<p>Colorectal cancer leads to more than 600,000 deaths worldwide per year. An abundance of research has shown that several non-steroidal anti-inflammatory drugs (NSAIDs) can exert chemotherapeutic and chemo-preventative effects in colorectal cancer patients. It is important to note, that use of many different NSAIDs carries a significant risk for cardiovascular and gastrointestinal (GI) complications. A recently developed group of NSAIDs, which release hydrogen sulfide (H₂S), has been shown to have greatly reduced these side effects as compared to conventional NSAIDs. This is likely attributable to the ability of H₂S to increase the resistance of the GI mucosa to injury, as well as to accelerate repair of injury when it occurs. Moreover, H₂S has been shown to be a vasodilator, and therefore may offset some of the hypertensive effects of NSAIDs.</p> <p>We assessed the chemotherapeutic actions of two of these newly developed NSAIDs. ATB-346 is an H₂S-releasing derivative of naproxen and ATB-352 is an H₂S-releasing derivative of ketoprofen. These drugs were tested in the azoxymethane mouse model and in the APC^Min/+mouse model of Colorectal cancer.</p> <p>In the azoxymethane model of colorectal cancer ATB-346 caused a significant reduction in number aberrant crypt foci (ACF), which are pre-neoplastic lesions used as markers of colorectal cancer. The reduction was superior to naproxen at all doses tested. ATB-352 also caused a significant reduction in the number of ACF, however the reduction was not superior to that produced by ketoprofen. In APC^Min/+mice treated with ATB-346 for 14 days (14.5 mg/kg) we observed a complete inhibition of the formation of colonic polyps/tumours and a 97.5% reduction in total polyp score. Shorter treatment with ATB-346 also produced similar reduction in total polyp score. We found that ATB-346-treated mice had lower levels of b-catenin and cmyc without significant changes in APC or p53 levels. <strong></strong></p> <p>These results demonstrate ATB-346 can exert superior chemo-preventative effects in mice models of colon cancer while leading to no gastric or intestinal damage.</p> / Master of Science (MSc)

No description available.

Syftet med denna undersökning är att ta reda på hur barns föreställning om matens väg genom kroppen skiljer sig åt. I undersökningen jämförs barn i tre- till fyra och fem- till sex årsåldern, närmare beskrivet barn födda 2010 och 2012. För att undersöka detta användes kvalitativa semistrukturerade intervjuer samt siluetter av en människokropp som barnen fick illustrera sina föreställningar på. Valet av metod gjorde det möjligt att fördjupa sig i barnens tankar och resonemang vilket är fördel när man undersöker något utifrån barns perspektiv. Resultatet i undersökningen visar att majoriteten av samtliga respondenter hade föreställningen om att maten transporteras till magen. I resultatet framgår olika beskrivningar på hur maten transporteras ned till magen men även vad som sker därefter. Resultatet visar även att femåringarna har en mer detaljerad beskrivning och bredare kunskap om matens väg genom kroppen. / The purpose of this study is to find out the difference between five- and three-years old children’s conception about the digestive system. The study was conducted by using qualitative semi-structured interviews and silhouettes of a human body so that the children could illustrate their conception on. The choice of method made it possible to immerse the children’s thoughts and reasoning which is a benefit when you base your study on children’s perspective. The results show that majority of the respondents thought that the food end up in the stomach, even though the descriptions was different. The results also show that the five- years old had more detailed description and more knowledge about the digestive system then the three-year-old children.

Thesis (MSc)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: Six groups each with six Friesian bull calves were used in this investigation and slaughtered at 20 weeks of age. Calves received a low- (LD) or high (HD) degradable protein diet, each with or without rumen inert fat supplementation. Two commercial fat sources were used, Morlae (m) and Golden Flake (gf), included at 2.5% of the diet. A commercial milk replacer (Denkavit) was fed at 4L for 42 days, followed by 2L until weaning at 49 days of age. The starter diets were fed ad lib. from day 14 to 10 weeks of age and finishing diets ad lib. from 11 to 20 weeks of age. There were no significant differences in body mass gain or dry matter intake over the entire 20 week period. The feed conversion ratio (FCR) was improved significantly (P=0.0032) when fat was supplemented to LD, but not to HD diets. The FCR (kg dry matter/ kg gain) ofLD, HD, LDm, HDm, LDgfand HDgfdiets were 3.45,3.44,3.07, 3.81, 3.02 and 3.43, respectively. All 36 calves were used in a digestibility trial, using chromium oxide (Cr203) as a marker, during week 18 of the investigation. Digestibility values (%) for the six diets (LD, HD, LDm, HDm, LDgfand HDgf) were 61.74, 65.91, 75.44, 69.00, 75.54 and 67.15 for dry matter, 61.44, 61.60, 71.33, 68.23, 75.44 and 66.12 for crude protein and 58.56, 66.45, 75.98, 70.92, 78.43 and 70.79 for fat, respectively. The dry matter (P=O.OOOl)and fat (P=O.OOOl) digestibilities were only significantly higher when fat was added to LD diets. The crude protein (CP) digestibilities were significantly higher when fat was added to either the LD (P=0.0001) or the HD (P=0.0488) diets. All the calves were slaughtered at 20 weeks of age and the fatty acid content of the meat (m. longissimus) and subcutaneous fat layer adjacent to the 12th rib as well as the meat colour, was determined. The fatty acid composition of the longissimus muscle was changed by feeding the rumen inert fat sources. The three predominant fatty acids found were palmitic, stearic and oleic acids. The palmitic acid (CI6:0) content of the muscle and diet was 24.44 & 20.47,25.97 & 22.57,31.06 & 33.23, 30.98 & 37.91, 34.94 & 31.77 and 29.71 & 32.88 of the total fat for the LD, HD, LDm, HDm, LDgf and HDgf diets, respectively. The C16:0 content was significantly higher in the muscle of the calves receiving the LD diets supplemented with fat (P=0.0008). There was also a significant interaction between the two fat sources and protein degradability (P=0.0065), but only in the LD diets. The stearic acid (CI8:0) content of the muscle and diet was 14.35 & 5.22, 19.65 & 8.61, 17.29 & 4.68, 22.59 & 5.78, 22.27 & 15.54, and 26.48 & 20.15 of the total fat for the LD, HD, LDm, HDm, LDgfand HDgfdiets, respectively. The C18:0 content was significantly higher in the muscle of calves receiving the HD (P=O.OOOl)compared to LD diets. The stearic acid content was also significantly higher when fat was added to LD (P=0.0042) or HD (P=0.0073) diets. The oleic acid (CI8:1) content of the muscle and diet was 36.06 & 21.51,39.99 & 21.11,32.21 & 23.67, 29.13 & 24.59, 25.23 & 18.68 and 35.93 & 16.02 of the total fat for the LD, HD, LDm, HDm, LDgf and HDgf diets, respectively. The linolenic acid (CI8:3) content of the muscle was significantly higher (P=0.0038) when fat was added to LD diets compared to no fat supplementation (0.87 vs. 0.15). The CIELAB values indicated that LD diets resulted in more pink meat. Mean values ofL* =-32.61, 34.19; a* = 7.08, 7.91 and b* = 3.18 and 4.07 were observed for the LD and HD diets, respectively. Meat from the LD diets had significantly lower L*-(P=0.0252), a*-(P=0.0283) and b*-(P=0.0109) values compared to meat from the HD diets. It was concluded that there was a positive response in CP digestibility when rumen inert fats were supplemented to LD or HD diets, although a greater response was shown in the LD diets. The FCR, dry matter and fat digestibility were only increased when fat was added to the LD and not to the HD diets. Similarly, the fatty acid contents of the longissimus muscle of veal calves can be manipulated with the supplementation of rumen inert fat sources, but only when combined with a low protein degradable diet. The low degradable protein diets also produce a more attractive meat colour for the potential veal consumer. / AFRIKAANSE OPSOMMING: Ses behandelings, lae- (LD) of hoë (HD) degradeerbare diëte, elk met of sonder rumeninerte vetsupplementering, is geëvalueer met ses kalwers in elke groep. Twee kommersiële vetbronne is gebruik, nl. Morlae (m) en Golden Flake (gf) teen 'n 2.5% insluitingspeil. 'n Kommersiële melksurrogaat (Denkavit) is aangebied teen 4L1dag tot 42 dae ouderdom, gevolg deur 2L/dag tot speenouderdom op 49 dae. Aanvangsdiëte is ad lib. aangebied vanaf 14 dae tot 10 weke ouderdom en die groeidiëte ad lib. vanafweek 11 tot 20. Daar was geen betekenisvolle verskille in die totale massatoename of die droëmateriaalinname nie. Die voeromsettingsverhouding is betekenisvol verbeter (P=0.0032) in die behandelings waarin rumeninerte vette by LD diëte ingesluit is, maar nie by die HD diëte nie. Die voeromsettingsverhouding (kg droëmateriaalinname / kg massatoename) van die LD, HD, LDm, HDm, LDgf en HDgf diëte was 3.45, 3.44, 3.07, 3.81, 3.02 en 3.43, onderskeidelik. Al 36 kalwers is in 'n verteringsproef gebruik gedurende week 18 van die proef. Chroomoksied (Cr203) is as merker gebruik. Verteerbaarheidswaardes vir die ses diëte was 61.74, 65.91, 75.44,69.00,75.54 en 67.15 vir droëmateriaal, 61.44, 61.60, 71.33, 68.23, 75.44 en 66.12 vir ruproteïen en 58.56, 66.45, 75.98, 70.92, 78.43 en 70.79 vir vet, onderskeilik. Die droëmateriaal- (P=O.OOOl) en vetverteerbaarheid (P=O.OOOI) was slegs betekenisvol hoër wanneer vet by LD diëte gevoeg is en nie by HD nie. Die ruproteïen (RP) verteerbaarheid Stellenbosch University http://scholar.sun.ac.za VI was betekenisvol hoër (P=0.0002) by LD en HD (P=0.0488) diëte met vet supplementering, teenoor geen vet insluiting. Die kalwers is op 20 weke ouderdom geslag en die vetsuursamestelling van die vleis (m. longissimus) en die subkutane vetlaag teenaan die 12de rib, asook en die vleiskleur, is bepaal. Die vetsuursamestelling van die longissimus spier is deur die supplementering van rumeninerte vet verander. Die drie primêre vetsure wat in die vleis voorgekom het, was palmitiensuur, steariensuur en oleïensuur. Die palmitensuur (CI6:0) inhoud van die spier en diëte was 24.44 & 20.47, 25.97 & 22.57, 31.06 & 33.23, 30.98 & 37.91, 34.94 & 31.77 en 29.71 & 32.88 van die totale vet van die LD, HD, LDm, HDm, LDgf en HDgf diëte, onderskeilik. Die C16:0 was betekinisvol hoër in die spiere van kalwers wat die LD diëte met vet supplementering (P=0.0008) ontvang het. Die steariensuur (CI8:0) inhoud van die spier en diëte was 14.35 & 5.22, 19.65 & 8.61, 17.29 & 4.68, 22.59 & 5.78, 22.27 & 15.54, en 26.48 & 20.15 van die totale vet vir die LD, HD, LDm, HDm, LDgf en HDgf diëte, onderskeidelik. Die C18:0 inhoud was betekinisvol hoër in die spiere van die kalwers wat die HD (P=O.OOOI),teenoor LD diëte ontvang het. Die steariensuur inhoud was ook betekenisvol hoër wanneer vet by LD (P=0.0042) of HD (P=0.0031) diëte gevoeg word. Die oleïensuur (CI8:1) inhoud van die spier en diëte was 36.06 & 21.51, 39.99 & 21.11, 32.21 & 23.67, 29.13 & 24.59, 25.23 & 18.68 en 35.93 & 16.02 van die totale vet vir die LD, HD, LDm, HDm, LDgf en HDgf diëte, onderskeidelik. Die linoleensuur (CI8:3) inhoud van die spier was betekinisvol hoër (P=0.0038) in die LD diëte met vet teenoor LD met geen vet supplementering (0.87 vs. 0.15). Die CIELAB waardes van die LD diëte dui op 'n pienker vleiskleur. Gemiddelde waardes van L* = 32.61 & 34.19, a* = 7.08 & 7.91 en b* = 3.18 & 4.07 is vir die LD en HD diëte, onderskeidelik, waargeneem. Die vleis van die LD diëte het 'n betekenisvol laer L*-(P=0.0252), a*-(P=0.0283) en b*-(P=0.0109) waarde in vergelyking met die HD diëte getoon. Die resultate dui daarop dat daar 'n positiewe respons in die ruproteïenverteerbaarheid by die supplementering van rumeninerte vette by LD en HD diëte voorkom, maar die response op die LD diëte is groter. Die voeromsettingsverhouding, droëmateriaal- en vetverteerbaarheid is egter net bevoordeel in die LD met vet en nie in die HD diëte nie. Die vetsuursamestelling van die longissimus spier in die kalf kan gemanipuleer word met die supplementering van rumeninerte vetbronne, maar slegs wanneer dit gekombineer word met lae degradeerbare proteïen diëte. Die lae degradeerbare proteïen diëte produseer ook die mees aanloklike vleiskleur vir die potensiële kalfsvleisverbruiker.

Thesis (PhD)--Stellenbosch University, 2005. / ENGLISH ABSTRACT: This study describes the use of a gastro-intestinal model to screen lactic acid bacteria isolated from the gastro-intestinal tract of post-weaned piglets (raised on six different diets) for probiotic properties. Intestinal bacteria were isolated from ,the stomach, duodenum, jejunum, caecum, ileum and colon. The highest cell numbers (6 x 107 cfulg) were isolated from the ileum. No significant differences in viable cell counts were recorded for piglets raised on the six diets. Isolates with the best overall probiotic properties were identified as members of Lactobacillus salivarius and Lactobacillus fermentum. The two strains selected for further studies were Lactobacillus plantarum 423 (originally isolated from sorghum beer) and Lactobacillus salivarius 241 (isolated from pig intestine). Enterococcus faecalis FAIR E 92 was originally isolated from pig intestine and was included in this study as a non-pathogenic challenge strain. L. plantarum 423 produces a bacteriocin plantaricin 423, active against E. faecalis FAIRE 92. L. plan/arum 423 and L. salivarius 241 were included in the gastro-intestinal model and their adhesion to the mucus of porcine ileum studied with fluorescent-in-si/u-hybridization (FISH). A decrease in viable cell numbers of L. plan/arum 423 was recorded in the duodenum, jejunum and ileum in the presence of bile and pancreatic juice. However, higher cell numbers were recorded in the caecum and anterior colon, which suggested that strain 423 recovered from these stress factors. Plantaricin 423 was detected for up to 28 hours in the duodenum, jejunum, ileum and middle colon. Lower cell numbers (one log unit) of L. salivarius 241 were recorded in the gastro-intestinal model over seven days, compared to strain 423. Piglets of one, 14 and 28-days-old were dosed with L. plan/arum 423 and L. salivarius 241, separately and in combination (1: 1). In a separate experiment, 14-day-old piglets were challenged twice with E. faecalis FAIRE 92, followed by dosage with strains 423 and 241. New-borne piglets dosed with L. plantarum 423 gained more weight (4 kg over 19 days) compared to piglets dosed with L. salivarius 241 (2.2 kg over 19 days), or a combination of the two strains (2 kg over 19 days). Piglets of 14 and 28-days-old, on the other hand, gained more weight when dosed with a combination of strains 423 and 241. The cell numbers of E. faecalis FAIR E 92 and other enterococci decreased drastically (two log units) when the piglets were dosed with the latter two strains. Overall, piglets of various ages reacted differently when administered L. plantarum 423 and L. salivarius 241, separately or in combination. Fluorescent-in-situ-hybridization (FISH) was used to study the in vivo adhesion of L. plantarum and L. salivarius to mucus in the stomach, duodenum, jejunum, ileum, caecum and colon. The highest number of L. plantarum cells was recorded in the ileum, whereas L. salivarius favoured adhesion to the duodenum. A decrease in cell numbers of E. faecalis in the ileum mucus was recorded when a combination of the probiotic strains 423 and 241 was administered. This study provided a reliable estimation of the presence and/or adhesion of L. plantarum and L. salivarius to various parts of the porcine gastro-intestinal tract, without the use of expensive cultivation techniques. Insight was gained into the co-evolution existing between probiotic bacteria and the porcine gastro-intestinal tract, emphasizing the use of gastro-intestinal models to study the dynamics of the gastro-intestinal tract. / AFRIKAANSE OPSOMMING: Hierdie studie beskryf die gebruik van 'n gastro-intestinale model, om melksuurbakterieë wat geïsoleer is uit die spysverteringskanaal (SVK) van reeds gespeende varkies (gevoed op ses verskillende diëte) vir probiotiese eienskappe te toets. Ingewandsbakterieë is uit die maag, duodenum, jejunum, caecum, ileum en kolon geïsoleer. Die hoogste aantal selle (6 x 107 kve/g) is geïsoleer uit die ileum. Geen betekenisvolle verskille in lewensvatbare seltellings, vir varkies gevoed op ses verskillende voere is aangeteken nie. Isolate met die beste algehele probiotiese eienskappe is as Lactobacillus salivarius en Lactobacillus fermentum geïdentifiseer. Vir verdere studie is twee isolate Lactobacillus plantarum (oorspronklik uit sorghum-bier geïsoleer) en Lactobacillus salivarius (uit die varkdermkanaal geïsoleer) geselekteer. Enterococcus faecalis FAIRE 92, oorspronklik uit die varkdermkanaal geïsoleer, is in hierdie studie as 'n nie-patogeniese indikator gebruik. L. plantarum 423 produseer 'n bakteriosien plantarisien 423 wat aktief is teen E. faecalis FAIR E92. L. plantarum 423 en L. sa/ivarius 241 is ingesluit in die gastro-intestinale model, en vashegting van die bakterieë aan die mukus van vark-ileum is met fluoresensie-in-si/uhibridisasie (FISH) bestudeer. 'n Afname in lewende selgetalle van L. plan/arum 423 in die duodenum, jejunum en ileum is aangetoon in reaksie tot die byvoeging van gal en pankreatiese sappe. Hoër selgetalle is nietemin aangeteken in die caecum en voorste gedeelte van die kolon, wat 'n aanduiding gee dat isolaat 423, ten spyte van hierdie stres-faktore, oorleef. Plantaricin 423 is vir 'n tydperk (28 uur) in die duodenum, jejunum, ileum en sentrale kolon gevind. Laer selgetalle (een logaritmiese eenheid) van L. salivarius 241 is in die gastro-intestinale modeloor 'n tydperk van sewe dae aangetoon, in vergelyking met isolaat 423. Een, 14 en 28 dag oud varkies is met L. plantarum 423 en L. salivarius 241 (afsonderlik en in kombinasie 1:1) twee keer gedaag met E. faecalis FAIR E 92, opgevolg met dosering van 423 en 241. Pasgebore varkies het die hoogste gewigstoename getoon (4 kg oor 19 dae) na dosering met L. plantarum 423 in vergelyking met varkies gedoseer met L. salivarius 241 (2.2 kg oor 19 dae) of 'n kombinasie van die twee isolate (2 kg oor 19 dae). Daarenteen het veertien- en 28 dag oud varkies beter gewigstoename getoon na dosering met 'n kombinasie van isolate 423 en 241. Die selgetalle van E. faecalis FAIRE 92 en ander enterococci het drasties afgeneem (twee logaritmiese eenhede) nadat die varkies met laasgenoemde twee isolate gedoseer is. Varkies van onderskeie ouderdom het verskillend gereageer na dosering met L. plantarum 423 en L. salivarius 241 afsonderlik of in kombinasie. Fluoresensie-in-situ-hibridisasie (FISH) is gebruik om die in vivo vashegting van L plantarum en L. salivarius tot die vark mukus in die maag, duodenum, jejunum, ileum, caecum en kolon te bestudeer. Die hoogste telling van L. plantarum selle is aangeteken in die ileum, terwyl L. salivarius aanhegting tot die duodenum verkies het. 'n Afname in seltellings van E. faecalis in die ileum mukus was aangeteken na toediening met 'n kombinasie van probiotiese isolate 423 en 241. Hierdie studie het 'n betroubare bepaling van die voorkoms en/ofvashegting van L. plantarum en L. sa/ivarius isolate in verskeie gedeeltes van die varkspysverteringskanaal voorsien, sonder die hulp van duur kwekings tegnieke. Probiotiese bakterieë is in 'n gastro-intestinale model, wat die natuurlike omgewing verteenwoordig, bestudeer. Insig oor die ko-evolusie tussen probiotiese bakterieë en die SVK van die vark is verkry. Die gebruik van 'n gastro-intestinale model om die dinamika van die SVK te bestudeer is met hierdie studie beklemtoon.

published_or_final_version / abstract / Pathology / Doctoral / Doctor of Philosophy

Dans le cadre de l'évaluation de la qualité des écosystèmes aquatiques, les réponses en lien avec l'énergie semblent particulièrement pertinentes pour appréhender l'état physiologique des êtres vivants et prévoir des effets populationnels. Dans ce contexte, l'objectif de ce travail a été de proposer un biomarqueur de l'assimilation de l'énergie chez un amphipode d'eau douce Gammarus fossarum. La caractérisation des activités enzymatiques digestives (amylase, cellulase et trypsine) et des réserves énergétiques (glycogène, lipides et protéines) soumises à l'influence de divers facteurs biotiques et abiotiques (sexe, stade reproducteur, température, conductivité et quantité de nourriture) a permis d'identifier l'amplitude des réponses étudiées afin d'améliorer l'interprétation des réponses obtenues en milieu naturel. Ces résultats ont également validé l'utilisation d'organismes mâles standardisés et nourris ad libitum dans le cadre d'approche active. D'autre part, la pertinence écologique des enzymes digestives en tant que biomarqueur a été évaluée en étudiant le lien existant entre une perturbation des réponses enzymatiques digestives au niveau sub-individuel et ses conséquences au niveau individuel sur le succès reproducteur des gammares. Par ailleurs, dans le cadre de deux campagnes de terrain, les mesures des activités enzymatiques digestives réalisées chez les organismes transplantés ont permis de discriminer les sites en fonction de leur qualité chimique et de proposer pour chaque marqueur une valeur référence et seuil traduisant un effet. Ainsi les activités enzymatiques digestives peuvent être utilisées comme outil pour établir un diagnostic de risque environnemental. / For the assessment of the aquatic ecosystems health, responses related to energy metabolism seem particularly relevant to understanding the physiological state of living organisms and provide population- effects. In this context, the objective of this work was to propose a biomarker of energy assimilation in a freshwater amphipod Gammarus fossarum. The characterization of digestive enzymes activities ( amylase , cellulase and trypsin) and energy reserves (glycogen , lipids and proteins) submited to the influence of various biotic and abiotic factors (sex, reproductive stage , temperature, conductivity and amount of food ) determine the range of responses studied to improve the interpretation of data obtained in the field. These results can also validate the use of standardized male fed ad libitum in caging approach. On the other hand, the ecological relevance of digestive enzymes was assess, the link between digestive enzyme impairments at sub -individual level and its consequences on the fitness of the gammarid population at individual level was established. Furthermore, in the context of two field campaigns, measures of digestive enzyme activities in organisms transplanted allowed to discriminate sites according to their chemical quality and to propose for each marker a benchmarck and a threshold value reflecting an effect. Thus, digestive enzyme activities can be used as diagnostic tool for environmental risk assessment.

Orientador: Adriana Camargo Ferrasi / Banca: Maria Inês de Campos Moura Pardini / Banca: Juliana Garcia de Oliveira / Resumo: O câncer gástrico é um dos tipos de câncer mais comum e está associado com uma alta frequência de mortalidade. Embora nas últimas décadas tenha ocorrido um decréscimo na incidência mundial, o prognóstico desta patologia permanece ruim, principalmente quando o diagnóstico é feito em estágios avançados. A etiologia é complexa e multifatorial, onde a combinação de fatores genéticos e ambientais parecem estar envolvidos na carcinogênese e progressão da doença. Desta forma, o objetivo deste estudo foi determinar a frequência do polimorfismo rs12979860 C/T no gene IL28B em amostras de carcinoma gástrico provenientes de dois estados brasileiros, São Paulo e Ceará, associando às características clínicas e epidemiológicas desses pacientes. Os resultados demonstraram que 29,7% dos pacientes apresentam o genótipo CC, enquanto a frequência dos indivíduos heterozigotos e (CT) e homozigotos (TT) foram 56,3% e 9%, respectivamente. Em comparação com dados descritos na literatura e do dBSNP de populações usadas como controle, a frequência do alelo T foi mais alta nos pacientes acometidos por câncer gástrico (tumor difuso: 45,5%, tumor intestinal: 39%, literatura: 11 a 35 e dBSNP: 36%). Ainda neste contexto, encontramos uma significante menor frequência do genótipo CC entre os pacientes acometidos por tumores difusos (22,7%), quando comparados aos indivíduos saudáveis (44 a 51%). Nos casos provenientes da população cearense o alelo C foi significativamente mais elevado no tipo intestinal (67%) quando comparado ao tipo difuso (50%), ressaltando a maior frequência do alelo T nos casos difusos. Analisando-se a frequência do polimorfismo rs12979860 C/T e a genotipagem de Helicobacter pylori foi observada uma forte associação entre o genótipo e a infecção pela bactéria CagA+, onde pacientes portadores da cepa mais patogênica (CagA+) apresentaram frequência maior do genótipo menos protetor (TT), principalmente no... / Abstract: Gastric cancer is one of the most common types of cancer and is associated with a high mortality rate. Although in recent decades there has been a decrease in the global incidence, prognosis of this condition remains poor, especially when the diagnosis is made in advanced stages. The etiology is complex and multifactorial, where the combination of genetic and environmental factors appear to be involved in carcinogenesis and progression of the disease. Thus, the aim of this study was to determine the frequency of the polymorphism rs12979860 C / T in the IL28B gene in gastric carcinoma samples from two Brazilian states, São Paulo and Ceará, associating the clinical and epidemiological characteristics of these patients. The results showed that 29.7% of patients present with the CC genotype, while the frequency of heterozygotes and (TC) and homozygous (TT) were 56.3% and 9%, respectively. Compared to data reported in the literature and used as control populations of dbSNP, the frequency of the T allele was higher in patients affected by gastric cancer (diffuse tumor: 45.5%, intestinal tumor: 39%, literature: 11-35 and dbSNP: 36%). Also in this context, we found a significant lower frequency of the CC genotype among patients affected by diffuse tumors (22.7%) when compared to healthy subjects (44-51%). In cases from Ceará population the C allele was significantly higher in the intestinal type (67%) compared to the diffuse type (50%), emphasizing the greater frequency of the T allele in diffuse cases. Analyzing the frequency of polymorphism rs12979860 C / T and genotyping of Helicobacter pylori was observed a strong association between genotype and infection with CagA + bacteria, where patients with the most pathogenic strain (CagA +) had a frequency higher than genotype less protective (TT), particularly in diffuse histotype (p = 0.001). In addition, this same allele was significantly associated with the occurrence of metastases in ... / Mestre

Este trabalho consiste em um estudo detalhado do sistema digestivo de Phibalosoma phyllinum (Phasmida, Phasmatidae) num enfoque morfofuncional. Do ponto de vista anatômico, o seu sistema digestivo é constituído por um intestino anterior, um intestino médio e um intestino posterior. O intestino anterior é composto por uma cavidade bucal (onde se abre o ducto da glândula salivar bilobada), uma faringe, que se continua para o esôfago, terminando em um proventrículo, que forma uma válvula em seu interior. O intestino médio é formado por um ventrículo tubular, dividido em três regiões: ventrículo anterior (VA), ventrículo médio (VM) e ventrículo posterior (VP). Este último foi, ainda, subdividido em proximal (VPI) e distal (VPII). Na região do VPI, foi constatada a presença de estruturas denominadas de apêndices ventriculares, formadas por protuberâncias, que se inserem no tubo digestivo, e canalículos, que se projetam da região apical de cada uma delas e terminam em fundo cego. O intestino posterior é dividido em um íleo e um reto que termina no ânus. A análise histológica mostrou que o intestino anterior é formado por um epitélio simples, composto por células pavimentosas, revestido por uma cutícula, que se modifica na forma de pequenas espículas ao longo desta região. O proventriculo é composto por uma válvula muscular simples, que separa o intestino anterior e o intestino médio. Este último é formado por um epitélio simples, constituído por células do tipo colunar, chamadas de enterócitos, principais responsáveis pela secreção de enzimas digestivas e pela absorção dos nutrientes, por células regenerativas, que se apresentam reunidas em ninhos na base do epitélio, e por células endócrinas. Os apêndices ventriculares, por sua vez, possuem um epitélio simples e contínuo com o epitélio ventricular. As células das protuberâncias apresentam-se grandes e arredondadas, enquanto que os canalículos são compostos por um epitélio de células achatadas, semelhante ao dos túbulos de Malpighi. Na região de transição entre o epitélio ventricular e o intestino posterior se inserem os túbulos de Malpighi. O epitélio do intestino posterior é do tipo cúbico, simples, revestido por cutícula. A luz do intestino médio (ou ventrículo) é revestida por uma estrutura tubular, denominada de membrana peritrófica, cuja existência foi comprovada por microscopia de fluorescência com a utilização da técnica WGA-FITC (aglutinina do gérmen do trigo conjugada à fluoresceína). Com a finalidade de identificar regiões específicas do ventrículo onde ocorre a absorção ou secreção de água através do epitélio, foram realizados experimentos fisiológicos de ingestão e injeção do corante amaranto em solução. Verificou-se que, quando o corante é ingerido pelos insetos, o VA apresenta-se corado, tanto em animais em jejum quanto alimentados, indicando ser este o possível sítio de absorção de água no ventrículo. Por sua vez, a partir de experimentos com a injeção do mesmo corante na hemolinfa, foi constatada a sua tomada pelos túbulos de Malpighi, indicando serem estes os principais sítios de tomada de água da hemolinfa e de sua secreção para a luz do tubo digestivo. Tanto em animais em jejum, quanto alimentados, o corante injetado é encontrado na luz do intestino posterior. Entretanto, nos animais em jejum, o corante é capaz de se difundir, também, pelo ventrículo até a sua região anterior. A análise ultraestrutural do intestino médio revelou que a superfície apical dos enterócitos constituintes apresenta-se modificada na forma de microvilosidades. Nas membranas laterais, observam-se especializações juncionais na forma de desmossomos apicais, seguidos por junções septadas lisas. A membrana plasmática basal, por sua vez, exibe diversas invaginações, formando uma rede complexa de canais com mitocôndrias associadas. Nas células do VA e VM, essa rede exibe um número limitado de aberturas para a lâmina basal, o que indica um maior potencial de absorção de água pelo epitélio ventricular, a partir de sua luz. Nas células da região do VP, o número de aberturas é bem maior, o que sugere que pode ocorrer secreção de água e íons através desta região, não detectável nos experimentos com corantes. Por outro lado, as células regenerativas exibem características típicas de células indiferenciadas, com núcleo grande e poucas organelas. Células endócrinas, por sua vez, são, eventualmente, detectadas na base do epitélio, sem se prolongarem até a superfície apical do epitélio. Foi observado, ainda, que tanto os canalículos ventriculares, quanto os túbulos de Malpighi são formados por células achatadas, com microvilosidades apicais modificadas portando mitocôndrias em seu interior e membrana plasmática basal formando um labirinto complexo, com muitas aberturas para a lâmina basal e com muitas mitocôndrias associadas. Com relação a atividade secretora do epitélio ventricular, verifica-se a existência de grandes quantidades de retículo endoplasmático rugoso e diversas áreas de Golgi concentradas, principalmente, no citoplasma perinuclear. Nas regiões VA e VM, é detectado um grande número de vesículas secretoras, cujo mecanismo de secreção parece ser o merócrino. Ao longo de todo VP, por outro lado, vesículas secretoras estão, aparentemente, ausentes no citoplasma apical, mas pode ser constatada a presença de dilatações nas microvilosidades, algumas das quais apresentem pequenas vesículas em seu interior. Esta observação é sugestiva da ocorrência de secreção do tipo microapócrina nesta região. Devem ocorrer, portanto, dois mecanismos de secreção diferentes ao longo do ventrículo de P. phyllinum: o merócrino (no VA e no VM) e o microapócrino (no VP) O local de produção e secreção das enzimas amilase e tripsina pôde ser localizado através de experimentos de imunomarcação ultraestrutural com a utilização de anticorpos heterólogos. Ambas as enzimas foram detectadas na região do VA e do VM, podendo ser traçadas nas áreas de Golgi, nas vesículas secretoras e na luz do epitélio, junto às microvilosidades, seguindo, pois, a rota secretora. Ambas as enzimas foram identificadas no interior do mesmo tipo de vesículas de secreção, indicando que devem ser eliminadas conjuntamente através da mesma via secretora. Medidas de pH luminal revelaram grandes diferenças de pH ao longo do tubo digestivo nesta espécie. Enquanto que na região anterior o pH é mais ácido (5,3 no intestino anterior e 5,6 no VA), ele vai se tornando mais alcalino nas regiões mais posteriores do ventrículo (6,3 no VM anterior, 8,0 no VM posterior, 9,1 no VPI e 8,5 no VPII) e é neutro no intestino posterior (7,3). Foi determinado, ainda, o efeito do pH sobre a atividade das enzimas tripsina e amilase. A maior atividade para tripsina foi observada no pH 9,0. Já para amilase, a maior atividade foi observada em pH 5,0. No que diz respeito a atividade de enzimas digestivas, ficou evidente que a atividade de amilase e tripsina se concentra no conteúdo do intestino anterior e VA. / This work presents a detailed morphofunctional study of the digestive system of Phibalosoma phyllinum (Phasmida, Phasmatidae). From an anatomical point of view, the digestive system of this insect is formed by a foregut, a midgut and a hindgut. The foregut is composed by the buccal cavity (where the salivary gland\'s duct open), a pharynx and an aesophagus that ends in the proventriculus. The midgut consists of a tubular ventriculus and it can be divided in three regions: the anterior ventriculus (AV), the middle ventriculus (MV), and the posterior ventriculus (PV) which can be further subdivided in its proximal (PVI), and distal (PVII) sub-regions. The presence of a complex system of ventricular appendices was observed in the PVI sub-region, which are formed by protuberances, in the external ventricular surface, and by blind-ended canaliculi, which are connected to the protuberances. The hindgut is divided into an ileum and a rectum that ends in the anus. Seen by light microscopy, the foregut is made up by a simple epithelium, which is composed of squamous cells and covered by a cuticle layer. Through the whole foregut the cuticle forms spines. The proventriculus is formed by a simple muscular valve that separates the foregut from the midgut. The midgut itself is formed by a simple epithelium, which is made up of three different cell types: columnar cells (enterocytes), which are the main site of enzyme production and secretion, and nutrient absorption, the regenerative cells, which are clustered in nidi at the basal portion of the epithelium, and the endocrine cells. The ventricular appendices, in turn, are formed by a simple epithelium that is continuous with the ventricular epithelium. Cells from the protuberances are large showing a round shape, while canalicular cells are short, and very similar to the ones presented in the Malpighian tubules. In the transition between the midgut and the hindgut, several Malpighian tubes branch off. The hindgut is formed by a simple epithelium, lined by cuticle. The ventricular lumen is covered by a tubular structure called peritrophic membrane whose presence was confirmed by fluorescence microscopy, using chitin-binding lectin WGA (wheat germ agglutinin) coupled with FITC (fluorescein isothiocyanate). In order to identify specific ventricular regions where water absorption and secretion through the epithelium take place, physiological experiments, using amaranth dye solution were performed. This solution was either orally administered, or injected in the hemolinph of both fed and starved insects. These experiments revealed that, both in fed and starved animals, the AV is the main site of water absorption, whereas the Malpighian tubules are the main sites of water secretion into the ventricular lumen. Ultrastructural analysis showed that enterocytes present an apical surface modified into well-developed microvilli. In their lateral surface, the adjacent plasma membranes are linked by desmosomes and smooth septate junctions. The basal plasma membrane shows several infoldings, forming a labyrinth of channels with associated mitochondria. In the AV and MV, these infoldings present a limited number of openings to the basal lamina, which indicates a greater water absorption potential of these regions from the gut lumen. In the PV cells, the number of openings is greater, indicating that these regions may be involved in water secretion (although occurring in levels that are undetectable through dye experiments). Regenerative cells nidi, in turn, can be observed in the basal portion of the epithelium throughout the midgut. This cell type shows characteristics of undifferentiated cells, such as large nuclei and few organelles. Endocrine cells are confined to the basal portion of the epithelium. Both the ventricular canaliculi and the Malpighian tubules are made up by cuboidal cells with modified apical microvilli bearing mitochondria in their interior, and basal plasma membrane forming a labirinth with many well developed infolds and openings to the basal lamina, as well as many associated mitochondria. An intense secretory activity was observed along the entire midgut. Large amounts of rough endoplasmic reticulum, well developed Golgi areas were observed in the enterocytes. In the AV and MV a large number of secretory vesicles could be observed concentrated in the apical portion of the cells, and their contents are probably eliminated by a merocrine mechanism. Along the PV, secretory vesicles are apparently absent but microvilli display dilated tips frequently showing small vesicles in their interior. This observation may indicate the occurrence of a microapocrine secretory mechanism in this ventricular region. The enzymes amylase and trypsin were immunolocalized in the cells from the AV and MV. In these regions, the two enzymes were detected in Golgi areas, secretory vesicles and in the the ventricular lumen, between the microvilli. Thus, both enzymes seem to be produced and eliminated through the same secretory pathway. Luminal pH measurements revealed a great variation of the pH along the intestine of this species. While in its anterior region the pH is acid (5,3 in the foregut and 5,6 in the AV), it becomes gradually alkaline towards the posterior regions (6,3 in the anterior MV, 8,0 in the posterior MV, 9,1 in the PVI and 8,5 in the PVII); in the hindgut the pH is neutral (7,3). The effect of the pH on amylase and trypsin activities was also measured. It was determined that trypsin\'s optimal pH was 9,0, and amylase\'s optimal pH was 5,0. Biochemical assays of digestive enzymes revealed the presence of amylase and trypsin in the luminal contents, mainly in the foregut and AV. In spite of that, it is possible that trypsin\'s activity is greater in the MV and PV, where the alkaline luminal pH matches the optimal pH for this enzyme. Chymotrypsin is also present in the lumen of the foregut and AV, but, unlike amylase and trypsin, its major activity is found in the AV. Aminopeptidase is found in the ventricular epithelium, mainly in the MV and PV, and maltase is detected associated with the microvillar glicocalix in the AV and PV regions. The hindgut showed low levels of digestive enzyme excretion, both in fed and starved animals, suggesting that these enzymes are recovered during the digestive process. Thus, these results point to the occurrence of an endo-ectoperitrophic circulation of digestive enzymes in both fed and starved animals, in which the AV is the main absorption site of water and the Malpighian tubules, the main secretion site. Initial carbohydrates digestion should occur in the foregut and in the AV, in the endoperitrophic space, whereas its final digestion should take place in the epithelial surface. Protein digestion should take place in the MV and in the PV; initial digestion should occur in the luminal endoperitrophic space, whereas final digestion takes place in the epithelial surface of these regions. Carbonic anhydrase assays revealed a high activity of this enzyme in the ventricular appendices. This fact, along with the morphological similarity between ventricular canaliculi and Malpighian tubules suggest that the canaliculi and the Malpighian tubules are homologous structures. Along the evolutionary process, the canaliculi probably acquired the capability to promote the alkalization of the PV lumen (mainly in the PVI region) thus affecting the digestive process

Tripsinas são enzimas comuns à maioria dos insetos e de fundamental importância para a digestão inicial de proteínas. Desta forma, tornam-se alvos importantes para orientar a construção de plantas transgênicas resistentes a insetos. As tripsinas são serina endopeptidases que clivam cadeias protéicas na porção carboxílica de resíduos de aminoácidos básicos como lisina e arginina, sendo que a hidrólise da ligação peptídica formada por um resíduo de arginina é de duas a dez vezes mais eficiente que a hidrólise da ligação peptídica formada por lisina. A purificação das tripsinas de insetos de diferentes ordens e o estudo da especificidade de seus subsítios utilizando substratos de fluorescência apagada servem de base para a seleção de um método mais eficiente de inibição da sua atividade, assim como para desvendar as tendências evolutivas da especificidade destas enzimas. O trabalho dessa dissertação levou ao desenvolvimento de processos de purificação das tripsinas de Periplaneta americana, Tenebrio molitor, Musca domestica e Diatraea saccharalis. O estudo da especificidade dos subsítios S1, S2, S3 e S1\' das tripsinas demonstraram que, diferentemente das tripsinas dos outros insetos e das tripsinas de mamíferos, a tripsina de Diatraea saccharalis hidrolisa com maior eficiência substratos que apresentem lisina em P1, demonstrando uma diferença na especificidade primária desta enzima. Além disso, é possível verificar ao longo da evolução dos grupos de insetos estudados uma tendência a tornar os subsítios cada vez mais hidrofóbicos. / Trypsins are serine endopeptidases that hydrolyze peptide bonds at the carboxyl side of positively charged residues: arginine and lysine. Mammalian trypsin preferentially cleaves the peptide bond formed by arginine. Site directed mutagenesis has shown that trypsin specificity is related to residues present at the primary specificity site and to structural determinants like two surface loops. Differences in trypsins specificity may be the cause of some insects be resistant to serine endopeptidases plant inhibitors and to Bacillus thuringiensis toxins. Trypsins are usual enzymes in insects and are very important to protein digestion. There are few studies dealing with insect trypsin specificity. They generally consist in analyses of fragments formed by the action of the enzyme on peptide chains like insulin &#946; chain. As these studies were semi-quantitative, insect trypsin specificity requires a better characterization. This dissertation describes the purification of trypsins from Periplaneta americana, Tenebrio molitor, Musca domestica and Diatraea saccharalis and the characterization of the specificity of the subsites S1, S2, S3 e S1\' by the use of quenched fluorescence peptide substrates. The results showed that trypsins from the mentioned insects have different specificities, including the primary specificity. Thus, Diatraea saccharalis trypsin cleaves at Lys more efficiently than at Arg, whereas the eontrary is true for the other insects. The data also showed that trypsin subsites tend to beeome more hydrophobic as the insects are more evolved.