Thesis (M.S.)--University of North Carolina at Wilmington, 2005. / Includes bibliographical references (leaves: 60-61)

Dissertation (Ph.D.)--University of Toledo, 2010. / "Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Sciences." Title from title page of PDF document. "A Dissertation entitled"--at head of title. Bibliography: p. 63-71, 126-140.

Orientador : Profª. Drª. Célia Regina Cavichiolo Franco / Coorientador : Prof. Dr. Edvaldo da Silva Trindade / Autor não autorizou a divulgação de impresso e digital / Dissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e Molecular. Defesa: Curitiba, 29/02/2012 / Inclui referências : f. 105-109 / Área de concentração: Biologia celular e molecular / Resumo: Sete milhões e seiscentos mil pessoas no mundo morreram de câncer em 2008. Câncer é uma doença que apresenta controle anormal da divisão celular, resultando em crescimentos invasivos, ou formação de tumores, que podem se difundir por todo o corpo. Desde tempos antigos há uma busca por compostos naturais para a cura de doenças, sendo associado à medicina popular, assim pesquisas científicas têm sido intensificadas por todo o mundo. Drogas extraídas de diferentes fontes naturais podem ser utilizadas na quimioterapia para o câncer e no combate a outras doenças. O presente trabalho teve por objetivo avaliar o potencial biológico de diferentes polissacarídeos, obtidos de cogumelos dos gêneros Pleurotus, Lentinus e Agaricus, em células de melanoma murino, linhagem B16F10. Quatro diferentes polissacarídeos foram empregados nesta pesquisa, denominados de P1, P2, P5 e P6. Após os experimentos realizados, os resultados demonstram que o P1 não apresenta citotoxicidade, não altera a proliferação celular, aumenta o espraiamento celular e reduz a capacidade invasiva das células. Já o polissacarídeo P2, também reduz a capacidade invasiva das células e aumentou a capacidade de adesão celular em laminina. O P6 modificou a morfologia celular visualizada em microscopia eletrônica de varredura. Já o P5 foi o menos promissor, pois modificou apenas o espraiamento celular. Evidências na literatura atual demonstram corroborar com nossos resultados, visto que muitos polissacarídeos apresentam as mesmas alterações em células tumorais, sendo promissores também em análises in vivo. Assim podemos concluir que os polissacarídeos P1, P2 e P6 serão possíveis agentes antitumorais. Estes resultados alcançados com os respectivos polissacarídeos, de forma promissora, nos dão motivação para seguir com estudos posteriores, que possam comprovar a eficiência desses compostos em modelos tumorigênicos in vivo. Palavras-chave: Melanoma. Polissacarídeos. Cogumelos. Pleurotus. Lentinus. Agaricus. B16F10. / Abstract: Seven and six hundred thousand million people worldwide died from cancer in 2008. Cancer is a disease that shows abnormal control of cell division, resulting in invasive growth or tumor formation which can spread throughout the body. Since ancient times there is a search for natural compounds for curing diseases being associated to popular medicine as well scientific research have intensified worldwide. Drugs extracted from different natural sources can be used in chemotherapy for cancer and to combat other diseases. This study aimed to assess the biological potential of different polysaccharides obtained from mushrooms of the genera Pleurotus, Lentinus and Agaricus; in murine melanoma cells, B16F10 line. Four different polysaccharides were used in this study, called P1, P2, P5 and P6. After the experiments, the results show that P1 non-cytotoxic, does not alter cell proliferation, increased cell spreading and reduces the invasiveness of cells. Since P2 polysaccharide also reduces the invasiveness of the cells and increase cell adhesiveness laminin. The P6 modified cell morphology displayed in scanning electron microscopy. But the P5 was the least promising because only modified cell spreading. Evidence in the literature demonstrates corroborate our results, as many polysaccharides have the same changes in tumor a cell, also promising and in vivo analyzes. Thus we conclude that the polysaccharides P1, P2 and P6 are possible antitumor agents. These results achieved with the respective polysaccharides, promisingly, give us motivation to continue with further studies that can demonstrate the effectiveness of these compounds in vivo tumorigenic models. Keywords: Melanoma. Polysaccharides. Mushrooms. Pleurotus. Lentinus. Agaricus. B16F10.

The typical response of cancer patients to treatment is only temporary, and is often followed by relapse. The failure of various therapeutic strategies is commonly attributed to the emergence of drug resistance. The response patterns for patients under such treatments indicate that complex dynamics regulate the response of the tumour to the therapy. The environment in which the tumour lives (the stroma) is known to be a modulator of multiple mechanisms that lead to drug resistance and seems to be a likely candidate for explaining some of this complexity. Understanding the role of stromal cells in the promotion of drug resistance is critical for the design of optimal treatment strategies, and for the development of novel therapies that selectively target both the tumour and the stroma. In this thesis we design two novel mathematical models that describe cancer growth within its environment and the evolution of drug resistance within spatially complex and temporally dynamic tumours. A compartment model captures clinically observed dynamics and allows direct comparison with experimental data, facilitating model parametrisation and the understanding of inter-tumour heterogeneity. An individual cell-based model highlights the key role of local interactions, determining heterogeneity at the tissue scale, that will eventually determine treatment outcome. A non-spatial approximation of this second model allows us to find analytic guidelines for the design of effective therapy. These tools allow the simulation of a range of treatment strategies (including combination of different drugs and variation of schedule) as well as the investigation of therapy response based on patient- or organ-specic parameters. The work developed in this dissertation is based on the paradigmatic biology of melanoma and non-small cell lung cancer. Its results are therefore applicable to a variety of cancer treatments that target similar processes, and whose therapeutic failure can be attributed to environment-mediated drug resistance.

Orientador: Marlus Chorilli / Coorientador: Valéria Valente / Banca: Rose Mary Zumstein Georgetto Naal / Banca: Gislaine Ricci Leonardi / Banca: Janice Rodrigues Perussi / Banca: Marcos Antonio Correa / Resumo: O trans-resveratrol (RES) atua na prevenção e no tratamento de desordens cutâneas, porém apresenta baixa biodisponibilidade e rápida metabolização quando administrado por via oral em humanos. As nanopartículas lipídicas sólidas (NLSs) têm sido exploradas para a administração cutânea de fármacos, em virtude das características de interação com a pele. Assim, o objetivo deste capítulo foi à avaliação in vitro e in vivo da atividade do RES incorporado em NLSs para administração cutânea, bem como a avaliação da atividade antinociceptiva e antiedematogênica in vivo e dos efeitos celulares em células HaCat e B16F10 dos sistemas nanoestruturados. As formulações desenvolvidas foram selecionadas através de design fatorial, as quais eram compostas por ácido esteárico (AE), como lipídeos sólidos e fosfatidilcolina de soja (FS), polissorbato 80 (T80) e poloxamer 407 (P407) como tensoativos (F1). A esta formulação foi acrescido brometo de cetrimônio (BC) como tensoativo catiônico para promoção de carga superficial positiva (F2). O RES foi adicionado na fase lipídica da formulação, obtendo-se as formulações F1.RES e F2.RES, respectivamente. O diâmetro hidrodinâmico médio das formulações foi de 195,0 ± 3,34 nm, 241,3 ± 48,33 nm, 159,15 ± 4,78 nm e 158,25 ± 33,92 nm para F1, F1.RES, F2 e F2.RES, respectivamente. O potencial zeta (mV) das formulações foi -25,5 ± 1,01; -26,0 ± 1,67; 30,6 ± 1,13 e 30,0 ± 1,85 mV para F1, F1.RES, F2 e F2.RES, respectivamente. O RES livre foi quantificado de acor... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Not available / Doutor

Nas últimas décadas, é intensa a procura de uma explicação genética sobre a origem, crescimento e progressão do melanoma cutâneo. A tentativa de encontrar uma ligação direta entre as mutações gênicas e a origem da doença tem sido o objetivo dos pesquisadores dedicados ao estudo dessa neoplasia. Diversos métodos são utilizados na busca de uma avaliação prognóstica para a progressão do melanoma, citando-se, entre eles, a pesquisa do linfonodo sentinela, a imunoistoquímica, as técnicas moleculares e a técnica de microarray. A necessidade de estabelecer um método, com excelente sensibilidade e especificidade, tem levado os pesquisadores a buscarem melhores evidências. É importante para esses estudos a obtenção de dados confiáveis sobre as técnicas, progressão e sobrevida livre de doença. Por meio da imunoistoquímica, técnica relativamente simples e de baixo custo, a expressão da proteína p16 pode ser analisada e correlacionada com o prognóstico da doença. No melanoma cutâneo, a expressão da proteína diminui, à medida que aumenta sua agressividade, ou seja, é forte nos nevos e melanomas in situ, e fraca ou ausente nos melanomas metastáticos. Em alguns estudos, a comparação com outros marcadores é analisada. A finalidade deste estudo é fazer uma revisão da literatura internacional sobre o uso da proteína p16 como fator prognóstico para o melanoma, bem como avaliar a importância das alterações do gene p16INK4a, co-responsáveis pela gênese e evolução do melanoma.

Le projet concerne des molécules hétérocycliques, de faible poids moléculaire, présentant des activités cytotoxiques comparables à celles des meilleurs anticancéreux actuellement sur le marché. Ces molécules sont originales, protégées par un brevet international et un brevet de sélection déposé en décembre 2014. La synthèse des premières molécules leaders est maîtrisée et l’exemplification de la diversité moléculaire est en cours. Les études menées pour définir leur profil d’activité permettent d’identifier des caractéristiques tout à fait originales. Le projet, en phase de développement préclinique académique, a permis l’identification de composés leaders présentant des potentiels de développement en tant qu’anticancéreux. Le mécanisme exact des molécules développées est encore en cours d’étude et permettra de définir s’il s’agit d’un mode d’action unique ou multiple. Plusieurs têtes de séries ont pu être identifiées avec visiblement des modes d’actions différents. En effet, les composés EAPB0203 et EAPB0503 montrent un effet dose à partir de 1 µM sur la polymérisation de la tubuline mais la molécule EAPB02303, la plus active sur la lignée A375 (CI50 = 10 nM, de dix fois à cent fois plus active que les deux précédentes), ne montre aucune fixation à la tubuline à la dose de 1µM suggérant un mécanisme d’action différent et original. Le sujet de recherche présenté concerne le développement des études précliniques de ces molécules à visée anticancéreuse. Le premier axe de travail a été de mettre au point une formulation galénique de l'EAPB0503 sous forme de nanocapsules lipidiques. Afin d’optimiser la biodisponibilité des composés, sans perdre leur activité intrinsèque, nous avons ensuite réalisé une modulation chimique sur la structure la plus active des Imiqualines : l’EAPB02303. Afin d’améliorer la balance globale hydrophilie/lipophilie (HLB) des composés dérivés de l’EAPB02303, nous avons greffé un résidu acide aminé en position 4. Enfin, la mise au point d’une méthode de dosage de l’EAPB02303 et l’EAPB02302 en milieu plasmatique en vue d’une étude pharmacocinétique a été le dernier axe du travail de thèse. / The project relates to heterocyclic molecules, low molecular weight, having cytotoxic activities similar to those presented by the best anticancer agents currently on the market. These molecules are originals, protected by an international patent and a selection patent filed in December 2014. The synthesis of the first molecules leader is under control and the exemplification of molecular diversity is underway. Studies to define their activity profile allow to identify quite original features. The project, in its academic preclinical development phase, enabled the identification of leader’s compounds with potential for development as anticancer. The exact mechanism of the molecules developed is still under study and will determine whether there is a single or multiple mode of action. Several series leads were identified with apparently different modes of action. Indeed, compounds EAPB0203 and EAPB0503 show a dose effect from 1 µM on tubulin polymerization but compound EAPB02303, the more active on A375 cell line (IC50 = 10 nM, ten times to hundred times more active than the preceding two), shows no binding to tubulin at a dose of 1 µM suggesting a different and original mechanism of action. The research topic presented concerns the preclinical development of these anticancer molecules. The first line of work was to develop a galenic formulation of EAPB0503 in the form of lipid nanocapsules. To optimize the bioavailability of the compounds, without losing their intrinsic activity, we achieved chemical modulation on the most active structure Imiqualines: EAPB02303. To improve the overall balance hydrophilic / lipophilic (HLB) of compounds derived from EAPB02303, we considered grafting amino acids on position 4. Finally, the development of a method for assaying EAPB02303 and EAPB02302 in plasma for a pharmacokinetic study has been the final focus of the thesis work.

PURPOSE: The purpose of this study was to compare the rate of grade 3-4 immune related adverse events (irAEs) in patients with advanced metastatic melanoma treated with the combined anti-CTLA-4 and anti-PD-immune-therapy at the Dana Farber Cancer Institute(DFCI), to that of the published rate of grade 3-4 irAEs among patients treated with the same combination of check-point therapy in the pivotal phase II and phase III trials that led to the FDA approval of the combination regimen. This study also measures the tumor response with the Ipi-Nivo combination therapy and overall-survival of patients in the study cohort at DFCI. METHODS/PROCEDURES: This is a retrospective cohort study conducted at DFCI during 2014 to 2016 among stage III/IV melanoma patients treated outside of the clinical trials with the Ipi-Nivo combination therapy. Chart review of the electronic medical record(EMR) was conducted to abstract the data for this study. irAEs were graded and classified as per the NCI-CTCAE v.4.0 guidelines. The comparison of the rate of grade 3 4 toxicity in the clinical settings at DFCI and the clinical trials was performed using a one sample proportion hypothesis test. For efficacy assessment of tumor response, RECIST1.1 criterion was used to ascertain the best clinical response. RESULTS: During an overall follow-up period of 600 days, 52 patients were treated on expanded access protocol (EAP) and commercial Ipi-Nivo combination therapy at DFCI. The rate of grade 3-4 immune mediated toxicity for this cohort of patients treated outside of clinical trials was 32.6%. The average rate of grade 3-4 irAEs reported in phase II/III clinical trials was approximately 55%. The results from the one-proportion hypothesis test [(P-value: 0.002) (95% C.I: 19.14-46.23)], prove that patients in the “real world” clinical settings have a different safety profile than patients treated in the clinical trials. The rate of grade 3-4 irAEs was found to be lower (19.14% to 46.23%) in the population treated with Ipi/Nivo combination therapy at the DFCI, compared to the check-mate clinical trials (approximately 55%) CONCLUSION: The results from the study indicate a lower rate of grade 3-4 irAEs in patients treated at DFCI, in comparison with the patients treated in the clinical trials for the Ipi-Nivo combination group. The results support the need for preemptive safety signal detection of symptoms of irAEs to improve patient’s safety. However, larger database studies are required for the generalizability of this results to a wider patient population treated outside of DFCI.

Developing personalized cancer therapies based on cancer genomics methodologies forms the basis for future cancer therapeutics. A genomics platform was developed based on canine cancer to produce a proof-of-concept for personalized genomics led therapeutic choices but also developing personalized therapeutics for canine cancer patients themselves. The platform identified the genetic state of a canine cancer patient within two drugable pathways; p53 and HSP90/IRF1. The former gene was wild-type p53 thus directing the use of p53 activating molecules. The latter mutations in both HSP90 and IRF1 suggested an investigation into HSP90 and interferon signalling molecules as drug leads. Drugs that target both of these pathways were subsequently used to measure drug effects in cell line models but also to identify novel biomarkers of drug responses. My study focused on the effect of the HSP90-inhibitor Ganetespib had on its client proteins, particularly IRF-1. Briefly my results indicated the following:(i) Ganetespib downregulated IRF-1 protein levels in A375 cell lines and this attenuation was not mediated by either MDM2 or CHIP (E3 ligase). IFNγ- induced IRF-1 was also observed to be downregulated when Ganetespib was used in combination therapy.(ii) Insitu proximity ligation assay showed induced HSC70 upregulation upon HSP90 inhibition by Ganetespib and HSC70/MDM2 complexes were seen to be stabilized compared to the usage of MDM2/p53 inhibitor-nutlin. I hypothesize that MDM2/HSC70 complex might chaperon IRF-1 into lysosome for degradation via chaperon mediated autophagy pathway. (iii) My results also indicate that Ganetespib can downregulate IFN γ- induced PDL-1 expression in melanoma cell lines. Pre-sensitizing the cells with Ganetespib prior to the addition of IFNγ could attenuate PDL-1 to basal levels. (iv) My results also showed that the downregulation of PDL-1 by Ganetespib is an IRF-1 dependent mechanism. Therefore, my results suggest that HSP90 represents an important emerging target for cancer therapy because its inactivation results in the simultaneous blockade of multiple signalling pathways and can also sensitize tumor cells to other anticancer agents. Targeting HSP90 could also help to disrupt PD1/PDL- 1 interaction and activate immune system to recognise tumor cells. I conclude that HSP90 and IRF-1 play a critical role in types II interferon pathways and these findings establish a novel basis for the design of future Ganetespib-based combinatorial approaches to improve patient outcomes in this disease. These approaches finally demonstrate that cancer genomics can stratify choice of cancer drugs used on patients but also provide evidence that cancer patient samples can be used for the specific stratification of cancer drug choice based on cancer genomics data.

L'utilisation d'un agent déméthylant induit la réexpression de la protéine RIP3, une sérine-thréonine kinase, dans un modèle leucémique murin exprimant BCR-ABL humain. La réexpression de RIP3 conduit rapidement les cellules vers la nécroptose. Le mutant délété du domaine kinase est de façon surprenante plus "apoptogène" et induit le clivage de p65/RelA sur le résidu d'acide aspartique D361 par la caspase 6. Pour déterminer l'impact de ce clivage, nous avons construit un mutant non clivable p65/RelA D361E, ainsi que des plasmides exprimant chacun des fragments p65/RelA 1-361 ou p65/RelA 362-549, ou un plasmide exprimant simultanément p65/RelA 1-361 + p65/RelA 362-549. Ces différents plasmides codant pour les différentes formes de la protéine p65/RelA sont incorporés par transfection dans les cellules leucémiques ou de mélanome pour lesquels le gène RIP3 est respectivement méthylé ou exprimé. In vivo, nous mettons en évidence une différence de tumorigénicité entre les deux modèles. Elle est accrue par la présence de p65/RelA D361E par rapport à celle de p65/RelA WT et de p65/RelA 1-361 + p65/RelA 362-549 dans le modèle leucémique. Elle est au contraire faible dans le modèle du mélanome pour lequel la surexpression des fragments p65/RelA 1-361 +362-549 induit la tumorigenèse la plus forte. L'agressivité du mutant non clivable in vivo n'est pas corrélée à l'activité de NF-kB mesurée in vitro. Les fragments comme le mutant p65/RelA D361E induisent des profils d'expression différents dans le modèle murin de leucémie avec la modulation notable d'expression génique de la famille d'inhibiteurs de protéases à cystéine Stefins, ainsi que le transporteur de bicarbonate de sodium SLC4A5 qui joue un rôle majeur dans la régulation du pH intracellulaire. Le mutant p65/RelA D361E induit une expression importante du transporteur de bicarbonate de sodium SLC4A5 dans le modèle leucémique responsable de l'augmentation du pH intracellulaire qui participe au développement tumoral. Par contre, ce sont les deux fragments p65/RelA 1-361 + p65/RelA 362-549 qui induisent simultanément une expression plus forte de la molécule d'immunoéchappement PDL1, vraisemblablement par un mécanisme post-traductionnel. L'étude de la "souchitude" des modèles montre une différence d'activité du mutant p65/RelA D361E selon le modèle. On observe une augmentation de l'activité ALDH dans le modèle leucémique et une diminution de la formation de sphères dans le modèle de mélanome. En conclusion, ces résultats indiquent que les fragments issus du clivage de p65/RelA par l'activité de RIP3 indépendante de la kinase possèdent un rôle différent de celui de la forme sauvage sur la souchitude des cellules cancéreuses, et qu'elle dépend du modèle étudié. Ils confirment que le mutant non clivable possède la plus forte activité tumorigénique. Ils laissent également supposer que les fragments Nter et Cter puissent avoir une activité dans des cellules tumorales possédant une protéine RIP3 fonctionnelle et active, probablement par des mécanismes inflammatoires ou autres qui doivent être caractérisés. / The receptor-interacting protein kinase 3 (RIPK3) can induce necroptosis, apoptosis, or cell proliferation, and is silenced in several hematological malignancies. We previously reported that RIPK3 activity independent of its kinase domain induces p65/RelA caspase-mediated cleavage resulting in N-terminal 1-362 and C-terminal 362-549 fragments. We show here that a non-cleavable p65/RelA D361E mutant expressed in DA1-3b leukemia cells decrease mouse survival and that coexpressed p65/RelA fragments increase tumoriginicty of B16/F1 melanoma cells that did not correlated with in vitro measured Nf-kB activity. Fragments and p65/RelA fragments display different expression profiles in DA1-3b leukemic cells, with the notable modulation of gene expression of the Stefin cysteine protease inhibitor family and of SLC4A5, a Na+-coupled HCO−3 transporter. DA1-3b cells expressing p65/RelA D361E mutant showed more basic intracellular pH. p65/RelA fragments induced ovexpression of PD-L1 immunoescape molecule in DA1-3b cells. Markers of stemness were also affected: p65/RelA D361E induced increased ALDH activity in DA1-3b cells and fragments expression resulted in increased melanoma sphere formation in B16/F1 cells. Thus, far from being neutral, p65/RelA cleavage initiated by kinase independent activity of RIPK3 induced a pleiotropic range of effects in vitro and in vivo in cancer cells, that may vary across tumor types.