This thesis deals with the phylogeny of the lichen genus Usnea (Parmeliaceae, Ascomycetes). The relationships and the morphological variation among Usnea species has been studied, as well as the relationship of Usnea to allied genera. Two species, U. florida and U. subfloridana, which earlier were regarded to form two separate species have been synonymized. In an analysis based on sequence data these two taxa formed a monophyletic group of intermixed specimens. Usnea florida and U. subfloridana have earlier been regarded to form a species pair, but the species pairs concept cannot be applied in this case. The morphological characters traditionally used for species recognition of a number of European Usnea species have been analyzed regarding their reliability. The evolution and distribution of the morphological characters was studied in relation to a phylogeny based on sequence data. Most characters proved to be homoplastic in relation to the phylogeny. Few characters were consistent in a clade, and the same character could be inconsistent in another clade. Therefore a combination of several characters is recommended for species recognition. The relationship of Neuropogon to Usnea was investigated based on sequence data. Neuropogon showed to be closely related to Usnea subg. Usnea. The subgenera Eumitria and Dolichousnea formed the sister group to the clade comprising subg. Usnea and Neuropogon. Usnea is paraphyletic in this investigation. Eumitria is treated as a genus and the subgenus Dolichousnea is elevated to generic rank. The position of Usnea, Neuropogon, Eumitria, and Dolichousnea in the family Parmeliaceae was investigated based on a phylogeny obtained by sequence data. Protousnea probably forms the sister group to the clade of Usnea, Neuropogon, Eumitria, and Dolichousnea. Several monophyletic groups in the family Parmeliaceae were identified.

We developed new methods for controlling and studying microtubules (MTs) outside the complex workings of the living cell. Several surface treatments for preventing MT fouling on surfaces were analyzed and, for the first time, a self-assembled monolayer (SAM) was developed which prevented MT adsorption in the absence of passivating proteins. The morphology and thickness of the SAM was measured to determine the mechanism of formation and origin of the MT-resistant behavior. The SAM was integrated into electron beam lithography for patterning and manipulating MTs using electrophoresis. Reversible MT adsorption and patterning and alignment of single MTs were achieved. We characterized the mechanism for the MT migration under electric field with a focus on the electrodynamics of the flow cell and the forces acting on the MT, along with the time dependence of the process.

Resistance to broad spectrum of chemotherapeutic agents in cancer cell lines and tumors has been called multiple drug resistance (MDR). In this study, the molecular mechanisms of resistance to two anticancer agents (paclitaxel and vincristine) in mammary carcinoma cell line MCF-7 were investigated. MCF-7 cells were selected in the presence of paclitaxel and vincristine by stepwise dose increments. The cell viability and growth profiles of resistant sublines were examined. As the resistance indices increased, the growth rates of sublines were found to decrease. Gene and protein expression levels of the basic drug resistance proteins P-gp and MRP1 were studied in sensitive and drug resistant MCF-7 cells. It was shown that P-gp overexpression is significantly contributing to the developed drug resistance phenotype. Mutation analysis of beta tubulin gene which encodes the target of paclitaxel and vincristine was performed. Single histidine to proline mutation was identified near GTP binding site of beta tubulin in vincristine resistant subline which was not reported before. Apoptosis related BCL-2 and BAX were examined at both gene and protein expression levels and they were not found to be significantly related to the developed resistance in the sublines. The reversal of drug resistance by various inhibitory agents of P-gp and MRP1 was investigated by using flow cytometry. Synthetic silicon compounds were found to be the most effective MDR reversal agents. The effects of various combinations of anticancer drugs and reversal agents on cell proliferation were examined by checkerboard microplate method. ALIS409-paclitaxel and paclitaxel-doxorubicin pairs seem to have highest antiproliferative effects on resistant sublines. The microarray expression profiling of sensitive and resistant MCF-7 cells was performed for a much detailed and comprehensive analysis of drug resistance. The results indicated that the upregulation of MDR1 gene is the dominating mechanism of paclitaxel and vincristine drug resistance. Additionally up regulation of the genes encoding the detoxifying enzymes (i.e. GSTP1) was observed. Significant down regulation of apoptotic genes (i.e. PDCD2/4/6/8) and alterations in expression levels of genes related to invasion and metastasis (MMPs, ADAMs, COL4A2, LAMA etc.) were detected. Upregulation of some oncogenes (i.e. ETS, RAS) and cell cycle regulatory genes (CDKN2A, CCNA2 etc.) was seen which may be in close relation to MDR in breast cancer. Further studies will demonstrate the relationship between the components contributing to drug resistance phenotype in breast cancer cells.

The genetic manipulation of perennial woody tree species presents a range of additional challenges compared to that of annual weedy crop species. These include long generation times and reproductive cycle, the heterogeneity of plants under investigation and, when investigating xylogenesis, a number of physical and biochemical limitations to microscopic and molecular experimentation. Efforts have been made to understand molecular aspects of xylogenesis and have involved functional gene testing using transgenic approaches. These methods involve the production of plantlets from a variety of plant tissues using in vitro full plant regeneration techniques. Although these systems are effective, the time taken from transformation event, to plant establishment and growth, then finally to secondary wood production can take up to several years and requires high labor and technical inputs. (For complete abstract open document)

Le cytosquelette d'une cellule eucaryote est constitué de trois types de polymères différents qui sont l'actine, les filaments intermédiaires et les microtubules. Ces éléments confèrent à la cellule l'essentiel de ses propriétés mécaniques telles que le maintien de l'architecture ou la modification de sa forme pour permettre le déplacement cellulaire. Ils sont également impliqués dans le transport d'organites ou de nutriments d'un bout à l'autre de la cellule, dans la ségrégation des chromosomes lors de la mitose ou encore dans le processus de division cellulaire. Pour répondre aux différents besoins de la cellule, ces filaments sont extrêmement dynamiques et peuvent se désassembler pour se réassembler à un autre endroit de la cellule. Cette dynamicité est régulée par de nombreuses protéines accessoires qui vont être capables de modifier les propriétés intrinsèques des différents filaments (dynamique, mécanique et organisatrice). Parmi ces protéines régulatrices, l'on distingue tout particulièrement les MAPs, pour Microtubule Associated Proteins, capables de modifier la dynamique et la structure des microtubules. MAP6, ou encore STOP pour Stable Tubule Only Peptide, est une MAP neuronale qui fut initialement décrite pour sa capacité à protéger les microtubules d'une exposition au froid ou encore de drogues dépolymérisantes comme le nocodazole. Des souris délétées pour le gène MAP6 montrent des troubles cognitifs et comportementaux proches des patients atteints de schizophrénie, impliquant au moins en partie des défauts de stabilisation des microtubules. Cependant, les effets de la protéine sur les microtubules restaient encore à déterminer. Dans ce contexte, à l'aide de diverses approches biochimiques et vidéomicroscopiques, nous avons montré que la protéine MAP6 est capable d’interagir de façon directe avec les microtubules in vitro et permet leur stabilisation. Elle permet aussi de réguler la dynamique des microtubules en augmentant la vitesse de polymérisation de l'extrémité (+), de diminuer la fréquence de catastrophe et l'apparition d’événements de sauvetage, de façon similaire à d'autres MAPs comme Tau ou MAP2. Cependant, contrairement aux autres MAPs, nous avons montré que MAP6 présente une dualité d'action sur le bout (-) des microtubules en diminuant et figeant très rapidement la dynamique de cette extrémité. Cette dualité pourrait ainsi conférer à MAP6 un rôle essentiel de nucléateur de microtubules en figeant l'extrémité (-) du microtubule et en favorisant la polymérisation et la stabilisation de l'extrémité (+). De plus, la protéine MAP6 est capable de modifier fortement la structure des microtubules. De part leur composition et leur rôle, les microtubules sont les éléments les plus rigides du cytosquelette et forment naturellement un tube creux linéaire. Or en présence de MAP6, les microtubules perdent cet aspect linéaire et adoptent une structure hélicoïdale (avec un pas d'environ 4,5 μm et une hauteur d'environ 1 μm) qui n'avait encore jamais été observée jusqu'à présent. La présence d'une telle population de microtubules dans la cellule pourrait ainsi apporter une certaine résistance mécanique ou encore permettre le maintien de l'architecture de l'axone. Enfin, nous avons montré que MAP6 peut aussi interagir de façon directe avec les filaments d'actines et les associer entre eux pour former des faisceaux. Dans les neurones, de nombreuses molécules ont été identifiées comme étant des régulateurs clés dans le « crosstalk » entre les filaments d'actines et les microtubules. L'interaction et la coordination entre les différents éléments du cytosquelette jouent un rôle essentiel dans la transmission et le relais du message synaptique. MAP6 pourrait être importante pour l'ensemble de ces mécanismes ce qui expliquerait les défauts de plasticité synaptique ainsi que les défauts cognitifs observés chez les souris KO MAP6. / The eukaryotic cell's cytoskeleton is constitued by three types of different polymers which are the actin filaments, the intermediate filaments and the microtubules. These elements confer on the cell the main part of its mechanical properties such as the architecture preservation or the modification of its shape to allow the cellular movement. They are also involved in the organelles or nutrients transport throughout the cell, in the chromosomes segregation during mitosis or still in the cellular division process. To answer the cell's various needs, these filaments are extremly dynamics and are able to dis-assemblate to re-assemblate in another place of the cell. Tis dynamic is regulated ny numerous proteins which are going to be capable of modifiying the intrinsic properties of the different filaments (dynamic, mechanic and structure). Among them are present the MAPs, for Microtubule-Associated Proteins, which will be able to influence the microtubule dynamics and structure. MAP6, also known as STOP for Stable Tubule Only Peptide, is a neuronal MAP which was initially described for its capacity to protect microtubule from cold or nocodazole exposure. KO MAP6 mice display cognitive and behavioral disorders close to patient with schyzophrenia, involving at least partially microtubules stabilization defects. However, the effects of the protein on the microtubules still remained to determine. In this context, using diverse biochemical and cideomicroscopy technics, we showed that MAP6 is able to directly interact in vitro with the microtubules and stabilizes them. It also regulates the microtubule dynamics by increasing the microtubule growth rate of the plus end extremity, decreases the shrinkage frequency and allows rescue of shrinking microtubules, similarly to other MAPs like Tau or MAP2. However, contrary to the other MAPs, we showed that MAP6 has another effect on the microtubule (-) end by decreazing and freezing its dynamics. This dual effect could confer to MAP6 an essential role of microtubules nucleation by stabilizing the new formed microtubule (-) end and by stabilizing and increasing the (+) end microtubule growth rate. Furthermore, MAP6 is also able to strongly modify the microtubule structure. Microtubules are the stiffest elements of the cytoskeleton and naturally form due to their composition linear hollow tubes. Yet in presence of MAP6, microtubules lose their usual shape and adopt a helical structure (4,5 μm pitch and approximatly 1 μm thickness) which had never been observed until now. The presence of such a population of microtubules in the neuron could thus provide a mechanical strength and allow the preservation of the axon architecture. Finally, we showed that MAP6 can also directly interact with the actin filaments to associate them and form bundles. In neurons, several molecules have been identified as key regulators in the " crosstalk " between actin filaments and microtubules. The interaction and coordination between the different cytoskeletal elements play a vital role in the synaptic transmission. MAP6 may be important for all these mechanisms which would explain the synaptic plasticity and cognitive defects observed in KO MAP6 mice.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Cultivation of Walnut-pecan (Carya illinoinensis [Wangenh.] K. Koch) has intensified in recent years in the state of Rio Grande do Sul. However, the research related to plant pathology has not developed to the same degree, so producing pecan nuts have faced many unknown diseases in the state. The main objective of this study was: a) identify and characterize causal agents of new diseases on Walnut-pecan in Rio Grande do Sul, and as specific objectives: b) to collect and to identify pathogens in different locations in Rio Grande do Sul, c) evaluate the pathogenicity of isolates collected in plantations of Walnut-pecan from different locations in Rio Grande do Sul, d) perform the isolates morphological characterization collected from diseased plants of Walnut-pecan, and e) to identify the isolates species. For this purpose, samples were taken in seven cities in the state for isolating potentially pathogenic fungi which were also collected and analyzed soil samples to characterize the area and georeferencing. Pathogens, Fusarium spp. and Pestalotiopsis spp. which were not yet reported were tested for their pathogenicity. The first genus was tested by inoculation on substrate, and the second by leaf inoculation with spore suspension. The pathogenic isolates were characterized by mycelial growth, sporulation, conidial dimensions, colony pigmentation and formation of specific structures to each genus. The same isolates were also identified by molecular sequencing of the ITS and TEF-1α genes for Fusarium spp. and ITS and β-tubulin genes for Pestalotiopsis spp. Twelve pathogenic isolates of Fusarium spp. and eleven of Pestalotiopsis spp. were identified. The variables used on morphological characterization were able to differentiate the isolates, especially the width of conidia for Fusarium spp. and diameter of the colonies for Pestalotiopsis spp. The sequencing of the ITS regions and TEF-1α to Fusarium spp. confirmed the separation of isolates through morphological characteristics and identified five species: F. chlamydosporum, F. oxysporum, F. equiseti, Giberella fujikuroi species complex and F. graminearum species complex and for Pestalotiopsis spp. sequencing of the ITS regions and β-tubulin could identify some species, such as P. clavisora and P. cocculi, and other isolates remained without precise identification of the species, since the phylogeny of the genus is still poorly known. / O cultivo da nogueira-pecan (Carya illinoinensis [Wangenh.] K. Koch) tem se intensificado nos últimos anos no estado do Rio Grande do Sul. Entretanto, as pesquisas relacionadas aos problemas fitossanitários da espécie não se desenvolveram na mesma intensidade, de modo que muitos produtores do estado têm enfrentado enfermidades desconhecidas. Diante disto, o objetivo geral do presente trabalho foi identificar e caracterizar os agentes causais de novas doenças que atacam a nogueira-pecan no Rio Grande do Sul. Como objetivos específicos estabeleceram-se: a) coletar e identificar agentes patogênicos em diferentes localidades no Rio Grande do Sul; b) avaliar a patogenicidade de isolados coletados em plantios de nogueira-pecan de diferentes localidades no Rio Grande do Sul; c) caracterizar morfofisiologicamente os isolados coletados de plantas doentes de nogueira-pecan; e d) identificar, em nível de espécie, os isolados provenientes de plantas doentes de nogueira-pecan. Para tanto, foram realizadas coletas em sete municípios do estado, para isolamento de fungos potencialmente patogênicos. Também foram coletadas e analisadas amostras de solo para caracterização da área e georreferenciamento dos pontos. Os patógenos, ainda não relatados, Fusarium spp. e Pestalotiopsis spp., foram testados quanto a sua patogenicidade. O primeiro foi testado com inoculação em substrato, e o segundo com inoculação foliar por suspensão de esporos. Os isolados patogênicos foram caracterizados morfofisiologicamente através das variáveis crescimento micelial, esporulação, dimensões de conídios, pigmentação das colônias e formação de estruturas específicas de cada gênero. Os mesmos isolados também foram identificados molecularmente através de sequenciamento dos genes ITS e TEF-1α, para Fusarium spp., e ITS e β-tubulina, para Pestalotiopsis spp. Foram identificados doze isolados patogênicos de Fusarium spp. e onze de Pestalotiopsis spp. As variáveis utilizadas na caracterização morfofisiológica foram suficientes na diferenciação dos isolados, especialmente a largura dos conídios, para Fusarium spp. e o diâmetro das colônias, para Pestalotiopsis spp. O sequenciamento das regiões ITS e TEF-1α, para Fusarium spp., confirmou a separação dos isolados por meio das características morfofisiológicas e identificou cinco espécies, sendo elas F. chlamydosporum, F. oxysporum, F. equiseti, Giberella fujikuroi species complex e F. graminearum species complex. Para Pestalotiopsis spp., o sequenciamento das regiões ITS e β-tubulina permitiu que se identificassem algumas espécies, como é o caso de P. clavisora e P. cocculi. Outros isolados permaneceram sem identificação precisa da espécie, já que a filogenia do gênero ainda é pouco conhecida.

Submitted by Izabel Franco (izabel-franco@ufscar.br) on 2016-09-20T14:33:37Z No. of bitstreams: 1 TeseDSC.pdf: 4230414 bytes, checksum: d505357e4ed13e446578eb00507beec7 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-21T12:36:04Z (GMT) No. of bitstreams: 1 TeseDSC.pdf: 4230414 bytes, checksum: d505357e4ed13e446578eb00507beec7 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-21T12:36:19Z (GMT) No. of bitstreams: 1 TeseDSC.pdf: 4230414 bytes, checksum: d505357e4ed13e446578eb00507beec7 (MD5) / Made available in DSpace on 2016-09-21T12:36:27Z (GMT). No. of bitstreams: 1 TeseDSC.pdf: 4230414 bytes, checksum: d505357e4ed13e446578eb00507beec7 (MD5) Previous issue date: 2015-06-16 / Não recebi financiamento / Malaria is an acute febrile disease caused by protozoan parasites of the genus Plasmodium, being the species P. falciparum responsible for the most severe forms and deaths caused by the disease. These parasites have developed resistance to commonly used drugs and therefore there is a need to develop new antimalarial agents. Aryltetralone lignans are compounds that show antiplasmodial activity in vitro against P. falciparum, but its mechanism of action is still not fully understood. In this work, we postulate a plausible mode of action of some aryltetralone lignans and according to the obtained results we suggest modifications to the ligands for a better biological activity. In order to achieve our objectives we first performed a search for similar chemical compounds, for which their macromolecular targets were known. From the results obtained, P. falciparum tubulin was selected as a potential target for these lignans. Since there is no experimentally determined three-dimensional structure for this protein, we performed a molecular homology modeling of P. falciparum tubulin and the structure of bovine tubulin complexed with colchicine was selected as template. The analysis of the obtained model showed that the three dimensional structure of Plasmodium tubulin is conserved in relation to the bovine tubulin with some important substitutions occurring in the colchicine binding site region: Ala250B by Ser248B, Ala316B by Cys314B and Ile318B by Met316B. Then, molecular docking of the aryltetralone lignans, colchicine and podophyllotoxin was performed in the modeled P. falciparum tubulin. The docking calculations results allowed to conclude firstly that, although the amino acid substitutions in the binding site, the colchicine binding mode in the P. falciparum tubulin is exactly the same as that already described in the literature for bovine tubulin. As for podophyllotoxin, a different binding mode from that described in the literature for bovine tubulin was obtained due to the replacement of Ala250B by Ser248B and the Val318B by Met316B. For the aryltetralone lignans studied, three different binding modes were obtained: one exhibited by compounds 1, 2 and 3, another by 4 and 6, and a third one by 5. The lignans 1, 2 and 3 are oriented in a way so that the C ring containing the dimethoxy or methylenedioxy group is positioned in the same region obtained for the ring containing the trimethoxy group in the case of colchicine and podophyllotoxin, performing a C-H...π interaction with Leu246B. Lignans 4 and 6 orient themselves with the aromatic ring C between Ala180A and Leu246B and being held in this position by C-H...π interactions. Lignan 5 is oriented with the aromatic ring C between Leu246B and Leu253B, performing C-H...π interactions with these residues, in a similar way to what was obtained with colchicine in this site. So the likely mechanism of action of the aryltetralone lignans studied here would be their binding to the same colchicine binding site in the tubulin protein of P. falciparum and thereby interrupting the divisions and other cellular functions. / A malária é uma doença febril aguda causada por protozoários parasitas pertencentes ao gênero Plasmodium, sendo a espécie P. falciparum a responsável pela maioria das formas severas e mortes pela doença. Estes parasitas desenvolveram resistência aos fármacos comumente utilizados e, portanto, existe a necessidade de se desenvolver novos agentes antimaláricos. Lignanas ariltetralônicas são compostos que apresentam atividade antiplasmodial in vitro contra o P. falciparum, porém seu mecanismo de ação ainda não é totalmente compreendido. Neste trabalho, conseguimos postular o modo de ação de algumas lignanas ariltetralônicas e, a partir dos resultados obtidos, sugerimos modificações nestes compostos de modo a obter uma melhoria na sua atividade biológica. Para isso, primeiramente foi realizada uma busca por compostos químicos semelhantes, cujos alvos macromoleculares eram conhecidos. A partir dos resultados obtidos, selecionou-se a tubulina do P. falciparum como potencial alvo para estas lignanas. Como não há estrutura tridimensional determinada experimentalmente para esta proteína, foi realizada a modelagem molecular por homologia da tubulina do P. falciparum, selecionando como molde a estrutura da tubulina bovina complexada com colchicina. A partir da análise do modelo construído, verificou-se que a estrutura tridimensional da tubulina do Plasmodium é conservada em relação à tubulina bovina e que ocorrem algumas substituições importantes na região do sítio de ligação da colchicina: Ala250B por Ser248B, Ala316B por Cys314B e Ile318B por Met316B. Em seguida, foi realizado o docking molecular das lignanas ariltetralônicas, da colchicina e da podofilotoxina na tubulina do P. falciparum. Os resultados do docking permitiram concluir primeiramente que, embora ocorram algumas substituições de aminoácidos no sítio, o modo de ligação da colchicina na tubulina do P. falciparum é exatamente o mesmo ao já descrito na literatura para a tubulina bovina. Já para a podofilotoxina, foi obtido um modo de ligação diferente do descrito na literatura para a tubulina bovina, devido à substituição da Ala250B pela Ser248B e da Val318B pela Met316B. Para as lignanas ariltetralônicas estudadas, foram obtidos três modos de ligação diferentes: um exibido pelos compostos 1, 2 e 3, outro para 4 e 6 e um terceiro modo exclusivo para 5. As lignanas 1, 2 e 3 orientam-se de modo que o anel C, que contém o grupo dimetóxi ou metilenodióxi, se posiciona na mesma região do sítio obtida para o anel contendo o grupo trimetóxi da colchicina e da podofilotoxina, realizando uma interação C–H...π com a Leu246B. As lignanas 4 e 6 orientam-se com o anel aromático C entre a Ala180A e a Leu246B, sendo mantido nesta posição por interações C–H...π. No caso da lignana 5, esta se orienta com o anel aromático C entre a Leu246B e Leu253B, realizando interações C– H...π com estes resíduos, semelhante ao que foi obtido para a colchicina neste sítio. Assim, o mecanismo provável de ação das lignanas ariltetralônicas aqui estudadas passaria pela sua ligação ao mesmo sítio de ligação da colchicina na proteína tubulina do P. falciparum e, com isso, interrompendo as divisões e outras funções celulares.

O planejamento de moduladores da dinâmica dos microtúbulos, a partir da ligação à αβ-tubulina, constitui importante estratégia para a terapia do câncer. Os efeitos de inibição da polimerização da tubulina ou de estabilização dos microtúbulos são promovidos pela interação de compostos em cavidades específicas da proteína alvo. A interferência com a dinâmica dos microtúbulos nas células em rápida multiplicação provoca o bloqueio do ciclo celular, disparando sinais bioquímicos que culminam em apoptose. Os taxanos são os agentes antimitóticos mais importantes dentre os estabilizadores de microtúbulos, ao passo que os alcalóides da vinca e a colchicina são membros representativos dos inibidores da polimerização. Entretanto, o aparecimento de resistência, baixa biodisponibilidade e reações adversas graves são fatores que têm impulsionado a pesquisa por novos agentes anticâncer. Dentre os ligantes identificados recentemente para o sítio do taxol, o discodermolídeo e a dictiostatina, produtos naturais de origem marinha, são potentes estabilizadores de microtúbulos que apresentam maior solubilidade que o taxol e atividade contra células de câncer resistentes aos taxanos. Por essa razão, o modo de ligação desses compostos à cavidade de interação da β-tubulina tem sido objeto de investigação. Modelos preditivos de HQSAR foram desenvolvidos para derivados sintéticos do discodermolídeo e utilizados em conjunto com estudos baseados na estrutura do receptor, em concordância com evidências experimentais, para a proposição de modelos de interação para os análogos na cavidade do taxol. Os modelos de conformação bioativa foram utilizados no alinhamento estrutural do conjunto de dados para estudos de QSAR 3D CoMFA e comparação com o alinhamento baseado nas estruturas minimizadas dos ligantes. A caracterização de padrões de reconhecimento molecular foi útil para a proposição de um modelo farmacofórico baseado nas estruturas dos produtos naturais estabilizadores de microtúbulos. Um modelo farmacofórico simplificado foi integrado ao processo de triagem virtual consistindo na aplicação de filtros hierárquicos sucessivos para a identificação de novos estabilizadores de microtúbulos. Caseobliquinas foram ensaiadas como estabilizadoras de microtúbulos, enquanto que intermediários sintéticos da dictiostatina foram avaliados quanto aos seus efeitos na polimerização da tubulina. Nos últimos anos, tem sido intensificada a busca por novos ligantes do sítio da colchicina, dentre os quais os indóis estão entre os mais relevantes. Para quatro conjuntos de dados totalizando 170 derivados de indóis, modelos de HQSAR foram desenvolvidos para a avaliação virtual de uma base de dados. Estudos de QSAR 3D CoMFA e CoMSIA baseados nos farmacóforos foram comparados para 3 conjuntos de dados. Os resultados sugerem que o núcleo indólico pode interagir de maneiras diferentes na cavidade de interação da proteína. Chalconas do tipo 1, inspiradas em ligantes clássicos do sítio da colchicina, foram avaliadas em ensaios celulares e de polimerização da tubulina, levando a síntese e avaliação de uma nova série de chalconas com propriedade antiproliferativa significativa. Por outro lado, derivados de N-acil-hidrazonas apresentaram atividade antimitótica semelhante à colchicina, enquanto as tiosemicarbazonas citotóxicas não interagiram diretamente com a tubulina. / Inhibition of microtubule function is one of the most important approaches to anticancer therapy. Two main effects can be elicited by tubulin/microtubule-interactive agents: inhibition of tubulin assembly or microtubule stabilization. Interference with either the assembly or disassembly of microtubules within the mitotic spindle in rapidly dividing cells disrupts the normal process of cell division and provokes chemical signals that induce apoptosis. On one hand, taxanes are the most prominent among the microtubule-stabilizing antimitotic agents, while on the other hand, colchicine and the vinca alkaloids are representative members of the tubulin polymerization inhibitors. However, due to poor pharmacokinetic properties, high toxicity and resistance, their clinical utility has been limited, generating new opportunities for the development of novel anticancer agents. In recent years, structurally diverse taxoid-site ligands have been identified, including the potent microtubule-stabilizers discodermolide and dictyostatin. These marine sponge-derived natural products have higher water solubility than taxol and exhibit activity against taxane-resistant cell lines. Therefore, the elucidation of their binding modes is important in drug design. Predictive conformation-independent hologram QSAR models were developed for a series of synthetic discodermolide analogs as antiproliferative agents. Receptor-based studies were integrated with molecular recognition patterns, in agreement with experimental evidences, leading to ligand-binding conformations for discodermolide analogs in the taxol-site. The bioactive conformation models were used to the structural alignment of the data set for the development of 3D QSAR CoMFA models, and for comparison with the models constructed with the rigid-body alignment based on minimized structures of the ligands. A set of structural features related to the interaction with the taxol cavity was identified and the molecular recognition patterns were employed to the construction of pharmacophore models based on the microtubule-stabilizing natural products. A final simplified pharmacophore model was integrated in a virtual screening procedure consisting of consecutive hierarchical filters targeting the identification of novel microtubule-stabilizers. Caseobliquins were screened as microtubule-stabilizers, while intermediates for the synthesis of dictyostatin were evaluated by their effects on tubulin assembly. Recently, the search for more simple anti-tubulin agents has renewed the interest in the development of colchicine analogs, often discarded for their high toxicity. Considering the structurally diverse ligands of the colchicine site, the indole derivatives are among the most important ones. Hologram QSAR models were developed for four data sets consisting of 170 indole derivatives, and used to evaluate a data set of commercially available compounds. Pharmacophore-based 3D QSAR CoMFA and CoMSIA models were constructed and compared to three of the individual data sets. The results indicated that the indole nucleus bind to the protein cavity in different ways. Type 1 chalcones, designed based on classical colchicine site ligands, have been screened for their anti-proliferative activity and tubulin assembly inhibition, leading to the synthesis and assessment of a novel series of chalcone analogs with substantial antiproliferative properties. Some N-acylhydrazone derivatives behaved as mitotic arresters in a way similar to that of colchicine, while cytotoxic thiosemicarbazones did not exhibit tubulin-interacting properties.

Câncer é a denominação de um conjunto de mais de cem doenças causadas pelo crescimento e multiplicação desordenados de células anormais capazes de invadir e se disseminar por diversos tecidos e órgãos. É considerado um problema de saúde mundial, sendo uma das maiores causas de morte. Dados da Organização Mundial da Saúde (OMS) indicam que 15% das mortes no mundo serão causadas por câncer em 2015. No Brasil, o Instituto Nacional do Câncer (INCA) estima 580 mil novos casos da doença para 2014. Apesar da vasta quimioterapia disponível, os tratamentos possuem alta toxicidade e estão sujeitos à resistência. Nesse contexto, a presente dissertação de mestrado tem como foco principal a identificação e caracterização de novos compostos com propriedades anticâncer. Os estudos foram realizados com base em dois alvos principais. O primeiro foi a proteína tubulina, um alvo anticâncer validado que é modulado por moléculas importantes como o taxol, a vimblastina e a colchicina. O segundo alvo foi a migração celular, característica relacionada ao processo de metástase, que é responsável por 90% das mortes por câncer. Uma série de acridinonas sintéticas foi avaliada in silico frente à proteína tubulina empregando métodos de modelagem molecular. Para tanto, fez-se uso de estruturas cristalográficas da proteína disponíveis no PDB (Protein Data Bank). Os resultados das análises de docagem molecular indicaram que as moléculas poderiam interagir com o sítio da colchicina e, dessa forma, atuariam como inibidoras da polimerização dos microtúbulos. Ensaios wound healing e em câmara de Boyden permitiram a identificação de quatro compostos com potente efeito de inibição da migração celular (valores de IC50 variando entre 0,294 e 1,7 μM) em uma linhagem metastática (MDA-MB-231). Estes compostos foram submetidos a ensaios de citotoxicidade frente à mesma linhagem tumoral e também apresentaram boa potência, com valores de IC50 variando entre 0,110 e 3 μM. Além disso, ensaios de citotoxicidade em células saudáveis mostraram que estes compostos inviabilizaram seletivamente células tumorais. Para a validação dos estudos in silico, ensaios de polimerização da tubulina foram conduzidos. Os resultados mostraram que as quatro moléculas ativas nos ensaios celulares atuam como inibidores de polimerização dos microtúbulos, com valores de IC50 variando entre 0,9 e 13 μM. Estudos das relações entre a estrutura e atividade (SAR) revelaram alguns aspectos interessantes nesta série de acridinonas, como, por exemplo, a perda de atividade, que se deu tanto nos ensaios celulares quanto nos ensaios bioquímicos, causada pela substituição de um grupo nitro da posição meta- por para- em duas moléculas da série. A progressão do ciclo celular foi analisada por citometria de fluxo e os resultados mostraram que os compostos estudados são capazes de interromper o ciclo celular entre as fases G2 e M. A citometria fluxo também permitiu verificar a indução da apoptose celular devida à ação das moléculas. Em resumo, este trabalho possibilitou a identificação e caracterização de quatro compostos com propriedades antitumorais promissoras que serão utilizados como compostos líderes para posterior desenvolvimento como agentes anticâncer. / Cancer is a set of diseases with high diversity and aggressiveness. It is one of the major causes of death according to the World Health Organization (WHO), being responsible for 15% of worldwide deaths in 2015. In Brazil, the National Institute of Cancer (INCA) estimates 580,000 new cases of cancer for the year of 2014. Despite the vast availability of cancer chemotherapy, the treatments cause high toxic effects and are liable to resistance. In this context, the main goal of the present master\'s dissertation is the identification and the characterization of new compounds having anticancer properties. These studies were conducted based on two main targets. The first one was the protein tubulin, a validated anticancer target that is modulated by important molecules such as taxol, vinblastine and colchicine. The second target was the cellular migration, a feature related to the metastasis process, which causes 90% of the cancer deaths. A series of synthetic acridinones was studied in silico employing molecular modeling methods. For this, crystallographic structures of tubulin were collected from PDB (Protein Data Bank). The docking results indicated that the molecules could interact with the colchicine site and, thereby, could act as tubulin polymerization inhibitors. The series was assessed by the wound healing and Boyden chamber\'s assays using the metastatic cell line MDA-MB-231. In these assays, four compounds were identified as good inhibitors of cellular migration (IC50 values varying between 0.294 and 1.7 μM). These compounds were evaluated in cytotoxicity assays using the same cell line and presented good potency, with IC50 values varying between 0.110 and 3 μM. Furthermore, cytotoxicity assays using a healthy cell line showed that these compounds act selectively in tumor cells. For the validation of the in silico studies, tubulin polymerization assays were conducted. The results showed that the four active molecules in the cellular assays act as tubulin polymerization inhibitors, with IC50 values varying between 0.9 and 13 μM. Structure-activity relationship (SAR) studies revealed interesting structural aspects in this series of acridinones, for instance, the exchange of the nitro group substituent from the meta- to the para- position in two molecules of the series led to a lack of activity in both cellular and biochemical assays. The cell cycle progression was evaluated by flow cytometry, and the results showed that the four compounds are capable of arrest cells in the G2/M phase. Moreover, the apoptosis induction was verified using flow cytometry. In summary, this work provided the identification and characterization of four new compounds with promising antitumor properties. These molecules will be used as lead compounds for further development as anticancer agents.

As concepções de moduladores da dinâmica dos microtúbulos, que levam ao bloqueio do ciclo celular, e de bloqueadores de canais de cálcio tipo L (Cav), tais como o 1,4-di-hidropiridinas e análogos, que diminuem a resistência do organismo humano aos tratamentos quimioterápicos através da inibição da proteína de transmembrana P-gp, são estratégias importantes tanto para terapias antitumorais quanto para leishmanicida. Esta abordagem tem mostrado resultados interessantes na diminuição da resistência à quimioterapia em câncer chamada de MDR (do inglês Multi Drug Resistence), além de também serem uma estratégia importante para controlar a fase inicial da leishmaniose. Diante desse contexto, e baseado no estudo de Ueki 2013 e colaboradores que, a partir de estudos anteriores, os quais relatam a superexpressão das enzimas estona deacetilase (HDAC) e catepsina L (CTSL) em células tumorais, propuseram um pró-fármaco seletivo, planejado a partir de um espaçador de lisina acetilada, que garante a liberação do fármaco seletivamente nas células tumorais, trabalhamos no desenvolvimento de uma nova proposta de pró-fármaco trifuncional. Nossa proposta foi desenvolvida a partir de estudos de triagem virtual, baseados em ligantes e em estrutura, predição das propriedades farmacocinéticas e toxicológicas (ADME/Tox) e também técnicas de bioinformática para a construção de um modelo de canal de cálcio, devido à inexistência de estruturas, do mesmo, que estivessem depositadas no banco de dados de proteínas PDB (Protein Data Bank). Paralelamente, nosso grupo de síntese colaborador sintetizou, através de técnicas de \"Click Chemistry\" e reações de Mitsunobu multicomponentes, uma biblioteca de novos híbridos trifuncionais, os quais, após estudos de atividade biológica, foram avaliados (in silico) frente à estrutura da tubulina, e os compostos mais promissores desta biblioteca serviram de base para novos estudos de triagem virtual. Para a obtenção dos nossos hits, executamos 4 estratégias de triagem virtual, separadas em 2 tarefas. Ao final, selecionarmos um total 59 hits, dos quais, 9 hits apresentam promissoras atividades bloqueadoras do canal de cálcio e 65 hits apresentam promissoras atividades moduladoras da tubulina. Estes hits seguem em estágio de compra e ensaios in vitro e após comprovada a eficácia dos mesmos, estes futuramente farão parte de uma nova proposta de pró-farmaco trifuncional. / The concepts of modulating microtubule dynamics, and calcium channel L-types (CAV) blockers are important strategies for anticancer and antileishmanial therapies. Microtubule modulators that blocks the cell cycle and the calcium channel blockers, such as, 1,4-dihydropyridines and analogues, reduce the resistance of the human body to chemotherapeutic treatments by inhibiting transmembrane P-gp protein. This approach has shown interesting results in reduced resistance to chemotherapy in cancer called MDR (Multi Drug Resistance), and an important strategy for controlling the early stage of leishmaniasis. In this context, we work to develop a new proposal for trifunctional prodrug. We have based on the study of Ueki 2013 and collaborators, which, from earlier studies with reported overexpression of both deacetylase estona enzymes (HDACs) and cathepsin L (CTSL) in tumor cells, proposed a selective prodrug. We considering an acetylated lysine link/spacer, which ensures the release of the drug selectively in tumor cells, have now designed this selective prodrug. Our proposal was developed from virtual screening studies, based on ligands and structure, prediction of pharmacokinetic and toxicological properties (ADME / Tox) and also bioinformatics techniques for the construction of a calcium channel model, due to the inexistence of Structures of the same that were deposited in the database of proteins PDB (Protein Data Bank). At the same time, our collaborating synthesis group synthesized, through Click Chemistry techniques and Mitsunobu multicomponent reactions, a library of new trifunctional hybrids, which, after studies of biological activity, were evaluated (in silico) against the structure of tubulin , And the most promising compounds from this library served as the basis for further virtual screening studies. To obtain our hits, we performed 4 virtual screening strategies, separated into 2 tasks. In the end, we selected 59 hits, of which 9 hits show promising calcium channel blocking activities and 65 hits show promising tubulin modulating activities. These hits follow in vitro purchase and testing, and after proven effectiveness, they will be part of a new tri prodrug proposal.