A identificação de genes relacionados com resistência a compostos antiparasitários tem contribuído para um melhor entendimento do mecanismo de ação de compostos antileishmania. Pouco se sabe sobre o mecanismo de ação do análogo de purina tubercidina (TUB) em Leishmania. Utilizando a estratégia de superexpressão após transfecção gênica, isolamos um locus de Leishmania (Leishmania) major, de 31 kb, capaz de conferir níveis de resistência quatro vezes maior que o parasita selvagem. Várias deleções desse locus foram geradas e a construção de 3 kb (pSNBR/3kbClaI-EcoRI) também conferiu níveis de resistência quando comparado ao parasita elvagem. Através de análises no genoma de L. (L.) major, localizamos esse locus no cromossomo 31 e, no fragmento de 3 kb, um gene que codifica para uma proteína com função desconhecida até o momento (LmjF.31.2010). Esta proteína foi relacionada com resistência a TUB em todas as linhagens transfectadas analisadas (cosTUB2 e pSNBR/3kbClaI-EcoRI), assim denominamos LmjF.31.2010, de proteína relacionada com resistência a TUB (PRRT). A quantificação relativa de transcritos de mRNA na construção pSNBR/3kbClaI-EcoRI apresentou níveis altos de transcritos da PRRT. Foram gerados ainda mutantes de L. (L.) major e L. (L.) amazonensis resistentes a TUB e estes se apresentaram bem adaptados a concentrações altas de TUB, apresentando razão de resistência maior que 200 vezes, quando comparado com os respectivos parasitas selvagens. A PRRT também foi relacionada na resistência a TUB nos mutantes gerados, pois houve amplificação gênica de prrt. Os resultados obtidos neste trabalho fornecem dados para inferir a importância da PRRT no mecanismo relacionado com resistência a TUB. / The identification of genes associated with resistance to antiparasitic compounds has contributed to a better understanding of the mechanism of action of compounds against Leishmania. Little is known about the mechanism of action of purine analog tubercidin (TUB) in Leishmania. Using a strategy of gene overexpression after transfection, we isolated a locus of Leishmania (Leishmania) major, 31 kb, capable of conferring fold resistance four times greater than the wild type parasite. A set of deletions of this locus were generated and a 3 kb construction (pSNBR/3kbClaI-EcoRI) conferred fold resistance twice than the wild type. Analysis of L. (L.) major genome, located this locus on chromosome 31 and on 3 kb fragment we identified a gene encoding a protein with unknown function (LmjF.31.2010). This protein has been related to TUB resistance in all strains analyzed (cosTUB2 and pSNBR/3kbClaI-EcoRI), so we named mjF.31.2010 of protein related with resistance to TUB (PRRT). Relative quantification of mRNA transcripts in the construction pSNBR/3kbClaI-EcoRI showed high levels of PRRT transcripts.Mutants of L. (L.) major and L. (L.) amazonensis resistant to TUB were also generated and these were well adapted to high TUB concentrations, presenting fol resistance greater than 200 times when compared with their respective wild type. The PRRT was also related to TUB resistance mutants generated by PRRT gene amplification. Despite the high fold resistance presented by TUB resistant mutants, the ratio of expression of these mutant PRRT transfected and wild was similar to the wild type.

A identificação de genes relacionados com resistência a compostos antiparasitários tem contribuído para um melhor entendimento do mecanismo de ação de alguns desses compostos. Utilizando a estratégia que permite a indução de super-expressão após transfecção gênica, isolamos dois loci relacionados com resistência ao análogo tóxico de purina, tubercidina (TUB). Em um desses locus identificamos um ortólogo do gene TOR (TOxic nucleoside Resistance) em L. (L.) major (TOR-Lm), capaz de conferir altos níveis de resistência a TUB. A identificação e localização cromossomal do segundo locus foi obtida, mas os testes funcionais em presença de TUB não foram tão significativos quanto os obtidos após a transfecção do TOR-Lm. Na segunda parte desta dissertação avaliamos a eficácia da associação de TUB com um inibidor específico do transporte de nucleosídeos em mamíferos, nitrobenziltioinosina (NBMPR), visando reverter a toxicidade de TUB apenas no hospedeiro. Demonstramos que TUB tem uma potente ação anti-parasitária em culturas de Leishmania spp., e que o inibidor NBMPR é capaz de proteger células mamíferas de camundongos infectados da ação tóxica de TUB. / Gene identification associated with drug resistance has contributed to a better understanding of the mechanism of action of anti parasitic compounds. Using transfection and over-expression selection strategy we isolated two loci related with the resistance of tubercidin (TUB), a toxic analog purine. In the first locus we identified an ortholog of the TOR gene (TOxic nucleoside Resistance) in L. (L.) major (TOR-Lm), capable to render wild cells resistance to TUB after transfection and over-expression. Chromosomal location and identification of the second locus was done, but functional tests in the presence of TUB were not as significant as those obtained after TOR-Lm transfection. In the second part of this work, we evaluate the effectiveness of the association of TUB with an inhibitor specific to the mammals nucleoside transport, as nitrobenzylthioinosine (NBMPR), aimed at reversing the TUB toxicity only on the host. We first demonstrate that TUB has a potent anti-parasitic action in cultures of Leishmania spp. Then, we discuss the capacity of the NBMPR inhibitor to protect infected macrophages from the toxic effects of TUB.

A leishmaniose é causada por protozoários do gênero Leishmania e no Brasil, as principais espécies causadoras da leishmaniose cutânea são Leishmania (V.) braziliensis e Leishmania (L.) amazonenses. O tratamento da leishmaniose apresenta diversas dificuldades, portanto é fundamental a descoberta de novos fármacos ativos, podendo ser detectada em células cultivadas in vitro e também em animais íntegros, através da técnica de bioimageamento. Neste trabalho, propusemo-nos a produzir linhagens de L. (V.) braziliensis e L. (L) amazonenses expressoras de luciferase e caracterizar o comportamento das linhagens mutantes em testes de sensibilidade a fármacos e de infecção in vitro e in vivo. Foi confirmada a emissão de luz pelas linhagens mutantes das duas espécies de Leishmania, em promastigotas e amastigotas. O comportamento das linhagens mutantes obtidas em relação a curvas de crescimento, sensibilidade aos fármacos tamoxifeno e anfoterina B em promastigotas, perfil de infectividade e sobrevivência em macrófagos e sensibilidade de amastigotas à anfotericina B foi comparado ao comportamento das linhagens parentais, não sendo observadas diferenças significativas. Camundongos BALB/c infectados com a linhagem expressora de luciferase de L. (L.) amazonenses desenvolveram lesões comparáveis aos animais infectados com a cepa selvagem, sendo possível quantificar a carga parasitária nesses animais por bioimageamento. Os resultados obtidos neste trabalho indicam que os parasitas mutantes expressores de luciferase obtidos podem ser utilizados em testes de sensibilidade a fármacos tanto in vitro como in vivo, representando um avanço metodológico nessa área de pesquisa. / Leishmaniasis is caused by protozoan parasites in Brazil, the main causative species of cutaneous leishmaniasis are Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonenses. The treatment of leishmaniasis presents several difficulties, and the discovery of new active drugs for the treatment of leishmaniasis is therefore fundamental. The enzyme luciferase is a reporter widely used in screening tests for new drugs. This enzyme catalyzes the oxidation of luciferase in the presence of ATP emitting light that can be detected in cultured cells in vitro as well as in intact animals, using the technique of bioimaging. In this work, we sought to produce strains of L. (V.) braziliensis and L. (L) amazonenses expressing luciferase and characterize the behavior of these mutant strains in drug susceptibility tests and in in vitro and in vivo infections. Production of light was detected in mutants of both species, in all life cycle stages. Mutant strains were compared to their corresponding parental lines as to their growth pattern, infectivity and survival profile in macrophages and sensitivity to amphotericin B and tamoxifen. No significant differences were observed for these parameters. BALB/c mice infected with the luciferase expressing line of L. (L.) amazonenses developed lesions comparable to those in animals infected with the wild-type strain. The parasite load in these animals was quantified through bioimaging. The results obtained of this study indicate that the mutant parasites expressing luciferase can be used for drug susceptibility testing in vitro and in vivo, representing a methodological advance in this area of research.

Respiratory Syncytial Virus (RSV) is a potentially life-threatening respiratory pathogen that infects approximately 64 million children and immunocompromised adults globally per year. Currently, there is a need for prophylactic and therapeutic approaches effective against primary and secondary RSV infections. This project focuses on the development of a simple, smart, and scalable anti-RSV nanotherapeutic that combines novel cellular antiviral defense mechanisms targeting the inhibition of viral fusion and replication. An ICAM-1 targeted liposomal nanocarrier will be synthesized and coated with a layer of chitosan containing the anti-fusion HR2-D peptide as an extracellular defense mechanism. Additionally, chitosan complexed to dual expressing short hairpin RNA (shRNA) recombinant plasmids will be encapsulated within the nanocarrier, and provide an intracellular defense mechanism that will interfere with the expression of the NS1 and P proteins. In combination, both defense mechanisms are expected to induce a synergistic anti-RSV effect that will surpass those of conventional therapeutics. Through this research, the NS1 and P containing plasmid (pSH-NS1-P) was cloned, and the nanotherapeutic was successfully synthesized. Based on the acquired results, pSH-NS1-P was shown to express anti-RSV effects, and it was also concluded that both inserts were producing active shRNA. Additionally, the anti-RSV efficiency of HR2-D was confirmed. Overall, this research will lead to development of a dual-mechanistic anti-viral nanotherapeutic.

The interactions of biomolecules and cells at solid-liquid interfaces play a pivotal role in a range of biomedical applications and have hence been studied in detail. An improved understanding of these interactions results in the ability to manipulate biomolecules and concurrently cells spatially and temporally at surfaces with high precision. Spatial control can be achieved using patterned surface chemistries whilst temporal control is achieved by switchable surfaces. The combination of these two surface properties offers unprecedented control over the behaviour of biomolecules and cells at the solid-liquid interface. This is particularly relevant for cell microarray applications, where a range of biological processes must be duly controlled in order to maximise the efficiency and throughput of these devices. Of particular interest are transfected cell microarrays (TCMs), which significantly widen the scope of microarray genomic analysis by enabling the high-throughput analysis of gene function within living cells Initially, this thesis focuses on the spatially controlled, electro-stimulated adsorption and desorption of DNA. Surface modification of a silicon chip with an allylamine plasma polymer (ALAPP) layer resulted in a surface that supported DNA adsorption and sustained cell attachment. Subsequent high density grafting of poly(ethylene glycol) (PEG) formed a layer resistant to biomolecule adsorption and cell attachment. PEG grafted surfaces also showed significantly reduced attachment of DNA with an equilibrium binding constant of 23 ml/mg as compared with 1600 ml/mg for ALAPP modified surfaces. Moreover, both hydrophobic and electrostatic interactions were shown to contribute to the binding of DNA to ALAPP. Spatial control over the surface chemistry was achieved using excimer laser ablation of the PEG coating which enabled the production of patterns of re-exposed ALAPP with high resolution. Preferential electro-stimulated adsorption of DNA to the ALAPP regions and subsequent desorption by the application of a negative bias was observed. Furthermore, this approach was investigated for TCM applications. Cell culture experiments demonstrated efficient and controlled transfection of cells. Electro-stimulated desorption of DNA was shown to yield enhanced solid phase transfection efficiencies with values of up to 30%. The ability to spatially control DNA adsorption combined with the ability to control the binding and release of DNA by application of a controlled voltage enables an advanced level of control over DNA bioactivity on solid substrates and lends itself to biochip applications. As an alternative approach to surface patterning, the fabrication and characterisation of chemical patterns using a technique that can be readily integrated with methods currently used for the formation of microarrays is also presented. Here, phenylazide modified polymers were printed onto low fouling ALAPP-PEG modified surfaces. UV irradiation of these polymer arrays resulted in the crosslinking of the polymer spots and their covalent attachment to the surface. Cell attachment was shown to follow the patterned surface chemistry. Due to the use of a microarray contact printer it was easily possible to deposit DNA on top of the polymer microarray spots. A transfected cell microarray was generated in this way, demonstrating the ability to limit cell attachment to specific regions and the suitability of this approach for high density cell assays. In order to allow for the high-throughput characterisation of the resultant polymer microarrays, surface plasmon resonance imaging was utilised to study the adsorption and desorption of bovine serum albumin, collagen and fibronectin. This analysis enabled insights into the underlying mechanisms of cell attachment to the polymers studied. For the system analysed here, electrostatic interactions were shown to dominate cellular behaviour.

Receptor tyrosine kinases (RTKs) are integral membrane proteins that regulate many functions including cell proliferation, cell survival, and cell death. They have been shown to be responsible for the uncontrolled growth of several cancers. RTKs phosphorylate downstream targets such as phosphatidylinositol 3 kinase (PI3K), a lipid kinase that is made up of two major subunitsp85 and p110. Receptor-mediated endocytosis delivers RTKs from the plasma membrane to late endosomes and lysosomes for degradation. This process is controlled by ESCRT proteins and Rab7. PI3K associates with PDGFR during endocytosis, and PI3K binding sites are necessary for the lysosomal trafficking of PDGFR. The smaller isoforms of the ankyrin 3 (Ank3) proteins bind p85. Ank3 overexpression was shown to increase PDGFR degradation, perhaps by controlling the targeting of PDGFR to late endosomes and lysosomes. Ank3 overexpression also reduced the RTK levels and cell proliferation rates of NIH 3T3 cells. We sought to investigate if cancer cells with RTK overexpression might be deficient in Ank3, and if overexpression of ankyrin 105 (Ank105), one of the smaller isoforms of Ank3, would reduce RTK levels and the tumorigenic properties of cancer cells. Two brain cancer cell lines showed reduced Ank105 levels associated with high RTK levels, while high levels of Ank105 associated with low RTK levels were found in normal brain cells. This suggested a loss of Ank105 in the cancer cells, which may have played a role in the cancer development process. We observed reduced RTK levels and anchorage-independent growth in cancer cells overexpressing HA-Ank105, however, most cells overexpressing a blank vector also showed the same results. An independent effect of the overexpression process was thought to play a role in influencing cell behavior. In the lung cancer cell line HCC827, however, there was significant reduction of anchorage-independent growth that was specific for HA-Ank105. There also appeared to be a significant reduction in the cell proliferation rate of T98G brain cancer cells following transfection with HA-Ank105. Furthermore, those cells overexpressing HA-Ank105 tended to die early in tissue culture, with those that survived losing their HA-Ank105 expression. Overall our results suggest a possible role for Ank105 in downregulating RTK levels and growth properties of cancer cells.

Receptor tyrosine kinases (RTKs) are integral membrane proteins that regulate many functions including cell proliferation, cell survival, and cell death. They have been shown to be responsible for the uncontrolled growth of several cancers. RTKs phosphorylate downstream targets such as phosphatidylinositol 3 kinase (PI3K), a lipid kinase that is made up of two major subunitsp85 and p110. Receptor-mediated endocytosis delivers RTKs from the plasma membrane to late endosomes and lysosomes for degradation. This process is controlled by ESCRT proteins and Rab7. PI3K associates with PDGFR during endocytosis, and PI3K binding sites are necessary for the lysosomal trafficking of PDGFR. The smaller isoforms of the ankyrin 3 (Ank3) proteins bind p85. Ank3 overexpression was shown to increase PDGFR degradation, perhaps by controlling the targeting of PDGFR to late endosomes and lysosomes. Ank3 overexpression also reduced the RTK levels and cell proliferation rates of NIH 3T3 cells. We sought to investigate if cancer cells with RTK overexpression might be deficient in Ank3, and if overexpression of ankyrin 105 (Ank105), one of the smaller isoforms of Ank3, would reduce RTK levels and the tumorigenic properties of cancer cells. Two brain cancer cell lines showed reduced Ank105 levels associated with high RTK levels, while high levels of Ank105 associated with low RTK levels were found in normal brain cells. This suggested a loss of Ank105 in the cancer cells, which may have played a role in the cancer development process. We observed reduced RTK levels and anchorage-independent growth in cancer cells overexpressing HA-Ank105, however, most cells overexpressing a blank vector also showed the same results. An independent effect of the overexpression process was thought to play a role in influencing cell behavior. In the lung cancer cell line HCC827, however, there was significant reduction of anchorage-independent growth that was specific for HA-Ank105. There also appeared to be a significant reduction in the cell proliferation rate of T98G brain cancer cells following transfection with HA-Ank105. Furthermore, those cells overexpressing HA-Ank105 tended to die early in tissue culture, with those that survived losing their HA-Ank105 expression. Overall our results suggest a possible role for Ank105 in downregulating RTK levels and growth properties of cancer cells.

Surface Biological Modification and Cellular Interactions of Magnetic Spinel Nanoparticles Eva Liang-Huang Heintz 191 Pages Directed by Dr. Z. John Zhang The interest in magnetic nanoparticles is multi-dimensional. Fundamentally, it is important to be able to control their magnetic properties and to correlate to specific applications. In biology, magnetic nanoparticles offer promising potential as magnetic carriers or chaperones for magnetic localization and manipulation of therapeutic reagents. The synthesis of superparamagnetic CoFe2-xSmxO4 nanoparticles and the tunability of their magnetic properties by size and composition variations are discussed. An increase in size of CoSm0.19Fe1.81O4 nanoparticles produced an increase in blocking temperature and saturation magnetization, but a non-linear coercitivity response was observed with change in size. By varying the composition, the saturation magnetization of CoFe2-xSmxO4 decreased dramatically while the coercitivity increased when compared to native cobalt spinel ferrite (CoFe2O4) nanoparticles. These results demonstrate how the magnetic properties of cobalt spinel ferrite nanoparticles can be tailored to specific applications. Surface modifications of cobalt spinel ferrite nanoparticles facilitated the conjugation of oligonucleotides. Using a transfection reagent, CoFe2O4 ??igonucleotide conjugates were delivered into mammalian cells. Post transfection, synchronized movement of cells in response to an external magnetic field was observed. This demonstrated the possibility of magnetic manipulation and localization of therapeutic reagents coupled to CoFe2O4 magnetic nanoparticles. Results from this thesis demonstrate the potential role of magnetic spinel nanoparticles in cell biology and will facilitate the progress towards in vivo testing.

The unique gene, pleiotrophin (ptn) was identified in the expressed sequence taq (EST) derived from the developing tilapia brain in our lab. The cDNA full length of ptn was cloned. ptn play a role in the differentiation of nerve cell. In this study, bioinformatics were searched for EE723939.1 (ptn), which is a gene with 1026 bp of cDNA sequence, open reading frame(ORF) is 483bp, and deduced as 160 amino acids. The protein of PTN was expressed in the prokaryotic system, BL21(E.coli), and purified with Ni-NTA affinity chromatography. In the present studies, ptn, cloned from tilapia, Oreochromis mossambicus. The influence of ptn on the proliferation of Neuro-2a cell was also investigated.The ORF of ptn was cloned, and the pEGFP-ptn plasmid was constructed. The distribution of ptn in the pEGFP-ptn transfected Neuro-2a cell was identified by fluorescence and laser confocal microscopy.

Reproduction de : Thèse de doctorat : Sciences de la vie et de la santé : Lille 1 : 2004. / N° d'ordre (Lille 1) : 3494. Résumé en français. Publications en anglais reproduites dans le texte. Titre provenant de la page de titre du document numérisé. Bibliogr. f. 160-173.