Dissertation (Ph.D.)--University of Toledo, 2009. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Title from title page of PDF document. Bibliography: p. 70-80, p. 28-158.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Diss. (sammanfattning) Umeå : Univ., 2007. / Härtill 4 uppsatser.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (doctoral)--Rijksuniversiteit te Utrecht.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on July 15, 2010). Includes bibliographical references.

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O câncer colorretal é o terceiro em mortalidade, e apesar das classificações moleculares, entre 30% e 40% dos pacientes apresentam recidiva após quimioterapia, o que aponta a necessidade de novas estratégias terapêuticas. É crescente o estudo de vetores virais para a terapia gênica do câncer, e dados do nosso laboratório mostraram que a entrega combinada dos genes Arf (supressor tumoral e parceiro funcional de p53) e interferon-beta (IFNbeta, citocina imunomodulatória), via vetores adenovirais sob o controle do promotor responsivo à p53 causou indução de morte celular massiva e um efeito imunomodulatório importante. Entretanto, esses resultados foram observados em modelos murinos. O presente trabalho avalia como a combinação da modulação das vias de p53 e IFNbeta impacta células de carcinoma colorretal humano em ensaios in vitro. Para isto, utilizamos nosso adenovírus para transferir p53 (p53) e IFN? como uma estratégia combinada para induzir a morte celular na presença de um modulador imunológico. As linhas celulares HCT116, HCT116p53-/- toleraram uma MOI (multiplicidade de infecção) de 25, enquanto uma MOI de 100 foi suportada por HT29 (mutante p53 R273H). O promotor PG, dependente de p53, como esperado, proporcionou níveis de expressão de GFP reduzidos em HCT116 p53 - / - e HT29, mas a expressão foi aumentada em todas as linhas celulares quando co-transduzida com o vetor codificando p53, mas não com IFNbeta. Em geral, HCT116 e HCT116p53-/- foram mais sensíveis à transferência de p53, enquanto o HT29 foi particularmente afetado pela combinação de p53 + IFNbeta. Esta tendência reflectiu-se na viabilidade celular (curva de crescimento, MTT), formação de colónias e acumulação de células hipodiplóides. Os níveis de morte celular correlacionaram-se com a atividade da caspase 3/7 e coloração com o Anexina V. Os marcadores de morte imunogênico ATP e calreticulina também foram aumentados, especialmente para o HT29 tratado com INFbeta sozinho ou em combinação com p53. Além disso, observamos aumento da expressão de TP63, TP73, bem como alvos transcricionais de p53, como p21 e NOXA, enquanto SESTRIN foi reduzido em ambas as linhagens HCT, mas aumentou em HT29. Adicionalmente, testamos a combinação p53 + IFNbeta em associação com quimioterápicos, revelando cooperação entre a transferência gênica e a doxorrubicina ou 5-FU, mesmo nas menores doses testadas. Ensaios em andamento incluem a avaliacao da ativacao de celulas dentriticas humanas expostas para as celulas tumorais tratadas com os vetores portadores de p53 e/ou IFNbeta. Com este projeto, nossa abordagem de transferência gênica revelará a resposta aos transgenes em modelo humano e também abrirá o caminho para estudar o envolvimento do sistema imune humano / Colorectal cancer is the third leading cause of cancer death and despite new molecular classifications, 30% to 40% of all patients will relapse after chemotherapy, pointing the need for new and innovative therapeutic strategies. The number of studies about viral vectors for gene therapy is growing, and previous data from our laboratory indicates that the combined delivery of Arf (a tumor suppressor gene and functional partner of p53) and interferon beta (IFNbeta, an immunomodulator cytokine), under a p53 responsive promoter, induced massive cell death and an important immunomodulatory effect. However, these results were only observed in murine models. Here we present an RGD-modified adenovirus whose transgene is under the control of a p53 responsive promoter (PG), used to transfer p53 (p53) and IFNbeta as a combined strategy to induce cell death in the presence of an immune modulator. Cell lines HCT116, HCT116 p53 -/- tolerated a MOI (multiplicity of infection) of 25, while a MOI of 100 was supported by HT29 (mutant p53 R273H). The p53-dependent PG promoter, as expected, provided reduced GFP expression levels in HCT116 p53 -/- and HT29, but the expression was increased in all cell lines when co-transduced with the p53 vector, but not with IFNbeta . In general, HCT116 and HCT116p53-/- were more sensitive to p53 gene transfer while HT29 was particularly affected by the combination of p53 + IFNtbeta. This trend was reflected in cell viability (growth curve, MTT), colony formation and accumulation of hypodiploid cells. Levels of cell death correlated with caspase 3/7 activity and staining with Annexin V. Immunogenic markers were also increased, especially for HT29 treated with INFbeta and p53/IFNbeta, as we detected exposure of calreticulin and release of ATP. Also, we observed increased expression of TP63, TP73 as well as p53 transcriptional targets, such as p21 and NOXA; SESTRIN was reduced in both HCTs but increased in HT29. Additionally, we tested the p53+IFNb combination in association with chemotherapeutics, revealing cooperation between gene transfer and doxorubicin or 5-FU even at the lowest doses tested. Ongoing studies include the evaluation of human dendritic cells exposed to tumor cells treated with the vectors for p53/IFNbeta. In conclusion, our combined gene transfer approach has potentiated the killing of colorectal cancer cell lines and may provide an immunomodulatory effect

<p>Uvod: Virusni gastrointestinalni sindrom je aktuelni zdravstveni problem u celom svetu. To važi kako u razvijenim zemljama, tako i u zemljama u razvoju, a posebno u nerazvijenim zemljama, gde je drugi po redu uzrok mortaliteta. Nagli početak bolesti, praćen pojavom velikog broja tečnih stolica, mukom, povraćanjem, bolovima u stomaku, temperaturom, malaksalo&scaron;ću, ima za posledicu dehidrataciju. U svim starosnim grupama obolelih, a naročito kod sasvim male dece, starih i imunodeficitarnih osoba može da dođe do smrtnog ishoda, ukoliko se brzo ne postavi tačna etiolo&scaron;ka dijagnoza bolesti i ne pristupi se odmah nadoknadi vode i elektrolita, kao i primeni svih ostalih mera simptomatske terapije. Brzo postavljena tačna dijagnoza, &scaron;to se najbolje postiže real-time PCR testom, sprečava pojavu komplikacija, pa i fatalnog ishoda bolesti. Istovremeno, omogućava primenu odgovarajućih epidemiolo&scaron;kih mera da se spreči nastanak epidemija i njihovo &scaron;irenje. Cilj ovog istraživanja bio je da se tačno utvrdi incidenca virusnog gastrointestinalnog sindroma u Vojvodini i učestalost pojave epidemijskog i sporadičnog javljanja ove bolesti. Cilj je bio i postavljanje algoritma za primenu real-time PCR testa u dijagnostici virusnog gastrointestinalnog sindroma u budućem radu. Isto tako, cilj je bio da se molekularnom analizom, sekvenciranjem delova genoma pozitivnih uzoraka stolice, izvr&scaron;i genetska tipizacija i odredi filogenetska pripadnost virusa. Materijal i metode: Tokom petogodi&scaron;njeg istraživanja molekularnim real-time PCR testom pregledane su 1003 obolele osobe sa simptomima virusnog dijarealnog sindroma, starosti od mesec dana do preko 90 godina. Pregledani su na rota, noro, astro i enterične adenoviruse. Na osnovu podataka iz anketnih upitnika i istorija bolesti, detaljno su analizirani svi klinički pokazatelji (javljanje bolesti tokom godine, trajanje bolesti, simptomi). Procena težine kliničke slike vr&scaron;ena je prema Vesikari skali. Svi podaci su upoređivani prema vrsti virusnog uzročnika, prema starosti obolelih, godinama trajanja istraživanja i epidemijskom i sporadičnom javljanju oboljenja. Dobijeni podaci su statistički obrađeni, tabelarno i grafički prikazani. Rezultati: U petogodi&scaron;njem periodu real-time PCR testom pregledan je uzorak od 1003 obolele osobe različite starosti na 4 virusna uzročnika dijarealnog sindroma (rota, noro, astro i enterične adenoviruse). Virusni dijarealni sindrom dokazan je kod 709 obolelih (70,69%). Najče&scaron;će su dokazane rotavirusne infekcije u 28,81%. Statistički značajno najče&scaron;će rotavirusi su bili utvrđeni kod dece do 5 godina (38,90%), ali u visokom procentu i kod dece uzrasta 6 do 14 godina (24,83%). Deca mlađa od 5 godina imala su statistički značajno najtežu kliničku sliku, bila su če&scaron;će hospitalizovana i imala su statistički značajno vi&scaron;u temperaturu. Pored vi&scaron;e temperature kod obolelih od rotavirusa, klinička slika je kod ovih bolesnika bila teža i bolest je duže trajala nego kod obolelih od drugih virusa. Norovirusna infekcija je dokazana u 23,03% obolelih i to statistički značajno če&scaron;će kod odraslih osoba, starijih od 20 godina. Od kliničkih simptoma kod ovih bolesnika statistički značajno če&scaron;će su dokazani muka, povraćanje i bolovi u stomaku, nego kod obolelih od drugih virusa. Norovirusi su značajno če&scaron;će bili uzročnici epidemijskog javljanja bolesti. Astrovirus je dokazan kod znatno manjeg broja obolelih (u 2,29%) i to samo kod dece do 5 godina i dece uzrasta 6 do 14 godina. Infekcija izazvana enteričnim adenovirusima dokazana je kod 13,36% bolesnika. Njače&scaron;će je utvrđena kod dece uzrsta do 5 godina i 6 do 14 godina. Oboleli od adenovirusa imali su statistički značajno blažu kliničku sliku bolesti. Dva virusna uzročnika u uzorku stolice dokazana su u 3,19% osoba, obično u toku epidemijskog javljanja bolesti. Ovi bolesnici su imali bitno težu kliničku sliku. Najvi&scaron;e obolelih od dijarealnog sindroma bilo je u hladnim mesecima, mada su dijagnostikovani i tokom cele godine. U petogodi&scaron;njem periodu utvrđene su 22 epidemije u kolektivima i 9 porodičnih epidemija. Epidemijsko javljanje bolesti bilo je statistički značajno najče&scaron;će kod najstarijih bolesnika (starijih od 50 godina), a sporadično javljanje bilo je statističko značajno najče&scaron;će kod dece. U cilju potvrde tačnosti dijagnostike virusa u ispitivanim uzorcima real-time PCR testom, genotipizacije, kao i detaljnije molekularne analize, izabrani su reprezentativni uzorci pozitivni na rota, noro, astro ili adenoviruse. Delovi genoma ovih uzoraka su amplifikovani, a zatim sekvencirani. Sekvencirani izolati rotavirusa pripadali su grupi A i tipovima G1P[8], G2P[4], G3P[8] i G9P[8]. Sekvencirani izolati norovirusa pripadali su genogrupi I tipu 2, zatim genogrupi II tipovima 1, 2, 4 i 17. Sekvencirani izolati astrovirusa pripadali su grupi klasičnih astrovirusa i tipovima 1, 4 i 5. Sekvencirani izolati adenovirusa pripadali su grupi F i tipovima 40 i 41, kao i grupi C tipu 2. Pripadnost dobijenih sekvenci u ovom istraživanju, dodatno je potvrđena izradom filogenetskog stabla za sekvence pozitivne na rota, noro, astro ili adenoviruse. Zaključak: Incidenca virusnog dijarealnog sindroma u Vojvodini (70,69%) vrlo je visoka i vi&scaron;a je nego &scaron;to je bilo pretpostavljeno prilikom prijave teze (u hipotezi). Real-time PCR test treba da bude redovno kori&scaron;ćen u budućem dijagnostičkom radu, jer dovodi do brze dijagnostike, čak i ako su virusi prisutni u malom broju u uzorcima tečnih stolica, &scaron;to je utvrđeno tokom ovog dijagnostičkog rada. Ispitivani virusi treba da budu redovno dijagnostikovani kod obolelih od dijarealnog sindroma i to u svim starosnim grupama, tokom epidemijskog i sporadičnog javljanja oboljenja.</p> / <p>Introduction: Viral gastrointestinal syndrome is a current ongoing health problem worldwide. This is true of both developed and developing countries, especially underdeveloped ones where it is the second leading cause of mortality. Sudden onset of the disease&mdash;accompanied by the occurrence of large numbers of liquid stools, nausea, vomiting, abdominal pain, fever, and exhaustion&mdash;leads to dehydration. A fatal outcome can occur in all age groups of patients, especially very young children, the elderly, and the immuno-deficient, unless an accurate etiological diagnosis of the disease is quickly established, followed by a prompt institution of fluid and electrolyte placement, and implementation of other symptomatic therapy measures. Quick establishment of an accurate diagnosis, which is best achieved using the real-time PCR test, prevents the onset of complications, including a potentially fatal outcome of the disease. Simultaneously, it enables the implementation of appropriate epidemiological measures to prevent epidemic outbreaks and their spread. The aim of this study was to accurately determine the incidence of viral gastrointestinal syndrome in Vojvodina and the frequency of epidemic and sporadic occurrence of this disease. The aim was also to set up an algorithm for the application of the real-time PCR test in diagnostics of viral gastrointestinal syndrome in future work. Likewise, the aim was to carry out genetic typing and determine phylogenetic affiliation of the virus using molecular analysis and sequencing of parts of genomes from positive stool samples. Material and Methods: During a five-year study, 1003 patients with symptoms of viral diarrheal syndrome, aged from one month to more than 90 years old, were examined using molecular real-time PCR test. They were screened for rota, noro, astro, and enteric adenoviruses. Based on the data from survey questionnaires and medical case history, all clinical indicators were meticulously analyzed (disease occurrence during the year, disease duration, symptoms). The assessment of the clinical severity was carried out according to the Vesikari Clinical Severity Scoring scale. All data were compared according to the type of the viral causing agent, age of the patients, duration of research in years, and epidemic and sporadic occurrence of the disease. Obtained data were statistically analyzed, tabulated, and graphically displayed. Results: In a five-year period, a sample of 1003 patients of different ages was screened for four different viral causing agents of diarrheal syndrome (rota, noro, astro, and enteric adenoviruses) using the real-time PCR test. Viral diarrheal syndrome was confirmed in 709 patients (70.69%). The most commonly found were rotavirus infections in 28.81% of the cases. Rotaviruses were statistically significantly most common in children younger than 5 years old (38.90%), but were also found in high percentage in children aged 6-14 years old (24.83%). Children under 5 years of age had statistically significantly highest clinical severity and fever, and were more frequently hospitalized. In addition to higher fever in patients with rotavirus, clinical severity in these patients was also higher, and the disease lasted longer than in patients with other viruses. Norovirus infections were reported in 23.03% of the subjects, statistically significantly more frequently in adults over 20 years of age. Regarding the clinical symptoms in these patients, nausea, vomiting, and abdominal pain were statistically significantly more common than in patients with other viruses. Noroviruses were significantly more common as causing agents of epidemic disease outbreaks. Astrovirus was found in a significantly smaller number of patients (in 2.29%), and only in children under 5 years of age and children aged 6-14 years old. Enteric adenovirus infections were reported in 13.36% of the subjects. They were most commonly found in children younger than 5, and those aged 6- 14 years old. Adenovirus sufferers had statistically significantly milder clinical disease. Two viral causing agents in the stool sample were found in 3.19% of the subjects, usually during an epidemic disease outbreak. These patients had a significantly more severe clinical disease. Highest numbers of sufferers from diarrheal syndrome occurred during the cold months, although they were diagnosed throughout the year. In a five-year period, 22epidemics in collective groups and 9 family epidemics were identified. Epidemic outbreaks of the disease were statistically significantly most frequent in the elderly patients (older than 50), while sporadic occurrences were statistically significantly most frequent in children. Representative samples positive for rota, noro, astro, or adenoviruses were selected in order to confirm the accuracy of virus diagnostics in samples tested by the real-time PCR test, and perform genotyping as well as more detailed molecular analyses. Parts of the genomes of these samples were amplified and then sequenced. Sequenced rotavirus isolates belonged to group A and types G1P[8], G2P[4], G3P[8], and G9P[8]. Sequenced norovirus isolates belonged to genogroup I type 2, and genogroup II types 1, 2, 4, and 17. Sequenced astrovirus isolates belonged to the group of classical astroviruses and types 1, 4, and 5. Sequenced adenovirus isolates belonged to group F and types 40 and 41, as well as group C type 2. The affiliation of the obtained sequences in this study was further confirmed by creating a phylogenetic tree for sequences positive for rota, noro, astro, or adenoviruses. Conclusion: The incidence of viral diarrheal syndrome in Vojvodina (70.69%) is very high&mdash;higher than what was assumed at the time of the thesis submission (in the hypothesis). The real-time PCR test should be regularly used in future diagnostic work, since it leads to rapid diagnostics even if viruses are present in small numbers in liquid stool samples, as determined in the course of this diagnostic study. The investigated viruses should be regularly tested in patients with diarrheal syndrome belonging to all age groups during both epidemic and sporadic occurrences of the disease.</p>