There was demonstrated with Raman spectroscopy that in the hyperosmotic environment induced crystallization of the intracellular PHB granules. Transmission electron microscopy indicated that strain Cupriavidus necator H16/PHB-4 is subject to plasmolysis during hyperosmotic stress. As a consequence the hyperosmomotic stress occurs to the aggregation intracellular PHB granules in strain Cupriavidus necator H16 but there is no plasmolysis or is much less intensive.
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