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Role of intestinal circadian clock in epithelial transport, proliferation, and tumourigenesis
Soták, Matúš ; Pácha, Jiří (advisor) ; Bendová, Zdeňka (referee) ; Herichová, Iveta (referee)
AABBSSTTRRAACCTT The molecular circadian clock enables anticipation of environmental changes. In mammals, clocks are ubiquitously present in almost all tissues and they are comprised of transcriptional-translational feedback loops of the so-called clock genes. The central clock represents the intrinsic pacemaker which is located in suprachiasmatic nuclei (SCN) of hypothalamus and synchronizes peripheral clocks. Clockwork system in alimentary tract and its regulatory link to intestinal functions are poorly understood. Therefore the objective of the thesis was to characterize molecular clock in particular parts of the rat intestine and to elucidate its link to the intestinal transport, regulation of cell cycle and neoplastic transformation in colonic tissue. We used quantitative RT-PCR (qPCR) to determine circadian profiles of mRNA expression of clock genes in the epithelium of duodenum, jejunum, ileum, and colon of rat. Furthermore, we analysed the expression of genes coding sodium chloride transporters and channels as well as cell cycle regulators in colon. To focus more precisely on different structures of intestinal epithelia we used laser capture microdissection. In addition, we performed Ussing chamber measurements to determine the colonic electrogenic transport. To study the contribution of circadian...
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Role of intestinal circadian clock in epithelial transport, proliferation, and tumourigenesis
Soták, Matúš
AABBSSTTRRAACCTT The molecular circadian clock enables anticipation of environmental changes. In mammals, clocks are ubiquitously present in almost all tissues and they are comprised of transcriptional-translational feedback loops of the so-called clock genes. The central clock represents the intrinsic pacemaker which is located in suprachiasmatic nuclei (SCN) of hypothalamus and synchronizes peripheral clocks. Clockwork system in alimentary tract and its regulatory link to intestinal functions are poorly understood. Therefore the objective of the thesis was to characterize molecular clock in particular parts of the rat intestine and to elucidate its link to the intestinal transport, regulation of cell cycle and neoplastic transformation in colonic tissue. We used quantitative RT-PCR (qPCR) to determine circadian profiles of mRNA expression of clock genes in the epithelium of duodenum, jejunum, ileum, and colon of rat. Furthermore, we analysed the expression of genes coding sodium chloride transporters and channels as well as cell cycle regulators in colon. To focus more precisely on different structures of intestinal epithelia we used laser capture microdissection. In addition, we performed Ussing chamber measurements to determine the colonic electrogenic transport. To study the contribution of circadian...
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Role of intestinal circadian clock in epithelial transport, proliferation, and tumourigenesis
Soták, Matúš
AABBSSTTRRAACCTT The molecular circadian clock enables anticipation of environmental changes. In mammals, clocks are ubiquitously present in almost all tissues and they are comprised of transcriptional-translational feedback loops of the so-called clock genes. The central clock represents the intrinsic pacemaker which is located in suprachiasmatic nuclei (SCN) of hypothalamus and synchronizes peripheral clocks. Clockwork system in alimentary tract and its regulatory link to intestinal functions are poorly understood. Therefore the objective of the thesis was to characterize molecular clock in particular parts of the rat intestine and to elucidate its link to the intestinal transport, regulation of cell cycle and neoplastic transformation in colonic tissue. We used quantitative RT-PCR (qPCR) to determine circadian profiles of mRNA expression of clock genes in the epithelium of duodenum, jejunum, ileum, and colon of rat. Furthermore, we analysed the expression of genes coding sodium chloride transporters and channels as well as cell cycle regulators in colon. To focus more precisely on different structures of intestinal epithelia we used laser capture microdissection. In addition, we performed Ussing chamber measurements to determine the colonic electrogenic transport. To study the contribution of circadian...
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Role of intestinal circadian clock in epithelial transport, proliferation, and tumourigenesis
Soták, Matúš ; Pácha, Jiří (advisor) ; Bendová, Zdeňka (referee) ; Herichová, Iveta (referee)
AABBSSTTRRAACCTT The molecular circadian clock enables anticipation of environmental changes. In mammals, clocks are ubiquitously present in almost all tissues and they are comprised of transcriptional-translational feedback loops of the so-called clock genes. The central clock represents the intrinsic pacemaker which is located in suprachiasmatic nuclei (SCN) of hypothalamus and synchronizes peripheral clocks. Clockwork system in alimentary tract and its regulatory link to intestinal functions are poorly understood. Therefore the objective of the thesis was to characterize molecular clock in particular parts of the rat intestine and to elucidate its link to the intestinal transport, regulation of cell cycle and neoplastic transformation in colonic tissue. We used quantitative RT-PCR (qPCR) to determine circadian profiles of mRNA expression of clock genes in the epithelium of duodenum, jejunum, ileum, and colon of rat. Furthermore, we analysed the expression of genes coding sodium chloride transporters and channels as well as cell cycle regulators in colon. To focus more precisely on different structures of intestinal epithelia we used laser capture microdissection. In addition, we performed Ussing chamber measurements to determine the colonic electrogenic transport. To study the contribution of circadian...
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Úloha osmotických a iontových činidel ve fyziologii rybích spermií
BONDARENKO, Olga
The present study focused on clarification of the roles of environmental osmolality and ion composition in the initiation of spermatozoa motility in different freshwater fish species. Additionally, the role of osmotic and ionic agents in sturgeon spermatozoa maturation was investigated. A first aim of the present study focused on studies of osmotic regulation of spermatozoa volume alterations in different fish species. This led to the discovery that spermatozoa swelling are species-specific: fish sperm with ionic mode of motility activation (sturgeon, trout) has no capacity for swelling, In contrast to carp sperm (osmotic mode of activation) for which volume correlates with environmental osmolality. A second aim was to apply the capacity for swelling in carp spermatozoa to improve the cryopreservation procedure relative to post-thaw sperm quality. Post-thaw motility parameters and ability to fertilize eggs were measured and compared with non-treated sperm. As a result, higher post-thaw motility rate, spermatozoa velocity, and fertilization rate were observed in sperm pretreated with hypotonic solutions compared to non-treated sperm. Thus, our results demonstrate for the first time, that spermatozoa ability for hypotonic changes could be used for improvement of cryopreservation procedure. A third aim of this study was the investigation of the role of environmental osmolality and ion composition in fish sperm motility. Sturgeons and salmonids sperm was activated in media of differing ionic and osmotic composition. Even though environmental Ca2+ concentration is not crucial in conditions of Na+ presence, a minimal intracellular free Ca2+ concentration should be present in sturgeon spermatozoa for initiation of motility. Alteration of osmolality does not seem to play a major regulatory role in trout spermatozoa activation. Alteration of sperm sensitivity to Ca2+ was detected during spawning, according to seasonality. Low motility rate was detected in brook trout spermatozoa at the end of the spawning period. However, a Ca2+ concentration increase up to 10 mM in activating media led to activate motility in at least 85% of the spermatozoa, which suggests a decrease of sperm sensitivity to Ca2+ at the end of the spawning season. The fourth aim was the study of the role of osmotic and ionic agents in sterlet spermatozoa maturation. Our results demonstrate that sturgeon testicular spermatozoa lack capability for motility activation and fertilization. During spawning, sperm is released from testis, pass through the kidney where it is mixed with urine, into the Wolffian duct. We demonstrated for the first time, that testicular sperm incubation with urine controls in vivo maturation, but can be performed in vitro as well (Chapter 6). We detected that sperm maturation does not occur under conditions of Ca2+ deficiency, suggesting that Ca2+ plays an essential role in this process (Chapter 7). During this maturation process, we observed a gradual decrease of spermatozoa sensitivity to environmental Ca2+. We found that testicular spermatozoa motility could be activated only when the Ca2+ concentration is high. Thus, we hypothesize that maturation process might be controlled by Ca2+ influx into the cell with subsequent loading of some Ca2+ stores. To our knowledge, these results provide, for the first time, evidence for the presence of Ca2+ stores in sturgeon spermatozoa. As a conclusion, the results of this study shed light on osmotic and ionic regulation of sperm motility in different fish species. The involvement of specific ions together with osmotic shock in fish spermatozoa maturation and aging were clarified. These findings broaden the possibilities of in vitro sperm manipulation in fisheries practice such as the use of sturgeon testicular spermatozoa, improvement of poor quality sperm in salmonids at the end of spawning season, and increase of carp sperm cryoresistance.
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