Transport of polymeric immunoglobulin A (plgA) in rat salivary glands has been investigated by combined morphological and biochemical techniques in vivo and in vitro. The distribution of IgA and its cellular receptor secretory component (SC) was observed by immunoperoxidase staining of cryosections from parotid and submaxillary gland, showing serous acinar cells are the site of IgA transport into saliva. Binding of horse radish peroxidase specific IgA to parotid serous acinar cells in vitro, observed by electron microscopy, shows that only the basolateral domain of acinar cells possesses exposed SC. A combination of new cell fractionation methods and standard western blotting techniques shows that SC present on basolateral plasma membrane of parotid acinar cells has a molecular weight (mwt) >100,000 and shows a high affinity for plgA in vitro. The existence of a 73,000 mwt SC occurring with plgA in cellular fractions of parotid gland suggest cleavage of SC occurs prior to secretion. The kinetics of plgA trancytosis was studied using isolated parotid acini. Bound plgA was secreted into the incubation medium as slgA, within thirty minutes of incubation at 37°C. Secretion of plgA was initially rapid but slowed over a 2hr period of incubation at 37°C. In addition to facilitating plgA transport serous acinar cells also synthesise and secrete a diverse range of other salivary proteins which are packaged into secretion granules and secreted directly through the apical plasma membrane. It is improbable that one complex secretory pathway facilitates both bulk secretion of salivary protein and transport of plgA. Therefore secreted proteins must be selectively segregated during secretion into saliva. Secretion of proteins from acinar cells in vitro shows proteins are released at two distinct rates.

The aim of the present study was to characterize the phenomenon of resistance in putative preneoplastic hepatocyte nodules. These hyperplastic nodules are generated during the development of liver cancer in response to chemical carcinogens, and comprise a population of cells from which hepatocellular carcinoma can develop. As hepatocyte nodules grow in an environment that is otherwise toxic they possess a resistant phenotype. To understand this resistance phenomenon at the biochemical level, several phase I and II drug-metabolizing enzymes in the nodules were examined. Initial experiments were carried out in rats with nodules produced by initiation with diethylnitrosamine, followed by selection with 2-acetylaminofluorene and carbon tetrachloride. These nodules showed a large decrease in phase I enzymes and enzymic activities, such as the cytochromes P-450, cytochrome b[5], total microsomal haem, aminopyrine N-demethylase and ethoxyresorufin 0-deethylase, but glutathione and the phase II enzymes, namely, glutathione S-transferase, UDP-glucuronyl transferase, DT-diaphorase and gamma-glutamyltransferase were significantly increased. The pattern of changes of these drug-metabolizing enzymes of the nodules was similar when the nodules were produced by different initiation-promotion treatments, including diethylnitrosamine plus a choline/methionine-deficient diet, 2-acetamidofluorene plus phenobarbi-tone, or diethylnitrosamine plus orotic acid. In addition, the resistance phenotype was maintained when these nodules were transferred into the spleen of a rat not exposed to chemical carcinogens, and allowed to grow for several months, thus indicating that the newly acquired biochemical pattern in the nodules had become constitutive. Unlike the hepatic nodules generated by previous initiation-pro-motion treatments, nodules generated by the hypolipidemic agent, ciprofibrate, exhibited only a decrease in phase I components of the drug-metabolizing enzymes, with no increase in the phase II components. Similarly, hyperplastic nodules in liver mouse showed a decrease in phase I components, but no increase in phase II components. In addition to cytochrome P-450 and cytochrome b5, the total haem and two other haem containing proteins, namely, catalase and tryptophan 2,3-dioxygenase were also decreased in the nodules. A deficiency in hepatic iron, and a decrease in the activity of delta-ALA-synthetase, the first rate limiting enzyme in haem synthesis, were also apparent. Characterization of the phase II components revealed the presence of a new glutathione-S-transferase polypeptide, which has been shown to be identical to a placental form of the transferase. This polypeptide, although present to a minimal extent, or absent, in normal rat liver, is present in normal male mouse liver. Administration of lead nitrate to rats induces a biochemical pattern in the liver similar to that seen in the hepatocyte nodules, including a decrease of phase I components and an increase in phase II components of the drug-metabolizing enzymes, and the induction of the novel glutathione S-transferase. Further studies with lead nitrate may yield new insights into the mechanisms of production of the biochemical changes induced in the nodules, as this agent generates the same changes within 30 hours. Furthermore, the lead nitrate-induced changes in phase I and phase II enzymes are reversible, while the changes seen in the hyperplastic nodules are not. Thus this study has characterized one pattern of biochemical changes exhibited by the resistant phenotype of hyperplastic hepatic nodules, and a model system has been developed which induces the same changes, more rapidly and in a reversible fashion. One of the important questions yet to be answered however is the biological significance of the resistant phenotype in cancer development. Is the acquisition of resistance only important in expanding the initiated cell population to generate nodules or does it also have a more direct role in the progression of nodules to cancer? This is highly relevant to the clarification of the carcinogenic process in the liver and perhaps in other organs as well.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Anna Beránková Supervisor: PharmDr. Jana Pourová, Ph.D. Title of diploma thesis: The in vitro effects of selected substances on isolated rat aorta Flavonoids are a numerous group of secondary plant metabolites. Flavonoid compounds are substances widely extended in nature and many of them have a positive influence on human health, primarily for their vasodilatory, antioxidant and anti- inflammatory effects. Three substances were selected for this diploma theses: two substances of isoflavonoid group, genistin and genistein, and the end product of genistein metabolism, 4-ethylphenol. The aim of this work is examination of vasorelaxant effects of this substances in vitro. Vasorelaxing potential of tested substances was tested in vitro in isolated aortic rings of Wistar rat. The effect of increasing doses of individual substances in precontracted aortic rings with intact endothelium was measured. From the obtained values of vessel tension, the DRC curves and EC50 values were created. The results were evaluated. The results analysis shows, that genistein (EC50 2,903.10-5 M) had the most significant activity. Also genistin (EC50 4,045.10-4 M) and high doses of 4-ethylphenol (EC50 1,509.10-3 M) caused...

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1. In these studies the role of the sympathetic nervous system (SNS) has been investigated in two models of renovascular hypertension in the rat and after surgical reversal. 2. Plasma noradrenaline (NA), heart rate and blood pressure (BP) were measured simultaneously in conscious rats with one-kidney, one-clip (1K1C) (4-6 weeks) and two-kidney, one-clip (2K1C) (4-6 and >16 weeks) hypertension, and parallel loose clip controls, before and 48 hours after unclipping. BP in all hypertensive groups fell to normal after unclipping. 3. Plasma NA was elevated in 1K1C hypertension and fell on unclipping. Conversely, in early 2K1C hypertension plasma NA was unaltered before and rose after unclipping. Plasma NA did not change with unclipping in chronic 2K1C hypertension and was not different from controls. Heart rate showed a similar pattern. Unclipping loose clip control rats produced no change in BP, plasma NA or heart rate. 4. Heart rate was correlated with plasma NA in 1K1C hypertension, and changes in these variables on unclipping were correlated in all three models. BP was only correlated with plasma hypertensive rats. 5. In contrast, renal and cardiac NA levels showed a remarkably similar pattern in 1K1C and 2K1C hypertension of 4-6 weeks duration. Ipsilateral renal NA was reduced in loose clip rats compared to sham-operated controls. This is most likely due to renal denervation during clipping. Hypertension produced a further reduction in ipsilateral renal NA and a reduction in cardiac and contralateral renal (2K1C) NA. 6. It is concluded that the SNS may have a minor role in 1K1C, but not in 2K1C, renovascular hypertension. However, changes in SNS activity upon reversal of hypertension do not explain the BP fall in either model. Changes in renal and cardiac levels of NA in renovascular hypertension are primarily secondary to sustained elevation of BP.

In the studies described in this thesis the ability of muscarinic and ?1-adrenoceptor, as well as depolarising stimuli to initiate phosphoinositide metabolism in various regions of rat brain were examined. Furthermore, the ability of these stimuli to initiate phosphoinositide hydrolysis in developing brain was observed. Both muscarinic and ?1-adrenoceptor-induced phosphoinositide hydrolysis have marked regional distributions in rat brain. This regional distribution of functional responsiveness seems to correlate reasonably well with measurements of known receptor density. It is also clear that there is no variability in the coupling of both of these receptors in rat brain, thus implying a relationship between the functional responses and receptor occupancy. Pirenzepine appears to be able to differentiate between muscarinic receptor-induced phosphoinositide responses in the hindbrain from those in the forebrain regions. Both elevated K+ and veratrine can initiate phosphoinositide hydrolysis in rat brain. The regional responses to elevated K+ seem, at least, in part to be due to transmitter release, although a role for voltage-sensitive Ca++ channels in such responses is indicated by the effects produced by dihydropyridine Ca++ channel antagonists and activators. Muscarinic and ?1-adrenoceptors show different developmental patterns of phosphoinositide responsiveness. The ?1-adrenoceptor seems to be more efficiently coupled during the first two weeks of postnatal development whereas the muscarinic receptor shows no variability in coupling. Instead, carbachol produces supramaximal responses in young rats. Lithium also potentiates [3H]-InsP1 and [3H]-InsP2 accumulations more in young rats. Moreover lithium produces a time-dependent inhibition of [3H]-InsP3 and [3H]-InsP4 in both young and adult rats. Physostigmine produces no enhancement of the response to elevated K+ in young rats. Furthermore, brain slices from young rats seem to be more sensitive to the Ca++ channel activator BAY-K8644. The significance of these results are discussed in the text.

Orientador: Fernanda Klein Marcondes / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-15T12:47:47Z (GMT). No. of bitstreams: 1 Costa_Rafaela_M.pdf: 755140 bytes, checksum: 92fa87f3ac79484f4ed67811cdc9d446 (MD5) Previous issue date: 2010 / Resumo: Problemas emocionais como ansiedade e depressão, relacionados ao estresse, estão cada vez mais presentes na sociedade moderna, e o suporte social, mais especificadamente suporte familiar, pode exercer um importante papel em atenuar os efeitos de diversos estressores. Em modelos animais o enriquecimento ambiental tem sido utilizado para melhora do bem estar animal. O objetivo deste estudo foi avaliar os efeitos do enriquecimento ambiental por meio da estimulação tátil em ratos submetidos ou não a estresse crônico. No Capítulo I, foi evidenciado que a estimulação tátil diminuiu a ansiedade e aumentou as respostas indicadoras de aprendizado e memória em ratos jovem-adultos. No Capítulo II, foram avaliados os efeitos do estresse crônico moderado e imprevisível e da estimulação tátil sobre respostas comportamentais (ansiedade, anedonia, aprendizado e memória) e sobre o perfil lipídico. O estresse aumentou a secreção de corticosterona avaliada quinze dias após o fim do estresse; induziu anedonia evidenciada pela diminuição da preferência pela sacarose 1%; aumentou a atividade locomotora; teve efeito negativo sobre o aprendizado e memória; e aumentou a concentração sérica de triglicerídeos, colesterol total e lipoproteína de baixa densidade (LDL). A manipulação diminuiu a ansiedade em animais submetidos ou não ao estresse crônico; diminuiu a secreção de corticosterona induzida pelo estresse e cancelou a redução do aprendizado e retenção de memória induzida pelo estresse crônico. Os resultados obtidos mostram que a estimulação tátil de ratos adultos jovens produziu efeitos comportamentais positivos que podem melhorar o bem-estar animal e diminuir efeitos deletérios induzidos pelo estresse crônico. / Abstract: Emotional problems such as stress related anxiety and depression, are becoming increasingly present in modern society, and social support, more specifically familiar support, can play an important role in attenuating the effects of various stressors. In animal models environmental enrichment has been used to improve animal welfare. The aim of this study was to evaluate the effects of environmental enrichment by handling, in rats submitted and those not submitted to chronic stress. In chapter 1 it was shown that handling diminished anxiety, and enhanced learning abilities and memory indicating response in young-adult rats. In chapter 2 the effects of handling on behavioral (anxiety, ahnedonia, learning and memory) and metabolic responses induced by chronic mild unpredictable stress. Whereas stress raised the corticosterone secretion evaluated fifteen days after the end of stress; induced ahnedonia evidenced by a 1% decrease in sucrose preference; increased locomotor activity; had negative effects on learning and memory; and raised the serum concentration of triglycerides, total cholesterol and low density lipoprotein (LDL). Handling reduced the anxiety in animals both when they were and were not submitted to chronic stress; diminished the corticosterone secretion induced by the stress and cancelled the reduction of learning and memory retention induced by the chronic stress. The results obtained showed that the handling of young-adult rats produced positive behavioral effects capable of improving the animal's welfare and diminishing the deleterious effects induced by chronic stress. / Mestrado / Fisiologia Oral / Mestre em Odontologia

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The consequence of a folate deficiency during organogenesis has been investigated in the rat embryo. In vitro culture of 9.5 day embryos in serum from dietary induced folate deficient rats frequently resulted in abnormal embryos. Many were growth retarded and exhibited a defect in the turning mechanism which inverts the embryo from ventrally to dorsally convex. Affected embryos displayed abnormal twisting or kinking of the neural tube. Gross anaemia was also frequently observed and the protein content of the embryos was markedly less than that of embryos grown in normal rat serum. Supplementation of the deficient serum with folic acid improved growth and greatly reduced the occurrence of deformities. It virtually eliminated the incidence of gross anaemia but only partially restored the protein content to the level observed in embryos cultured in normal rat serum. The effects of the folate deficiency could not be reversed by supplementation with multivitamins or by increasing the volume of culture serum. They were, however, eliminated by supplementing the deficient culture serum with normal rat serum. The effects could also be overcome by in vivo folate supplementation; rats which had previously been so folate deficient that culture in their serum would have resulted in malformed embryos, after restoration to a folate supplemented diet produced serum which supported completely normal embryonic growth. The results indicate that embryos undergoing organogenesis require adequate folate in order for normal growth and differentiation to take place. They also suggest that some of the embryopathic effects of maternal folate deficiency are mediated by secondary effects. These may involve complex growth or metabolic factors which can be corrected in vivo but are not readily reversed in vitro.

Immobility causes joint contractures, loss in range of motion (ROM), notably in elderly and bed-ridden patients. In a rat knee immobilization flexion contracture (FC) model, the posterior capsule contributes to irreversible limitation of ROM. Through microarray, extracellular matrix and collagen pathways were identified as differentially expressed in the posterior capsule of knees with FC. We hypothesized that intra-articular injection of collagenases in rats with knee FC will interfere with collagen in the capsule and allow increased ROM. After four weeks of hind-limb immobilization, rats develop knee FC; two weeks of remobilization with collagenase treatment showed increased ROM compared to buffer injected knees of 8.043° (p-value=0.046). Histological analysis of knee sections revealed changes in collagen content of the extracellular matrix in posterior capsule. In vitro incubation of rat capsules with collagenases confirmed changes in collagen. Along with current rehabilitation methods, treatment with collagenase may augment ROM recovery from knee joint contractures.