A Doença de Parkinson (DP) é a segunda doença neurodegenerativa mais comum. Atinge 3,3% das pessoas com mais de 64 anos. Com o envelhecimento populacional, sua prevalência deve dobrar. A doença classicamente se caracteriza por degeneração dos neurônios da substantia nigra, afetando principalmente a transmissão dopaminérgica. O tratamento mais eficaz na DP continua sendo a levodopa, um precursor dopaminérgico com excelente resposta motora. Entretanto, à medida que a doença progride, aparece uma série de complicações motoras e não motoras que limitam o tratamento. Para facilitar o reconhecimento destas complicações, foram criados questionários que aumentam a possibilidade de diagnóstico, abordando aspectos motores e não motores. O principal deles é um questionário de 19 itens. Ele consiste em 19 manifestações que o paciente deve assinalar, caso tenha determinado sintoma, e se ele melhora com a próxima dose da medicação. Quando existem pelo menos duas respostas positivas, o questionário tem ótima sensibilidade e especificidade. A Doença de Parkinson tem evolução heterogênea, sendo que uma das causas atribuídas para tal é genética. Tem se estudado muitos genes da rota de dopamina por sua relação íntima com a fisiopatologia e tratamento da doença. O transportador de dopamina (DAT), que realiza a retirada da dopamina da fenda sináptica, desempenha papel fundamental nesta rota. Existe um polimorfismo VNTR com cópias de uma unidade de repetição variando de 3 a 11 com as repetições 9 e 10 sendo os alelos mais comuns. Diversos estudos correlacionaram esse polimorfismo com transtorno do déficit de atenção e hiperatividade quando há 10 repetições e com a Doença de Parkinson quando há 9 repetições. Seria um candidato ideal para avaliação com relação às complicações do tratamento e progressão. Assim, avaliamos pacientes com Doença de Parkinson longitudinalmente para comparar a progressão da doença com polimorfismo do DAT e verificar as complicações associadas ao tratamento. Inicialmente, realizamos uma revisão sistemática das propriedades clinimétricas dos questionários de wearing off. Esta revisão mostrou que o questionário de 9 itens tem sensibilidade de 0,87-1 e especificidade de 0,1-0,69. Já o questionário de 19 itens tem sensibilidade de 0,81-0,9 e especificidade de 0,63-0,8 com ponto de corte igual a 2 itens positivos. Realizamos a validação deste último para português, com boa estabilidade no teste-reteste com correlação intraclasse de 0,87 (IC 95% 0,69-0,95 p < 0,01) e sensibilidade de 0,97 (IC 95% 0,94-1 p < 0,01) e especificidade de 0,71 (IC 95% 0,56-0,85 p < 0,01). Para progressão, foi demonstrada uma associação de sexo feminino e a presença de alelo com 9 repetições como fatores de risco para progressão mais rápida. A progressão em homens medida pelo UPDRS 3 era menor em 1,277 (IC 95% 2,18-0,38 p < 0,01) e pelo UPDRS total era menor em 1,50 (IC 95% 2,92-0,11 p = 0,031). Com presença de alelo com 9 repetições, a progressão do UPDRS 3 era menor em 1,92 (ICC 95% 0,04-1,01 p = 0,0317), e presente mesmo controlando para sexo. Não houve diferença na escala de discinesias ou questionário wearing off. Assim, os resultados obtidos mostraram que o questionário de 19 itens é uma boa ferramenta diagnóstica, sendo validado para português. Além disso, o polimorfismo DAT está associado à progressão mais rápida da DP com 9 repetições. / Parkinson’s disease (PD) is the second most common neurodegenerative disease. It affects 3.3% of people over 64 years. With population aging, its prevalence should double. The disease is classically characterized by degeneration of substantia nigra neurons, mainly affecting dopaminergic transmission. The most effective treatment for PD continues to be levodopa, a dopaminergic precursor with excellent motor response. However, as the disease progresses, there is a number of motor and non-motor complications that limits the treatment. To facilitate the recognition of these complications, questionnaires were created to increase the possibility of diagnosis, addressing motor and non-motor aspects. The main one is a questionnaire of 19 items. It consists of 19 symptoms that the patients should indicate, if they feel particular symptom, and if they get better with the next dose of medication. When there are at least two positive responses, the questionnaire has optimal sensitivity and specificity. Parkinson’s disease has a heterogeneous evolution, and one of the reasons attributed for that is genetic. Many genes of the dopamine route have been studied for their intimate relationship with the pathophysiology and treatment of the disease. The dopamine transporter, with synaptic cleft reuptake, plays a key role in this route. It has a VNTR polymorphism with copies of a repeating unit ranging from 3 to 11 with repetitions 9 and 10 being the most common alleles. Several studies correlated this polymorphism with Attention Deficit Hyperactivity Disorder with 10 repetition allele and Parkinson’s disease with 9 repetition alleles. It would be an ideal candidate for evaluation regarding treatment complications and progression. Thus, we evaluated patients with Parkinson’s disease longitudinally to compare disease progression with DAT polymorphism and to verify treatment-related complications. Initially we performed a systematic review of the clinimetric properties of the wearing off questionnaires. That showed that the questionnaire of 9 items has sensitivity of 0.87-1 and specificity of 01-0.69. The questionnaire of 19 items has a sensitivity of 0.81-0.9 and a specificity of 0.63-0.8 with a cut-off point of 2 items. We performed the validation of the latter for Portuguese, with good stability in the testretest with intraclass correlation of 0.87 (95% CI 0.69-0.95 p < 0.01) and sensitivity of 0.97 (CI 95% 0.94-1 p < 0.01) and specificity of 0.71 (95% CI 0.56-0.85 p < 0.01). We demonstrated a female association and presence of 9 DAT allele repetition as risk factors for faster progression. The progression in men ofwith UPDRS 3 was lower in 1.277 (95% CI 2.18-0.38 p < 0.01) and total UPDRS was lower in 1.50 (95% CI 2.92-0.11 p = 0.031). With the presence of allele with 9 repetitions the progression of UPDRS 3 was lower in 1.92 (ICC 95% 0.04-1.01 p = 0.0317), and present even correcting sex. There was no difference in the dyskinesia scale or wearing off questionnaire. Thus, the results obtained showed wearing off questionnaire is a good tool for clinical and research, and it is validated to Portuguese. Also, DAT polymorphism is associated with faster PD progression with 9 repetition allele.

This diploma thesis deals with international shipping. Transportation in international trade holds, despite unstable developments during the last few years, is of vital importance. The main factor that affects most of the weaknesses associated with the current issue of transportation, the effect of the impact of the global economic crisis. The thesis also focuses on detail to frequent damage of cargo and proposes measures for their elimination.

A Doença de Parkinson (DP) é a segunda doença neurodegenerativa mais comum. Atinge 3,3% das pessoas com mais de 64 anos. Com o envelhecimento populacional, sua prevalência deve dobrar. A doença classicamente se caracteriza por degeneração dos neurônios da substantia nigra, afetando principalmente a transmissão dopaminérgica. O tratamento mais eficaz na DP continua sendo a levodopa, um precursor dopaminérgico com excelente resposta motora. Entretanto, à medida que a doença progride, aparece uma série de complicações motoras e não motoras que limitam o tratamento. Para facilitar o reconhecimento destas complicações, foram criados questionários que aumentam a possibilidade de diagnóstico, abordando aspectos motores e não motores. O principal deles é um questionário de 19 itens. Ele consiste em 19 manifestações que o paciente deve assinalar, caso tenha determinado sintoma, e se ele melhora com a próxima dose da medicação. Quando existem pelo menos duas respostas positivas, o questionário tem ótima sensibilidade e especificidade. A Doença de Parkinson tem evolução heterogênea, sendo que uma das causas atribuídas para tal é genética. Tem se estudado muitos genes da rota de dopamina por sua relação íntima com a fisiopatologia e tratamento da doença. O transportador de dopamina (DAT), que realiza a retirada da dopamina da fenda sináptica, desempenha papel fundamental nesta rota. Existe um polimorfismo VNTR com cópias de uma unidade de repetição variando de 3 a 11 com as repetições 9 e 10 sendo os alelos mais comuns. Diversos estudos correlacionaram esse polimorfismo com transtorno do déficit de atenção e hiperatividade quando há 10 repetições e com a Doença de Parkinson quando há 9 repetições. Seria um candidato ideal para avaliação com relação às complicações do tratamento e progressão. Assim, avaliamos pacientes com Doença de Parkinson longitudinalmente para comparar a progressão da doença com polimorfismo do DAT e verificar as complicações associadas ao tratamento. Inicialmente, realizamos uma revisão sistemática das propriedades clinimétricas dos questionários de wearing off. Esta revisão mostrou que o questionário de 9 itens tem sensibilidade de 0,87-1 e especificidade de 0,1-0,69. Já o questionário de 19 itens tem sensibilidade de 0,81-0,9 e especificidade de 0,63-0,8 com ponto de corte igual a 2 itens positivos. Realizamos a validação deste último para português, com boa estabilidade no teste-reteste com correlação intraclasse de 0,87 (IC 95% 0,69-0,95 p < 0,01) e sensibilidade de 0,97 (IC 95% 0,94-1 p < 0,01) e especificidade de 0,71 (IC 95% 0,56-0,85 p < 0,01). Para progressão, foi demonstrada uma associação de sexo feminino e a presença de alelo com 9 repetições como fatores de risco para progressão mais rápida. A progressão em homens medida pelo UPDRS 3 era menor em 1,277 (IC 95% 2,18-0,38 p < 0,01) e pelo UPDRS total era menor em 1,50 (IC 95% 2,92-0,11 p = 0,031). Com presença de alelo com 9 repetições, a progressão do UPDRS 3 era menor em 1,92 (ICC 95% 0,04-1,01 p = 0,0317), e presente mesmo controlando para sexo. Não houve diferença na escala de discinesias ou questionário wearing off. Assim, os resultados obtidos mostraram que o questionário de 19 itens é uma boa ferramenta diagnóstica, sendo validado para português. Além disso, o polimorfismo DAT está associado à progressão mais rápida da DP com 9 repetições. / Parkinson’s disease (PD) is the second most common neurodegenerative disease. It affects 3.3% of people over 64 years. With population aging, its prevalence should double. The disease is classically characterized by degeneration of substantia nigra neurons, mainly affecting dopaminergic transmission. The most effective treatment for PD continues to be levodopa, a dopaminergic precursor with excellent motor response. However, as the disease progresses, there is a number of motor and non-motor complications that limits the treatment. To facilitate the recognition of these complications, questionnaires were created to increase the possibility of diagnosis, addressing motor and non-motor aspects. The main one is a questionnaire of 19 items. It consists of 19 symptoms that the patients should indicate, if they feel particular symptom, and if they get better with the next dose of medication. When there are at least two positive responses, the questionnaire has optimal sensitivity and specificity. Parkinson’s disease has a heterogeneous evolution, and one of the reasons attributed for that is genetic. Many genes of the dopamine route have been studied for their intimate relationship with the pathophysiology and treatment of the disease. The dopamine transporter, with synaptic cleft reuptake, plays a key role in this route. It has a VNTR polymorphism with copies of a repeating unit ranging from 3 to 11 with repetitions 9 and 10 being the most common alleles. Several studies correlated this polymorphism with Attention Deficit Hyperactivity Disorder with 10 repetition allele and Parkinson’s disease with 9 repetition alleles. It would be an ideal candidate for evaluation regarding treatment complications and progression. Thus, we evaluated patients with Parkinson’s disease longitudinally to compare disease progression with DAT polymorphism and to verify treatment-related complications. Initially we performed a systematic review of the clinimetric properties of the wearing off questionnaires. That showed that the questionnaire of 9 items has sensitivity of 0.87-1 and specificity of 01-0.69. The questionnaire of 19 items has a sensitivity of 0.81-0.9 and a specificity of 0.63-0.8 with a cut-off point of 2 items. We performed the validation of the latter for Portuguese, with good stability in the testretest with intraclass correlation of 0.87 (95% CI 0.69-0.95 p < 0.01) and sensitivity of 0.97 (CI 95% 0.94-1 p < 0.01) and specificity of 0.71 (95% CI 0.56-0.85 p < 0.01). We demonstrated a female association and presence of 9 DAT allele repetition as risk factors for faster progression. The progression in men ofwith UPDRS 3 was lower in 1.277 (95% CI 2.18-0.38 p < 0.01) and total UPDRS was lower in 1.50 (95% CI 2.92-0.11 p = 0.031). With the presence of allele with 9 repetitions the progression of UPDRS 3 was lower in 1.92 (ICC 95% 0.04-1.01 p = 0.0317), and present even correcting sex. There was no difference in the dyskinesia scale or wearing off questionnaire. Thus, the results obtained showed wearing off questionnaire is a good tool for clinical and research, and it is validated to Portuguese. Also, DAT polymorphism is associated with faster PD progression with 9 repetition allele.

The aim of the work with subject Logistical management of transport of the chosen company is making of suggestion of better solution to the system of sale and transportation from the manufactory to the customer. The chosen company deals with woodworking and production of wooden products. The introductory literature search interprets the single logistical concepts and it introduces us this work. Next comes the characteristics of the chosen company, its{\crq} history and structure. Following chapter describes the production process and products which are produced by the company. In further part I{\crq}m concerned with analysis of present condition of the sale which is realized by company stores to the customer, and with system of supply and transportation to the links of this distribution chain. The final part of this work presents proposed changes in solution of this system and information how could this system function.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2017-07-26T18:47:21Z No. of bitstreams: 1 2014 - Marcela Jacques de Lemos Neves.pdf: 434472 bytes, checksum: c9817e97c5d136299acb936ad4b87b6f (MD5) / Made available in DSpace on 2017-07-26T18:47:21Z (GMT). No. of bitstreams: 1 2014 - Marcela Jacques de Lemos Neves.pdf: 434472 bytes, checksum: c9817e97c5d136299acb936ad4b87b6f (MD5) Previous issue date: 2014-05-30 / FAPERJ / The main goal of this study was evaluate the effect of downregulation of the ammonium transporter OsAMT1.3 in the ammonium uptake through the high affinity system, as well as the effects on nitrogen metabolism. To perform the OsAMT1.3 gene silencing, it was used the artificial micro RNA (amiRNA) technology. In this system, the OsAMT1.3 coding region sequence (cds) is inserted in W marcelaMD3 website and putatives amiRNAs to silencing OsAMT1.3 were made. The amiRNA was inserted by PCR using the pNW55 vector as template. The amiRNA was inserted in the IRS154 vector using the T4 DNA ligase. The IRS154 plus amiRNA was cloned in the E. coli by electroporation. After rice transformation of Nipponbare variety through Agrobacterium and Hygromycin selection, 14 lineages were obtained. Six lineages showed high seed production and only one lineage with abnormal growth. After seed production in a greenhouse, the lineages L1, L2 and L6 were selected to further experiments evaluating the effects of OsAMT1.3 downregulation. First, the lineages selected were evaluated about the levels of OsAMT1.3 downregulation. The rice lineages transformed with amiRNA showed lower level of OsAMT1.3 expression compared to control plants, however, different levels of OsAMT1.3 downregulation was observed. The lineage L1 showed lower levels of OsAMT1.3 downregulation, L2 and L6 showed higher levels of OsAMT1.3 downregulation. The lineages L1, L2 and L6 as well as IRS control plant (transformed with empty vector) were grown in growth chamber at 30 days after germination, and the treatments used were: N starvation for three days, resupply with 0.15 mM of NH4+-N (low level) and 2.0 mM of NH4+-N (high level). The lineages L1, L2 and L6 showed lower NH4+ uptake with 0.15 mM of NH4+-N compared to control plants (IRS), on the other hand, the plants grown with 2.0 mM of of NH4+-N did not show NH4+ uptake differences, except L1. The expression of OsAMT1.1, OsAMT1.2 and OsAMT1.3 ammonium transporters were upregulated with 0.15 mM of NH4+-N in the control plants (IRS); in the lineages L1, L2 and L6 showed downregulation of the OsAMT1.1, OsAMT1.2 and OsAMT1.3 genes. The lower NH4+ uptake with 0.15 mM of NH4+-N resulted in lower levels of NH4+-N and Amino-N in the roots in the lineages, while the NH4+-N and Amino-N in the plants grown with 2.0 mM of NH4+-N was minimally changed. The results indicate that OsAMT1.3 downregulation leads to OsAMT1.1 and OsAMT1.2 downregulation as well, decreasing the NH4+ uptake. Despite the OsAMT1.3 lower expression compared to OsAMT1.1 and OsAMT1.2, the OsAMT1.3 transporter may be involved in the nitrogen uptake efficiency in low levels of NH4+. / O objetivo deste trabalho foi estudar o efeito do silenciamento do gene do transportador OsAMT1.3 em arroz sobre a habilidade das plantas em absorver o N-NH4+ atrav?s do sistema de alta afinidade, bem como os reflexos sobre o metabolismo de nitrog?nio. Para o silenciamento do gene OsAMT1.3 foi usada a tecnologia do miRNA artificial (amiRNA). Para tanto, a sequ?ncia codante do gene (cds) OsAMT1.3 ? inserida no sistema WMD3 que indica sequ?ncias de amiRNA?s para silenciar o pr?prio OsAMT1.3. A inser??o do amiRNA foi feita por PCR usando o vetor pNW55 como molde. O miRNA foi inserido no vetor IRS154 por corte e liga??o usando a enzima T4 DNA ligase. O produto da liga??o foi introduzido em E. coli por eletropora??o. Ap?s a transforma??o de arroz da variedade Nipponbare mediada por Agrobacterium. A sele??o das plantas transformadas foi feita com o antibi?tico Higromicina. No total foram obtidas 14 linhagens transformadas. Para confirmar que as plantas mutantes possuiam a constru??o, foi realizado o teste com a folha bandeira em solu??o de higromicina. Seis linhagens apresentaram boa produ??o de sementes e houve uma linhagem com crescimento anormal. Ap?s a multiplica??o das linhagens em casa de vegeta??o, foram selecionadas as linhagens L1, L2 e L6 para os experimentos de an?lise dos efeitos do silenciamento do gene OsAMT1.3. Primeiramente, as linhagens selecionadas foram avaliadas quanto ao n?vel de silenciamento do gene OsAMT1.3. As linhagens de arroz transformadas apresentaram maior n?vel de silenciamento do gene OsAMT1.3 quando comparadas ?s plantas controle, no entanto, diferentes n?veis de silenciamento foram observados. A linhagem L1 apresentou menor n?vel de silenciamento do gene OsAMT1.3, enquanto L2 e L6 apresentaram maior silenciamento. As linhagens L1, L2 e L6 e a planta controle IRS (transformada com o vetor vazio) foram cultivadas em c?mara de crescimento at? os 30 dias ap?s a germina??o, com a aplica??o dos seguintes tratamentos: sem N por tr?s dias, ressuprimento com 0,15 mM de N-NH4+ ap?s tr?s dias de priva??o de N (baixa dose) e 2,0 mM de N-NH4+ constante (alta dose). As linhagens L1, L2 e L6 apresentaram menor absor??o de NH4+ com 0,15 mM de N-NH4+ quando comparadas com as plantas controle (IRS), enquanto as plantas cultivadas com 2,0 mM de N-NH4+ n?o apresentaram diferen?as na absor??o de NH4+. A express?o dos genes dos transportadores de NH4+ OsAMT1.1, OsAMT1.2 e OsAMT1.3 foi induzido pelo tratamento com 0,15 mM de N-NH4+ nas plantas controle (IRS), enquanto as linhagens transformadas apresentaram repress?o dos genes OsAMT1.1, OsAMT1.2 e OsAMT1.3. A menor absor??o de NH4+ com 0,15 mM de N-NH4+ causou menor n?vel de N-NH4+ e N-amino nas ra?zes das linhagens transformadas, enquanto nas plantas com 2,0 mM de N-NH4+ houve pouca altera??o nos conte?dos de N-NH4+ e N-amino. Os resultados indicam que o silenciamento do gene OsAMT1.3 provoca regula??o negativa dos transportadores OsAMT1.1 e OsAMT1.2, alterando a absor??o de NH4+. Apesar do gene OsAMT1.3 ser menos expresso que os genes OsAMT1.1 e OsAMT1.2, o transportador OsAMT1.3 pode estar envolvido na efici?ncia de absor??o em baixas doses de NH4+.

O parálogo 3 do trocador Na+/H+ (NHE3) é essencial para a reabsorção de HCO3- nos túbulos proximais renais e sua expressão e função adaptam-se às diferentes condições ácido-base do organismo. O objetivo desta tese foi avaliar quais as diferenças entre os efeitos da acidose metabólica (AM) e respiratória (AR) sobre a regulação do NHE3 e identificar variáveis responsáveis pelas respostas adaptativas observadas. Em células OKP, a AM foi simulada diminuindo a [HCO3-] do meio de cultura e a AR aumentando a pCO2 ambiente por 24 h. Foram observados os efeitos das acidoses sobre o RNAm-Nhe3, a presença da proteína-NHE3 na membrana celular e a atividade promotora do gene do Nhe3. Concluiu-se que o pH extracelular não é a variável físico-química responsável por estimular a expressão do NHE3, contudo é um importante candidato à variável responsável por regular o tráfego da proteína para a membrana. Além disso, a região de -471 a -153 pb em relação ao sítio de início de transcrição do promotor do gene do Nhe3 contém prováveis reguladores positivos que atuam em resposta à AM. / The Na+/H+ exchanger 3 (NHE3) is essential for HCO3- reabsorption in renal proximal tubules and its expression and function must adapt to acid-base conditions. The goal of the presente study was to evaluate whether there are differences between metabolic (MA) and respiratory acidosis (RA) with regard to NHE3 modulation and to identify variables that may trigger these distinct adaptive responses. In OKP cells, MA was achieved by lowering [HCO3-] in the cell culture medium and RA by increasing pCO2 in the incubator chamber for 24 h. The effects of both acidosis on Nhe3 mRNA levels, cell-surface NHE3 expression and promoter activity were evaluated. In summary, it was concluded that low extracellular pH is not the physical-chemical variable that up-regulates NHE3 expression, however, extracellular pH is a candidate for the variable related to the NHE3 displacement to the apical membrane. Moreover, the Nhe3 gene promoter region spanning from -471 to -153 base pairs upstream from the transcriptional start site contains putative enhancers regulated in response to MA.

A creatina (Cr) é uma reserva de fosfato de alta energia, sendo a fonte mais rápida de restauração do ATP intracelular. O hormônios tireoideano participa de forma importante na manutenção da taxa metabólica, aumentando a síntese e consumo de ATP, por meio da regulação de diferentes genes-alvo. Neste sentido, avaliamos o efeitos do HT sobre a expressão gênica do transportador de Cr nos músculos esqueléticos e cardíaco de ratos. O tratamento com o hormônio regula estes processos, porém de forma distinta nos diferentes tipos de músculos. / Creatine (Cr) is a high-energy phosphate reservoir and the fastest source for intracellular ATP regeneration. The thyroid hormone plays a key role on the maintenance of basal metabolic rate, increasing the synthesis and the degradation of ATP through regulation of target-genes. In this study, we explore the effects of thyroid hormone on Cr transporter gene expression and regulation of intracellular pool of Cr in skeletal and cardiac muscles in rats. The hormone can regulate these processes in distinct ways in different muscle types.

Pouco se sabe sobre o mecanismo de ação da pentamidina, um composto utilizado no tratamento das leishmanioses. Para uma melhor compreensão do mecanismo de ação assim como o mecanismo de resistência à pentamidina, foi isolado um gene que codifica um membro da família de transportadores ABC, chamado PRP1 capaz de conferir resistência à pentamidina em formas promastigotas e amastigotas de Leishmania spp. O tratamento dos transfectantes que superexpressam a PRP1 com pentamidina em presença de concentrações não tóxicas de verapamil, um inibidor de transportadores ABC, foi capaz de reverter a resistência mediada por esse transportador. Foram ainda isolados nesse estudo duas linhagens de L. amazonensis resistentes à pentamidina. A análise molecular dos parasitos indicou que esses mutantes não apresentaram nenhuma amplificação de DNA, inclusive do gene PRP1 que também não se mostrou superexpresso em ambas as linhagens. As duas linhagens resistentes à pentamidina tiveram sua resistência revertida quando tratadas com verapamil, indicando que o mecanismo de resistência nesses mutantes pode estar associado a um transportador ABC. Os resultados obtidos nesse estudo fornecem dados para uma melhor compreensão do mecanismo de resistência à pentamidina e sugerem um provável potencial da associação pentamidina e verapamil no tratamento da doença. / Little it is known about the mechanism of action of pentamidine, an compound used for leishmaniases chemotherapy. To understand the mechanism of action and resistance of pentamidine, it was isolated a gene that codifies a member of ABC transporter family, named as PRP1 able to confer pentamidine resistance in promastigotes and amastigotes of Leishmania spp. Treatment of transfectants overexpressing PRP1 with pentamidine in presence of non toxic concentration of verapamil, an inhibitor of ABC transporters, was able to reverse the drug resistance mediated by this transporter. Two lines of L. amazonensis resistant to pentamidine were selected. Molecular analysis of parasites indicated that these mutants do not contain amplified DNA, including the PRP1 gene either not associated with overexpression in both lines. The two lines resistant to pentamidine had their resistance reversed when treated with verapamil, indicating that the mechanism of resistance may be associated to an ABC transporter. The results of this work lead to new insights for a better understanding of the mechanism of of resistance suggesting a probably potencial of pentamidine and verapamil association in the chemotherapy.

SUMMARYThe diversity of cancer molecular origins associated with the genetic variability of patients has encouraged the development of chemotherapeutic treatments adapted not only to the target tumor, but also to a specific patient. This personalized strategy is based on cancer biomarkers allowing a better identification and characterization of each tumor where predictive biomarkers provide the distinction between various factors indicative of the response to the treatment. In this context, several studies highlighted the role of the solute carrier transporter family 22 (solute carriers 22 or SLC22) in the uptake of platinum anticancer drugs. This mechanism being not well understood, our work intends to establish the potential role of SLC22 member A18 (SLC22A18) as predictive biomarker in the aim to help to a better targeted chemotherapeutic strategy for each patient. We optimized a system overexpressing SLC22A18 stably or transiently in HeLa cancer cell line. SLC22A18 expression was confirmed by qRT-PCR, western blotting, microscopy and flow cytometry. The cell lines were treated with taxane, anthracyclin, vinca alkaloid and nitrosoureas anticancer drug families. We showed that doxorubicin, camptothecin, chloroquine, tetracycline and carmustin had no effect on the cell viability assays suggesting that they are not substrates of SLC22A18. Interestingly, the cell line was sensitized in the presence of antimitotic drug with a sensitivity factor of 2.7 in the presence of paclitaxel, 1.4 with docetaxel, 1.8 with vinblastin and 2.2 in the presence of vincristine. To confirm these results, we elaborated a SLC22A18 knockdown cell line in HS683 cells using siRNA technology. The downexpression of SLC22A18 was correlated to a tendency to resist to the accumulation of paclitaxel thereby confirming the previous results. Simultaneously, a knockout cell line was established using the transcription activator-like effectors nuclease (TALEN) technology in U373 cell line. Our studies constitute a robust base of knowledge for further investigation on SLC22A18 transporter as a predictive biomarker promoting antimitotic treatment in tumors where this transporter is detected. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Chez les plantes supérieures, la distribution des sucres entre organes sources et puits requiert l'activité de transporteurs membranaires de sucre. Les flux de sucre variant au cours du développement de la plante et en réponse à des stress, il est logique de penser que les transporteurs de sucres sont impliqués dans ces changements. Le but du travail était de suivre la répartition des sucres et l'expression des transporteurs correspondants dans les racines de la plante modèle Arabidopsis thaliana, en réponse à un stress osmotique. Afin de pouvoir récolter des racines, un système de culture en hydroponie a été mis en place. Après avoir vérifié l'homogénéité des plantes cultivées dans ce système, une étude de l'expression des gènes de transporteurs de sucre a été effectuée au cours du développement des plantes et de d'une journée (24h), en utilisant la technique de macroarray. Cette étude a révélé l'expression dans les racines de 3 transporteurs de saccharose (AtSUC1, AtSUC2 et AtSUC5), 2 transporteurs de polyols (AtPLT5 et AtPLT6) et 2 transporteurs d'hexoses (AtSTP7 et AtSTP13). Le suivi de la teneur en sucres solubles et en amidon ainsi que du transport à longue distance de [U-14C]-saccharose a permis d'émettre des hypothèses quant au rôle des transporteurs de sucres exprimés dans la racine. Afin de mimer un stress hydrique en hydroponie, un protocole d'application progressif d'un agent osmotique (polyéthylène glycol 6000) dans le milieu de culture a été élaboré. Ce système a permis de mettre en évidence que 5 des gènes de transporteurs de sucre identifiés dans la racine ont une expression qui varie dans ces conditions. Trois d'entre eux sont fortement réprimés : AtSUC1 / In plants, sugar allocation between source and sink organs is based on the activity of membrane transporters for sugars. As sugar fluxes are changing during development and in response to stress, sugar transporters are supposed to be involved in those changes. The aim of this work was to study sugar allocation and the corresponding transporters gene expression in the roots of the model plant Arabidopsis thaliana during an osmotic stress. In order to have access to the roots, an hydroponic culture system was developped. The homogeneity of the plants obtained with this system was checked and the expression of sugar tranporter genes was followed during development and during a 24h period was studied by a macro-array technique. The expression in the roots of the following genes was found: 3 sucrose transporters (AtSUC1, AtSUC2 and AtSUC5), 2 polyol transporters (AtPLT5 and AtPLT6) and 2 hexose transporters (AtSTP7 and AtSTP13). The sugar and starch content and the long distance of 14C-sucrose were measured and used to build some hypothesis on the role of sugar transporters in the roots. To mimick a water stress, an osmotic stress due to the progressive addition of Polyethylene-glycol was applied. This system demonstrated that 5 of the identified transporter genes display a change in expression: 3 are repressed (AtSUC1, AtSUC5 and AtPLT6) and 2 are over expressed (AtSUC2 and AtSTP13). Moreover, soluble sugar and starch accumulate in the leaves and 14C-sucrose transport to the roots is decreased in plants subjected to an osmotic stress. The respective role of transporters is discussed. The gene expression data were also confirmed with plants grown in rhizoboxes.