O Orlistat é um membro de uma classe de drogas usadas como tratamento para obesidade. No entanto, a segurança de seu uso em longo prazo ainda não é conhecida. O Orlistat exerce sua atividade no lúmen do trato gastrintestinal inibindo a enzima lipase pancreática, responsável pela hidrólise dos triacilglicerídeos normalmente ingeridos com a dieta. Esse medicamento, quimicamente sintetizado, é um derivado da lipstatina, inibidora natural da lipase produzida pelo Streptomyces toxytricini. Assim, a excreção de gordura fecal fica significativamente aumentada com o uso do Orlistat. Estudos epidemiológicos e em modelos experimentais, sugerem que o aumento das dietas hipergordurosas tem efeito promotor para o câncer colorretal. Tal efeito deve ser relacionado, pelo menos parcialmente, às mudanças intra-colônicas causadas pela ação direta da gordura nas células da mucosa do cólon, os colonócitos. O estudo atual tem como objetivo verificar os efeitos do Orlistat na formação colônica de focos de criptas aberrantes (FCA) e na proliferação celular epitelial da mucosa gastrintestinal. Ratos Wistar machos receberam dieta padrão ou dieta com aumento de gordura, suplementada ou não com Orlistat (200 mg/kg), e duas doses semanais do carcinógeno químico Dimetilhidrazina (DMH) (25 mg/kg). Após 30 dias, nos animais tratados com DMH, o Orlistat foi associado a um aumento significativo no número de FCAs colônicos e na proliferação celular epitelial da mucosa do cólon, independentemente da dieta. Os achados obtidos neste trabalho permitem concluir que o aumento do teor de gordura na luz do cólon distal, em decorrência da ação do Orlistat, pode potencializar a ação da DMH na formação de FCAs e no aumento da proliferação celular epitelial da mucosa colônica. / Orlistat is a member of a drug class used as obesity treatment. However, the security of its use in a long period of time is not known yet. Orlistat has its activity in the lumen of the gastrointestinal tract inhibiting the pancreatic lipase enzyme responsible for the hydrolyze of the triglycerides that usually are swallowed with the diet. This drug chemically synthesized, is a derived from lipstatina, that inhibit naturally the lipase produced by Streptomyces Toxytricini. So, the excretion of fat excrement stays significantly increased with the use of Orlistat. Epidemiologic studies and in experimental pattern, suggest that the increase of the high-fat diets facilitate the appearance of the colorectal cancer. Such effect must be related, at least partially to the changes intracolonic caused by the direct action of the fat in the cells of the colon mucous, the colonocytes. The current study has as objective to check the effects of the Orlistat on the formation of rat colonic aberrant crypt foci (ACF) and in the epithelial cell proliferation of the gastrointestinal mucous. Male Wistar rats received either a standard diet or a high fat diet (HFD), supplemented or not with Orlistat (200 mg/kg chow) and two doses of the carcinogen dimethyl-hydrazine (25 mg/Kg). After 30 days, Orlistat was associated to a significant increase in the number of colonic ACFs and cell proliferation in DMH-treated animals, independently of the HFD. The find got in this study permit to conclude that the increase in the level of adiposity inside the distal colon, due to Orlistat action, can potencializar the DMH action in the formation of ACFs and in the increase of epithelial cell proliferation of the colônica mucous.

O Orlistat é um membro de uma classe de drogas usadas como tratamento para obesidade. No entanto, a segurança de seu uso em longo prazo ainda não é conhecida. O Orlistat exerce sua atividade no lúmen do trato gastrintestinal inibindo a enzima lipase pancreática, responsável pela hidrólise dos triacilglicerídeos normalmente ingeridos com a dieta. Esse medicamento, quimicamente sintetizado, é um derivado da lipstatina, inibidora natural da lipase produzida pelo Streptomyces toxytricini. Assim, a excreção de gordura fecal fica significativamente aumentada com o uso do Orlistat. Estudos epidemiológicos e em modelos experimentais, sugerem que o aumento das dietas hipergordurosas tem efeito promotor para o câncer colorretal. Tal efeito deve ser relacionado, pelo menos parcialmente, às mudanças intra-colônicas causadas pela ação direta da gordura nas células da mucosa do cólon, os colonócitos. O estudo atual tem como objetivo verificar os efeitos do Orlistat na formação colônica de focos de criptas aberrantes (FCA) e na proliferação celular epitelial da mucosa gastrintestinal. Ratos Wistar machos receberam dieta padrão ou dieta com aumento de gordura, suplementada ou não com Orlistat (200 mg/kg), e duas doses semanais do carcinógeno químico Dimetilhidrazina (DMH) (25 mg/kg). Após 30 dias, nos animais tratados com DMH, o Orlistat foi associado a um aumento significativo no número de FCAs colônicos e na proliferação celular epitelial da mucosa do cólon, independentemente da dieta. Os achados obtidos neste trabalho permitem concluir que o aumento do teor de gordura na luz do cólon distal, em decorrência da ação do Orlistat, pode potencializar a ação da DMH na formação de FCAs e no aumento da proliferação celular epitelial da mucosa colônica. / Orlistat is a member of a drug class used as obesity treatment. However, the security of its use in a long period of time is not known yet. Orlistat has its activity in the lumen of the gastrointestinal tract inhibiting the pancreatic lipase enzyme responsible for the hydrolyze of the triglycerides that usually are swallowed with the diet. This drug chemically synthesized, is a derived from lipstatina, that inhibit naturally the lipase produced by Streptomyces Toxytricini. So, the excretion of fat excrement stays significantly increased with the use of Orlistat. Epidemiologic studies and in experimental pattern, suggest that the increase of the high-fat diets facilitate the appearance of the colorectal cancer. Such effect must be related, at least partially to the changes intracolonic caused by the direct action of the fat in the cells of the colon mucous, the colonocytes. The current study has as objective to check the effects of the Orlistat on the formation of rat colonic aberrant crypt foci (ACF) and in the epithelial cell proliferation of the gastrointestinal mucous. Male Wistar rats received either a standard diet or a high fat diet (HFD), supplemented or not with Orlistat (200 mg/kg chow) and two doses of the carcinogen dimethyl-hydrazine (25 mg/Kg). After 30 days, Orlistat was associated to a significant increase in the number of colonic ACFs and cell proliferation in DMH-treated animals, independently of the HFD. The find got in this study permit to conclude that the increase in the level of adiposity inside the distal colon, due to Orlistat action, can potencializar the DMH action in the formation of ACFs and in the increase of epithelial cell proliferation of the colônica mucous.

This research was conducted to investigate the expression of porcine intestinal nutrient transporters along the neonatal crypt-villus axis and during the postnatal development. First, we examined the transport kinetics of Na+-glucose co-tranporter 1 (SGLT1) and Na+-dependent neutral amino acid (AA) transporter B0AT1 and then the protein and mRNA abundances of SGLT1, B0AT1 and Na+-dependent neutral AA exchanger ASCT2 along the jejunal crypt-villus axis in the neonatal pig and the potential mechanisms associated with their regulations. Our results suggested that: 1) high levels of apical maximal SGLT1 and B0AT1 uptake activities were shown to exist along the entire jejunal crypt-villus axis in the neonatal pig; 2) there were no significant differences in the SGLT1, B0AT1 and ASCT2 protein abundances in spite of their different mRNA abundances among the crypt-villus axis, suggesting unique posttranscriptional regulatory mechanisms; and 3) global protein translational efficiency, as assessed by examining some of the key protein translational initiation and elongation factors, was higher in the crypt cells than in the upper villus cells, likely playing a regulatory role for maintaining apical nutrient transporter abundances in crypt cells of the neonate. Second, we further examined the protein and mRNA abundances of jejunal neutral AA transporters B0AT1 and ASCT2 and acidic AA transporter EAAC1 during the postnatal development in pigs at the ages of d 1, 4, 6, 12, 20, 28 (1-wk post-weaning), and 70 (mature gut at grower phase), respectively. Our results showed that the jejunal apical B0AT1, ASCT2 and EAAC1 protein abundances were dramatically decreased during the postnatal development and were likely regulated at both the transcriptional and post-transcriptional levels. These substantial decreases in the small intestinal apical Na+-dependent AA transporter abundances may contribute to increased intestinal microbial catabolism of AA, which may be partially responsible for the reduced whole body efficiency of nitrogen utilization during the postnatal growth in pigs. Collectively, our results suggest that apical nutrient transporters SGLT1, B0AT1 and ASCT2 are abundantly expressed along the entire jejunal crypt-villus axis in the neonatal pig, whereas abundances of jejunal apical AA transporters EAAC1, B0AT1 and ASCT2 declined substantially during the postnatal growth in pigs.

Hormone replacement therapy and estrogen replacement therapy have shown the ability to reduce risk of colon cancer development in clinical and animal studies, but in vitro research has been unable to reproduce an estradiol (E2) induced response in colon cancer cell lines. We demonstrated that young adult mouse colonocytes (YAMC, non-malignant colonocytes) exhibit an anti-proliferative response to E2 treatment. These cells demonstrate reduced cell culture growth and increased apoptosis in response to E2. YAMC cells containing an activated Ras mutation are considered to be malignantly transformed, and lose the ability to respond to E2 treatment. Fulvestrant (ICI) was used as an estrogen receptor antagonist to determine that these results were estrogen receptor mediated. Furthermore, this effect was demonstrated to require the presence of ER? through the use of a transgenic ERbeta knockout mouse. In these mice, the presence of E2 significantly reduced the formation of azoxymethane induced premalignant lesions. Since YAMC cells exhibit an anti-proliferative response to E2 treatment, we utilized isogenic YAMC cell lines with and without a dominant negative p53 mutation to demonstrate that this E2 induced action involves p53 activity. E2 treatment results in increased p53 transcriptional activity and a pro-apoptotic change in expression of p53 downstream targets. Presence of the dominant negative p53 mutant nullifies these effects of E2 treatment. The involvement of p53 in the previously described protection against AOM induced premalignant lesions, was investigated using wild type and heterozygous p53 knockout (Het p53KO) mice. The reduction in p53 protein corresponded to reduced effectiveness of E2 treatment on the prevention of premalignant lesion formation in Het p53KO mice. In summary, our data indicate that E2 treatment induces anti-proliferative responses in non-malignant colonocytes and protects against the formation of carcinogen-induced premalignant lesions. These effects require the presence of functional ER? and p53. Further studies are required to more thoroughly elucidate the specific interactions and downstream effects of ER? and p53 in response to E2 stimulation.

Destaca-se a Salmonella como um dos mais importantes patógenos veiculados por alimentos, devido ao fato de estar amplamente distribuído na natureza, possuir um grande número de reservatórios, apresentar sorotipos inespecíficos quanto aos hospedeiros e apresentar cepas multiresistentes aos antimicrobianos. Salmonella Enteritidis tornou-se um grande problema de saúde pública no Brasil, a partir da década de 80, quando foi associada a produtos avícolas. Desde 1962, a Salmonella Heidelberg tem sido isolada de aves e produtos derivados e aves no Brasil e tem reconhecida importância para saúde humana e em poedeiras comerciais na América do Norte. O presente trabalho teve por objetivo o estudo de diferentes cepas de Salmonella Heidelberg (SH) em comparação com Salmonella Enteritidis (SE) em pintinhos e frangos de corte no que diz respeito à capacidade de invasibilidade de órgãos, persistência de excreção fecal e alterações na morfometria intestinal. Para o estudo in vivo, 65 isolados de SH foram avaliados quanto à presença dos genes de virulência invA, lpfA e agfA e diferenciados por eletroforese em campo pulsado (PFGE). Outro objetivo foi testar uma mistura de ácidos orgânicos e óleos essenciais no controle de excreção fecal de SE. Duas amostras de SH não relacionadas pelo método de PFGE (SH23 e SH35) e uma cepa de SE padrão foram selecionadas para a inoculação das aves. As três cepas promoveram alteração na medida de vilosidades e criptas dos pintinhos, frente a grupo controle. A cepa SH35 promoveu maior reação inflamatória nos cecos. Ambas as cepas testadas foram capaz de atingir o fígado de pintinhos às 6 horas pós-inoculação. Tanto a SE como a SH demonstram persistência e intermitência de excreção fecal em frangos de corte até os 21dias pós-inoculação. A mistura de ácidos e óleos essenciais foi eficaz na redução da excreção fecal de SE, porém somente com o uso contínuo da mistura na ração. As cepas apresentaram comportamentos diferentes quando inoculadas em frangos de corte e devido à persistência destas nos intestinos dos frangos, prejuízo para as aves podem ocorrer, enquanto a subseqüente contaminação de carcaças, representa risco para a saúde humana. / Salmonella, one of the most important food borne pathogen due to the widely distribution in nature, large number of reservoirs, to show unspecific host serotypes and provide multiresistant strains to antibiotics. Salmonella Enteritidis became a big public health problem in Brasil, since 80’s, when it was correlated with poultry products. Since 1962, Salmonella Heidelberg has been isolated from birds and poultry products in Brazil, with importance in human health and layers in North America. The present work aimed to the study of different strains of Salmonella Heidelberg (SH) and compared with Salmonella Enteritidis (SE) in chicks and broiler chickens with regard to the ability of invasibilidade of organs, persistence of fecal excretion and changes in intestinal morphometry. For the in vivo study, 65 isolates of SH were evaluated for the presence of virulence genes invA, lpfA and agfA and differentiated by pulsed field gel electrophoresis (PFGE). Another objective was to test a organic acids and essential oils mixture for controlling SE fecal excretion. Two not related SH samples by the method of PFGE (SH23 and SH35) and a standard strain of SE were selected to the poultry inoculation. The three strains promoted changes in the measures of villi and crypts of the chicks intestine, when compared to the control group. The SH35strain promoted greater inflammatory reaction in the caeca. Both strains tested were able to achieve the chicks liver at 6 hours post inoculation. Both strains, SE and SH, showed persistence and intermittence of fecal excretion in broiler chickens until the 21days post-infection. The organic acids and essential oils mix was effective in reducing the SE fecal excretion, but only with the continuous use of the mix added to the feed. The strains showed different behavior when inoculated in broiler chickens, and because of the persistence of these strains in the chickens intestines, injury to birds may occur, while the subsequent carcasses contamination represents risk to human health.

Destaca-se a Salmonella como um dos mais importantes patógenos veiculados por alimentos, devido ao fato de estar amplamente distribuído na natureza, possuir um grande número de reservatórios, apresentar sorotipos inespecíficos quanto aos hospedeiros e apresentar cepas multiresistentes aos antimicrobianos. Salmonella Enteritidis tornou-se um grande problema de saúde pública no Brasil, a partir da década de 80, quando foi associada a produtos avícolas. Desde 1962, a Salmonella Heidelberg tem sido isolada de aves e produtos derivados e aves no Brasil e tem reconhecida importância para saúde humana e em poedeiras comerciais na América do Norte. O presente trabalho teve por objetivo o estudo de diferentes cepas de Salmonella Heidelberg (SH) em comparação com Salmonella Enteritidis (SE) em pintinhos e frangos de corte no que diz respeito à capacidade de invasibilidade de órgãos, persistência de excreção fecal e alterações na morfometria intestinal. Para o estudo in vivo, 65 isolados de SH foram avaliados quanto à presença dos genes de virulência invA, lpfA e agfA e diferenciados por eletroforese em campo pulsado (PFGE). Outro objetivo foi testar uma mistura de ácidos orgânicos e óleos essenciais no controle de excreção fecal de SE. Duas amostras de SH não relacionadas pelo método de PFGE (SH23 e SH35) e uma cepa de SE padrão foram selecionadas para a inoculação das aves. As três cepas promoveram alteração na medida de vilosidades e criptas dos pintinhos, frente a grupo controle. A cepa SH35 promoveu maior reação inflamatória nos cecos. Ambas as cepas testadas foram capaz de atingir o fígado de pintinhos às 6 horas pós-inoculação. Tanto a SE como a SH demonstram persistência e intermitência de excreção fecal em frangos de corte até os 21dias pós-inoculação. A mistura de ácidos e óleos essenciais foi eficaz na redução da excreção fecal de SE, porém somente com o uso contínuo da mistura na ração. As cepas apresentaram comportamentos diferentes quando inoculadas em frangos de corte e devido à persistência destas nos intestinos dos frangos, prejuízo para as aves podem ocorrer, enquanto a subseqüente contaminação de carcaças, representa risco para a saúde humana. / Salmonella, one of the most important food borne pathogen due to the widely distribution in nature, large number of reservoirs, to show unspecific host serotypes and provide multiresistant strains to antibiotics. Salmonella Enteritidis became a big public health problem in Brasil, since 80’s, when it was correlated with poultry products. Since 1962, Salmonella Heidelberg has been isolated from birds and poultry products in Brazil, with importance in human health and layers in North America. The present work aimed to the study of different strains of Salmonella Heidelberg (SH) and compared with Salmonella Enteritidis (SE) in chicks and broiler chickens with regard to the ability of invasibilidade of organs, persistence of fecal excretion and changes in intestinal morphometry. For the in vivo study, 65 isolates of SH were evaluated for the presence of virulence genes invA, lpfA and agfA and differentiated by pulsed field gel electrophoresis (PFGE). Another objective was to test a organic acids and essential oils mixture for controlling SE fecal excretion. Two not related SH samples by the method of PFGE (SH23 and SH35) and a standard strain of SE were selected to the poultry inoculation. The three strains promoted changes in the measures of villi and crypts of the chicks intestine, when compared to the control group. The SH35strain promoted greater inflammatory reaction in the caeca. Both strains tested were able to achieve the chicks liver at 6 hours post inoculation. Both strains, SE and SH, showed persistence and intermittence of fecal excretion in broiler chickens until the 21days post-infection. The organic acids and essential oils mix was effective in reducing the SE fecal excretion, but only with the continuous use of the mix added to the feed. The strains showed different behavior when inoculated in broiler chickens, and because of the persistence of these strains in the chickens intestines, injury to birds may occur, while the subsequent carcasses contamination represents risk to human health.

Made available in DSpace on 2014-06-11T19:28:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-06-10Bitstream added on 2014-06-13T20:18:12Z : No. of bitstreams: 1 augusto_rmn_me_botfmvz.pdf: 475859 bytes, checksum: c7eb29be32951647006a8bc33ef784a0 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Universidade Estadual Paulista (UNESP) / Foram realizados dois experimentos: no primeiro utilizaram-se 96 leitões desmamados com idade média de 21 dias, num delineamento em blocos com arranjo fatorial dos tratamentos 2 x 2 ( duas fontes de carboidratos e duas classes de peso ao desmame). No segundo foram utilizados 48 leitões, num delineamento em blocos com arranjo fatorial 2 x 2 x 2 x 3 (duas classes de pesos ao desmame x duas fontes de carboidratos x duas porções do intestino delgado x três épocas de abate). Portanto, no modelo estatístico foram usados os efeitos fixos de peso, fontes de carboidratos, porções do intestino delgado, épocas de abate e suas respectivas interações; e o efeito aleatório de blocos. O objetivo deste estudo foi avaliar os efeitos da substituição da lactose pela maltodextrina nas rações de leitões desmamados, com diferentes pesos, sobre o consumo diário de ração, ganho diário de peso, conversão alimentar, incidência de diarreia, altura das vilosidades, profundidade das criptas e espessura de mucosa. No primeiro experimento, nos períodos de 0 a 17 e 0 a 29 dias após o desmame constatou-se efeito da classe de peso, no ganho diário de peso e no consumo diário de ração dos leitões, ou seja, os animais mais pesados ao desmame apresentaram maiores consumo diário de ração e ganho diário de peso em relação aos animais mais leves. No segundo experimento, verificou-se que a maltodextrina determinou aumentos na espessura média da mucosa intestinal dos leitões, independente do peso, e na profundidade média das criptas apenas nos animais leves. A maltodextrina pode ser usada como uma alternativa para substituir a lactose nas rações de leitões desmamados, pois não afeta negativamente o desempenho e a maioria dos parâmetros morfométricos do intestino. / Two trials were conducted: in the first trial, were used, 96 weaning pigs at the age of about 21 days, in a 2 × 2 factorial arrangement (two sources of carbohydrate: lactose and maltodextrin and two weaning weight classes: light, from 4,50 to 5,20 kg, and heavy, from 5,90 to 6,60 kg). In the second trial, 48 pigs, in a 2 x 2 x 2 x 3 factorial arrangement (two weaning weight classes x two sources of carbohydrate x two portions of the small intestine x three slaughter times). Therefore, in the statistical model the fixed effect of weight, sources of carbohydrate, portions of the small intestine, times of slaughter and its respective interactions; and the random effect of blocks. The objective of this study was to evaluate the effects of maltodextrin diets in weaning pigs of different weights on feed intake, feed:gain ratio, weight gain, diarrhea incidence, villus height, crypt depth and mucous membrane height. In the first trial, from 0-17 days and from 0-29 days after the weaning effects of weight classes, were detected on daily weight gain, and daily feed intake. The heaviest animals at weaning showed the best results. In the second trial, we verified that maltodextrin determined an increase in the average thickness of the intestinal mucosa, regardless of weight classes, and the increase in the average depth of the crypt, only in the highter animals. Maltodextrin can be used as an alternative to lactose in diets of weaning pigs, as it doesn’t affect negatively the performance neither the morfometric parameters of the intestine.

Destaca-se a Salmonella como um dos mais importantes patógenos veiculados por alimentos, devido ao fato de estar amplamente distribuído na natureza, possuir um grande número de reservatórios, apresentar sorotipos inespecíficos quanto aos hospedeiros e apresentar cepas multiresistentes aos antimicrobianos. Salmonella Enteritidis tornou-se um grande problema de saúde pública no Brasil, a partir da década de 80, quando foi associada a produtos avícolas. Desde 1962, a Salmonella Heidelberg tem sido isolada de aves e produtos derivados e aves no Brasil e tem reconhecida importância para saúde humana e em poedeiras comerciais na América do Norte. O presente trabalho teve por objetivo o estudo de diferentes cepas de Salmonella Heidelberg (SH) em comparação com Salmonella Enteritidis (SE) em pintinhos e frangos de corte no que diz respeito à capacidade de invasibilidade de órgãos, persistência de excreção fecal e alterações na morfometria intestinal. Para o estudo in vivo, 65 isolados de SH foram avaliados quanto à presença dos genes de virulência invA, lpfA e agfA e diferenciados por eletroforese em campo pulsado (PFGE). Outro objetivo foi testar uma mistura de ácidos orgânicos e óleos essenciais no controle de excreção fecal de SE. Duas amostras de SH não relacionadas pelo método de PFGE (SH23 e SH35) e uma cepa de SE padrão foram selecionadas para a inoculação das aves. As três cepas promoveram alteração na medida de vilosidades e criptas dos pintinhos, frente a grupo controle. A cepa SH35 promoveu maior reação inflamatória nos cecos. Ambas as cepas testadas foram capaz de atingir o fígado de pintinhos às 6 horas pós-inoculação. Tanto a SE como a SH demonstram persistência e intermitência de excreção fecal em frangos de corte até os 21dias pós-inoculação. A mistura de ácidos e óleos essenciais foi eficaz na redução da excreção fecal de SE, porém somente com o uso contínuo da mistura na ração. As cepas apresentaram comportamentos diferentes quando inoculadas em frangos de corte e devido à persistência destas nos intestinos dos frangos, prejuízo para as aves podem ocorrer, enquanto a subseqüente contaminação de carcaças, representa risco para a saúde humana. / Salmonella, one of the most important food borne pathogen due to the widely distribution in nature, large number of reservoirs, to show unspecific host serotypes and provide multiresistant strains to antibiotics. Salmonella Enteritidis became a big public health problem in Brasil, since 80’s, when it was correlated with poultry products. Since 1962, Salmonella Heidelberg has been isolated from birds and poultry products in Brazil, with importance in human health and layers in North America. The present work aimed to the study of different strains of Salmonella Heidelberg (SH) and compared with Salmonella Enteritidis (SE) in chicks and broiler chickens with regard to the ability of invasibilidade of organs, persistence of fecal excretion and changes in intestinal morphometry. For the in vivo study, 65 isolates of SH were evaluated for the presence of virulence genes invA, lpfA and agfA and differentiated by pulsed field gel electrophoresis (PFGE). Another objective was to test a organic acids and essential oils mixture for controlling SE fecal excretion. Two not related SH samples by the method of PFGE (SH23 and SH35) and a standard strain of SE were selected to the poultry inoculation. The three strains promoted changes in the measures of villi and crypts of the chicks intestine, when compared to the control group. The SH35strain promoted greater inflammatory reaction in the caeca. Both strains tested were able to achieve the chicks liver at 6 hours post inoculation. Both strains, SE and SH, showed persistence and intermittence of fecal excretion in broiler chickens until the 21days post-infection. The organic acids and essential oils mix was effective in reducing the SE fecal excretion, but only with the continuous use of the mix added to the feed. The strains showed different behavior when inoculated in broiler chickens, and because of the persistence of these strains in the chickens intestines, injury to birds may occur, while the subsequent carcasses contamination represents risk to human health.

This thesis is about encrypted filesystems and is aimed mainly for Linux solutions. At first there is explained why is it necessary to encrypt your data at all. Next is written about pros and cons of encrypting methods such as whole disk encryption, partition encryption, container encryption and file encryption. After the introduction there are mentioned most common security issues which can help attacker to decrypt your data or modify them. Furthermore this thesis contains short description about the most often used encrypted filesystems. There is also small how-to for the most important encrypted filesystems available in Linux and comparison of their speed. Next part of this thesis contains dm-crypt and LUKS description. In the last chapter all information are concluded. There is also mentioned benefit of this work and possibilities, what can be done in future.

The expression of calcium sensing receptor (CaSR) in the human colonic crypt epithelium is linked to cellular differentiation while its lack of expression is associated with undifferentiated and invasive colon carcinoma. Recent studies show that CaSR suppresses the malignant phenotype through a variety of pathways that inhibits growth and promotes differentiation. CaSR also promotes cytotoxic response to fluorouracil. These studies, taken together, have led me to formulate the following working hypotheses: (i), CaSR is a robust inducer of differentiation by virtue of its ability to activate and integrate diverse growth and differentiation control signals; (ii), loss of CaSR expression enable cellular escape from CaSR control and (iii), loss of CaSR expression is an underlying mechanism of malignant transformation, progression and drug resistance in colon cancer. Previous studies showed that there are endogenous small subpopulations that do not express CaSR in colon carcinoma cell lines. These cells are highly drug resistant. Indeed, immunocytochemical analyses of CaSR showed that the expression of CaSR in both the CBS and HCT116 colon carcinoma cell lines are heterogeneous. Human colon carcinoma cell lines contain small subpopulations (10-20%) that do not express CaSR (termed CaSR null cells). In order to further test my hypotheses, the isolation and characterization of CaSR null cells are required. Here, I report on the isolation, propagation, maintenance and characterization of CaSR null cells from the CBS and HCT116 human colon carcinoma cell lines. CaSR null cells grew as three-dimensional non-adherent spherical clusters with increased propensity for anchorage independent growth, cellular proliferation and invasion of matrigels. CaSR null cells were highly resistant to fluorouracil and expressed abundant amount of thymidylate synthase and survivin. Molecular profiling showed a high level of expression of the previously reported cancer stem cell markers CD133, CD44 and Nanog in CaSR null cells. A significant increase in the expression of epithelial-mesenchymal transitional (EMT) molecules and transcription factors was also observed. These include N-cadherin, β-catenin, vimentin, fibronectin, Snail1, Snail2, Twist and FOXC2. The expression of the tumor suppressive E-cadherin and miR145, on the other hand, was greatly reduced while the expression of oncogenic micro RNAs: miR21, miR135a and miR135b was significantly up-regulated. CaSR null cells possess a myriad of cellular and molecular features that drive and sustain the malignant phenotype. I conclude that CaSR null constitutes a highly malignant and drug resistant phenotype of colon cancer. I discovered that CaSR null cells, cultured in defined human embryonic stem cell culture medium, can be induced to differentiate and acquire CaSR expression when the medium of the null cells was changed to conventional cell culture medium containing fetal bovine serum. I hypothesize that expression of CaSR can alter the phenotype of the CaSR null cells. The objectives of this study were then three folds: (i), determine if induction of CaSR expression could circumvent the molecular phenotype of the CaSR null cells; (ii), determine if CaSR was required in altering the null phenotype and (iii), determine the underlying mechanism of CaSR induction. I hypothesize that if CaSR is a strong promoter of differentiation, then without CaSR, the constraint exerted by CaSR will not be functional and pathways normally inhibited by CaSR will be activated. I found that induction of CaSR expression led to a more indolent phenotype which includes the acquisition of epithelial morphology, down-regulated expression of cancer stem cell markers, down-regulated expression of thymidylate synthase and survivin and increased sensitivity to fluorouracil. Molecular profiling also revealed that the induction of CaSR expression was linked to a down-regulated expression of EMT molecules, EMT associated transcription factors and oncogenic miRNAs with a concurrent up-regulated expression of tumor-suppressive molecules. With the exception of the cancer stem cell markers, the reversal of molecular features, upon the induction of CaSR expression, was directly linked to the expression and function of CaSR because blocking CaSR induction by shRNA circumvented such reversal. I further report that demethylation of the CaSR gene promoter underlie CaSR induction. I conclude that induction of CaSR expression in CaSR null cells resulted in a more indolent phenotype concurrent with a variety of molecular changes and that these changes (with the exception of stem cell markers) are dependent on the expression and function of CaSR. I further conclude that methylation of the CaSR gene promoter is an underlying mechanism of maintaining the CaSR null phenotype while promoter demethylation is an underlying mechanism responsible for CaSR induction. CaSR null is a phenotype of the rapidly proliferating, undifferentiated crypt stem cells at the base of colonic crypts. Differentiation of crypt stem cells toward the apex of a crypt (in the direction of the lumen), on the other hand, is tightly linked to CaSR expression. What induces CaSR expression as the crypt stem cells migrate up the crypts is unknown. I hypothesize that as the colonic crypt stem cells migrate up the crypt, they become increasingly exposed to the colonic fluid in the lumen and components in the colonic fluid can trigger the induction of CaSR expression. Both Ca2+ and vitamin D are good candidates because either Ca2+ or vitamin D can stimulate CaSR expression in the parental CBS and HCT116 human colon carcinoma cells. Certainly, Ca2+ and vitamin D are not the only components involved in regulating CaSR expression. A variety of minerals in the colonic fluid may also serve as good candidates in the induction of CaSR. Of interest is Aquamin, a calcium-rich mineralized extract from the red marine algae, Lithothamnion calcareum, which has been shown to induce differentiation in colon carcinoma cells and possess chemopreventive properties against colon polyp formation in mice fed a high fat diet. CaSR null cells cultured in defined human embryonic stem cell culture medium were used to test this hypothesis because they offer an in vitro model in determining the triggers and the underlying mechanisms of CaSR induction that may resemble that of the colonic crypt stem cells in vivo. I found that all three agonists (Ca2+, vitamin D and Aquamin) induced CaSR mRNA and protein expression and inhibited cellular proliferation in the parental cells which express a heterogeneous mixture of cells with different level of CaSR expression. These agonists also induced CaSR mRNA and protein expression and inhibited cellular proliferation in the homogeneous isolated CaSR null cells. In both cases, Aquamin was found to be most potent in this regard. Induction of CaSR expression by these agonists in the CaSR null cells resulted in demethylation of the CaSR gene promoter with a concurrent increase in CaSR promoter reporter activity. Induction of CaSR expression resulted in a down-regulated expression of tumor inducers and up-regulated expression of tumor suppressors in the CaSR null cells. Again, Aquamin was found to be most potent in this regard. Taken together, I conclude that nutrients are good candidate in the induction of CaSR and differentiation in colonic epithelia cells. Similar to CaSR, transforming growth factor β (TGFβ) is also a robust promoter of differentiation in the colonic epithelium. The expression profile of both CaSR and TGFβ in the colonic epithelium is tightly linked to differentiation. Both CaSR and TGFβ expression progressively increases as the undifferentiated crypt stem cells migrate and differentiate toward the apex of a crypt in the direction of the lumen. Similar to the loss of CaSR in cancer cells, loss of TGFβ responsiveness has long been considered an underlying mechanism of early colon carcinogenesis. I hypothesize that there is functional linkage between CaSR and TGFβ function. Human colonic epithelial CBS cells originally developed from a differentiated human colon tumor, retain CaSR expression and function, TGFβ responsiveness and TGFβ receptor expression. Thus, these cells offer an opportunity to determine the functional linkage (if any) between CaSR and TGFβ. I found that knocking down CaSR expression in the CBS cells abrogated TGFβ-mediated cellular responses and attenuated the expression of TGFβ receptors. Ca2+ or vitamin D treatment induced CaSR expression with a concurrent up-regulation of TGFβ receptor expression. Ca2+ or vitamin D, however, did not induce CaSR in CaSR knocked down cells and without CaSR, there was no up-regulation of TGFβ receptor. I conclude that TGFβ receptor expression and TGFβ mediated responses requires CaSR expression and function. In summary, my research has revealed the important role of CaSR in controlling differentiation. CaSR also function as a robust tumor suppressor. My study clearly discerns the multifarious molecular signaling cascades involved in CaSR function and that methylation and demethylation regulates CaSR expression. My work has also established the importance of CaSR in the chemoprevention of colon cancer. My thoughts in regard to future studies and the potential role that CaSR could play in the management of colon cancer are given in the perspective section of this dissertation.