Transcriptomics is the large-scale study of RNA molecules produced by the genome, in single cells or population of cells using high-throughput methods. With the advances in transcriptomic analysis, the monitoring of genome-wide gene expression provides a powerful approach for determining the action of drugs. In this study, we analyzed the transcriptional responses of cells treated with drugs either alone or in combinations to explore their effects in two different applications: breast cancer therapy and cell conversion. In the first part of this thesis, we aim at modeling the relationship between single and multidrug breast therapy at the transcriptome level. We monitored the effects of three drugs, and their combinations in human breast cancer MCF-7 cells using the cap analysis of gene expression method. We are the first to explore the impact of single and combinatorial treatment on promoter and enhancer expression on a genome-wide scale. After applying and customizing a broad spectrum of regression algorithms, we showed that the transcriptional response to combinatorial drug treatment at both promoters and enhancers is accurately described by a linear combination of the responses to the individual drugs. Our analysis is promising for eliciting the transcriptional reaction to multidrug therapies in an unbiased genome-wide way, which may minimize the need for exhaustive combinatorial screens. Following the drug combination analysis, we explored the possibility to systematically identify drugs that either alone or in combinations facilitate cell conversion. To date, no computational approach prioritizes or suggests chemical compounds promoting cell reprogramming. Using transcriptomic data of human primary cells and drug response expression profiles, we developed a computational framework which accurately predicts single drugs or drug cocktails driving any source cell type towards the desired lineage. Experimental and in-silico validation on human pluripotent stem cells confirms the ability of the top predicted drugs to enhance reprogramming. The introduced method has countless applications in regenerative medicine and can significantly speed up the research in this field.

Thesis (M.Sc. (Pathobiology))--Mahidol University, 1983.

Thesis (M.C.T.M. (Clinical Tropical Medicine))--Mahidol University, 2003.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Diversos sÃo os relatos de resistÃncia in vitro de cepas de Candida spp. a fÃrmacos antifÃngicos, em especial a derivados azÃlicos. Deste modo, a prospecÃÃo de novos compostos com propriedade antifÃngica se faz necessÃria. Assim, o objetivo deste estudo foi avaliar a atividade antifÃngica do sesquiterpeno farnesol, sua aÃÃo sinÃrgica com antifÃngicos fluconazol (FLC), itraconazol (ITC), voriconazol (VRZ), anfotericina B (AMB) e caspofungina (CASP), bem como avaliar sua aÃÃo sob a atividade enzimÃtica de fosfolipases e proteases, consideradas importantes fatores de virulÃncia em Candida spp. Para tanto, foram avaliadas cepas de C. albicans (n=23), C. parapsilosis (n=16) e C. tropicalis (n=6). A concentraÃÃo inibitÃria mÃnima (CIM) foi determinada por meio da tÃcnica de microdiluiÃÃo, preconizada pelo Clinical Laboratory Standards Institute (CLSI). Em adiÃÃo, foi investigado o efeito do farnesol sobre a atividade de fosfolipase utilizando Ãgar Sabouraud suplementado com gema de ovo e a atividade de protease por tÃcnica espetrofotomÃtica. Paralelamente 14 cepas escolhidas aleatoriamente, foram prÃ-incubadas em trÃs concentraÃÃes sub-CIM de farnesol, afim de avaliar se o farnesol era capaz de causar algum dano na sensibilidade da cepa. Para as 45 cepas avaliadas, os valores de CIM para farnesol variaram de 9,37 a 150 ÂM para Candida spp. Para os derivados azÃlicos, o intervalo dos valores de CIM para fluconazol foi de 0,03125 a > 64 Âg/ml, observando-se a presenÃa de 18 cepas resistentes (CIM > 64 Âg/mL). Em relaÃÃo ao itraconazol, os valores de CIM variaram de 0,03125 a >16 Âg/mL e 35 cepas apresentaram resistÃncia a esse antifÃngico (CIM ≥1 Âg/mL). Constatou-se que das 45 cepas em estudo, 19 apresentaram resistÃncia a anfotericina B, com CIM > 1 Âg/mL. Quanto a caspofungina, os valores de CIM variaram de 0,0625 a 2 Âg/mL, com apenas seis cepas exibido resistÃncia, com CIM de 2 Âg/mL. A combinaÃÃo anfotericina B e farnesol apresentou sinergismo em 94,7% (18/19) das cepas de Candida spp. resistentes, evidenciado pelos valores de FICI ≤ 0,5. Na associaÃÃo fluconazol e farnesol, observou-se uma interaÃÃo sinÃrgica frente a todas as cepas de Candida spp. avaliadas, apresentando FICI ≤ 0,5. Trinta e seis cepas (18 C. albicans, 12 C. parapsilosis e 6 C. tropicalis) foram testadas frente à associaÃÃo de itraconazol e farnesol, observando a ocorrÃncia de sinergismo (FICI ≤ 0,5) em 94,4% dos isolados. Quanto à caspofungina todas as cepas testadas (C. parapsilosis, n=4; C. albicans, n=2) tiveram FICI ≤ 0,5, caracterizando associaÃÃo sinÃrgica com o farnesol. ApÃs constataÃÃo de que 17 das 45 cepas expressavam a enzima fosfolipase, as mesmas foram incubadas em concentraÃÃes sub-CIM de farnesol, por 24 horas, quando observouâse uma medida de Pz mÃdia de 0,73 para as cepas testadas sem contato com farnesol e valores de 0,71, 0,61, e 0,54 para aquelas incubadas em doses sub-inibitÃria de farnesol. Em relaÃÃo à produÃÃo de protease, observou-se que todas as 14 cepas produziam a enzima, em baixas concentraÃÃes, variando de 0,002 a 0,02 U/mL, e que a prÃ-incubaÃÃo com farnesol nÃo interferiu na produÃÃo enzimÃtica. No tocante a prÃ-incubaÃÃo das cepas com farnesol constatou-se que o mesmo interferi direta e indiretamente no fenÃmeno de resistÃncia das cepas. Estes resultados demonstram, em especial, o efeito do composto farnesol sobre a sensibilidade antifÃngica de Candida spp., abrindo a perspectiva de novos estudos com o intuito de determinar os mecanismos de aÃÃo desses compostos no metabolismo celular dos fungos. / There are several reports of in vitro resistance to antifungal drugs, especially azole derivatives, in strains of Candida spp. For this reason, the pursuit for new compounds that present antifungal properties is necessary. Thus, this study aimed at evaluating the antifungal activity of farnesol and its interaction with classical antifungal drugs against Candida spp., as well as evaluating its effect on the production of phospholipase and protease, which are important virulence factors for Candida species. Fourty-five strains of Candida spp. (23 C. albicans, 16 C. parapsilosis and 6 C. tropicalis) were tested through broth microdilution as described by the Clinical Laboratory Standards Institute (CLSI), document M27A2, and the minimum inhibitory concentrations (MICs) for amphotericin B (AMB), fluconazole (FLC), itraconazole (ITC), caspofungin (CAS) and farnesol (FAR) were individually determined. In addition, the effect of FAR on phospholipase activity was assessed by growing the strains on 2% Sabouraud agar, supplemented with egg yolk, and protease activity was determined through spectrophotometry. Then, 13 strains were randomly chosen, pre-incubated at three sub-inhibitory concentrations of FAR for 24 hours and re-submitted to microdilution assay. For the 45 evaluated Candida spp. strains the MIC values for FAR varied from 9.37 to 150 ÂM. For the classical antifungal drugs, MICs ranged from 0.0625 to 4 Âg/mL for AMB, with 19 resistant strains (MIC>1 Âg/mL); from 0.0125 to >64 for FLC, with 18 resistant strains (MIC> 64 Âg/mL); from 0.03125 to >16 Âg/mL for ITC, with 35 resistant strains (MIC ≥1 Âg/mL), and from 0.0625 to 2 Âg/mL for CAS, with six resistant strains (MIC≥2 Âg/mL). All resistant strains were tested against the combination of FAR with the drug to which they presented resistance. The combination of AMB and FAR was synergistic against 94.7% (18/19) of the Candida spp. isolates, as shown through the obtention of FICI≤0.5. FAR and FLC interacted synergistically against all tested strains (n=18), exhibiting FICI values of ≤0.5. The combination of FAR and ITC presented synergistic interactions (FICI≤0.5) against 94.4% of the tested isolates (33/35). Finally, FAR and CAS showed to interact synergistically (FICI≤0.5) against all tested strains. Concerning virulence factors, it was observed that 17/45 isolates produced phospholipase, with a mean Pz of 0.71. After incubating these strains at three different concentrations of FAR the mean Pz values of 0.71, 0.61 and 0.54 were obtained, after incubation at the lowest, the intermediate and the highest concentrations of FAR, respectively. In addition, it was observed that the 14 randomly chosen strains that were screened for protease activity produced low concentrations of these enzymes, varying from 0.002 to 0.02 U/mL and that FAR presented no effect on enzymatic production. Finally, it was observed that pre-incubating strains at the highest sub-inhibitory concentration of FAR reduced the MIC range of the tested antifungal drugs. These results especially show the effects of FAR on the susceptibility of Candida species to classical antifungal drugs, providing perspectives for the development of researches on the mechanisms of this compound on fungal cell metabolism

Les réponses adaptatives fonctionnelles (secondaires à des modulations de la signalisation cellulaire) peuvent contribuer à la résistance des tumeurs humaines aux traitements ciblés. Ce travail vise à caractériser les changements induits par la chimiothérapie dans le phosphokinome de cellules de cancer du côlon, et à sélectionner des combinaisons de médicaments synergiques en ciblant les kinases dont l’activation est la plus importante. Nous avons créé des scripts informatiques pour l’analyse sur le logiciel de calcul R de données provenant de tests cytotoxiques avec des matrices de combinaison de doses à deux ou trois médicaments. Nous avons profilé les changements induits par différents médicaments sur des cellules de la lignée HT29 xénogreffées dans des souris immunodéprimées. Nous avons testé le 5FU, l’oxaliplatine, l’irinotecan, le cetuximab ainsi que les combinaisons de ces médicaments. Nous avons sélectionné les kinases activées par le traitement par irinotecan, notamment AKT et MEK1, et nous avons testés in vitro et in vivo des combinaisons à deux médicaments d’inhibiteurs de ces deux kinases et l’irinotecan sur 6 lignées de cancer du côlon. Enfin nous avons testés in vitro la combinaison des trois médicaments. Nous avons observé que la combinaison d’un inhibiteurs d’AKT et de MEK ainsi que la combinaison des trois médicaments étaient caractérisées par un synergisme significatif Cette étude a démontré que la chimiothérapie induit une reprogrammation des voies de signalisation intracellulaires, et fournit le rationnel pour une évaluation du profilage de la reprogrammation du phosphokinome comme outil pour développer de nouvelles combinaisons de médicaments. / Functional (i.e. due to cellular machinery modulations) adaptive responses could also contribute to human tumor resistance to targeted drugs. We hypothesized that the activation of tumor cell kinases in response to chemotherapy treatment could render the cell depending on them, and that the inhibition of these activated kinases could achieve a synergistic effect with chemotherapy agents. We compiled R scripts for analysis of data from cytotoxic tests with dose matrix combinations of 2 or 3 drugs. We evaluated phosphokinome rewiring induced by 5FU, irinotecan, oxaliplatin and cetuximab when these drugs were used alone or in combination on mice xenografted with HT29 cell line. We observed an activation of AKT and MEK1 after irinotecan treatment, and we tested two- and three drug combinations of BKM120, an AKT inhibitor, MEK162, a MEK inhibitor, and irinotecan. We showed that BKM120 and MEK162 are synergistic, as well as the combination of the three drugs. Our study shows that chemotherapy induces a significant rewiring of intracellular signaling pathways, and that profiling phosphokinome remodeling after chemotherapy treatment is useful in order to design synergistic drug combinations.

Purpose: To show the necessity of distinguishing several patterns of drug prescribing that may lead to co-medication. It is demonstrated how these different patterns can be investigated using large databases containing pharmacy data or reimbursement data. Methods: Two examples illustrate how the particular pattern of co-medication studied will influence the reported proportion of patients having co-medication, the use of antidepressants among people using anticonvulsants, and the use of antihistamines among people receiving penicittines. Results: Depending on definition and period considered, the percentage of anticonvulsant users co-medicated with antidepressants ranged from 5.8% (95%CI 5.0%, 6.8%) to 14.5% (95%CI 13.2%, 15.9%) in 2000. Comparing 2002 with 2000, the ratio of proportions ranged from 1.3 to 2.1. The percentage of people who received penicillines and were co-medicated with antihistamines ranged from 0.5% (95%CI 0.4%, 0.6%) to 9.7% (95%CI 9.3%,10.2%) in 2000. Comparing 2002 with 2000, the ratio of proportions ranged from 1.2 to 1.6. Conclusion: The co-medication patterns investigated yielded clinical as well as statistically significant different estimates. The estimates differed up to a factor 2.5 for the drugs usually prescribed for long periods, and a factor 12 for drugs prescribed for short periods. Hence, we propose to distinguish the patterns 'co-prescribing', 'concomitant medication,' and 'possibly concurrent medication.' The research question determines the co-medication pattern of interest, and the drug and disease under study determine the time window.

Yes / Background: Discovery of novel gametocytocidal molecules is a major pharmacological strategy in the elimination and eradication of malaria. The high patronage of the aqueous root extract of the popular West African anti-malarial plant Cryptolepis sanguinolenta (Periplocaceae) in traditional and hospital settings in Ghana has directed this study investigating the gametocytocidal activity of the plant and its major alkaloid, cryptolepine. This study also investigates the anti-malarial interaction of cryptolepine with standard anti-malarials, as the search for new anti-malarial combinations continues. Methods: The resazurin-based assay was employed in evaluating the gametocytocidal properties of C. sanguinolenta and cryptolepine against the late stage (IV/V) gametocytes of Plasmodium falciparum (NF54). A fixed ratio method based on the SYBR Green I fluorescence-based assay was used to build isobolograms from a combination of cryptolepine with four standard anti-malarial drugs in vitro using the chloroquine sensitive strain 3D7. Results: Cryptolepis sanguinolenta ( IC50 = 49.65 nM) and its major alkaloid, cryptolepine ( IC50 = 1965 nM), showed high inhibitory activity against the late stage gametocytes of P. falciparum (NF54). In the interaction assays in asexual stage, cryptolepine showed an additive effect with both lumefantrine and chloroquine with mean ΣFIC50s of 1.017 ± 0.06 and 1.465 ± 0.17, respectively. Cryptolepine combination with amodiaquine at therapeutically relevant concentration ratios showed a synergistic effect (mean ΣFIC50 = 0.287 ± 0.10) whereas an antagonistic activity (mean ΣFIC50 = 4.182 ± 0.99) was seen with mefloquine. Conclusions: The findings of this study shed light on the high gametocytocidal properties of C. sanguinolenta and cryptolepine attributing their potent anti-malarial activity mainly to their effect on both the sexual and asexual stages of the parasite. Amodiaquine is a potential drug partner for cryptolepine in the development of novel fixed dose combinations.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2015-02-13T20:15:11Z No. of bitstreams: 2 Dissertação - Anna Carolina Galvão Ferreira - 2012.pdf: 1738587 bytes, checksum: b4a75ebc8af8f8a234573009292ed508 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2015-02-13T20:16:17Z (GMT) No. of bitstreams: 2 Dissertação - Anna Carolina Galvão Ferreira - 2012.pdf: 1738587 bytes, checksum: b4a75ebc8af8f8a234573009292ed508 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-02-13T20:16:17Z (GMT). No. of bitstreams: 2 Dissertação - Anna Carolina Galvão Ferreira - 2012.pdf: 1738587 bytes, checksum: b4a75ebc8af8f8a234573009292ed508 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2012-05-28 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / OBJECTIVE: To describe tuberculosis treatment rates of cure, failure and default of a self administered regimen, with rifampin, isoniazid, pyrazinamide and ethambutol in the first two months of treatment followed by isoniazid and rifampcina in the four last months (2RHZE/4RH) in four-drug fixed-dose combination (FDC), implemented in Brazil since 2010 to replace a regimen of separately administered rifampicin, isoniazid and pyrazinamide in the first two months of treatment followed by isoniazid and rifampcina for four months (2RHZ/4RH). METHODS: Descriptive study using prospectively collected data from medical records of TB cases, older than18 years of age undergoing treatment with 2RHZE/4RH in two units of primary health care in the metropolitan area of Goiânia. RESULTS: The study included 40 cases of TB. The cure rate was 67.5% (27/40), the abandonment was 17.5% (7/40) and there were no cases of failure. There was reports of adverse reactions in 47% (19/40) of the cases. Of these, 87% were mild and 13% were moderate. There was no need for change or suspension of the scheme. CONCLUSIONS: The cure rate in FDC 4RHZE/2RH scheme under self-administered regimen was similar to historical rates of cure with 2RHZ/4RH. The default rate in the sample studied was much higher than the rate recommended as appropriate (up to 5%). / INTRODUÇÃO: O esquema de tratamento da TB tem alta eficácia em torno de 95% e com possibilidade de cura de aproximadamente 100% dos casos e reduz rapidamente a transmissão, e assim pode-se reduzir a incidência da doença. Embora a distribuição da medicação seja gratuita em todo país pelo Sistema Único de Saúde, a efetividade do tratamento da TB varia muito nos diferentes locais. JUSTIFICAVA: Conhecer as taxas de sucesso de tratamento ,falência e abandono além de verificar a segurança do tratamento da TB com 4 medicamentos em apresentação dose fixa combinada. OBJETIVO: Descrever as taxas de cura, falência e abandono do tratamento da tuberculose com o esquema básico com rifampicina, isoniazida, pirazinamida e etambutol nos dois primeiros meses de tratamento seguidos de isoniazida e rifampcina por quatro meses (2RHZE/4RH), sob forma de comprimidos em dose fixa combinada (DFC), em regime auto administrado implementado no Brasil desde 2010, em substituição ao esquema utilizando cápsulas e comprimidos com rifampicina, isoniazida e pirazinamida nos dois primeiros meses de tratamento, seguidos de isoniazida e rifampcina por quatro meses (2RHZ/4RH). MÉTODOS: Estudo descritivo utilizando dados secundários coletados prospectivamente de prontuários de casos de TB, maiores de 18anos, submetidos ao tratamento com esquema básico para tuberculose, em duas Unidades Básicas de Saúde da região metropolitana de Goiânia. RESULTADOS: Foram incluídos no estudo 40 casos de TB. A taxa de cura foi de 67,5% (27/40), a de abandono de 17,5% (7/40) e a não ocorreram casos de falência. Houve relato de reações adversas em 47% (19/40) num total de 31 ocorrências. Dessas, 87% foram leves e 13% moderadas. Em nenhum caso houve necessidade de mudança ou suspensão do esquema. CONCLUSÕES: A taxa de cura do esquema 4RHZE/2RH em DFC sob regime autoadministrado foi semelhante às taxas históricas do tratamento com 2RHZ/4RH. A taxa de abandono na amostra estudada foi superior a taxa preconizada como adequada (até 5%).

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.