Neste trabalho, estudamos a interação de um peptídeo antimicrobiano com membranas modelo, por meio de dicroísmo circular (CD), fluorescência e microscopia óptica. Tal peptídeo, chamado de híbrido, foi sintetizado como uma mistura das regiões mais ativas de dois outros peptídeos antimicrobianos, chamados de pediocina A e plantaricina 149. Esse peptídeo híbrido possui carga de +8, em pH fisiológico, e as membranas estudadas foram compostas por uma mistura de fosfolipídeos zwiteriônicos (cabeça polar de fosfatidilcolina, PC) e aniônicos (cabeça polar de fosfatidilglicerol, PG), em diferentes razões molares. Os resultados de CD evidenciaram que este peptídeo se apresenta de forma desordenada em solução aquosa, porém adota uma conformação helicoidal na presença de grandes vesículas unilamelares carregadas negativamente (LUVs). A quantidade de componente helicoidal é dependente da quantidade de lipídeo negativo presente na bicamada lipídica. A fluorescência do triptofano revelou um deslocamento para o azul muito significativo, chegando a 20 nm para membranas compostas por 100 mol% de PG. Os dois resultados (CD e fluorescência) indicam que a região dos aminoácidos que contém o triptofano deve estar interagindo muito fortemente com a região hidrofóbica da membrana, numa conformação tipo-helicoidal. Experimentos de vazamento de carboxifluoresceína encapsulada em LUVs, por espectroscopia de fluorescência, demonstraram a ação lítica do peptídeo induzindo a formação de poros nas membranas, independentemente da composição das LUVs. Entretanto, a razão molar peptídeo:lipídeo necessária para induzir vazamento da sonda foi menor para membranas lipídicas compostas por bicamadas contendo altas quantidades de PG. Tal fato coloca em evidência o papel fundamental da interação eletrostática entre os peptídeos carregados positivamente com as membranas carregadas negativamente para o processo de ligação e mecanismo de ação deste peptídeo. Para estudar mais detalhadamente o mecanismo de ação, realizamos experimentos de microscopia óptica em vesículas unilamelares gigantes. Concluímos que o peptídeo provoca total desestabilização das vesículas unilamelares gigantes, com formação de poros, seguidos de ruptura da bicamada lipídica e sua transformação em pequenos e mal definidos complexos de peptídeos e fosfolipídeos. / In this work, we investigated the interaction between an antibacterial peptide with model membranes, by means of circular dichroism (CD), fluorescence and optical microscopy. Such a peptide was synthesized from the most active regions of two others antimicrobial peptides, namely pediocin A and plantaricin 149. The hybrid peptide has a net charge of ~ +8, at physiological pH, and the studied model membranes were composed of a mixture of zwitterionic phospholipids (phosphatidylcholine polar head) and anionic phospholipids (phosphatidylglycerol polar head), at differente molar ratio. The CD results evidenced that the peptide was essentially structureless in aqueous solution, but acquired an helical conformation in the presence of charged large unillamellar vesicles LUVs. The helical content is dependent on the negative charge amount on membrane surface. The tryptophan fluorescence revealed a significant blue shift of the maximium emission wavelength, up to 20 nm for the membranes composed of 100 mol% of PG in respect to the peptide fluorescence in the aqueous solution. This indicates that part of the aminoacid residues, that contains the tryptophan, must be buried into the hydrophobic medium of the lipid membrane. Leakage experiments using fluorescence spectroscopy of carboxyfluorescein encapsulated in LUVs demonstrated the lytic action of the peptide, inducing the pore formation in the membrane, regardless of lipid membrane composition. However, it should be stressed that the peptide:lipid molar ratio necessary to induce probe leakage was smaller for lipid membranes made up of large PG amounts. Such evidence points out the key role of the electrostatic interaction between a positively charged peptide and the negatively charged membrane, mediated by hydrophobic contribution. To gain further insight into the lytic mechanism of the peptide, we performed single vesicle experiments using giant unilamellar vesicles under optical microscopy observations. We conclude that the peptide provokes a total membrane desestabilization, with pore formation, followed by a membrane disruption and its transformation into smaller and not well defined complexes of phospholipids and peptides.

A demanda por combustíveis baseados em recursos renováveis é alta nos dias de hoje e tende a aumentar bastante no futuro. No Brasil, indústrias de biocombustíveis produzem principalmente etanol a partir cana-de-açúcar. A biomassa lignocelulósica, compreendendo resíduos de culturas, resíduos florestais, sólidos urbanos, é explorada como um elevado potencial secundário na produção de biocombustíveis, mesmo na categoria de subprodutos, eliminando assim os usos competitivos. Para tornar a produção de etanol de segunda geração a partir da cana-de-açúcar economicamente sustentável, é imprescindível utilizar fração hemicelulósica da biomassa, o que corresponde de 20% a 25%, sendo a xilose seu principal componente. Saccharomyces cerevisiae não fermenta xilose, entretanto, xilulose pode ser fermentada. Portanto a busca e o estudo de enzimas que procedem com a conversão de xilose em xilulose (em condições sinérgicas às da fermentação alcoólica) se torna de extrema importância no que se refere ao aproveitamento da hemicelulose para a geração de etanol de segunda-geração. Xilose isomerases (XI) de três microorganismos diferentes (de Xanthomonas campestris pv. Campestris [Xyl_Xcc], Bifidobacterium adolescentis [Xyl_Bad] e de Lactobacillus crispatus [Xyl_LCr]) são o objeto de estudo deste projeto. A partir do conteúdo genômico desses três microorganismos, foi realizada a amplificação do gene xylA (que codifica para XI), via Clonagem Independente de Ligação/Ligase (do inglês, LIC) e clonagem em vetor de expressão pPROEX HTa adaptado para LIC, e superexpressão em Escherichia coli BL21 (DE3). As XIs foram então extraídas e purificadas por cromatografia de afinidade com metal quelado, seguida de cromatografia de exclusão molecular. Nessa etapa, as massas moleculares e raios hidrodinâmicos (RH) foram estimados, tanto por cromatografia de exclusão molecular quanto em gel nativo, revelando que Xyl_Xcc e Xyl_Bad se apresentam diméricas enquanto Xyl_LCr monomérica. Subseqüentemente, foram realizados testes de atividade em diferentes condições (pHs e temperaturas), para mapear condições ótimas de reação. A atividade ótima de ambas Xyl_Xcc e Xyl_Bad foi ao redor do pH 5,5, com temperaturas ótimas girando em torno de 60°C. Xyl_LCr se mostrou sem atividade. Além disso, o monitoramento da estabilidade térmica das XIs foi realizado através de espalhamento de raios-X a baixo ângulo (SAXS) e espectroscopia de dicroísmo circular (CD). As estabilidades térmicas da estrutura secundária e da estrutura terciária como um todo parecem aumentadas com a elevação do pH. Entretanto, isso não condiz com perfil de atividade dessas enzimas, visto que a atividade ótima se apresentou deslocada para valores de pHs ácidos. Modelos de baixa resolução obtidos por SAXS foram alinhados e sobrepostos às estruturas de alta resolução de proteínas homólogas, revelando um bom ajuste da forma tetramérica para Xyl_Bad e Xyl_Xcc e monomérica para Xyl_LCr. Portanto, levanta-se a hipótese da dissociação do tetrâmero em dímeros, possivelmente causado pela interação (mecânica) com o sistema de emaranhados do gel nativo e com os poros da coluna de exclusão molecular. Foram obtidos cristais de Xyl_Bad e Xyl_Xcc, e esses foram submetidos à difração de raios-X, revelando a presença de um domínio conservado na maioria das XIs reportadas, formado por um barril α⁄β (N-terminal). As estruturas estão em fase avançada de refinamento. Ao final, são propostos estudos futuros que complementem os resultados apresentados, e que poderão comprovar as hipóteses criadas a partir deste trabalho. / The demand for fuels based on renewable resources is high these days and tends to increase considerably in the future. In Brazil, biofuels industries mainly produce ethanol from sugarcane. The lignocellulosic biomass, including crop residues, forest residues, urban solids, is explored as a secondary high potential for biofuels production, in the same category of products, thus eliminating the competing uses. To make the production of sugarcane secondgeneration ethanol economically sustainable, it is essential to use the hemicellulose fraction of the biomass, which corresponds from 20% to 25%, the main component represented by xylose. Saccharomyces cerevisiae doesnt ferment xylose, however, xylulose may befermented. Therefore the research and study of enzymes that carry out the conversion of xylose to xylulose (in synergistic fermentation conditions) become very important with regard to the use of hemicellulose in second-generation ethanol production. Xylose isomerases (XI) from three different microorganisms (Xanthomonas campestris pv. Campestris [Xyl_Xcc], Bifidobacterium adolescentis [Xyl_Bad] and Lactobacillus crispatus [Xyl_Lcr]) are the target of this project. From the genomic content of these three organisms, gene amplification of the xylA gene (encoding XI) was performed, via Ligand / Ligation Independent Cloning (LIC) and cloning in LIC adapted pPROEX HTA expression vector , with overexpression in Escherichia coli BL21 (DE3). The XIs were then extracted and purified by affinity metal quelate chromatography, followed by size exclusion chromatography. At that time, the molecular weight and hydrodynamic radius (RH) were estimated both by size exclusion chromatography and native gel, suggesting that Xyl_Xcc and Xyl_Bad were as dimers in solution, while Xyl_Lcr as monomer. Subsequently, activity assays were performed in different conditions (pH and temperature), to find out the optimum reaction conditions. The optimal activity of both Xyl_Xcc and Xyl_Bad was around pH 5.5, with optimum temperatures hovering around 60°C. Xyl_Lcr showed no activity. Furthermore, monitoring the thermalstability of XIs was performed by small angle X-ray scattering (SAXS) and circular dichroism spectroscopy (CD). The thermal stabilities of the secondary structure and tertiary structure as a whole appear increased with increasing pH. However, this does not match with the activity profiles of these enzymes, since they showed optimal activity shifted to acidic pHs. SAXS low-resolution models were aligned and superimposed on high resolution structures of homologous proteins, revealing a concordance of the tetrameric form of Xyl_Xcc and Xyl_Bad in solution and monomeric form of Xyl_Lcr. Thus arises the possibility of dissociation of tetramer into dimers, possibly caused by interaction (mechanical) system with the tangles of native gel and pores of molecular exclusion column. Crystals were obtained from Xyl_Bad and Xyl_Xcc, and these were subjected to X-ray diffraction to generate high resolution structures, revealing the presence of a conserved domain in the most reported XIs, consisting of a α⁄ β barrel (N-terminus). The structures are in an advanced stage of refinement. Finally, future studies are proposed to complement the results presented, which may prove the hypotheses generated from this work.

Orientador: Flavia Maria Netto / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-09-27T19:42:42Z (GMT). No. of bitstreams: 1 HozUrrejola_LuciadelCarmendela_D.pdf: 2021405 bytes, checksum: 7522596dbd78ae21dcc1d21542fc59d5 (MD5) Previous issue date: 2006 / Resumo: Amostras de ß-lactoglobulina (ß-LG) no estado sólido, com diferentes atividades de água (aw) (0,22; 0,53; 0,74), ou em solução, em concentração de proteína de 3 e 10 mg/mL, foram irradiadas (1-50 kGy) utilizando uma fonte de Cobalto-60. Espalhamento de raios-X a baixos ângulos (SAXS), dicroísmo circular (CD) e fluorescência foram as técnicas utilizadas para estudar as mudanças conformacionais da ß-LG por efeito da irradiação. A proteína irradiada foi também examinada por eletroforese em gel de poliacrilamida (PAGE) e desnaturante (SDS-PAGE) sob condições redutoras e não redutoras, cromatografia de exclusão molecular (SE-HPLC) e ¿SH livre. A ß-LG no estado sólido nas diferentes aw estudadas não foi afetada pela irradiação. As amostras não apresentaram diminuição na digestibilidade sob ação da pepsina e tripsina e não mudaram seu poder ligante de retinol quando irradiadas com doses até 10 kGy. Nas amostras irradiadas em solução, houve modificações estruturais, ao nível de estrutura terciária, enquanto a estrutura secundária parece ter sido preservada, como indicado pela quantificação das estruturas obtidas a partir da deconvolução dos espectros de CD no UV-longínquo. Os dados de SE-HPLC e espalhamento de raios-X mostraram que a irradiação promoveu a formação de oligômeros e agregados, que resultaram ser altamente solúveis, como mostrado pelas medidas de turbidez. Da análise dos dados de SAXS e a construção de modelos pode ser mostrado que os oligômeros eram múltiplos de dímeros de ß-LG, (tetrâmeros e hexâmeros, etc.), linearmente ordenados. Ligações cruzadas intermoleculares entre radicais tirosil teriam participação na agregação e as ligações dissulfeto, pelo contrário, não teriam sido relevantes para a formação dos agregados. O mecanismo de agregação da ß-LG mediante ligações cruzadas de bitirosil, contrário ao que acontece em outros tratamentos físicos, como o térmico ou de alta pressão hidrostática, poderia estar gerando agregados com características e propriedades diferentes. O efeito da irradiação na proteína foi maior nas amostras mais diluídas e com maior dose de radiação, indicação da ocorrência do chamado ¿efeito indireto¿ da irradiação. Considerando que não houve alterações estruturais, nem perda de funcionalidade ou de digestibilidade da ß-LG irradiada no estado sólido até com 10 kGy em diferentes aw, o tratamento com radiação gama poderia ser usado para melhorar a qualidade microbiológica da proteína e aumentar as condições de segurança se for aplicada uma radiação na faixa geralmente utilizada no processamento de alimentos (<10 kGy) / Abstract: ß-lactoglobulin (ß-LG) in solution at different protein concentrations (3 and 10 mg/mL) and in solid state, with different water activity (aw) (0.22; 0.53; 0.74), was irradiated using a Cobalt-60 radiation source at a dose level of 1-50 kGy. Small-angle X-ray scattering (SAXS), Circular Dichroism (CD) and Fluorescence spectroscopy were used to study the conformational changes of ß-LG due the irradiation treatment. The irradiated protein was also examined by polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS) PAGE under reducing and nonreducing conditions, size exclusion chromatography (SE-HPLC), and free SH. ß-LG in the solid state, at different aw, was not significantly affected by gamma radiation. The digestibility of these samples under pepsin or trypsin action was not diminished, and the retinol binding property was not changed by irradiation up to 10 kGy. The irradiation of ß-LG in solution promoted structural changes at the tertiary structure level but the secondary structure seemed to be maintained, as indicated by the quantification of structures from the deconvolution of the far-UV CD spectra. The SEHPLC, and X-rays scattering data showed that irradiation of ß-LG solutions promoted oligomers and aggregates formation, which proved to be highly soluble, as shown by turbidity measurements. From the SAXS data analyses and the model building it could be shown that the formed oligomers were linear molecules, built by linear combinations of ß- LG dimers (tetramers, hexamers, etc.). Formation of intermolecular cross-linking between tyrosil radicals are proposed to be at least partially responsible by this occurrence, and the disulphide bonds, in contrast, do not appear to be relevant to the formation of these aggregates. The mechanism of aggregation of ß-LG through bityrosil cross-links, differently from the mechanism occurring in other physical treatments as heating or high pressure, could form aggregates with special and different characteristics. The radiation effect was more significant at low protein concentration and higher irradiation dose, being consistent with the occurrence of the ¿indirect effect¿ when proteins are irradiated in solution. Considering that neither structural changes or loss of functionality and digestibility of ß-LG irradiated in the solid state at different aw, with dose up to 10 kGy, was detected, the gamma radiation treatment could be used for improving the microbiological quality of the protein, enhancing the safety conditions when applying the radiation range generally used in food processing (<10kGy) / Doutorado / Doutor em Engenharia de Alimentos

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-02-15T15:54:32Z No. of bitstreams: 1 Zenaide Severina do Monte.pdf: 2760660 bytes, checksum: c07a4369e12f4af72ee82c783db1000c (MD5) / Made available in DSpace on 2017-02-15T15:54:32Z (GMT). No. of bitstreams: 1 Zenaide Severina do Monte.pdf: 2760660 bytes, checksum: c07a4369e12f4af72ee82c783db1000c (MD5) Previous issue date: 2012-02-24 / This paper describes the qualitative and quantitative analysis by circular dichroism (CD) of paroxetine hydrochloride and qualitative analysis of biperiden hydrochloride. It was developed the method a simple, rapid, which uses the differential absorption of circularly polarized light right and left environment for the qualification and quantification of raw materials the drugs mentioned above using the technique by circular dichroism. The method developed followed the criteria of rules and recommended by ANVISA and ICH. The paroxetine hydrochloride and biperiden hydrochloride are chiral drug that can be used for the treatment of depression and Parkinson's, respectively. Paroxetine (PRX), a chiral drug, has two stereogenic carbon atoms in its chemical structure. The PRX, was dissolved in ultrapure water concentration 5,47.10-4 molL-1 DC analysis. Their spectrum showed an intense negative Cotton effect in mdeg - 37.4653. The standard curve the solution of reference a correlation coefficient of 0.9907 for the concentration range of the 0,91.10-4/ 5,47.10-4 molL-1. The limits of detection and quantification were 0,197.10-4 and 0,656.10-4 molL-1, respectively. The repeatability and precision intermediate had variance of 1.32. The results showed good accuracy for interval of 95%. The study showed that the recovery of the extraction method of PRX the drug was effective. The method is robust and the samples were stable to temperature variations. The method was selective for quantification of the drug by evaluating the interference of the excipients. Analyses with biperiden, a substance which has four chiral centers, were performed with the secondary standard BPR and the drug by the DC of molar concentrations 3,21.10-3 and 3,21.10-4 respectively. The samples had EC negative as well as the primary standard USP BPR. The spectrum obtained by theoretical calculation showed a positive Cotton effect of the enantiomers of BPR. These results indicate that one of the enantiomers can BPR is in excess or that the substance is pure or impurities which are chiral. / O presente trabalho descreve a análise qualitativa e quantitativa por dicroísmo circular (DC) do cloridrato de paroxetina e a análise qualitativa do cloridrato de biperideno. Foi desenvolvido um método simples, rápida, que faz uso da absorção diferenciada da luz circularmente polarizada a direita e a esqueda para a qualificação e quantificação da matéria-prima e os fármacos citados anteriormente empregando a técnica por dicroísmo circular. O desenvolvimento do método seguiu os critérios das normas vigentes e recomendadas pela ANVISA e ICH. O cloridrato de paroxetina e o cloridrato de biperideno são drogas quirais, que pode ser utilizadas para o tratamento da depressão e de parkinson, respectivamente. A paroxetina (PRX), um fármaco quiral, apresenta dois carbonos estereogênicos em sua estrutura química. A PRX, foi dissolvida em água ultrapura a concentração de 5,47.10-4 molL-1 para análise em DC. O seu espectro apresentou um Efeito Cotton intenso e negativo em -37,4653 mdeg. A curva da solução de referência apresentou um coeficiente de correlação de 0,9907 para o intervalo de concentração de 0,91.10-4 a 5,47.10-4 molL-1. Os limites de detecção e quantificação foram de 0,197.10-4 e de 0,656.10-4 molL-1, respectivamente. A repetitividade e precisão intermediária apresentaram variancia média 1,32. A exatidão apresentou resultados bons para o intervalo de confiança de 95%. O método foi seletivo para quantificação do fármaco avaliando a interferência dos excipientes. As análises com o biperideno, uma substância que tem quatro centros quirais, foram realizadas com o padrão secundário BPR e com o fármaco por DC nas concentrações molares de 3,21.10-3 e de 3,21.10-4 respectivamente. As amostras apresentaram EC negativos, assim como no BPR padrão primário da USP. O espectro obtido por cálculo teórico apresentou um Efeito Cotton positivo para um dos enantiômeros do BPR. Esses resultados indicam que um dos enantiômeros do BPR pode está em excesso ou que a substância está pura ou ainda que existem impurezas quirais.

Made available in DSpace on 2014-06-11T19:30:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-08-18Bitstream added on 2014-06-13T19:19:38Z : No. of bitstreams: 1 cabrera_mps_dr_sjrp.pdf: 1625766 bytes, checksum: 6073fec2bef34125e9db4700102a3680 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Neste trabalho estudamos a conformação em ambientes anisotrópicos e a atividade lítica em vesículas aniônicas e zwitteriônicas de um conjunto de peptídeos biologicamente ativos, extraídos de veneno de vespas solitárias, que se caracterizam por usar seus venenos para paralisar as presas com as quais alimentam suas larvas. Esses peptídeos que são desgranuladores de mastócitos, apresentam atividade antimicrobiana e a maioria deles não é hemolítica. Possuem entre 10 e 15 resíduos, são catiônicos, com alta proporção de resíduos carregados e polares, e são lineares e helicoidais em meios miméticos de membranas. Buscamos correlacionar a atividade lítica em vesículas de diferentes composições, analisada em experimentos de fluorimetria, às mudanças conformacionais, induzidas por diferentes ambientes miméticos, monitoradas por dicroísmo circular, complementando com a análise das características físico-químicas como comprimento da cadeia, amidação do terminal-C, carga líquida, influências no macrodipolo da hélice, hidrofobicidade, momento hidrofóbico e ângulo polar. Observamos que estes peptídeos apresentam intensa atividade em membranas modelo, interagem preferencialmente com bicamadas aniônicas, e sua atividade lítica acontece de modo cooperativo tanto em vesículas aniônicas como nas zwitteriônicas. Com exceção de Anoplin, todos os peptídeos com ação antimicrobiana apresentam curvas de dose-resposta que mostram uma dependência sigmoidal com a concentração do peptídeo. Isso sugere que esses peptídeos se acumulam na superfície da vesícula até atingir uma concentração crítica, além da qual o vazamento aumenta cooperativamente. De uma forma geral os peptídeos mais eficientes como antimicrobianos, são também aqueles caracterizados pela maior eficiência em permeabilizar vesículas aniônicas do tipo PCPG 7030 e por baixas razões limite P/L. / Solitary wasps use their venoms to paralise prays to feed their larvae. A set of biologically active peptides, obtained from these venoms, have been investigated in relation to the conformational changes they undergo in anisotropic environments and their lytic activity on zwitterionic and anionic vesicles. These peptides are mast cell degranulators, present antimicrobial activities and most of them are not hemolytic. They are cationic, their chain length are 10 to 15 residues long, with high hydrophilic / hydrophobic ratio; they are linear and helical in membrane mimetic environments. We searched correlation between the lytic activity in vesicles of different compositions, monitored in fluorimetric experiments, and conformational changes, induced by varied mimetic media, monitored by circular dichroism. The results have been also correlated with peptides' physical-chemical parameters such as chain length, amidated or carboxylated C-terminal, net charge, influences on the helix macrodipole, hydrophobicity, hydrophobic moment and polar angle. We observed that these peptides present intense activity on model membranes, they interact preferentially with anionic bilayers, and their lytic activity is a cooperative process either in anionic or in zwitterionic vesicles. Exception made to Anoplin, all the other peptides that have antimicrobial activity present in their dose-response curves a sigmoidal dependence with the peptide concentration. This fact suggests that these peptides accumulate on the vesicles surface until they reach a threshold concentration, beyond which leakage increases cooperatively. As a general rule, the most efficient antimicrobial peptides are also those characterized by efficient permeabilization of anionic vesicles, namely PCPG 7030 and by small threshold P/L ratios.

La chiralité est une propriété essentielle des molécules naturelles, intervenant dans de nombreux mécanismes biologiques du vivant. Elle peut être caractérisée grâce à l’activité chiroptique des molécules chirales, à travers le dichroïsme circulaire, le pouvoir rotatoire, la dispersion rotatoire optique ou encore la luminescence polarisée circulairement. Les molécules biaryles et hélicéniques possèdent des propriétés chiroptiques intenses et sont des molécules de choix pour leurs applications dans de nombreux domaines, de la catalyse à l’optoélectronique.Les travaux réalisés au cours de cette thèse traitent de la synthèse de nouvelles molécules biaryles et hélicénoïdes et de l’étude de leurs propriétés spectroscopiques en interaction avec leur environnement. Dans un premier temps, nous avons effectué la synthèse de différentes familles de molécules à géométrie hélicoïdale possédant des systèmes aromatiques étendus. Afin de cibler des applications biologiques, nous avons également mis au point leur hydrosolubilisation par introduction de chaînes PEG. Dans un deuxième temps, nous nous sommes consacrés à l’étude de leurs propriétés spectroscopiques et en particulier chiroptiques en fonction de leur structure et de la dépendance de ces propriétés en présence de protons, de métaux ou d’acide désoxyribonucléique (ADN). Au cours de cette étude, une réaction photochimique inattendue a permis d’obtenir un produit photogénéré inédit présentant des interactions sélectives avec des ADN G-quadruplexes. Enfin, afin d’exploiter la modularité contrôlée des propriétés chiroptiques, nous avons travaillé sur un projet de conception de nouvelles molécules chirales pouvant détecter sélectivement les sucres en modifiant le squelette de ces molécules avec des acides boroniques. / Chirality is an essential property of natural molecules, involved in many living biological mechanisms. It can be characterized by the chiroptical activity of chiral molecules, through circular dichroism, rotatory power, optical rotatory dispersion or circularly polarized luminescence. Biaryl and helicenic molecules are known for their intense chiroptical properties and they can be used for their applications in many fields, going from catalysis to optoelectronics.The research carried out during this thesis deals with the synthesis of new biaryl and helicenoid molecules and the study of their spectroscopic properties in interaction with their environment. First, different families of helicoidal molecules with extended aromatic systems have been synthesized. In order to target biological applications, their hydrosolubilization was achieved by the addition of PEG chain. Then, we focused on their spectroscopic and especially chiroptical properties and their modulation according to their structures, their interaction with protons, metals or deoxyribonucleix acid (DNA). During these studies, we experienced an unexpected photochemical reaction, which lead to a new photogenerated product revealing selective interactions with DNA G-quadruplexes. Finally, in order to exploit the controlled modularity of chiroptical properties, new chiral molecules that selectively detecting sugars by with boronic acids have been designed.

Gene transfer represents an important advance in the treatment of both genetic and acquired diseases. Topical gene therapy involves administration of the genetic material onto the surface of skin and mucosal membranes. Cationic gemini surfactants (m-s-m, where m represents the carbon atoms in the alkyl tail and s represents the carbon atoms in the spacer) are a novel category of delivery agents with especially high potential for polynucleotides. This is due to their structural versatility, ability to bind and condense DNA, and relatively low toxicity. <p>The objectives were to design, construct and characterize a cationic, non-viral gemini surfactant-based delivery system for an IFN-ã coding plasmid suitable for cutaneous gene therapy and to evaluate this novel therapeutic approach in a Tsk (tight-skin scleroderma) mouse model to determine its clinical feasibility. <p>The delivery systems were characterized by microscopy, dynamic light scattering (DLS), circular dichroism (CD) and small angle X-ray scattering (SAXS). <i>In vitro</i> gene expression was evaluated in PAM 212 keratinocyte culture. The extent of topical delivery of the plasmid using nanoparticle and nanoemulsion formulations was evaluated by measuring IFN-ã levels in CD1, IFN-ã-deficient and Tsk mice. The effect of transgene expression on collagen synthesis was evaluated in Tsk animals by real-time PCR.<p>The <i>in vitro</i> plasmidgeminilipid (PGL) system showed heterogeneous particle size (100-200 nm small particles and 300-600 nm aggregates). Electrostatic interactions between the DNA and PGL systems shifted the negative æ-potential of the DNA (-47 mV) to positive values (30-50 mV). At the same time, condensation of the DNA, and formation of Ø DNA was indicated by the increase of the overall negative signal in the CD spectra, due to the flattening of the 290 nm peak and shift of the 260 nm peak into the negative region in a structure-dependent manner. Lipid organization of the DNADOPE system, in the absence of gemini surfactants, shows hexagonal structure, while addition of gemini surfactant at +/- charge ratio of 10 caused lamellar phase organization. For short spacers (n=3-6), additional Pn3m cubic phase also appear to be present. <p><i> In vitro</i> transfection efficiency in the 12-n-12 series was found to be dependent on the length of the spacer between the two positively charged head groups, with the n=3 spacer showing the highest activity. The PGL systems with 12-3-12 and 12-4-12 led to significantly higher transgene expression compared to the other surfactants of the series. The transfection efficiency significantly correlated with the surface area occupied by one molecule (a). The effect of the tail length influenced the transfection efficiency, with longer tails being associated with higher protein expression. The highest <i>in vitro</i> transfection efficiency was recorded with the 18:1-3-18:1 surfactant (1.4±0.3 ng/5x10E4 cells). <p><i>In vivo</i>, high levels of IFN-ã expression were detected in the skin of animals treated with both nanoparticle (359±239 pg/cm2) and nanoemulsion (607±411 pg/cm2) formulations compared to topical naked DNA (136±125 pg/cm2). IFN-ã levels in the skin of animals injected with 5 ìg DNA were 256±130 pg/cm2. IFN-ã levels in the lymph nodes were higher for the nanoparticle formulation (433±456 pg/animal) compared to nanoemulsion (131±136 pg/animal) suggesting different delivery pathway of the two formulations.<p>IFN-ã expression was at high levels in the skin of Tsk mice after 4-day and 20-day treatments (472±171 and 345±276 pg/cm2). Both 4-day and 20-day treatments reduced the procollagen type I á1 mRNA levels for the topical treatment (64 and 70% reduction) and intradermal injection (58 and 72% reduction). Intercellular adhesion molecule-1 (ICAM-1) was upregulated by 50% in both topically treated and injected animals after 20-day treatment. <p>Here, it has been demonstrated that cationic gemini surfactant-based delivery systems are able to transfect epidermal cells <i>in vivo</i>, and the transgene IFN-ã expression is sufficient to cause significant reduction of collagen in an animal model of scleroderma. It has been shown for the first time that topical gene therapy is a feasible approach for the modulation of excessive collagen synthesis in scleroderma-affected skin.

Gene transfer represents an important advance in the treatment of both genetic and acquired diseases. Topical gene therapy involves administration of the genetic material onto the surface of skin and mucosal membranes. Cationic gemini surfactants (m-s-m, where m represents the carbon atoms in the alkyl tail and s represents the carbon atoms in the spacer) are a novel category of delivery agents with especially high potential for polynucleotides. This is due to their structural versatility, ability to bind and condense DNA, and relatively low toxicity. <p>The objectives were to design, construct and characterize a cationic, non-viral gemini surfactant-based delivery system for an IFN-ã coding plasmid suitable for cutaneous gene therapy and to evaluate this novel therapeutic approach in a Tsk (tight-skin scleroderma) mouse model to determine its clinical feasibility. <p>The delivery systems were characterized by microscopy, dynamic light scattering (DLS), circular dichroism (CD) and small angle X-ray scattering (SAXS). <i>In vitro</i> gene expression was evaluated in PAM 212 keratinocyte culture. The extent of topical delivery of the plasmid using nanoparticle and nanoemulsion formulations was evaluated by measuring IFN-ã levels in CD1, IFN-ã-deficient and Tsk mice. The effect of transgene expression on collagen synthesis was evaluated in Tsk animals by real-time PCR.<p>The <i>in vitro</i> plasmidgeminilipid (PGL) system showed heterogeneous particle size (100-200 nm small particles and 300-600 nm aggregates). Electrostatic interactions between the DNA and PGL systems shifted the negative æ-potential of the DNA (-47 mV) to positive values (30-50 mV). At the same time, condensation of the DNA, and formation of Ø DNA was indicated by the increase of the overall negative signal in the CD spectra, due to the flattening of the 290 nm peak and shift of the 260 nm peak into the negative region in a structure-dependent manner. Lipid organization of the DNADOPE system, in the absence of gemini surfactants, shows hexagonal structure, while addition of gemini surfactant at +/- charge ratio of 10 caused lamellar phase organization. For short spacers (n=3-6), additional Pn3m cubic phase also appear to be present. <p><i> In vitro</i> transfection efficiency in the 12-n-12 series was found to be dependent on the length of the spacer between the two positively charged head groups, with the n=3 spacer showing the highest activity. The PGL systems with 12-3-12 and 12-4-12 led to significantly higher transgene expression compared to the other surfactants of the series. The transfection efficiency significantly correlated with the surface area occupied by one molecule (a). The effect of the tail length influenced the transfection efficiency, with longer tails being associated with higher protein expression. The highest <i>in vitro</i> transfection efficiency was recorded with the 18:1-3-18:1 surfactant (1.4±0.3 ng/5x10E4 cells). <p><i>In vivo</i>, high levels of IFN-ã expression were detected in the skin of animals treated with both nanoparticle (359±239 pg/cm2) and nanoemulsion (607±411 pg/cm2) formulations compared to topical naked DNA (136±125 pg/cm2). IFN-ã levels in the skin of animals injected with 5 ìg DNA were 256±130 pg/cm2. IFN-ã levels in the lymph nodes were higher for the nanoparticle formulation (433±456 pg/animal) compared to nanoemulsion (131±136 pg/animal) suggesting different delivery pathway of the two formulations.<p>IFN-ã expression was at high levels in the skin of Tsk mice after 4-day and 20-day treatments (472±171 and 345±276 pg/cm2). Both 4-day and 20-day treatments reduced the procollagen type I á1 mRNA levels for the topical treatment (64 and 70% reduction) and intradermal injection (58 and 72% reduction). Intercellular adhesion molecule-1 (ICAM-1) was upregulated by 50% in both topically treated and injected animals after 20-day treatment. <p>Here, it has been demonstrated that cationic gemini surfactant-based delivery systems are able to transfect epidermal cells <i>in vivo</i>, and the transgene IFN-ã expression is sufficient to cause significant reduction of collagen in an animal model of scleroderma. It has been shown for the first time that topical gene therapy is a feasible approach for the modulation of excessive collagen synthesis in scleroderma-affected skin.

The study for surfaced-immobilized nucleic acid probes in nanometer region in response to hybridization and to discrimination ofdifferent target nuclei acids. The hairpin locked nucleic acid (LNA-HP) isselected to be the probe molecule, and target molecules include perfect complementary (PC) and single mismatch (1MM). The self-assembledLNA-HP molecular nanospot is successfully prepared by liquid phaseAFM (Atomic Force Microscope)-based nanolithography technique, then in situ hybridization is carried out by using different targets (PC/1MM).To obtain the information of structure change, we use AFM to analyze therelative heights in the process of hybridization. The experimental results point out that (1) the structure changes of surface probe molecules maycorrelate with the AFM signal when target sequence hybridizes to the probe, (2) miniaturization of the size of the nucleic acid probe may promote hybridization efficiency and enhance the discrimination between PC and 1MM. Studies on whether the different chemical impetus in solution can affect conformation of the human telomeric DNA of sequence is conducted. A human talomeric DNA composed of ( 5¡¦-TTAGGG-3¡¦:5¡¦-CCCTAA-3¡¦ ) repeats, with a 100-200 nt ( T2AG3 ) repetitive unit overhang at 3¡¦ ends is chosen. This extended single-stranded sequence is called G-rich DNA, which forms the special G-quadruplex structure in solution containing sodium ions or potassium ions. The single-stranded sequence composed of ( C3TA2 ) repetitive units called C-rich DNA displays the i-motif folded structure in the low pH environment. These biomimetic DNA¡¦s are thiol-modified to self-assemble on gold surfaces. Separate measurements with AFM (the molecular thickness and rootmean- square roughness of the self-assembly monolayer of DNA ) and CD( circular dichroism ) ( structure characterization ) confirm the conformational changes of G-rich and C-rich DNA¡¦s on gold surface are indeed dependent of the presence of cations and protons.

The aim of this thesis was the investigation of the mitochondrial response mechanisms upon apoptotic stimuli. The specific objectives were the biophysical characterization of membrane dynamics and the specific roles of lipids in the context of apoptotic regulation occurring at the mitochondrion and its complex membrane systems. The BH4 domain is an anti-apoptotic specific domain of the Bcl-2 protein. Solid phase peptide synthesis was used to produce large amount of the peptide for biophysical studies. A protocol has been established and optimized, guarantying the required purity for biophysical studies. In detail the purification by high performance liquid chromatography and the characterisation via mass spectroscopy are described. The secondary structure of BH4 changes significantly in the presence of lipid vesicles as observed by infrared spectroscopy and circular dichroism. The BH4 peptide aggregates at the membrane surface and inserts slightly into the hydrophobic part of the membrane. Using nuclear magnetic resonance (NMR) and calorimetry techniques, it could even be shown that the BH4 domain modifies the dynamic and organization of the liposomes which mimic a mitochondrial surface. The second study was on the first helix of the pro-apoptotic protein Bax. This sequence called Bax-α1 has the function to address the cytosolic Bax protein to the mitochondrial membrane upon activation. Once again a protocol has been established for the synthesis and purification of this peptide. The aim was to elucidate the key role of cardiolipin, a mitochondria-specific phospholipid, in the interaction of Bax-α1 with the mitochondrial membrane system. The NMR and circular dichroism studies showed that Bax-α1 interacts with the membrane models only if they contain the cardiolipin, producing a strong electrostatic lock effect which is located at the membrane surface. Finally, a new NMR approach was developed which allows the investigation of the lipid response of isolated active mitochondria upon the presence of apoptotic stimuli. The goal was there to directly monitor lipid specific the occurring changes during these physiological activities.