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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
71

Uso da tomografia por emissão de pósitrons (PET) para identificação precoce de metástases e investigação da eficácia terapêutica da combinação p19Arf e Interferon-Beta em melanoma murino / Positron Emission Tomography (PET) as a tool for early detection of metastases and evaluation of the therapeutic efficacy of the combination p19Arf and Interferon Beta using metastatic mouse model of melanoma

Maria Renata Valente Brandão Freire 12 September 2017 (has links)
O melanoma maligno é um tipo de câncer com grande risco de produzir metástases e com altas taxas de mortalidade resultantes de diagnósticos tardios e falta de tratamentos eficazes. Ao longo dos últimos anos, a terapia gênica voltada para o câncer e o desenvolvimento de métodos capazes de visualizar processos moleculares e celulares ao longo da terapia, tem recebido especial atenção. Diante deste quadro, nossos objetivos foram utilizar o sistema de Tomografia por Emissão de Pósitrons (PET) para diagnosticar precocemente tumores e investigar a eficácia terapêutica de uma nova imunoterapia em um modelo animal de melanoma metastático. Visando atingir esses objetivos, padronizou-se a síntese e realizou-se o controle de qualidade do 9- [4-18F-fluoro-3-hidroximetil-butil) guanina, [18F] FHBG, considerado o padrão-ouro em estudos clínicos, para acompanhamento de terapia gênica por PET. Métodos: Sintetizou-se o [18F] FHBG, por substituição nucleofílica tipo 2 do precursor tosilato com [18F-] fluoreto de potássio /Kryptofix 2.2.2, seguido de desproteção com HCl 1 M e purificação por HPLC. A identidade química, pureza radioquímica e atividade específica do [18F] FHBG foram determinadas por Cromatografia Líquida de Alta Eficiência (CLAE). Introduziu-se o gene de timidina quinase (TK) com o vetor retroviral pCL-TK nas linhagens B16F10 (melanoma murino) e LLC (carcinoma de pulmão murino). Os estudos de captação in vitro dos radiotraçadores [18F] FHBG e [18F] FDG foram realizados nas linhagens celulares tumorais murinas transduzidas ou não com a proteína TK. Para os estudos in vivo, camundongos C57BL6 previamente inoculados intravenosamente com células de melanoma expressando a enzima TK, foram imageados subsequentemente utilizando os radiotraçadores [18F] FDG e [18F] FHBG. A eficácia da imunoterapia foi testada em modelo profilático e terapêutico animal de melanoma metastático. Resultados: O tempo de síntese total do [18F] FHBG variou entre 80-150 minutos. O rendimento radioquímico variou entre 1-4%, (n = 19) decaimento corrigido. A pureza radioquímica foi superior a 99% e a atividade específica variou entre 0,14GBq/μmoL-0,21GBq/μmoL. Com a introdução do gene timidina quinase (TK), obtiveram-se as linhagens repórter B16F10-TK e LLC-TK, para os estudos in vitro. As células B16F10 e LLC, expressando GFP foram utilizadas como linhagens controles. Estudos in vitro com o [18F] FHBG revelaram uma captação cerca de 4 vezes maior em células que expressam TK (B16-TK e LLC-TK) em comparação com as células controle GFP. O [18F] FDG apenas captou cerca de duas vezes mais em células TK do que em células que expressam GFP. A detecção de tumores em modelo animal de metástase pulmonar com o [18F] FDG ocorreu a partir de 15 dias do estabelecimento das lesões. No entanto, nos estudos in vivo com [18F] FHBG, houve captação apenas na região intestinal, durante as três semanas em que os animais foram acompanhados. A imunoterapia com células tratadas pela combinação de p19Arf e IFNβ, em camundongos C57BL6 com metástase pulmonar, conferiu redução do tamanho dos focos metastáticos aos animais tratados. Conclusões: Neste trabalho padronizou-se a síntese manual do [18F] FHBG, o qual foi avaliado em estudos in vitro e in vivo. Os estudos in vitro confirmaram a especificidade do [18F] FHBG no monitoramento da expressão de HSV1-tk em linhagens celulares. No entanto, o [18F] FHBG não se acumulou nas lesões metastáticas in vivo e estudos posteriores serão necessários para uma melhor caracterização utilizando o [18F] FHBG. O resultado do tratamento combinado de p19Arf e IFNβ foi promissor para o tratamento de lesões metastáticas. / Malignant melanoma is a type of cancer with a great risk of producing metastases and with high mortality rates resulting from late diagnosis and lack of effective treatments. Over the past few years, directed gene therapy for cancer and the development of methods to visualize molecular and cellular processes throughout therapy, have received special attention. In this context, our aim was to use the Positron Emission Tomography (PET) system, as a tool, for early detection of tumors and investigate the therapeutic efficacy of a new immunotherapy in an animal model of metastatic melanoma. To achieving these goals, the synthesis of [18F] FHBG, the gold standard in clinical studies for monitoring gene therapy by PET, was standardized and the quality control was performed. We also present the results of in vitro uptake and in vivo evaluation of [18F] FHBG, compared to [18F] FDG, the most commonly used radiopharmaceutical for diagnosis in oncology by PET. The vaccine was derived from transduced B16F10-TK cells with the adenoviral vectors AdRGDPGp19Arf and AdRGDPGIFNβ. Methods: [18F] FHBG was synthesized by type 2 nucleophilic radiofluorination of a tosylate precursor with [18F-] potassium fluoride / Kryptofix 2.2.2, followed by deprotection with 1N HCl and purification by HPLC. The chemical identity, radiochemical purity and specific activity of [18F] FHBG were determined by High Performance Liquid Chromatography (HPLC). The thymidine kinase (TK) gene was introduced with the pCL-TK retroviral vector into the B16F10 (murine melanoma) and LLC (murine lung carcinoma) lines. In vitro uptake studies of [18F] FHBG and [18F] FDG were performed on cell lines transduced or not with TK protein. For in vivo studies, C57BL6 mice, previously injected with HSV1tk expressing tumors, were subsequently imaged using the [18F] FDG and [18F] FHBG radiotracers. The efficacy of immunotherapy was tested in a prophylactic and therapeutic animal model of metastatic melanoma. Results: The total synthesis time of [18F] FHBG ranged from 80-150 min. The radiochemical yield ranged from 1-4%, (n = 19) corrected decay. Radiochemical purity was greater than 99% and the specific activity ranged from 0.14GBq / μmoL- 0.21GBq / μmoL. With the introduction of the thymidine kinase (TK) gene, the B16F10-TK and LLC-TK reporter lines were obtained for in vitro studies, B16F10 cells and LLC, expressing GFP, were used as controls. In vitro studies with [18F] FHBG revealed about 4-fold uptake in TK-expressing cells (B16-TK and LLC-TK) compared to GFP control cells. [18F] FDG binds only about twice as much in TK cells as in cells expressing GFP. The detection of tumors in an animal model of pulmonary metastasis with [18F] FDG occurred 15 days after lesion establishment. However, the in vivo studies with [18F] FHBG, the uptake was only found in the intestinal region, over the 3 weeks in which the mice were followed. Immunotherapy with cells treated by the combination of p19Arf and IFNβ, in C57BL6 mice with pulmonary metastasis, reduced the size of the metastatic foci in treated animals. Conclusions: In this study we demonstrate the standardization of [18F] FHBG synthesis and its use in in vitro and in vivo. The in vitro studies have confirmed the specificity of [18F] FHBG to monitor HSV1-tk expression in cell lines. However, [18F] FHBG did not accumulate in the metastatic lesions in vivo and further studies will be required for a better characterization using [18F] FHBG. The outcome of the combined treatment of p19Arf and IFNβ was promising for the treatment of metastatic lesions.
72

STUDY OF VIBRATIONAL PROPERTIES OF THYMIDINE CRYSTAL IN EXTREME CONDITIONS OF PRESSURE AND TEMPERATURE. / ESTUDO DAS PROPRIEDADES VIBRACIONAIS DO CRISTAL DE TIMIDINA EM CONDIÃÃES EXTREMAS DE PRESSÃO E TEMPERATURA.

Felipe Moreira Barboza 20 February 2017 (has links)
CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / The unit of sugar and base connected by a N-β-glycosyl linkage is named a nucleoside. In the present work the nucleoside thymidine, whose molecular formula is C10N2O5H14, was studied by Raman spectroscopy, subjecting it extreme conditions of pressure and temperature, as well as X ray diffraction measurements. An auxiliary analysis of normal crystal vibration modes was performed using first principles calculations using the B3LYP functional together with the Gaussian bases 6-31G+(d) and potential energy distribution analysis (PED). These results, together with literature data and Raman spectroscopy measurements in several thymidine scattering geometries, allowed the identification of the various normal modes of crystal vibration. X-ray diffraction experiments were performed in the temperature range between 83 and 413 K. Experiments of Raman spectroscopy under extreme temperature conditions (20 to 380 K) were performed in the spectral range of 20 to 3400 cm-1. From the analysis of the results, it is possible to draw some conclusions. (i) The thymidine crystal remained stable throughout the investigated temperature range, indicating that the temperature effect is not sufficient to modify the hydrogen bonds present between the molecules in such a way as to modify the symmetry of the crystal. (ii) The material studied showed some slight changes in the vibrational spectra in the experiment performed at low temperatures, suggesting, if not a structural phase transition, at least some conformational modification of the thymidine molecules. Raman spectra of thymidine crystal were obtained for pressures up to 5.0 GPa in a diamond anvil cell. The results show the presence of anomaly in the Raman spectrum at pressures close to 3.0 GPa. This anomaly is characterized by disappearance of lattice modes, appearance of some internal modes, splitting of high wavenumbers modes, downshift of modes associated with hydrogen bonds, changes in the intensity of internal modes and discontinuities of the slopes of the wavenumbers versus pressure for several Raman modes. This set of modifications was interpreted as consequence of a phase transition undergone by thymidine close to 3.0 GPa. Further, decompression to atmospheric pressure generates the original Raman spectrum, showing that the pressure-induced phase transition undergone by thymidine crystals is reversible. A comparison with results on other nucleosides submitted to high pressure is also furnished. / Quando a pentose (glicose) e uma base nitrogenada unem-se por meio de uma ligaÃÃo N-β glicosÃdica forma-se uma molÃcula denominada de nucleosÃdeo. No presente trabalho o nucleosÃdeo timidina, cuja fÃrmula molecular à C10N2O5H14, foi estudado atravÃs de espectroscopia Raman, submetendo-o a condiÃÃes extremas de pressÃo e de temperatura, alÃm de medidas de difraÃÃo de raios X. Uma anÃlise auxiliar a respeito dos modos normais de vibraÃÃo do cristal foi realizada atravÃs de cÃlculos de primeiros princÃpios utilizando-se o funcional B3LYP em conjunto com as bases gaussianas 6-31G+(d) e anÃlise de distribuiÃÃo de energia potencial (PED). Esses resultados, juntamente com dados da literatura e medidas de espectrocopia Raman em diversas geometrias de esplalhamento na timidina permitiram uma identificaÃÃo dos vÃrios modos normais de vibraÃÃo do cristal. Os experimentos por difraÃÃo de raios X foram realizados no intervalo de temperatura entre 83 e 413 K. Experimentos de espectroscopia Raman sob condiÃÃes extremas de temperatura (20 a 380 K) foram realizados no intervalo espectral compreendido entre 20 e 3400 cm-1. Da anÃlise dos resultados, à possÃvel tirar algumas conclusÃes. (i) O cristal de timidina manteve-se estÃvel em todo o intervalo de temperatura investigado, indicando que o efeito de temperatura nÃo à suficiente para modificar as ligaÃÃes de hidrogÃnio presentes entre as molÃculas de tal forma que haja modificaÃÃo da simetria do cristal. (ii) O material estudado apresentou algumas leves mudanÃas nos espectros vibracionais no experimento realizado a baixas temperaturas, sugerindo, se nÃo uma transiÃÃo de fase estrutural, pelo menos alguma modificaÃÃo conformacional das molÃculas da timidina. Experimentos submetendo o cristal a pressÃes de atà 5 GPa foram realizados utilizando-se uma cÃlula de pressÃo a extremos de diamantes. Os resultados mostraram anomalias nos espectros Raman por volta de 3.0 GPa. Essas anomalias foram caracterizadas pelo desaparecimento de alguns modos de rede, surgimento de alguns modos internos, deslocamento para menores nÃmeros de onda de modos associados a ligaÃÃes de hidrogÃnio e descontinuidades dos coeficientes lineares de vÃrios modos nos grÃficos de nÃmero de onda em funÃÃo da pressÃo. Essa sÃrie de modificaÃÃes foram interpretadas como consequÃncia de uma transiÃÃo de fase sofrida pela timidina por volta de 3.0 GPa. AlÃm disso, a descompressÃo da amostra atà a pressÃo atmosfÃrica mostrou que a transiÃÃo de fase à reversÃvel. TambÃm fornecemos uma comparaÃÃo com resultados de outros nucleosÃdeos submetidos a altas pressÃes.
73

Targeting the nucleotide metabolism of the mammalian pathogen Trypanosoma brucei

Vodnala, Munender January 2013 (has links)
Trypanosoma brucei causes African sleeping sickness in humans and Nagana in cattle. There are no vaccines available against the disease and the current treatment is also not satisfactory because of inefficacy and numerous side effects of the used drugs. T. brucei lacks de novo synthesis of purine nucleosides; hence it depends on the host to make its purine nucleotides. T. brucei has a high affinity adenosine kinase (TbAK), which phosphorylates adenosine, deoxyadenosine (dAdo), inosine and their analogs. RNAi experiments confirmed that TbAK is responsible for the salvage of dAdo and the toxicity of its substrate analogs. Cell growth assays with the dAdo analogs, Ara-A and F-Ara-A, suggested that TbAK could be exploited for drug development against the disease. It has previously been shown that when T. brucei cells were cultivated in the presence of 1 mM deoxyadenosine (dAdo), they showed accumulation of dATP and depletion of ATP nucleotides. The altered nucleotide levels were toxic to the trypanosomes. However the salvage of dAdo in trypanosomes was dramatically reduced below 0.5 mM dAdo. Radiolabeled dAdo experiments showed that it (especially at low concentrations) is cleaved to adenine and converted to ATP. The recombinant methylthioadenosine phosphorylase (TbMTAP) cleaved methylthioadenosine, dAdo and adenosine into adenine and sugar-1-P in a phosphate-dependent manner. The trypanosomes became more sensitive to dAdo when TbMTAP was down-regulated in RNAi experiments. The RNAi experiments confirmed that trypanosomes avoid dATP accumulation by cleaving dAdo. The TbMTAP cleavage-resistant nucleoside analogs, FANA-A and Ara-A, successfully cured T. brucei-infected mice. The DNA building block dTTP can be synthesized either via thymidylate synthase in the de novo pathway or via thymidine kinase (TK) by salvage synthesis. We found that T. brucei and three other parasites contain a tandem TK where the gene sequence was repeated twice or four times in a single open reading frame. The recombinant T. brucei TK, which belongs to the TK1 family, showed broad substrate specificity. The enzyme phosphorylated the pyrimidine nucleosides thymidine and deoxyuridine, as well as the purine nucleosides deoxyinosine and deoxyguanosine. When the repeated sequences of the tandem TbTK were expressed individually as domains, only domain 2 was active. However, the protein could not dimerize and had a 5-fold reduced affinity to its pyrimidine substrates but a similar turnover number as the full-length enzyme. The expressed domain 1 was inactive and sequence analysis revealed that some active residues, which are needed for substrate binding and catalysis, are absent. Generally, the TK1 family enzymes form dimers or tetramers and the quaternary structure is linked to the affinity for the substrates. The covalently linked inactive domain-1 helps domain-2 to form a pseudodimer for the efficient binding of substrates. In addition, we discovered a repetition of an 89-bp sequence in both domain 1 and domain 2, which suggests a genetic exchange between the two domains. T. brucei is very dependent on de novo synthesis via ribonucleotide reductase (RNR) for the production of dNTPs. Even though T. brucei RNR belongs to the class Ia RNR family and contains an ATP-binding cone, it lacks inhibition by dATP. The mechanism behind the RNR activation by ATP and inactivation by dATP was a puzzle for a long time in the ~50 years of RNR research. We carried out oligomerization studies on mouse and E. coli RNRs, which belongs to the same family as T. brucei, to get an understanding of the molecular mechanism behind overall activity regulation. We found that the oligomerization status of RNRs and overall activity mechanism are interlinked with each other. / Targeting the nucleotide metabolism of the mammalian pathogen Trypanosoma brucei.
74

Studies on the antiproliferative action of interferon : effects on proteins synthesized in the G1 and S phase of the cell cycle in 2 anchorage-dependent cell lines

Lundblad, Dan January 1991 (has links)
Interferons (IFNs) are a class of structurally related proteins first discovered to be produced by virus-infected cells. By now, several other inducing agents have been described. IFNs exert multiple effects on cells exemplified by the establishment of an antiviral state, inhibition of cell proliferation and alteration of different immune reactions. In the present thesis the inhibition of cellular growth concentrated on effects in the early cell cycle have been studied. The human glioma cell line 251 MG was found to be blocked in the S phase of the cell cycle upon addition of IFN both to exponentially growing and growth-factor depleted, synchronized cells. Thymidine kinase and DNA-polymerase activities were reduced in parallel with the S phase effect. 2-5 oligo Anucleotides transfected into glioma cells lead to inhibition of cell growth, exponentially growing cells being blocked in the S phase as during IFN treatment. In contrast, synchronized, restimulated cells were blocked in the cellcycle phase where they resided at the time of transfection. As 2-5 oligo A synthetase activity was induced in the middle of the Gl phase, these results might indicate that the kinetics of expression of oligonucleotides after IFN additiondetermines the type of cell cycle block obtained in differenttumor cells. IFN inhibited preferentially proteins originating from newly synthesized mRNA in Sw 3T3 cells, c-mvc did not seem to be included among these proteins. In both cell systems c-myc expression was unaltered after IFN treatment. In clone T1 selected from the the Sw 3T3 cell line , c-mvc expression was uncoupled to growth and seemed to be growth factor independent. The change in c-myc expression in clone T1 compared to SW 3T3 cells did not render the cells sensitive to IFN. Hence, c-myc regulation does not seem to be the mechanism by which IFN regulates cell growth in this system. The proliferation marker KI-67 antigen was shown not to be causatively involved in growth inhibition of IFN. The reduced levels of the antigen was proposed to be a secondary effect caused by the G0/G1 arrest. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1991, härtill 6 uppsatser</p> / digitalisering@umu
75

Estudo das propriedades vibracionais do cristal de timidina em condições extremas de pressão e temperatura / Study of vibrational properties of thymidine crystal in extreme conditions of pressure and temperature

Barboza, Felipe Moreira January 2017 (has links)
BARBOZA, F. M. Estudo das propriedades vibracionais do cristal de timidina em condições extremas de pressão e temperatura. 2017. 187 f. Tese (Doutorado em Física) – Centro de Ciências, Universidade Federal do Ceará, Fortaleza, 2017. / Submitted by Giordana Silva (giordana.nascimento@gmail.com) on 2017-04-17T18:08:44Z No. of bitstreams: 1 2017_tese_fmbarboza.pdf: 14915098 bytes, checksum: 92c70871f92588cea1807a2552402d46 (MD5) / Approved for entry into archive by Giordana Silva (giordana.nascimento@gmail.com) on 2017-04-17T18:09:44Z (GMT) No. of bitstreams: 1 2017_tese_fmbarboza.pdf: 14915098 bytes, checksum: 92c70871f92588cea1807a2552402d46 (MD5) / Made available in DSpace on 2017-04-17T18:09:44Z (GMT). No. of bitstreams: 1 2017_tese_fmbarboza.pdf: 14915098 bytes, checksum: 92c70871f92588cea1807a2552402d46 (MD5) Previous issue date: 2017 / The unit of sugar and base connected by a N-β-glycosyl linkage is named a nucleoside. In the present work the nucleoside thymidine, whose molecular formula is C10N2O5H14, was studied by Raman spectroscopy, subjecting it extreme conditions of pressure and temperature, as well as X ray diffraction measurements. An auxiliary analysis of normal crystal vibration modes was performed using first principles calculations using the B3LYP functional together with the Gaussian bases 6-31G+(d) and potential energy distribution analysis (PED). These results, together with literature data and Raman spectroscopy measurements in several thymidine scattering geometries, allowed the identification of the various normal modes of crystal vibration. X-ray diffraction experiments were performed in the temperature range between 83 and 413 K. Experiments of Raman spectroscopy under extreme temperature conditions (20 to 380 K) were performed in the spectral range of 20 to 3400 cm-1. From the analysis of the results, it is possible to draw some conclusions. (i) The thymidine crystal remained stable throughout the investigated temperature range, indicating that the temperature effect is not sufficient to modify the hydrogen bonds present between the molecules in such a way as to modify the symmetry of the crystal. (ii) The material studied showed some slight changes in the vibrational spectra in the experiment performed at low temperatures, suggesting, if not a structural phase transition, at least some conformational modification of the thymidine molecules. Raman spectra of thymidine crystal were obtained for pressures up to 5.0 GPa in a diamond anvil cell. The results show the presence of anomaly in the Raman spectrum at pressures close to 3.0 GPa. This anomaly is characterized by disappearance of lattice modes, appearance of some internal modes, splitting of high wavenumbers modes, downshift of modes associated with hydrogen bonds, changes in the intensity of internal modes and discontinuities of the slopes of the wavenumbers versus pressure for several Raman modes. This set of modifications was interpreted as consequence of a phase transition undergone by thymidine close to 3.0 GPa. Further, decompression to atmospheric pressure generates the original Raman spectrum, showing that the pressure-induced phase transition undergone by thymidine crystals is reversible. A comparison with results on other nucleosides submitted to high pressure is also furnished. / Quando a pentose (glicose) e uma base nitrogenada unem-se por meio de uma ligação N-β glicosídica forma-se uma molécula denominada de nucleosídeo. No presente trabalho o nucleosídeo timidina, cuja fórmula molecular é C10N2O5H14, foi estudado através de espectroscopia Raman, submetendo-o a condições extremas de pressão e de temperatura, além de medidas de difração de raios X. Uma análise auxiliar a respeito dos modos normais de vibração do cristal foi realizada através de cálculos de primeiros princípios utilizando-se o funcional B3LYP em conjunto com as bases gaussianas 6-31G+(d) e análise de distribuição de energia potencial (PED). Esses resultados, juntamente com dados da literatura e medidas de espectrocopia Raman em diversas geometrias de esplalhamento na timidina permitiram uma identificação dos vários modos normais de vibração do cristal. Os experimentos por difração de raios X foram realizados no intervalo de temperatura entre 83 e 413 K. Experimentos de espectroscopia Raman sob condições extremas de temperatura (20 a 380 K) foram realizados no intervalo espectral compreendido entre 20 e 3400 cm-1. Da análise dos resultados, é possível tirar algumas conclusões. (i) O cristal de timidina manteve-se estável em todo o intervalo de temperatura investigado, indicando que o efeito de temperatura não é suficiente para modificar as ligações de hidrogênio presentes entre as moléculas de tal forma que haja modificação da simetria do cristal. (ii) O material estudado apresentou algumas leves mudanças nos espectros vibracionais no experimento realizado a baixas temperaturas, sugerindo, se não uma transição de fase estrutural, pelo menos alguma modificação conformacional das moléculas da timidina. Experimentos submetendo o cristal a pressões de até 5 GPa foram realizados utilizando-se uma célula de pressão a extremos de diamantes. Os resultados mostraram anomalias nos espectros Raman por volta de 3.0 GPa. Essas anomalias foram caracterizadas pelo desaparecimento de alguns modos de rede, surgimento de alguns modos internos, deslocamento para menores números de onda de modos associados a ligações de hidrogênio e descontinuidades dos coeficientes lineares de vários modos nos gráficos de número de onda em função da pressão. Essa série de modificações foram interpretadas como consequência de uma transição de fase sofrida pela timidina por volta de 3.0 GPa. Além disso, a descompressão da amostra até a pressão atmosférica mostrou que a transição de fase é reversível. Também fornecemos uma comparação com resultados de outros nucleosídeos submetidos a altas pressões.
76

Targets and strategies for drug development against human African sleeping sickness

Ranjbarian, Farahnaz January 2017 (has links)
Trypanosoma brucei is a causative agent of African sleeping sickness. It is an extracellular parasite which circulates in the blood, lymph and eventually invades the central nervous system. There is a great need for new medicines against the disease and specific properties of nucleoside kinases in the pathogen can be exploited as targets for chemotherapy.  T. brucei contains a gene where two thymidine kinase sequences are fused into a single open reading frame. These types of tandem thymidine kinases were found only in different types of parasites, which made us to believe that it might be beneficial for them. Each thymidine kinase sequence in these tandem enzymes are here referred to as a domain. By cloning and expressing each domain from T. brucei separately, we found that domain 1 was inactive and domain 2 was as active as the full-length enzyme. T. brucei thymidine kinase phosphorylated the pyrimidine nucleosides thymidine and deoxyuridine and to some extent purine nucleosides like deoxyinosine and deoxyguanosine. Human thymidine kinase increases the affinity to its substrates when it forms oligomers. Similarly, the T. brucei two thymidine kinase sequences, which can be viewed as a pseudodimer, had a higher affinity to its substrates than domain 2 alone.  T. brucei lacks de novo purine biosynthesis and it is therefore dependent on salvaging the required purine nucleotides for RNA and DNA synthesis from the host. Purine salvage is considered as a target for drug development. It has been shown that in the presence of deoxyadenosine in the growth medium, the parasites accumulate high levels of dATP and the extensive phosphorylation of deoxyadenosine leads to depleted ATP pools. Initially, we wondered if deoxyadenosine could be used as a drug against T. brucei. However, we found that T. brucei is partially protected against deoxyadenosine because it was cleaved by the enzyme methylthioadenosine phosphorylase (MTAP) to adenine and ribose-1-phosphate. At higher concentration of deoxyadenosine, 3 the formed adenine was not efficiently salvaged into ATP and started to inhibit MTAP instead. The deoxyadenosine was then instead phosphorylated by adenosine kinase leading to accumulation of dATP. The MTAP reaction makes deoxyadenosine itself useless as a drug and instead we focused on finding analogues of deoxyadenosine or adenosine that were cleavage-resistant and at the same time good substrates of T. brucei adenosine kinase. Our best hit was then 9-(2-deoxy-2-fluoro-ß-D-arabinofuranosyl) adenine (FANA-A). An additional advantage of FANA-A as a drug was that it was taken up by the P1 nucleoside transporter family, which makes it useful also against multidrug resistant parasites that often have lost the P2 transporter function and take up their purines solely by the P1 transporter. In parallel with our study of nucleoside metabolism in T. brucei, we also have a collaboration project where we screen essential oils from plants which are used in traditional medicine. If the essential oils are active against the trypanosomes, we further analyze the different components in the oils to identify new drugs against African sleeping sickness. One such compound identified from the plant Smyrnium olusatrum is isofuranodiene, which inhibited T. brucei proliferation with an IC50 value of 3 μM.
77

Vectorisation cérébrale de deux radiotraceurs d’intérêts pour l’imagerie, la m-iodobenzylguanidine (MIBG) et la 3’-désoxy-3’- fluoro-L-thymidine (FLT) / Targeting two radiotracers into the brain for brain imaging, m-iodobenzylguanidine (MIBG) and 3’-désoxy-3’-fluoro-L-thymidine (FLT)

Henry, Axelle 25 November 2016 (has links)
Ce travail de recherche s'intéresse à la vectorisation au système nerveux central de la MIBG d'une part et de la FLT d'autre part. Pour cela, nous utiliserons des systèmes disposant d'une structure 1,4- dihydoquinoléine comme vecteurs. La synthèse des systèmes de vectorisation a tout d'abord été réalisée en chimie non radioactive. Concernant la MIBG, les résultats obtenus lors de précédents travaux nous ont conduit à envisager l'élaboration d'un système de vectorisation comportant un groupement espaceur entre le vecteur et la MIBG. Dans le cas de la FLT, nous nous sommes consacrés au développement d'un système dans lequel la FLT serait directement liée au vecteur par une liaison ester. Afin de pouvoir moduler les propriétés rédox de nos systèmes de vectorisation, nous avons synthétisé des 1,4-dihydroquinoléines présentant en position 6 et/ou 7 des groupements électroattracteurs ou électrodonneurs. Une étude en milieu acétonitrile/tampon PBS, pour évaluer l'influence de ces groupements sur la libération de la FLT, a été réalisée par un suivi CLHP. La radiosynthèse de ces systèmes a ensuite été menée afin d'évaluer la capacité des vecteurs 1,4- dihydroquinoléines à transporter la MIBG ou la FLT à travers la barrière hématoencéphalique (BHE). Ainsi, nous avons radiomarqué au carbone-11 les systèmes de vectorisation pour valider le passage de la BHE. Des études ex vivo chez le petit animal ont permis de suivre la pénétration cérébrale ainsi que la cinétique d'oxydation au niveau cérébral et en périphérie. / This research work focuses on targeting MIBG or FLT to the central nervous system. The synthesis of 1,4-dihydroquinoline carriers was first carried out in a non-radioactive manner. Previous results led us to consider the use of a linker to connect MIBG to the carrier. Regarding FLT, we focused our interest in the development of a carrier system connected directly to FLT via an ester function. For modulating the redox properties of our delivery systems, we synthesized 1,4-dihydroquinolines having electron-donating or electron-withdrawing groups at position 6 and/or 7. A study in acetonitrile/PBS buffer to determine the influence of these groups on the release of FLT was performed by HPLC. The radiosynthesis of these targeting systems was then conducted to evaluate the ability of 1,4- dihydroquinolines to deliver MIBG or FLT across the blood brain barrier (BBB). Thus, using carbon-11, we radiolabeled the delivery systems to validate the BBB crossing. Small animais were used for ex vivo studies to monitor the brain penetration and the kinetics of oxidation in the brain and periphery.
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Regulation of T Cell Activation by the CD5 Co-Receptor and Altered Peptides, Characterization of Thymidine Kinase-Specific Antibodies, and Integrating Genomics Education in Society

Whitley, Kiara Vaden 10 August 2022 (has links)
Helper T cells (Th) are a vital component of the immune system responsible for directing other immune cells to eliminate pathogens and cancer. Specifically, Th cells facilitate B cell and cytotoxic T cell (Tc) activation and recruitment and enhance their function against cancer and infectious diseases. Th cells are a valuable resource for improving Tc responses in cancer treatment and have become a focus of immunotherapeutic research. While it is increasingly clear that helper T cells serve an important role, the details about which entities produce an effective Th cell response remain unclear. CD5 is a T cell co-receptor that negatively regulates T cell activation and helps fine-tune the TCR repertoire by altering TCR signaling during the selection process in the thymus. This work discusses the role of the co-receptor CD5 in influencing Th cell metabolism, as well as the study of two T cells called LLO118 and LLO56 that have different CD5 expression levels, and their functional response to altered peptides. Antibodies have revolutionized the world of cancer research and accelerated the development of therapies that trigger the immune system to target disease. In recent years, many antibody-based immunotherapies have emerged as effective candidates for combating cancer due to their refined specificity and ability to target a variety of epitopes. However, many therapies, such as those that target CD19 on B cell cancers, are also present on healthy cells, destroying both cancerous and healthy cells alike. Thymidine kinase 1 (TK1) is an enzyme involved in the DNA salvage pathway that converts thymidine into the nucleotide thymine. Recently, TK1 has been shown to be overexpressed on the surface of many cancers such as acute lymphoblastic leukemia. Importantly, TK1 is not expressed on the surface of healthy cells, making it an ideal cancer-specific antigen that can be targeted for cancer treatment. This work discusses our efforts to characterize TK1-specific single-chain antibodies from a yeast display library. According to the World Health Organization, genomics is defined as the study of all genes and their related functions. In contrast to genetics, genomics analyzes the entire DNA makeup of an organism rather than a single gene. In the past 20 years, the cost of genomic sequencing has decreased dramatically, making it affordable and accessible. A key area that must be addressed with genomic testing involves education about their promise, challenges, potential consequences, and ethical considerations. Genomic testing provides a powerful opportunity to educate everyone on scientific and ethical issues to increase understanding on the subject. This work discusses the influence of personal genomics in society and focuses on the importance, benefits, and consequences of genomics education in the classroom, clinic, and the public.
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Flavins and Their Analogues as Natural and Artificial Catalysts

Sichula, Vincent A. 02 March 2011 (has links)
No description available.
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MECHANISTIC STUDIES ON THE PHOTOTOXICITY OF ROSUVASTATIN, ITRACONAZOLE AND IMATINIB

Nardi, Giacomo 31 March 2015 (has links)
Photosensitizing effects of xenobiotics are of increasing concern in public health since modern lifestyle often associates sunlight exposure with the presence of chemical substances in the skin. An important number of chemicals like perfumes, sunscreen components, or therapeutic agents have been reported as photosensitizers. In this context, a considerable effort has been made to design a model system for photosafety assessment. Indeed, screening for phototoxicity is necessary at the early phase of drug discovery process, even before introducing drugs and chemicals into clinical therapy, to prevent undesired photoreactions in humans. In the case of new pharmaceuticals, their phototoxic potential has to be tested when they absorb in the regions corresponding to the solar spectrum, that is, for wavelengths >290 nm. So, there is an obvious need for a screening strategy based on in vitro experiments. The goal of the present thesis was the photochemical study of different photoactive drugs to investigate the key molecular aspects responsible for their photosensitivity side effects. In a first stage, rosuvastatin was considered in chapter 3 as representative compound of the statin family. This lipid-lowering drug, also known as “superstatin”, contains a 2-vinylbiphenyl-like moiety and has been previously described to decompose under solar irradiation, yielding stable dihydrophenanthrene analogues. During photophysical characterization of rosuvastatin, only a long-lived transient at ca. 550 nm was observed and assigned to the primary photocyclization intermediate. Thus, the absence of detectable triplet-triplet absorption and the low yield of fluorescence ruled out the role of the parent drug as an efficient sensitizer. In this context, the attention was placed on the rosuvastatin main photoproduct (ppRSV). Indeed, the photobehavior of this dihydrophenanthrene-like compound presented the essential components needed for an efficient biomolecule photosensitizer i.e. (i) a high intersystem crossing quantum yield (ΦISC =0.8), (ii) a triplet excited state energy of ca. 67 kcal mol−1 , and (iii) a quantum yield of singlet oxygen formation (Φ∆) of 0.3. Furthermore, laser flash photolysis studies revealed a triplet-triplet energy transfer from the triplet excited state of ppRSV to thymidine, leading to the formation of cyclobutane thymidine dimers, an important type of DNA lesion. Finally, tryptophan was used as a probe to investigate the Type I and/or Type II character of ppRSV-mediated oxidation. In this way, both an electron transfer process giving rise to the tryptophanyl radical and a singlet oxygen mediated oxidation were observed. On the basis of the obtained results, rosuvastatin, through its major photoproduct ppRSV, should be considered as a potential sensitizer. Then, itraconazole (ITZ), a broad-spectrum antifungal agent, was chosen as main character of chapter 4. Its photochemical properties were investigated in connection with its reported skin photosensitivity disorders. Steady state photolysis, fluorescence and phosphorescence experiments were performed to understand ITZ photoreactivity in biological media. The drug is unstable under UVB irradiation, suffering a primary dehalogenation of the 2,4-dichlorophenyl moiety that occurs mainly at the ortho-position. In poorly H-donating solvents, as acetonitrile, the major photoproduct arises from intramolecular attack of the initially generated aryl radical to the triazole ring. In addition, reduced compounds resulting from homolytic cleavage of the C-Cl bond in ortho or para positions and subsequent Habstraction from the medium are obtained to a lesser extent. In good H-donating solvents, such as ethanol, the main photoproducts are formed by reductive dehalogenation. Furthermore, irradiation of a model dyad containing a tryptophan unit and the reactive 2,4-dichlorophenyl moiety of itraconazole leads to formation of a new covalent link between these two substructures revealing that homolysis of the C-Cl bond of ITZ can result in alkylation of reactive amino acid residues of proteins, leading to formation of covalent photoadducts. Therefore, it has been established that the key process in the photosensitization by itraconazole is cleavage of the carbon-halogen bond, which leads to aryl radicals and chlorine atoms. These highly reactive species might be responsible for extensive free radical-mediated biological damage, including lipid peroxidation or photobinding to proteins. In chapter 5, photobehavior of imatinib (IMT) was addressed. This is a promising tyrosine kinase inhibitor used in the treatment of some types of human cancer, which constitutes a successful example of rational drug design based on the optimization of the chemical structure to reach an improved pharmacological activity. Cutaneous reactions, such as increased photosensitivity or pseudoporphyria, are among the most common nonhematological IMT side effects; however, the molecular bases of these clinical observations have not been unveiled yet. Thus, to gain insight into the IMT photosensitizing properties, its photobehavior was studied together with that of its potentially photoactive anilino-pyrimidine and pyridyl-pyrimidine fragments. In this context, steady-state and time resolved fluorescence, as well as laser flash photolysis experiments were run, and the DNA photosensitization potential was investigated by means of single strand breaks detection using agarose gel electrophoresis. The obtained results revealed that the drug itself and its anilino-pyrimidine fragment are not DNA-photosensitizers. By contrast, the pyridyl-pyrimidine substructure displayed a marked photogenotoxic potential, which was associated with the generation of a long-lived triplet excited state. Interestingly, this reactive species was efficiently quenched by benzanilide, another molecular fragment of IMT. Clearly, integration of the photoactive pyridyl-pyrimidine moiety in a more complex structure strongly modifies its photobehavior, which in this case is fortunate as it leads to an improved toxicological profile. Thus, on the bases of the experimental results, direct in vivo photosensitization by IMT seems unlikely. Instead, the reported photosensitivity disorders could be related to indirect processes, such as the previously suggested impairment of melanogenesis or the accumulation of endogenous porphyrins. Finally, a possible source of errors in the TEMPO/EPR method for singlet oxygen detection was analyzed. For many biological and biomedical studies, it is essential to detect the production of 1O2 and to quantify its production yield. Among the available methods, detection of the characteristic 1270 nm phosphorescence of singlet oxygen by time-resolved near infrared (TRNIR) emission constitutes the most direct and unambiguous approach. An alternative indirect method is electron paramagnetic resonance (EPR) in combination with trapping. This is based on the detection of the TEMPO free radical formed after oxidation of TEMP (2,2,6,6- tetramethylpiperidine) by singlet oxygen. Although the TEMPO/EPR method has been largely employed, it can produce misleading data. This was demonstrated by the present study, where the quantum yields of singlet oxygen formation obtained by TRNIR emission and by the TEMPO/EPR method were compared for a set of well-known photosensitizers. The results revealed that the TEMPO/EPR method leads to significant overestimation of singlet oxygen yield when the singlet or triplet excited state of the photosensitizers were efficiently quenched by TEMP, acting as electron donor. In such case, generation of the TEMP+• radical cation, followed by deprotonation and reaction with molecular oxygen gives rise to a EPR detectable TEMPO signal that is not associated with singlet oxygen production. This knowledge is essential for an appropriate and error-free application of the TEMPO/EPR method in chemical, biological and medical studies. / Nardi, G. (2014). MECHANISTIC STUDIES ON THE PHOTOTOXICITY OF ROSUVASTATIN, ITRACONAZOLE AND IMATINIB [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48535 / TESIS

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