Résumé : Les régimes hyperprotéiques sont couramment consommés mais les conséquences au niveau du gros intestin sont peu connues. L’objectif de la thèse était d’étudier les effets des régimes hyperprotéiques et des métabolites bactériens dérivés des acides aminés sur la muqueuse du gros intestinUne série d’expérimentations animales et in vitro a permis de montrer que deux métabolites bactériens dérivés des acides aminés (le sulfure d’hydrogène et le p-cresol) sont toxiques pour l’épithélium lorsqu’ils sont présents en concentration élevée. Les résultats obtenus lors d’une étude clinique montrent que la quantité et la qualité des protéines alimentaires n’ont pas d’effets marqués sur la composition du microbiote fécal mais modifient les concentrations fécales et urinaires en métabolites bactériens.Ces modifications de l’environnement luminal du gros intestin n’étaient pas associées à une augmentation de la cytotoxicité des eaux fécales in vitro. Néanmoins, dans la muqueuse rectale, l’augmentation de l’apport en protéines a régulé l’expression de gènes impliqués dans le maintien de l’homéostasie et ces effets étaient distincts en fonction de la source de protéines utilisée. Toutefois, le niveau d’apport en protéines n’avait pas d’effet sur les paramètres inflammatoires et histologiques dans la muqueuse. Ces résultats ont été complétés par une étude chez le rat montrant qu’un régime hyperprotéique modifie le transcriptome dans les colonocytes mais n’a pas d’effets délétères en termes d’intégrité de l’ADN, de renouvellement de l’épithélium et de fonction barrière. / Abstract: High-protein diets are frequently consumed but the consequences for the large intestine are not well described. The objective of this thesis was to evaluate the effects of high-protein diets and of amino-acid derived bacterial metabolites on the large intestine mucosa. Animal and in vitro studies showed that two amino acid derived bacterial metabolites (hydrogen sulfide and p-cresol) are toxic for the epithelium when present at high concentration. The results obtained in a clinical trial indicate that quantity and quality of dietary protein do not have major effects on the fecal microbiota composition but modify the fecal and urinary concentration of bacterial metabolites.These changes in luminal environment were not associated with an increase in fecal water cytotoxicity in vitro. Nevertheless, in the rectal mucosa, the increase in protein intake regulated the expression of genes implicated in homeostatic processes and these effects were modulated by the source of protein. However, the level of protein intake had no effect on immune and histological parameters in the mucosa. These results were completed with a study in rats showing a clear transcriptome profile in colonocytes induced by a high-protein diet but that was not associated with detrimental effects in terms of DNA integrity, epithelial renewal and barrier function.

No / The interleukin-2-deficient (IL-2¿/¿) mouse model of ulcerative colitis was used to test the hypothesis that colonic epithelial cells (CEC) directly respond to bacterial antigens and that alterations in Toll-like receptor (TLR)-mediated signaling may occur during the development of colitis. TLR expression and activation of TLR-mediated signaling pathways in primary CEC of healthy animals was compared with CEC in IL-2¿/¿ mice during the development of colitis. In healthy animals, CEC expressed functional TLR, and in response to the TLR4 ligand LPS, proliferated and secreted the cytokines IL-6 and monocyte chemoattractant protein-1 (MCP-1). However, the TLR-responsiveness of CEC in IL-2¿/¿ mice was different with decreased TLR4 responsiveness and augmented TLR2 responses that result in IL-6 and MCP-1 secretion. TLR signaling in CEC did not involve NF-B (p65) activation with the inhibitory p50 form of NF-B predominating in CEC in both the healthy and inflamed colon. Development of colitis was, however, associated with the activation of MAPK family members and upregulation of MyD88-independent signaling pathways characterized by increased caspase-1 activity and IL-18 production. These findings identify changes in TLR expression and signaling during the development of colitis that may contribute to changes in the host response to bacterial antigens seen in colitis.

No / Methionine dependence is unique to cancer cells and defined as the inability to grow in a methionine-deprived environment even if supplemented with the metabolic precursor homocysteine. Cobalamin-dependent methionine synthase (MS) catalyses the formation of methionine and tetrahydrofolate from homocysteine and methyltetrahydrofolate, thus linking the methionine and folate pathways. The apparent altered methionine metabolism in methionine-dependent cancer cells suggests a role for MS, although results to date are conflicting. We have analysed key metabolites of the MS-associated transmethylation, transsulphuration and folate pathways of the methionine-dependent MAC15A tumour model as a function of tumour progression over a 10-day period. MS activity increased 2-fold from day I to day 10. Cysteine, homocysteine, S-adenosylmethionine and S-adenosylhomocysteine levels in tumour cytosolic fractions decreased as a function of tumour progression. Plasma cysteine levels also decreased, whilst the distribution of folates in erythrocytes was altered, with a maximum increase in methyltetrahydrofolate observed by day 5. The increasing MS activity and decreasing cysteine levels suggest an increasing methionine requirement by the tumour, whilst the induction of enzyme activity indicates that MS is not defective in the methionine-dependent MAC15A tumour. The decrease in tumour S-adenosylmethionine and S-adenosylhomocysteine levels suggests that methionine is required for some function other than cellular methylation, e.g., incorporation into protein. Overall, the results support a theory of methionine conservation in response to tumour growth, where the methionine-dependent MAC15A tumour has a higher than normal methionine requirement.

<p>UVOD: Kolorektalni karcinom je na trećem mestu po učestalosti oboljevanja od svih karcinoma uz porast incidencije CRC u visoko razvijenim zemljama.70% obolelih od CRC je starije od 65 godina uz veću incidenciju proksimalnih karcinoma u odnosu na distalne u svim uzrasnim grupama i kod oba pola. Smatra se da bi 66-75% slučajeva CRC moglo biti izbegnuto zdravim načinom života. 75% CRC nastaje iz adenomatoznih preko polip kancer sekvence i da vi&scaron;e od 90% adenoma neće progredirati u karcinom. U studijama skrining kolonoskopija prijavljeno je 6-12% neviđenih velikih polipa i ko 5% CRC na inicijalnom kolonoskopskom pregledu. Postoperativne periodične kolonoskopije nakon operacije kolorektalonog karicinoma imaju za cilj otkrivanje metahronih carcinoma polipa kao pojavu bolesti na anastomozi ali nije dokazani benefit u preživljavanju bolesnika koji su imali učestalije postoperativne kolonoskopije (na godinu dana) u odnosu na one koji su praćenina 3 ili 5 godina. CILJEVI ISTRAŽIVANJA: Prospektivno ispitivanje pojave intervalnih lezija kolona (malignih i premalignih) u periodu od 2-7 godine od prve negativne kolonoskopije bez obzira na razlog pregleda. Ispitivanje razlike u životnim navikama između ispitanika u zavisnosti od nalaza na ponovljenoj kolonoskopiji. Retrospektivna analiza svih dijagnostičkih i kontrolnih kolonoskopija. MATERIJAL I METODE: Ponavljana je kolonoskopija kod ispitanika koj su na dijagnostičkim kolonoskopijama rađenim na Institutu za onkologiju Vojvodine u periodu 2005-2011. imali uredan kolonoskopski nalaz. Od 160 pozvanih ispitanika na ponovnu kolonoskopiju se odazvalo 64 ispitanika a 151 ispitanik je popunio upitnik o životnim navikama. Urađena je i retospektivna analiza 2750 dijagnostičkih kolonoskopija. Analizirani su rezultati 1064 prvih postoperativnih kolonoskopija kao i nalazi sa 1147 ponovljenih kolonoskopija kod ispitanika operisanih od kolorektalnog carcinoma koji su imali uredan nalaz na prvoj kolonoskopiji. REZULTATI: Od 160 pozvanih ispitanika,njih 64 (42,3%) se odazvalo na ponovni pregled (45 žena i 19 mu&scaron;karaca) prosečne starosti60,13 godina. Kod 15 ispitanika(24.3%) nađeno je ukupno 22 polipa (10 žena i 5 mu&scaron;karaca) bez statistički značajne razlike u pozitivnosti nalaza u odnosu na pol (x2test; x2=0,014; p=0,904) i pozitivnu porodičnu anamnezu (x2test; x2=0,125; p=0,724). 12 slucajeva (14,06%) su bili polipi visokog rizika: 5 (41.6%) lokalizovano u proksimalnom kolonu i 7 (58.3% ) u distalnom kolonu. Nije dijagnostikovan nijedan intervalni karcinom. Nije dokazana statistički značajna razlika u pozitivnosti nalaza na ponovljenoj kolonoskopiji u odnosu na razmak posmatran u grupama do 3 i do 5 godina od predhodne kolonoskpije (x2test; x2=0,020; p=0,887) niti ukoliko se posmatra po grupama do 5 i preko 5 godina od negative kolonoskopije (x2test; x2=3,082; p=0,079). Nema statistički značajne razlike u pozitivnosti nalaza na ponovljenoj kolonoskopiji u odnosu na to da li su pacijenti konzumiraju alkohol ili ne (x2test; x2=0,113; p=0,911) kao i u odnosu na to da li su pacijenti imali redovnu fizičku aktivnost (x2test; x2=0,476; p=0,490). Na dijagnostičkim kolonoskopijama je uočena statistički značajna razlika u uzrastu pacijenata u zavisnosti od razloga kolonoskopije (F=7,111; p=0,000) kod pacijenata kod kojih su dijagnostikovani polipi. Oni sa pozitivnom porodičnom anamnezom i polipima su statistički značajno mlađi u odnosu na ostale osim onih koji su se na pregled javili zbog bola u trbuhu poremećaja ritma stolice. Nije bilo statistički značajne razlike po polu, uzrastu, u razlogu kolonoskopije kod osoba sa dijagnostikovanim polipima. Statistički je značajniji broj žena sa lokalizacijom polipa u distalnom delu debelog creva u odnosu na proksimalni (x2test; x2=18,495; p=0,000). Kod mlađih uzrasnih grupa statistički značajnije su zastupljeni polipi u rektumu(x2test; x2=79,963; p=0,000). Ispitanici sa proksimalnom lokalizacijom polipa imaju 1,724 puta veću &scaron;ansu za adenome visokog rizika u odnosu na one sa distalnom lokalizacijom. Nema statistički značajne razlike u distribuciji karcinoma u odnosuna pol (x2test; x2=3,2110; p=0,201). Na 1064 prvih postoperativnih kolonoskopija je bilo ukupno 346 (32,5%) pozitivnih nalaza. Dijagnostikovano je 60 karcinoma od kojih je 43,3 % lokalizovano na anastomozi a kod 286 ispitanika nađeno je ukupno 546 polipa. Mu&scaron;karci statistički značajnije če&scaron;će imaju pozitiva nalaz (x2 test; x2=17,252; p=0,000). Bonferroni post hoc testom je utvrđeno da su polipi proksimalne lokalizacije statistički značajno veći od onih u rektumu (p=0,043). Na kontrolnim kolonoskopijama rađenim u cilju praćenja nakon resekcije kolorektalnog karcinoma multivarijatnom analizom ( pol, uzrast i vreme od operacije) utvrđeno je da mu&scaron;karci imaju 1,4 puta veću &scaron;ansu (OR=1,457) od žena za pojavu promena (polipa i karcinoma).Ispitanici kod kojih je od operacije pro&scaron;lo od 3 do 5 godina imaju 1,6 puta veću &scaron;ansu za pojavu promene u odnosu na one kod kojih je pro&scaron;la 1 godina (OR=1,605). ZAKLJUČAK: Kod 24.3% pregledanih ispitanika dijagnostikovani su polipi(jedan hipeplastičnii 21 adenoma ). 14,06% svih polipa je imalo karakteristike polipa visokog rizika bez statistički značajne razlike u pojavi polipa kod ispitanika kod kojih je pregled rađen 3,5 ili nakon 5 godina od prve negativne kolonoskopije. Nije dijagnostikovan niti jedan karcinom &scaron;to znači da nema potrebe za ponavljanjem kolonoskopija u kraćem vremenskom intervalu od unapred planirane kolonoskopije kod ispitanika koji su imali uredan inicijalni kolonoskopski nalaz &scaron;to se odnosi i na ponavljane kolonoskopije kod ispitanika operisanih od CRC-a. Na dijagnostičkim kolonoskopijama statistički značajniji broj žena sa lokalizacijom polipa u distalnom delu debelog creva u odnosu na proksimalni i nije zapažena razlika u distribuciji karcinoma u odnosu na pol i uzrast ispitanika.</p> / <p>INTRODUCTION:Colorectal cancer is the third most frequent illness of all carcinomas with an increase in the incidence of CRC in highly developed countries. 70% of patients with CRC are older than 65 years with higher incidence of proximal cancers compared to distal in all age groups and in both sexes. It is believed that 66-75% of CRC could be avoided through healthy lifestyle. 75% of CRC arise from adenomatous polyp cancer via sequences, and that more than 90% of adenoma will not progress to carcinoma. In studies of screening colonoscopy was reported 6-12% of unobserved large polyps and approximately 5% of the CRC on the initial colonoscopy.Postoperative periodic colonoscopy after colorectal cancer surgery aim to detect metachronous cancer and polyps and disease occurrence anastomoses but not proven survival benefit in subjects who had more frequent postoperative colonoscopy (per year) compared to those who were followed for 3 or 5 years. AIM:Prospective study of interval colon lesions occurrence (malignant and pre-malignant) in the period from 2-7 years after initial negative colonoscopy regardless of the reason for the check. Test of differences in lifestyle between subjects depending on the findings of the repeated colonoscopy.A retrospective analysis of all the diagnostic and control colonoscopy. METHODOLOGY: Repeated colonoscopy in subjects who are at-made diagnostic colonoscopy at the Oncology Institute of Vojvodina in the period 2005-2011 had normal colonoscopy findings. Of the 160 subjects invited to re colonoscopy for review responded 64 subjects and 151 subjects filled out a questionnaire about life habits. Retrospective analysis of 2750 and diagnostic colonoscopy has been done. Results of the 1064 first postoperative colonoscopy and results of the 1147 repeated colonoscopy in patients operated on for colorectal cancer that had normal findings on the first colonoscopy has been analyzed. RESULTS:Of the 160 invited subjects, 64 of them (42.3%) responded to the repeated review (45 women and 19 men), mean age 60.13 years. In 15 subjects (24.3%) found a total of 22 polyps (10 women and 5 men) with no statistically significant differences in positivity findings in relation to sex (x2test; x2 = 0.014; p = 0.904) and a positive family anamnesis (x2test; x2 = 0.125; p = 0.724).12 cases (14.06%) were high risk of polyps: 5 (41.6%) localized in the proximal colon, and 7 (58.3%) in the distal colon. Not a single interval cancer diagnosed. There was no statistically significant difference in positivity findings with repeated colonoscopy in relation to the distance observed in groups of 3 to 5 years from the previous colonoscopy (x2test; x2 = 0.020; p = 0.887) or when observed in groups up to 5 and over 5 years of negative colonoscopy (x2test; x2 = 3.082; p = 0.079). No statistically significant differences in positivity findings with repeated colonoscopy in relation to whether the patients consume alcohol or not (x2test; x2 = 0.113; p = 0.911) as well as in relation to whether patients are regularly exercising (x2test; x 2 = 0.476; p = 0.490). Statistically significant difference is confirmed in the age of patients at the diagnostic colonoscopy, depending on the reason for colonoscopy (F = 7.111; p = 0.000) in patients who were diagnosed polyps. Those with a family anamnesis and polyps were statistically significant younger in comparison to others except those who have come forward for review because of abdominal pain and bowel movement rhythm disturbances.There were no statistically significant differences by sex, age, the reason for colonoscopy in patients diagnosed with polyps.Statistically is more significant number of women with the localization of polyps in the distal part of the colon comparing to the proximal (x2test; x2 = 18,495; p = 0.000).In younger age groups are represented statistically significant polyps in the rectum (x2test; x2 = 79.963, p = 0.000). Subjects with proximal localization of polyps are 1,724 times more likely for high-risk adenomas compared to those with distal localization.No statistically significant differences in the distribution of cancer in relation to sex (x2test; x2 = 3.2110; p = 0.201).On the first postoperative colonoscopy in 1064 subjects there were a total 346 (32.5%) positive findings. 60 carcinoma diagnosed of which 43.3% is localized on the anastomosis and in 286 of the subjects had a total of 546 of the polyps.Men statistically significantly more likely to have positive findings (x2 test; x2 = 17,252; p = 0.000). Bonferroni post hoc test showed that polyps proximal localization significantly bigger than those in the rectum (p = 0.043). On the control colonoscopy-made for the purpose of monitoring after resection of colorectal cancer by multivariate analysis (sex, age and time of surgery) it has been found that men are 1.4 times more likely (OR = 1.457) than women for the occurrence of changes (polyps and cancers).Subjects having passed since the operation of 3 to 5 years are 1.6 times more likely to develop a change with respect to those in which the more than one year elapsed (OR = 1.605). CONCLUSION:In 24.3% subjects were diagnosed polyps (one hyperplastic and 21 adenomas).14.06% of all polyps had the characteristics of high-risk polyps with no statistically significant difference in the occurrence of polyps in subjects where the examination was done after 3,5 or 5 years since the first negative colonoscopy. No cancers diagnosed, meaning there is no need to repeat colonoscopy in a shorter period of time than pre-planned colonoscopy in subjects who had normal initial colonoscopy findings which refers to the repeated colonoscopies in subjects operated on for CRC.For diagnostic colonoscopy statistically significant number of women with the localization of polyps in the distal part of the colon compared to proximal and was not observed differences in the distribution of carcinoma in relation to sex and age of the subject.</p>

Immunohistological (IHC) stained images occupy a fundamental role in the pathologist's diagnosis and monitoring of cancer development. The manual process of monitoring such images is a subjective, time consuming process that typically relies on the visual ability and experience level of the pathologist. A novel and comprehensive system for the automated quantification of antibody inside stained cell nuclei in immunohistochemistry images is proposed and demonstrated in this research. The system is based on a cellular level approach, where each nucleus is individually analyzed to observe the effects of protein antibodies inside the nuclei. The system provides three main quantitative descriptions of stained nuclei. The first quantitative measurement automatically generates the total number of cell nuclei in an image. The second measure classifies the positive and negative stained nuclei based on the nuclei colour, morphological and textural features. Such features are extracted directly from each nucleus to provide discriminative characteristics of different stained nuclei. The output generated from the first and second quantitative measures are used collectively to calculate the percentage of positive nuclei (PS). The third measure proposes a novel automated method for determining the staining intensity level of positive nuclei or what is known as the intensity score (IS). The minor intensity features are observed and used to classify low, intermediate and high stained positive nuclei. Statistical methods were applied throughout the research to validate the system results against the ground truth pathology data. Experimental results demonstrate the effectiveness of the proposed approach and provide high accuracy when compared to the ground truth pathology data.

The longitudinal muscle layer in gut is the functional opponent to the circular muscle layer during the peristalsis reflex. Differences in innervation of the layers allow for the contraction of one layer that corresponds with the simultaneous relaxation of the other, enabling the passage of gut contents in a controlled fashion. Differences in development have given the cells of the two layers differences in receptor populations, membrane lipid handling, and calcium handling profiles/behaviors. The kinase signaling differences between the two layers is not as well characterized. Upon activation of cells from the circular muscle layer, it is known that Rho kinase and ERK1/2 promote contraction, while CaMKK/AMPK and CaMKII perform inhibitory/self-inhibitory roles. Such behaviors are poorly understood in the longitudinal muscle layer. In longitudinal muscle strips, we measured muscarinic receptor-mediated contraction following incubation with kinase inhibitors. Upon comparison to control, contributions of Rho Kinase and ERK1/2 were similar to those seen in circular muscle. Inhibition of both of these enzymes leads to diminished contraction. However, CaMKK/AMPK and CaMKII have effects in longitudinal muscle opposite to their regulation in circular muscle – their inhibition also diminishes the contractile response. These contractile data from strips were supported by immunokinase assay measurements of MLCK activity from strip homogenates with and without kinase inhibition. Therefore, we suggest that the activities of CaMKK/AMPK and CaMKII in longitudinal muscle are indeed different from their regulatory roles in circular muscle, perhaps a consequence of the different calcium handling modalities of the two muscle types.

<p>Žučne kiseline deluju kao signalni molekuli u organizmu i uključene su u regulaciju brojnih metaboličkih, inflamatornih i imunomodulatornih procesa. Ova endogena jedinjenja ostvaruju svoje efekte najvećim delom putem nuklearnih receptora. Farnezoid X receptor (FXR) je glavni regulator homeostaze žučnih kiselina, a pokazano je da je značajno uključen i u procese inflamacije i kancerogeneze, prevashodno u jetri i intestinalnom traktu. Aktivacija FXR receptora predstavlja značajnu farmakolo&scaron;ku strategiju za terapiju holestatskih bolesti jetre, inflamatorne bolesti creva i karcinoma kolona. Definisana je uloga žučnih kiselina u signalnim putevima koji reguli&scaron;u ćelijski ciklus i doprinose razvoju ili regresiji maligniteta, ali je malo poznat uticaj ovih jedinjenja na epigenetske mehanizme regulacije ključnih ćelijskih procesa. Imajući u vidu da su efekti žučnih kiselina determinisani njihovom polarno&scaron;ću, cilj istraživanja je bio da se ispita uticaj sintetski dobijenog keto derivata holne kiseline, 12-monoketoholne kiseline (MKH), u komparaciji sa prirodnim žučnim kiselinama, hidrofobnom henodeoksiholnom kiselinom (HDH) i hidrofilnom ursodeoksiholnom kiselinom (UDH), na ćelijske procese apoptoze, oksidativnog stresa i inflamacije, koji su od značaja za hemoprevenciju i terapiju karcinoma kolona, u in vitro i in vivo sistemima. Cilj istraživanja je takođe obuhvatao i ispitivanje uloge odabranih žučnih kiselina u epigenetskoj regulaciji ovih procesa u ćelijama karcinoma kolona. Na in vivo modelu intrahepatične holestaze kod eksperimentalnih životinja, pokazano je da UDH i MKH ispoljavaju antiapoptotski, antioksidativni i antiinflamatorni efekat u jetri i intestinumu. Utvrđeno je da UDH i MKH sprečavaju mitohondrijalni put aktivacije apoptoze u jetri, dok UDH ispoljava antiapoptotski efekat i u intestinumu eksperimentalnih životinja sa holestazom. Ove dve žučne kiseline su u značajnoj meri modulirale ekspresiju gena uključenih u antioksidativnu za&scaron;titu, kao i aktivnost antioksidativnih enzima, u jetri i intestinumu eksperimentalnih životinja sa holestazom, ka nivoima ekspresije i aktivnosti kod zdravih, netretiranih životinja. Dok su UDH i MKH u dozi od 4 mg/kg ispoljile antiinflamatorno dejstvo u jetri i intestinumu smanjenjem ekspresije gena za proinflamatorni transkripcioni faktor NF-&kappa;B, primena HDH i MKH u dozi od 20 mg/kg je imala suprotan efekat. Na modelu HT-29 ćelijske linije adenokarcinoma kolona, utvrđeno je da polusintetska žučna kiselina MKH ispoljava značajno manju citotoksičnost u odnosu na HDH i ne&scaron;to veću citotoksičnost u odnosu na UDH. Epigenetski lek vorinostat je ispoljio sinergističko citotoksično dejstvo sa sve tri ispitivane žučne kiseline. Vorinostat je ostvario proapoptotski i antiproliferativni efekat u HT-29 ćelijama, koji je bio najizraženiji u kombinaciji sa MKH, s obzirom da je do&scaron;lo do značajnog povećanja odnosa ekspresije BAX i BCL2 gena i smanjenja ekspresije gena za marker proliferacije ciklin D1. Vorinostat je, takođe, značajno smanjio antioksidativni kapacitet HT-29 ćelija smanjenjem ekspresije NRF2 gena i sledstvenim smanjenjem ekspresije gena za antioksidativne enzime. HDH je dodatno smanjila, a MKH pobolj&scaron;ala antioksidativni kapacitet HT-29 ćelija modulacijom ekspresije NRF2 gena. U in vitro i in vivo sistemu u okviru ove doktorske disertacije je pokazano da, pored HDH kao poznatog endogenog agoniste FXR receptora, MKH takođe povećava ekspresiju gena za FXR i njegovog ciljnog gena za transkripcioni korepresor SHP, &scaron;to ukazuje da ova polusintetska žučna kiselina može da aktivira FXR. Osim toga, utvrđeno je da žučne kiseline ispoljavaju različite efekte prema ekspresiji gena za histon deacetilaze HDAC1 i HDAC2 u jetri i intestinumu eksperimentalnih životinja, kao i u HT-29 ćelijama karcinoma kolona, a jedino je UDH značajno smanjila ekspresiju gena za oba ispitivana enzima uključena u epigenetsku regulaciju ćelijskih procesa, i u isptivanim tkivima i HT-29 ćelijama. Rezultati na&scaron;eg rada ukazuju da bi se UDH i MKH mogle koristiti u hemoprevenciji karcinoma kolona u niskim dozama, s obzirom na utvrđene efekte u modulaciji ekspresije gena uključenih u procese apoptoze, oksidativnog stresa i inflamacije. Takođe, s obzirom na ostvaren sinergistički efekat žučnih kiselina sa epigenetskim antitumorskim agensom vorinostatom, otvara se mogućnost kombinovane farmakolo&scaron;ke strategije u terapiji solidnih tumora, koji u najvećem procentu pokazuju rezistenciju prema samom vorinostatu.</p> / <p>Bile acids act as signaling molecules in the organism and they are involved in the regulation of numerous metabolic, inflammatory and immunomodulatory processes. These endogenous compounds exert their effects mostly by binding and activation of nuclear receptors. Farnesoid X receptor (FXR) is the main regulator of bile acid homeostasis, and has been shown to be significantly involved in processes of inflammation and carcinogenesis, primarily in the liver and intestinal tract. Activation of FXR receptor represents a significant pharmacological strategy for the treatment of cholestatic liver disease, inflammatory bowel disease, and colon carcinoma. The role of bile acids in signaling pathways regulating the cell cycle and contributing to the development or regression of malignancies is well determined, but the effects of these compounds on epigenetic mechanisms of key cellular processes regulation is yet to be elucidated. Given that the effects of bile acids are mostly determined by their polarity, the aim of our study was to investigate in vitro and in vivo effects of semi-synthetic keto derivative of cholic acid, 12-monoketocholic acid (MKC), in comparison to natural bile acids, hydrophobic chenodeoxycholic acid (CDC) and hydrophilic ursodeoxycholic acid (UDC), on processes of apoptosis, oxidative stress and inflammation, which are significant for both&nbsp; chemoprevention and therapy of colon cancer. Besides, the aim of our study was to examine the role of selected bile acids in the epigenetic regulation of these processes in colon cancer cells. In in vivo model of intrahepatic cholestasis in experimental animals, it has been demonstrated that UDC and MKC exhibit antiapoptotic, antioxidant, and antiinflammatory effects in the liver and intestine. It was shown that UDC and MKC prevent the mitochondrial pathway of apoptosis activation in the liver, while UDC exhibits an antiapoptotic effect in the intestine of experimental animals with cholestasis as well. These two bile acids significantly modulated the expression of genes involved in antioxidant protection, as well as the activity of antioxidant enzymes, in the liver and intestine of experimental animals with cholestasis, towards levels of expression and activity in healthy, untreated animals. While UDC and MKC at a low dose of 4 mg/kg exhibited an antiinflammatory effect in the liver and intestine by reducing the expression of the gene encoding the proinflammatory transcription factor NF-&kappa;B, the application of CDC and MKC at a high dose of 20 mg/kg exerted the opposite effect. In HT-29 human adenocarcinoma cell line, it has been demonstrated that semi-synthetic bile acid MKC exhibits significantly lower cytotoxicity than CDC and slightly higher cytotoxicity than UDC. The epigenetic drug vorinostat has exhibited a synergistic cytotoxic effect with all three investigated bile acids. Vorinostat exerted proapoptotic and antiproliferative effects in HT-29 cells, which were most pronounced in combination with MKC, as there was a significant increase in the ratio of BAX and BCL2 genes expression and a decrease of the proliferation marker cyclin D1 gene expression. Vorinostat also significantly reduced the antioxidant capacity of HT-29 cells by reducing the expression of NRF2 gene and consequently decreasing the expression of genes encoding antioxidant enzymes. CDC further reduced, while MKC improved the antioxidant capacity of HT-29 cells by modulating the expression of NRF2 gene. In both in vitro and in vivo systems, it was demonstrated that, in addition to CDC as a known endogenous FXR agonist, MKC also increased the expression of the gene encoding FXR, and FXR target gene encoding transcriptional co-repressor SHP as well, indicating that this semi-synthetic bile acid can also activate FXR. Besides, bile acids have been shown to exert distinct effects on the expression of the histone deacetylases HDAC1 and HDAC2 gene in the liver and intestine of experimental animals, and in HT-29 colon cancer cells. Only UDC significantly reduced the expression of the genes for both studied enzymes involved in the epigenetic regulation of cell processes, in both tissues and HT-29 cells. The results of our work indicate that UDC and MKC could be used in chemoprevention of colon cancer at low doses, considering determined effects in the modulation of expression of the genes involved in processes of apoptosis, oxidative stress and inflammation. Furthermore, synergistic effects of bile acids with the epigenetic antitumor agent vorinostat open up the possibility of a combined pharmacological strategy in the treatment of solid tumors, which are at the high percentage resistant to the effects of vorinostat alone.</p>

<p>Sve veća potreba za izvođenjem kolonoskopije u dijagnostičke ili terapijske svrhe nameće potrebu za usavr&scaron;avanjem ove endoskopske procedure. Izvođenje kolonoskopije u op&scaron;toj, intravenskoj anesteziji, moglo bi u značajnoj meri olak&scaron;ati njeno izvođenje, pobolj&scaron;ati podno&scaron;enje ove procedure od strane ispitanika i omogućiti otkrivanje većeg broja pacijenata sa potencijalno malignim bolestima debelog creva. Cilj ispitivanja je bio utvrditi da li primena op&scaron;te intravenske anestezije tokom kolonoskopije povećava broj totalnih kolonoskopija i skraćuje vreme intubacije cekuma, povećava broj viđenih patolo&scaron;kih procesa i smanjuje osećaj bola i učestalost neželjenih reakcija. Primena op&scaron;te intravenske anestezije značajno je povećala broj totalnih kolonoskopija u 94.3% ispitanika u odnosu na 78.7% totalnih kolonoskopija kontrolne grupe i skratila vreme intubacije cekuma, značajno je povećala broj viđenih patolo&scaron;kih promena u 46.7% ispitanika u odnosu na broj viđenih patolo&scaron;kih promena u 28.8% ispitanika kontrolne grupe i značajno je smanjila intenzitet bola i učestalost neželjenih reakcija. U kontrolnoj grupi ispitanika skalom bola nakon kolonoskopije ustanovljen je značajno veći intenzitet bola u poređenju sa ispitanicima ekperimentalne grupe. Na Likertovoj skali zadovoljstva ustanovljena je značajno bolja kontrola bola i lični stav lekara u ekperimentalnoj grupi, dok su poseta ustanovi i procedura, razumevanje procedure, tehnička ve&scaron;tina lekara, lični stav medicinskih sestara i drugog tehničkog osoblja značajno bolje ocenjeni u kontrolnoj grupi. Od svih ispitivanih faktora na zadovoljstvo obe grupe pacijenata značajno su uticali: način izvođenja procedure, bol, uočene patolo&scaron;ke promene i intubacija cekuma. U kontrolnoj grupi ispitanika između skale zadovoljstva i skale bola ustanovljena je značajna negativna korelacija. U kontrolnoj grupi se 80,1% pacijenata izjasnilo da bi ponovnu kolonoskopiju uradili u op&scaron;toj intravenskoj anesteziji u poređenju sa svim pacijentima eksperimentalne grupe koji ne bi menjali način izvođenja ponovne procedure. Primena op&scaron;te intravenske anestezije tokom kolonoskopije povećava broj totalnih kolonoskopija i uočenih patolo&scaron;kih promena, smanjuje učestalost i intenzitet neželjenih reakcija povećavajući zadovoljstvo pacijenata, &scaron;to bi prevashodno moglo imati značaja u skriningu karcinoma debelog creva. Potrebno je pro&scaron;iriti ispitivanje primene op&scaron;te intravenske anestezije u drugim endoskopskim procedurama kako bi bila uvedena u svakodnevnoj kliničkoj praksi.</p> / <p>An increasing need to perform colonoscopy for diagnostic or therapeutic purposes imposed the need for mastering this endoscopic procedure. Performing colonoscopy in general intravenous anesthesia could greatly ease the procedure, make it more comfortable for patients and it could enable detecting a higher number of patients with occult malignant diseases of the colon. The aim of this paper was to determine if the implementation of general intravenous anesthesia during colonoscopy increases the number of total colonoscopies and shortens the time of cecum intubation, increases the number of familiar pathological processes and decreases the sensation of pain as well as the frequency of side reactions. The implementation of general intravenous anesthesia has significantly increased the number of total colonoscopies in 94.3% of examined patients in relation to 78.7% of total colonoscopies of the control group and shortened the time of cecum intubation. It has significantly increased the number of familiar pathological changes in 46.7% of the patients in relation to the number of familiar pathological changes in 28.8% patients of the control group and significantly decreased pain intensity and the frequency of side reactions. A statistically greater pain intensity after colonoscopy was determined by the pain scale in the control group in comparison to the examinees of the experimental group. Likert satisfaction scale has shown that the experiment group assessed pain control and doctors&rsquo; opinion as significantly better, while the institution visits and the procedure, understanding the procedure, doctors&rsquo; technical skills, nurses&rsquo; and technical personnel&rsquo;s personal opinions were assessed as significantly better in the control group. Out of all the examined factors on the satisfaction of both groups, the following ones had a significant inluence: the way the procedure was done, the level of pain, detected pathological changes and cecum intubation. A significanlty negative correlation was determined between the scale of satisfaction and the scale of pain in the control group. 80.1% of the control group patients stated that they would undergo a general anesthesia colonoscopy again in comparison to all the patients of the experimental group who would not change the way the procedure was previously done. The implementation of general intravenous anesthesia in the course of colonoscopy increases the number of total colonoscopies and detected pathological changes, decreases the frequency and intensity of side-effects therefore it enhances patients&#39; sastisfaction, which could play a major role in colon cancer screening. It is necessary to extend the implementation of general intravenous anesthesia in other endoscopic procedures in order to introduce it in everyday clinical practice.</p>

Chan, Man Hung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 111-126). / Abstract also in Chinese. / Examination Committee List --- p.ii / Declaration --- p.iii / Acknowledgements --- p.iv / 摘要（Abstract in Chinese) --- p.vi / Abstract --- p.viii / List of Abbreviations --- p.x / List of Figures and Tables --- p.xii / Table of Content --- p.xiv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Background of photodynamic therapy (PDT) --- p.2 / Chapter 1.1.1 --- History of PDT --- p.2 / Chapter 1.1.2 --- Photochemistry --- p.3 / Chapter 1.1.3 --- Principal stages of PDT --- p.5 / Chapter 1.1.4 --- Light sources of PDT --- p.6 / Chapter 1.2 --- Anti-tumor effect of PDT --- p.8 / Chapter 1.2.1 --- Mode of cell death --- p.8 / Chapter 1.2.2 --- PDT-induced anti-tumor immunity --- p.9 / Chapter 1.3 --- Clinical applications of PDT --- p.11 / Chapter 1.3.1 --- Photofrin® --- p.11 / Chapter 1.3.2 --- Clinical applications of PDT --- p.13 / Chapter 1.3.3 --- Challenges of PDT for clinical applications --- p.15 / Chapter 1.4 --- The development of new photosensitizers --- p.16 / Chapter 1.4.1 --- Targeted PDT --- p.16 / Chapter 1.4.2 --- Phthalocyanine --- p.18 / Chapter 1.5 --- Objective of my study --- p.21 / Chapter Chapter 2 --- Materials and Methods --- p.23 / Chapter 2.1 --- Synthesis of glucosylated silicon(IV) phthalocyanine (SiPcGlu) --- p.24 / Chapter 2.2 --- In vitro studies --- p.24 / Chapter 2.2.1 --- Cell line and culture conditions --- p.24 / Chapter 2.2.2 --- Photodynamic treatment --- p.25 / Chapter 2.2.3 --- Cell viability assay --- p.27 / Chapter 2.2.4 --- Light dose effect on the photocytotoxicity of SiPcGlu-PDT --- p.27 / Chapter 2.2.5 --- Determination of reactive oxygen species (ROS) production by SiPcGlu-PDT --- p.29 / Chapter 2.2.6 --- Effect of antioxidants on the photocytotoxicity of SiPcGlu-PDT --- p.29 / Chapter 2.2.7 --- Determination of ROS production after SiPcGlu-PDT --- p.30 / Chapter 2.2.8 --- Glucose competitive assay --- p.30 / Chapter 2.2.9 --- Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay --- p.30 / Chapter 2.2.10 --- DNA fragmentation analysis by gel electrophoresis --- p.31 / Chapter 2.2.11 --- Annexin-V & propidium iodide staining assay --- p.32 / Chapter 2.2.12 --- Subcellular localization studies --- p.33 / Chapter 2.2.13 --- Detection of mitochondrial superoxide production --- p.34 / Chapter 2.2.14 --- Assessment of mitochondrial membrane potential --- p.34 / Chapter 2.2.15 --- Caspase-3 activity assay --- p.35 / Chapter 2.2.16 --- "Western blot analyses for cytochrome c, caspase-3, PARP and glucose-regulated protein 78 (GRP78)" --- p.36 / Chapter 2.2.17 --- Ca2+ release from endoplasmic reticulum (ER) --- p.37 / Chapter 2.3 --- In vivo studies --- p.37 / Chapter 2.3.1 --- HT29 tumor-bearing nude mice model --- p.37 / Chapter 2.3.2 --- In vivo photodynamic treatment --- p.39 / Chapter 2.3.3 --- Biodistribution of SiPcGlu --- p.39 / Chapter 2.3.4 --- Assay for plasma enzyme activities --- p.40 / Chapter 2.4 --- Statistical analysis --- p.41 / Chapter Chapter 3 --- Results --- p.42 / Chapter 3.1 --- In vitro studies --- p.43 / Chapter 3.1.1 --- SiPcGlu-PDT induced cytotoxicity on HT29 cells --- p.43 / Chapter 3.1.2 --- Light dose effect on cytotoxicity by SiPcGlu-PDT --- p.46 / Chapter 3.1.3 --- SiPcGlu-PDT induced ROS production --- p.48 / Chapter 3.1.4 --- SiPcGlu-PDT induced cell death through Type I and II photoreactions --- p.48 / Chapter 3.1.5 --- ROS production after SiPcGlu-PDT --- p.51 / Chapter 3.1.6 --- Glucose competitive Assay --- p.55 / Chapter 3.1.7 --- SiPcGlu-PDT induced apoptosis in HT29 cells --- p.57 / Chapter 3.1.8 --- Subcellular localization of SiPcGlu --- p.61 / Chapter 3.1.9 --- SiPcGlu-PDT induced mitochondrial changes --- p.66 / Chapter 3.1.10 --- SiPcGlu-PDT induced caspase activation --- p.68 / Chapter 3.1.11 --- SiPcGlu-PDT increased expression of ER chaperone GRP78 --- p.72 / Chapter 3.1.12 --- SiPcGlu-PDT induced release of Ca2+ from ER --- p.72 / Chapter 3.2 --- In vivo studies --- p.75 / Chapter 3.2.1 --- In vivo photodynamic activities --- p.75 / Chapter 3.2.2 --- Tissue distribution of SiPcGlu --- p.77 / Chapter 3.2.3 --- Analysis of intrinsic toxicity --- p.77 / Chapter Chapter 4 --- Discussion --- p.80 / Chapter 4.1 --- Physical Properties of SiPcGlu --- p.81 / Chapter 4.2 --- In vitro studies --- p.82 / Chapter 4.2.1 --- SiPcGlu-PDT exhibits a high potency in killing HT29 cells --- p.82 / Chapter 4.2.2 --- ROS production is responsible for the cytotoxic effect of SiPcGlu-PDT --- p.83 / Chapter 4.2.3 --- SiPcGlu-PDT induced apoptosis in HT29 cells --- p.85 / Chapter 4.2.4 --- SiPcGlu is localized in various membranous organelles --- p.87 / Chapter 4.2.5 --- SiPcGlu-PDT induced mitochondria-mediated apoptosis --- p.89 / Chapter 4.2.6 --- SiPcGlu-PDT induced ER stress --- p.93 / Chapter 4.3 --- In vivo studies --- p.96 / Chapter 4.3.1 --- SiPcGlu failed to target to tumor tissues --- p.96 / Chapter 4.3.2 --- SiPcGlu-PDT induced retardation in tumor growth --- p.99 / Chapter 4.3.3 --- SiPcGlu is a safe photosensitizer for PDT --- p.101 / Chapter Chapter 5 --- Conclusion and Future Perspectives --- p.103 / Chapter 5.1 --- Conclusion --- p.104 / Chapter 5.2 --- Future Perspectives --- p.106 / Chapter 5.2.1 --- In vitro studies --- p.106 / Chapter 5.2.1.1 --- Lysosomal pathway to cell death --- p.106 / Chapter 5.2.2 --- In vivo studies --- p.107 / Chapter 5.2.2.1 --- Pharmacokinetic studies --- p.107 / Chapter 5.2.2.2 --- Eradication of HT29 tumor by repeated dose of SiPcGlu --- p.108 / Chapter 5.2.2.3 --- SiPcGlu-PDT-induced anti-tumor immunity --- p.108 / Chapter 5.2.2.4 --- Enhancement of tumor selectivity by conjugating with biomolecules --- p.109 / References --- p.110

Natriumpicosulfat-Tropfen erlauben eine sehr genaue Dosierung. Die randomisierte, plazebokontrollierte, doppelblinde cross-over Studie mit 20 gesunden Probanden wurde durchgeführt, um weitere Informationen sowohl über eine Dosis-Wirkungsbeziehung als auch über dosisabhängige pharmakokinetische Parameter und Verträglichkeit zu erhalten. Das Ziel war die Messung der Kolontransitzeit und der Zeit bis zum Wirkungseintritt und der Überprüfung, ob es eine Beziehung zwischen den kinetischen und dynamischen Parametern gibt. Die Probanden nahmen jeweils 5, 10 oder 15 mg Natriumpicosulfat-Tropfen oder Plazebo in einer oralen Einmaldosis. Die Kolontransitzeit, die Zeit bis zum Wirkungseintritt, die Urinausscheidung von BHPM (aktiver Metabolit von Natriumpicosulfat) und die Verträglichkeit wurden untersucht. Die mittlere Kolontransitzeit und die mittlere Zeit bis zum Wirkungseintritt waren nach Einnahme von 5, 10 und 15 mg Natriumpicosulfat-Tropfen im Vergleich zu Plazebo signifikant verkürzt. Es konnte jedoch keine eindeutige Dosis-Wirkungsbeziehung gefunden werden. Die Ausscheidung von BHPM sinkt mit Erhöhung der Natriumpicosulfatdosis und einer beschleunigten Kolontransitzeit. Die Verträglichkeit war gut. Die Nebenwirkungen (Magenkrämpfe, Blähungen) sind das Resultat der motilitätswirksamen Eigenschaften von Natriumpicosulfat. / Sodium picosulfate drops allow a very accurate dosage. The randomized, placebo-controlled, double blind cross-over study in 20 healthy volunteers was to provide further information about a dose-response ratio as well as dose-dependent pharmacokinetic parameters and tolerance. The aim was to measure transit time and time to onset of action and to check whether there was a correlation between kinetic and dynamic data. The volunteers received 5, 10 and 15 mg sodium picosulfate drops or placebo orally in a single dose. Colon transit time, time to onset of defecation, urinary excretion of BHPM (the active principle of sodium picosulfate) and tolerability were tested. Mean transit time and mean time to onset were significantly reduced by 5, 10 and 15 mg of sodium picosulfate, compared to placebo. No clear dose-response ratio could be found. The urinary excretion of BHPM dropped with increase of administered dose and accelerated transit velocity. The tolerance was good. The adverse events (stomach cramps, flatulence) are the result of motility stimulating effect of sodium picosulfate.