A caracterização de vias de sinalização alteradas em células tumorais, e a validação de fármacos inibidores de transdução de sinal que interajam com as mesmas de modo a atuar como terapia principal ou adjuvante no tratamento de neoplasias sólidas e hematopoiéticas, é um campo de grande interesse em oncologia. A necessidade da caracterização de novos alvos moleculares advém da incidência considerável de recidivas, quadros de quimiorresistência e efeitos adversos associados ao tratamento com os agentes antitumorais clássicos. Nesta tese, desenvolvemos a hipótese de que o fator de transcrição NFκB poderia estar envolvido com processos de proliferação, antiapoptose e quimiorresistência em células neoplásicas, caracterizando-se como um potencial alvo para interferência farmacológica. Para isso, avaliamos: i) o papel de NFκB na resposta celular a ERO e no controle da decisão entre morte e proliferação celular; ii) o estado de ativação de NFκB em modelos tumorais in vitro e in vivo; iii) o potencial citotóxico e seletividade dos inibidores de NFκB, seus mecanismos de ação, atividade em células neoplásicas e em linhagens quimiorresistentes. Para isso, foram utilizadas abordagens in vitro (cultivos celulares), in vivo (modelo animal de implante tumoral), e análises de bancos de expressão gênica através de ferramentas de biologia de sistemas. Os resultados demonstraram que NFκB está superestimulado em linhagens de leucemia e de glioblastoma quando comparado com leucócitos e astrócitos não-transformados. O tratamento com inibidores farmacológicos de NFκB causou morte celular programada em um mecanismo seletivo para células tumorais, potenciando os efeitos de antitumorais clássicos tanto em linhagens selvagens quanto em células quimiorresistentes. Além disso, os inibidores BAY117082 e curcumina apresentaram atividade in vivo em modelo animal de glioma sem evidência de citotoxicidade aguda. Em suma, os dados aqui apresentados sugerem que o fator de NFκB constitui-se um potencial alvo para inibição farmacológica no tratamento de neoplasias. Neste contexto, a diversidade da expressão de NFκB em diferentes tipos tumorais e a segurança associada ao uso clínico de seus inibidores permanece por ser avaliada. / Characterization of new molecular targets is one of the most important challenges in oncology. In this context, there is a growing interest in characterization of deregulated cell signaling pathways in solid and hematopoietic cancer cells in order to leverage the development of specific cell signaling inhibitors to act as principal or adjuvant therapy, and to circumvent the frequently observed chemoresistance and relapse in cancer patients. In this study, we hypothesized that the transcription factor NFκB could be involved on proliferation, antiapoptosis response and chemoresistance in cancer cells thus being a potential target for pharmacological interference. Attempting to test this, we evaluated: i) the role of NFκB in cell response against reactive oxygen species and in control of cell death and proliferation signaling; ii) NFκB activation status in cancer and noncancerous cells in vitro and in vivo; iii) cytotoxicity, selectivity and mechanisms of action of NFκB inhibitors in glioma and leukemia cells besides its biological activity against chemotherapy-resistant cell lines. Experiments were performed based on in vitro (cancer cell lines and primary cultures) and in vivo (tumor implants) models of cell growth. Moreover, in some experiments, bench-directed analysis of public gene expression databases followed by bioinformatic approach was used to ensure the significance and reliability of experimental data. Our findings showed an overstimulation of NFκB in leukemic and glioblastoma cell lines compared to healthy leucocytes and non-transformed astrocytes, respectively. Treatment with pharmacological inhibitors of NFκB induced programmed cell death in a cancer cells selective manner and potentiated the effects of classical anticancer drugs in both wild-type and chemoresistant cell lines. Indeed, the pharmacological NFκB inhibitors BAY117082 and curcumin showed significant anticancer efficacy in a model of brain-implanted gliomas without evidence of acute toxicity. Overall, the herein presented data suggest that NFκB is a potential target for cell death induction in leukemia and glioblastomas. Evaluation of its expression profile in different type of cancers beyond testing efficacy and safety of NFκB pharmacological inhibitors for clinical usefulness remains to be investigated.

A caracterização de vias de sinalização alteradas em células tumorais, e a validação de fármacos inibidores de transdução de sinal que interajam com as mesmas de modo a atuar como terapia principal ou adjuvante no tratamento de neoplasias sólidas e hematopoiéticas, é um campo de grande interesse em oncologia. A necessidade da caracterização de novos alvos moleculares advém da incidência considerável de recidivas, quadros de quimiorresistência e efeitos adversos associados ao tratamento com os agentes antitumorais clássicos. Nesta tese, desenvolvemos a hipótese de que o fator de transcrição NFκB poderia estar envolvido com processos de proliferação, antiapoptose e quimiorresistência em células neoplásicas, caracterizando-se como um potencial alvo para interferência farmacológica. Para isso, avaliamos: i) o papel de NFκB na resposta celular a ERO e no controle da decisão entre morte e proliferação celular; ii) o estado de ativação de NFκB em modelos tumorais in vitro e in vivo; iii) o potencial citotóxico e seletividade dos inibidores de NFκB, seus mecanismos de ação, atividade em células neoplásicas e em linhagens quimiorresistentes. Para isso, foram utilizadas abordagens in vitro (cultivos celulares), in vivo (modelo animal de implante tumoral), e análises de bancos de expressão gênica através de ferramentas de biologia de sistemas. Os resultados demonstraram que NFκB está superestimulado em linhagens de leucemia e de glioblastoma quando comparado com leucócitos e astrócitos não-transformados. O tratamento com inibidores farmacológicos de NFκB causou morte celular programada em um mecanismo seletivo para células tumorais, potenciando os efeitos de antitumorais clássicos tanto em linhagens selvagens quanto em células quimiorresistentes. Além disso, os inibidores BAY117082 e curcumina apresentaram atividade in vivo em modelo animal de glioma sem evidência de citotoxicidade aguda. Em suma, os dados aqui apresentados sugerem que o fator de NFκB constitui-se um potencial alvo para inibição farmacológica no tratamento de neoplasias. Neste contexto, a diversidade da expressão de NFκB em diferentes tipos tumorais e a segurança associada ao uso clínico de seus inibidores permanece por ser avaliada. / Characterization of new molecular targets is one of the most important challenges in oncology. In this context, there is a growing interest in characterization of deregulated cell signaling pathways in solid and hematopoietic cancer cells in order to leverage the development of specific cell signaling inhibitors to act as principal or adjuvant therapy, and to circumvent the frequently observed chemoresistance and relapse in cancer patients. In this study, we hypothesized that the transcription factor NFκB could be involved on proliferation, antiapoptosis response and chemoresistance in cancer cells thus being a potential target for pharmacological interference. Attempting to test this, we evaluated: i) the role of NFκB in cell response against reactive oxygen species and in control of cell death and proliferation signaling; ii) NFκB activation status in cancer and noncancerous cells in vitro and in vivo; iii) cytotoxicity, selectivity and mechanisms of action of NFκB inhibitors in glioma and leukemia cells besides its biological activity against chemotherapy-resistant cell lines. Experiments were performed based on in vitro (cancer cell lines and primary cultures) and in vivo (tumor implants) models of cell growth. Moreover, in some experiments, bench-directed analysis of public gene expression databases followed by bioinformatic approach was used to ensure the significance and reliability of experimental data. Our findings showed an overstimulation of NFκB in leukemic and glioblastoma cell lines compared to healthy leucocytes and non-transformed astrocytes, respectively. Treatment with pharmacological inhibitors of NFκB induced programmed cell death in a cancer cells selective manner and potentiated the effects of classical anticancer drugs in both wild-type and chemoresistant cell lines. Indeed, the pharmacological NFκB inhibitors BAY117082 and curcumin showed significant anticancer efficacy in a model of brain-implanted gliomas without evidence of acute toxicity. Overall, the herein presented data suggest that NFκB is a potential target for cell death induction in leukemia and glioblastomas. Evaluation of its expression profile in different type of cancers beyond testing efficacy and safety of NFκB pharmacological inhibitors for clinical usefulness remains to be investigated.

O manejo clínico dos pacientes com pólipos colorretais é principalmente baseado na histologia das lesões removidas. Em conseqüência, o diagnóstico histológico tem um papel muito importante na decisão terapêutica e a uniformidade de interpretação dos diferentes laudos de patologia é essencial. Apesar destas relevantes implicações, poucos estudos existem avaliando a variabilidade interobservador na elucidação dessa doença e a concordância não é considerada satisfatória. Objetivo: avaliar a variabilidade interobservador no diagnóstico histológico dos pólipos colorretais. Metodologia: foram avaliados 230 pólipos colorretais no Serviço de Patologia do Hospital de Clínicas de Porto Alegre (HCPA). Quatro patologistas examinaram todas as lâminas de forma independente e “cega”, ou seja, sem conhecimento do diagnóstico elaborado pelo seu colega. As lesões colorretais foram classificadas em relação ao diagnóstico: pólipo e carcinoma invasivo e quanto ao tipo de pólipo: adenomatoso versus hiperplásico. Nos adenomas foi avaliado o tipo histológico (tubular, túbulo-viloso e viloso) e o grau de displasia (baixo e alto grau). Resultados: o Kappa médio, em relação ao tipo de lesão, foi de 0,794, considerado moderado. Quanto ao tipo de pólipo, o Kappa médio foi 0,852, ou seja, uma ótima concordância. Em relação aos adenomas, no que se refere ao tipo histológico, obteve-se um Kappa médio, fraco de 0,291, e na avaliação do grau de displasia o Kappa médio foi regular com valor de 0,420. Conclusão: o índice de concordância, entre os quatro observadores foi considerado de moderado a ótimo no tipo de lesão e de pólipo, porém a variabilidade foi grande na avaliação dos adenomas, tanto no que concerne ao tipo histológico quanto ao grau de displasia com um kappa de fraco a regular. / The clinical management of patients with colorectal polyps is mainly based on the histology of the removed lesions. Therefore, the histological diagnosis has a very important role in deciding the treatment and the uniform interpretation of the different pathology reports is essential. In spite of these relevant implications, there are only very few studies assessing the interobserver variability in such diagnosis and the concordance of reports among different examiners is not considered satisfactory. Objective: to assess interobserver variability in the pathology reports in the diagnosis of colorectal polyps. Method: at the Department of Pathology of HCPA [Hospital de Clínicas de Porto Alegre] 230 slides of colorectal polyps were examined by four independent pathologists “blindly”, that is, the diagnosis given by their colleagues was not known. Colorectal lesions were classified according to the diagnosis as polyp or invasive cancer and to the polyp type (adenomatous or hyperplasic). The histological type of the adenomas (tubular, tubulovillous and villous) and the grade of dysplasia (high or low) were also assessed. Results: mean kappa of the type of lesion was 0.794, which is considered moderate. The mean kappa of 0.852 for the type of polyp is considered excellent concordance. Regarding the histology of adenomas, the mean kappa was 0.291, considered weak. The assessment of the degree of dysplasia showed a regular Kappa of 0.420. Conclusion: the concordance rate among the four pathologists was considered to be moderate to excellent for the type of lesion and of polyp but there was great variability in the assessment of adenomas both for the histological type and for dysplasia, showing a weak to regular kappa.

Milk protein polymorphisms have a significant influence on milk quantity and composition. Kappa-casein is of special interest due to its known relationship with milk quality. In goats, a number of allelic variants have been identified, primarily classified into two groups. Group BIEF alleles (D, E, K, and M) have been shown to have a positive effect on milk yield and technological properties, while group AIEF, the remaining alleles, have a less positive influence on milk composition. The aim of this study was to investigate genetic variation in the kappa-casein genotype of South African goats. PCR-RFLP and DNA sequencing were performed on 68 and 77 samples, respectively. In addition, 84 milk samples were analyzed for milk composition. RFLP analysis revealed that the A and/or B alleles were the most frequent in the populations studied. A frequency of 0.00 was observed for the BIEF variants using DNA sequencing. In all goat types included, the B allele was the most common, with frequencies ranging from 60% in SA Boer goats to 100% in Saanens. The B’ allele had lower frequencies of 0.357 and 0.207 in SA Boer goats and local goat types, respectively. The H allele was present at low frequencies in local goat types (10.3%) and in SA Boer goats (3.6%), but was absent in Saanens. AMOVA results indicated that most of the total variation occurred within populations (80.66%) with the remainder of the variation (FST = 0.1934; p < 0.01) occurring due to genetic differences between populations. / Dissertation (MSc(Agric))--University of Pretoria, 2009. / Animal and Wildlife Sciences / unrestricted

No description available.

Loss of muscle due to aging is often associated with significant detrimental effects. Therefore, it is crucial to understand signaling molecules that may trigger the muscle loss or prevent the process. The transcription factor, Nuclear Factor Kappa-B (NF-κB), is associated with both catabolic and anabolic pathways of muscle metabolism and may be involved in age-related muscle loss. Leucine is an essential amino acid that is required for both protein synthesis and intracellular signaling pathways that regulate protein synthesis and degradation. The current study examined muscle NF-kB levels in male Sprague-Dawley rats, aged 6 (adult) and 21 months (old) fed a diet containing suboptimal leucine levels for 10-17 days. We found that old rats consumed less grams of food per body weight (BW) each day than adult rats (1.45% g diet/g BW vs. 2.4% g diet/g BW). Weight loss during the study was not significantly different between age groups. However the average mass of gastrocnemius and soleus muscles (g muscle/g BW) was significantly lower in old rats. Reduction in gastrocnemius (g muscle/g BW*10²) was associated with 1.8 fold higher muscle cell NF-κB in old vs. adult rats (p = 0.0443). There was also a higher level of ubiquitinated proteins in old gastrocnemius muscle cells relative to the adult gastrocnemius, however differences did not reach statistical significance. For tibialis anterior muscle, the average mass (g muscle/g BW*10²), NF-κB levels and ubiquitinated proteins were not significantly different between adult and old rats. Our findings suggest that aging affects muscle loss and NF-kB in a tissue-specific manner in rats fed a diet with suboptimal leucine levels.

ZUSAMMENFASSUNG Obwohl diverse Mutationen des NF-κB Systems in Myelomzelllinien und primären Myelomzellen eine pathogenetische Beteiligung andeuten, ist die Relevanz des IKK Komplexes als molekularer Angriffspunkt für die Entwicklung medikamentöser Therapieoptionen noch nicht ausreichend geklärt. Zwar führte die Applikation des IKK-β Inhibitors MLN120b dosisabhängig und längerfristig zu einer Reduktion der Zellviabilität in einer Vielzahl von Myelomzelllinien, doch kann nicht ausgeschlossen werden, dass bei höheren Konzentrationen unspezifische Wirkungen für die beobachteten Effekte (mit-) verantwortlich sind. Aus diesem Grund erfolgte in der vorliegenden Arbeit eine spezifische Suppression von IKK-α, IKK-β oder IKK-γ mittels transienter Transfektion von shRNA Expressionsvektoren oder Stealth-siRNA. Es folgte die Charakterisierung der verminderten Zielproteinspiegel mittels Western-Blot und die Messung der Viabilität der Zellen mittels FACS Analysen. Darüber hinaus wurde in TNF-α Stimulationsexperimenten der Effekt der Suppression von IKK-β mittels Stealth-siRNA auf (Phospho-)IκB-α analysiert. Schließlich erfolgte die Applikation des IKK-β Inhibitors TPCA, dessen Wirkung auf die Zellviabilität und auf die TNF-α-vermittelte Phosphorylierung und Degradation von IκB-α in MM.1S Zellen untersucht wurde. Die Experimente mit Stealth-siRNA zeigten, dass weder die Suppression von IKK-β, noch die Suppression von IKK-α oder IKK-γ in AMO-1, L363 oder MM.1S eine Verminderung der Zellviabilität bewirken konnte. Auch eine kombinierte Suppression von IKK-α zusammen mit IKK-β in L363 und MM.1S Zellen bewirkte keinen vermehrten Zelltod. Dagegen zeigte die Behandlung von MM.1S Zellen mit hohen Konzentrationen von TPCA einen geringen Effekt auf das Überleben dieser Zellen. Die Suppression von IKK-β mittels Stealth-siRNA in MM.1S konnte nicht die TNF-α vermittelte IκB-α Phosphorylierung und Degradation verhindern. Sowohl die hohe TNF-α Konzentration von 100ng/ml, als auch eine unvollständige Suppression von IKK-β könnte dazu beigetragen haben. In analogen Experimenten mit TPCA konnte die TNF-α vermittelte IκB-α Phosphorylierung und Degradation dagegen effektiv unterdrückt werden. In der Zusammenschau der Ergebnisse konnte somit eine potenzielle therapeutische Relevanz des IKK-Komplexes als molekularer Angriffspunkt für eine Myelomtherapie nicht gefunden werden. Eine noch detailliertere Analyse der Funktionalität des Signalwegs (insbesondere eine Messung der Aktivität der NF-κB Transkriptionsfaktoren im Zellkern) und die Etablierung stabiler und induzierbarer Expressionssysteme für längerfristige Untersuchungen der RNAi Wirkungen in Myelomzellen, stellen weiterführende Wege zu einer umfangreicheren Beurteilung der pathobiologischen und therapeutischen Bedeutung des NF-κB Systems dar. Darüber hinaus sind die das NF-κB System betreffenden Mutationen genauer hinsichtlich ihrer potenziellen Wirkung auf NF-κB unabhängige Signalwege zu untersuchen. / ABSTRACT Although the known mutations in NF-kB in imply to view the IKK complex to be involved in promoting survival of Multiple Myeloma cells, and IKK-beta inhibitors have been reported to show anti-Myeloma activity, the actual significance of IKK as a molecular target for treatment of the desease remains unclear. This is due to the fact that the influence on survival by pharmacological inhibitors on many Multiple Myeloma cells has been shown to be dependend on high concentration applications and on long-term incubation times, both inheriting the possibility of unspecific drug effects. Therefor we chose an alternative approach and performed a RNAi mediated knockdown of each of the three components of the IKK Complex (IKK-alpha, IKK-beta and IKK-gamma) and measured the survival rate of the Multiple Myeloma celllines AMO-1, L363 and MM.1s. None of the knockdowns led to a decrease of Multiple Myeloma survival rate, and even a combined knockdown of IKK-alpha and IKK-beta in L363 and MM.1S showed significant effects on the survival. Summarized, IKK is not crucially involved in promoting survival of the mentioned Multiple Myeloma celllines and thus the relevance of the complex as a therapeutic target for the treatment of Multiple Myeloma is not thoroughly clarified yet. Further investiations in different celllines and RNAi mediated knockdowns in longer time intervalls might resolve the true significance of IKK for Myeloma survival.

The aim of this study was to determine whether there is an influence of the active components of the fish oil, in particular polyunsaturated fatty acids n3 (PUFA n3) on expression of selected proteins which modulate the inflammatory response in model organisms pig. In the n3 PUFA is to assume that over signaling pathway PPARgamma, NFkappaB, increase adiponectin production, inhibit the production of proinflammatory cytokines, increase antiinflammatory cytokines production, thereby suppressing the inflammatory response in the body. This hypothesis was tested on 32 pigs, which were divided into two groups of 16 pieces in each group. The first experimental group was fed the basic feed mixture enriched with 2.5% fish oil (F) and the second control group was fed the basic feed with 2.5% palm oil (P). Last day of fattening pigs were 8 F and 8 P pigs intravenously application lipopolysaccharide (LPS) to induce an inflammatory response, the rest of group left without LPS stimulation, followed by intramuscular application of anesthetics and defeat. By Western blot was measured protein expression PPARgamma and NFkappaB in selected tissues, by ELISA concentration of adiponectin in plasma and by multiplex analysis plasma levels of cytokines. The results indicate that PPARgamma concentration in the F adipocytes after LPS stimulation tended to increase by 21% in comparison with P control stimulation with LPS, but the result was not statistically significant (P> 0.05; P = 0.11). Further, the F pigs stimulation with LPS as compared with intact F counterparts trend toward increased plasma levels of adiponectin by 18%, which was likely a reflection of the tendency to higher values in adipocytes by 18% (P> 0.05, p = 0.12), result was not statistically significant. Plasma adiponectin took on the same values of 21.1 ng x mL-1 in experimental and control groups (P> 0.05). Elevation (P <0.05) of NFkappaB in fatty tissue F pigs after LPS stimulation. In addition, the plasma level of anti-inflammatory IL4 and IL10 interleukins, as well as the proinflammatory cytokine TNFalfa, was increased (P <0.05) in the F group of pigs stimulated with LPS. The results of the present experiment are thus ambiguous. The hypothesis of the effect of fish oil or n3 PUFA to suppress the inflammatory response cannot this experiment conclusively confirmed.

Kappa-casein has the general function in process of cheese production. Understanding the effect of gene polymorphism of kappa-casein to the nutritional and physical properties of milk may lead to more efficient breeding of dairy cattle. Within this thesis was genotyped by 240 brown swiss and jersey cows determinated using the PCR-RFLP. Then was performed association analysis of the impact of various genotypes on milk production parameters in the program SAS. An association between gene CSN3 polymorphism and milk yield was not statistically significant.

Entzündliche Prozesse stellen einen zentralen Aspekt der Karzinogenese dar und können sowohl zur Induktion als auch zum Progress von Tumoren beitragen. Der NF-kB-Signalweg ist einer der wichtigsten Signaltransduktionswege der In- flammation und Tumorpromotion, was ihn zur plausiblen Zielstruktur für die pros- pektive klinische Tumortherapie machen könnte. In der vorliegenden Arbeit wur- den die Eigenschaften von vier unterschiedlich targetierenden NF-kB-pathway- Inhibitoren – Cortisol, MLN4924, QNZ und TPCA1 – auf die Inflammation, Zell- proliferation und proapoptotische Sensitivierung am in vitro Modell des HNSCC untersucht. Es konnte gezeigt werden, dass die spezifische Auswahl des Inhi- bitors bzw. seines targets entscheidend für den wirkungsvollen Einsatz dieser Wirkstoffgruppe in der antiproliferativen Therapie des HNSCC zu sein scheint. Beispielsweise vermittelte MLN4924 die Freisetzung von IL-8. Cortisol bewirkte die Resistenz der FasL-induzierten Apoptose von HNSCC-Zellen. QNZ wirkte in einigen Zelllinien antiproliferativ und sensitivierend für den FasL-induzierten Zell- tod, beeinflusste jedoch in diesem Zusammenhang kontraproduktiv die IL-8-Sek- retion. Dies disqualifizierte diese Wirkstoffe für die Anwendung in der Therapie von Kopf-Hals-Tumoren. Dahingegen qualifizierte sich TPCA-1 aufgrund folgen- der Eigenschaften als geeigneter Wirkstoff für den prospektiven klinischen Ein- satz: 1) TPCA-1 wirkte antiproliferativ, 2) hemmte die TNF-a-induzierte Inflammation, 3) regulierte die IL-8-Expression herab, 4) wirkte sensitivierend für den TNF-a-induzierten Zelltod, 5) interferierte kaum mit der FasL-vermittelten Apoptose und 6) induzierte Apoptose. / Inflammatory processes represent a central aspect of carcinogenesis and may contribute to both tumour induction and progression. The NF-kB signalling pathway is one of the most important signal transduction pathways in inflammation and tumour promotion, which could make it a plausible target for prospective clinical tumour therapy. In the present study, the properties of four different targeting NF-kB pathway inhibitors - cortisol, MLN4924, QNZ and TPCA1 - on inflammation, cell proliferation and proapoptotic sensitivity were investigated in an in vitro model of HNSCC. It was shown that the specific selection of the inhi- bitor or its target seems to be crucial for the effective use of this group of drugs in the antiproliferative therapy of HNSCC. For example, MLN4924 mediated the release of IL-8, and cortisol induced the resistance of FasL-induced apoptosis of HNSCC cells. QNZ had an antiproliferative effect in some cell lines and was sensitive to FasL-induced cell death, but counteracted IL-8 secretion in this context. This disqualified these agents for use in the therapy of head and neck tumours. In contrast, TPCA-1 qualified as a suitable agent for prospective clinical use due to the following properties: 1) TPCA-1 had an antiproliferative effect, 2) inhibited TNF-a-induced inflammation, 3) down-regulated IL-8 expression, 4) was sensitive to TNF-a-induced cell death, 5) hardly interfered with FasL-mediated apoptosis, and 6) induced apoptosis.