L'action ubiquitaire de l'interféron de type I (IFN- alpha/beta , ici IFN) dans la physiologie et la pathologie est aujourd'hui certaine. Une réponse dérégulée à l'IFN peut entraîner des interféronopathies ou des maladies auto-immunes. Dans mon travail de thèse j'ai étudié trois éléments de la voie de signalisation de l'IFN afin de comprendre comment une dérégulation peut se produire. TYK2 est une tyrosine kinase de la famille Janus impliquée dans la signalisation de cytokines immunorégulatrices (IFN de type I, IL-10, IL-12, IL-23). Selon le récepteur, TYK2 est co-activé avec JAK1 ou JAK2. L'interaction moléculaire entre les deux kinases juxtaposées est peu connue. J'ai caractérisé deux variants de TYK2 associés à des maladies auto-immunes, TYK2 I684S et TYK2 P1104A. J'ai démontré que ces deux variants ont un défaut catalytique, mais soutiennent la réponse à l'IFN. Mes résultats suggèrent un modèle d'activation réciproque des deux kinases. Par des études de signalisation dans les cellules EBV-B j'ai montré que l'homozygotie TYK2 P1104A a un impact différent selon la cytokine étudiée. L'analyse de deux autres polymorphismes de TYK2 associés à des maladies auto-immunes (rs12720270, rs2304256) a montré un impact sur la rétention de l'Exon 8, ce qui augmente l'expression de TYK2. J'ai aussi contribué à la dissection du mécanisme moléculaire contrôlant la réponse à l'IFN dans les cellules de patients déficients pour USP18 ou ISG15 et souffrant d'interféronopathies. Ces travaux ont démontré le rôle essentiel d'USP18 pour restreindre la réponse à l'IFN et ont mis en évidence ISG15 comme un nouvel inhibiteur de l'IFN chez l'humain mais pas chez la souris. / Today, the pervasive action of type I IFN (IFN-alpha/beta, here IFN) in human physiology and pathology has become evident. Dysregulated IFN response can lead to interferonopathies and auto-immune diseases (AID). My thesis work has focused on the study of three elements of the IFN response pathway, aiming to understand how dysregulation occurs. TYK2 belongs to the Janus tyrosine kinase family and is involved in signaling of several immunoregulatory cytokines, such as type I IFN, IL-10, IL-12 and IL-23. Depending on the receptor complex, TYK2 is co-activated with either JAK1 or JAK2. A detailed molecular characterization of the interplay between the two juxtaposed enzymes is missing. In my study, I characterized two rare AID-associated human variants TYK2 I684S and TYK2 P1104A. I found that both variants are catalytically impaired but rescue signaling in response to IFN in fibroblasts. My results support a model of reciprocal activation of Janus kinases. Through signaling studies I showed that TYK2 P1104A homozygosity has a cytokine-specific impact in EBV-B cells. My studies of two other AID-associated TYK2 SNPs (rs12720270 and rs2304256) suggest that they promote Exon 8 retention and increase TYK2 expression. In the second part of my thesis work, I contributed to dissecting the molecular mechanism that tunes down IFN response in cells from rare USP18- and ISG15-deficient patients that suffered of interferonopathies. This work substantiated the essential role of USP18 in downregulating the IFN response and highlighted ISG15 as a novel IFN inhibitor in humans, but not in mice.

Made available in DSpace on 2014-06-11T19:28:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-03-30Bitstream added on 2014-06-13T19:26:31Z : No. of bitstreams: 1 souza_jac_me_arafo.pdf: 631355 bytes, checksum: 9371a4a7027469c6d4a66de2870a71ed (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A expressão de citocinas inflamatórias é um processo estritamente regulado por mecanismos variados, incluindo o controle da sinalização intracelular e da atividade transcricional por inibidores endógenos, os quais são pouco estudados e compreendidos. Três grupos de proteínas: SHP, PIAS e SOCS inibem de maneira distinta e específica a transdução de sinais pela via JAK/STAT, bem como a atividade dos fatores de transcrição, eventos que modulam a expressão de diversas citocinas. As doenças periodontais estão associadas à inflamação persistente, com elevados níveis de citocinas proinflamatórias, no entanto praticamente não existem informações sobre a participação destes mecanismos de regulação nas diferentes condições clínicas periodontais. Os objetivos deste projeto incluíram avaliar a cinética de expressão das proteínas SOCS1 e SOCS3 e suas proteínas-alvo, STAT1 e STAT3, respectivamente, durante a evolução da doença periodontal. Foram utilizados 36 ratos Wistar divididos em 2 grupos: DP - doença periodontal induzida por 2 métodos: ligaduras ao redor dos 1os molares inferiores e injeções de 60 μg de LPS de E. coli no tecido gengival palatino dos molares superiores, 3x/semana; Grupo controle negativo - recebeu apenas injeções de PBS (veículo). Os ratos foram sacrificados 7, 15 e 30 dias após a indução da doença periodontal para avaliação histológica e análise macroscópica da perda óssea alveolar. A expressão de SOCS1 e SOCS3 e a ativação de STAT1 e STAT3 foram avaliadas nas biópsias gengivais por PCR em tempo real e Western blot. Ambos os modelos apresentaram significante e progressiva perda óssea dos 7 aos 30 dias. A inflamação foi evidente já no período de 7 dias em ambos os modelos, porém enquanto manteve-se similar nos demais períodos no modelo de indução por LPS, apresentou uma diminuição na severidade da inflamação... / Inflammatory cytokine gene expression is a process strictly regulated by various mechanisms, including the negative regulation of signaling of cytokine receptors and of the activity of transcription factors such as STATs. These mechanisms involve endogenous proteins and are largely unknown, especially in periodontal diseases. Three groups of proteins, SHP, PIAS and SOCS modulate in a fairly specific manner JAK/STAT signaling and/or STAT activity. Periodontal diseases are infectious-inflammatory conditions of the supporting tissues of the teeth associated with increased levels of proinflammatory cytokines, but there are no information regarding the role of these endogenous mediators of JAK/STAT during its course. The aims of this study included the evaluation of the expression kinetics of inducible negative regulators and their target proteins during the course of experimentally induced periodontal disease. 36 Wistar rats were divided into two groups: PD - experimental periodontal disease induced by two methods: ligature placement around the first mandibular molars and E. coli lipopolysaccharide (LPS) injections into the palatal gingival tissues of the maxillary molars, 3x/week, and Negative Control group. Rats were sacrificed 07, 15 and 30 days after disease induction for histological evaluation of periodontal inflammation and macroscopic analysis of alveolar bone loss. SOCS expression and the activation status of STAT1 and STAT3 were evaluated in gingival biopsies by real time PCR and Western Blot. Both disease models presented significant progressive bone loss from 7 to 30 days. Inflammation was evident and similar for all the periods in LPS injected sites; however, a decrease on severity at the end of the experimental period was observed in the ligature model. There was a significant (p<0.05) increase on SOCS1 and SOCS3 gene expression in PD compared to control... (Complete abstract click electronic access below)

As neoplasias mieloproliferativas (NMPs) Filadelfia negativo como a policitemia vera (PV), trombocitemia essencial (TE) e mielofibrose primária (MFP) são desordens clonais da célula tronco hematopoiética caracterizadas pela produção excessiva de células mielóides diferenciadas. Este fenômeno ocorre devido à uma mutação somática (JAK2V617F) que ativa a via JAK-STAT de transdução de forma constitutiva. Esta mutação é mais frequente na PV, ocorrendo em 95% dos casos, e em 50% dos casos de TE e MFP. Outro defeito genético que ocorre é a mutação no receptor de trombopoetina, MPL. As mutações em MPL podem ser germinativas ou somáticas e menos de 10% dos pacientes com TE e MFP apresentam essa alteração genética. Entretanto, grande parte dos pacientes com TE e MFP que não apresentam mutação em JAK2V617F ou MPL podem apresentar mutações somáticas no gene CALR. Em adição às mutações somáticas que causam mieloproliferação, outras alterações genéticas em genes que funcionam como reguladores epigenéticos são encontrados nas NMPs nos genes TET2, IDH1, IDH2 e ASLX1. Objetivo: Estabelecer um perfil genético em pacientes com NMP através da avaliação de mutações nos genes JAK2, CALR, MPL, IDH1, IDH2, TET2 e ASXL1 assim como estabelecer uma correlação laboratorial destas na PV, TE e MFP. Casuística e Métodos: Foram utilizadas amostras de sangue periférico de 104 pacientes que foram enviadas para o Laboratório de Biologia Tumoral do Serviço de Hematologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo para avaliação diagnóstica. Quinze dos 104 pacientes são de pacientes com PV (14,4%), 20/104 (19,2%) com MFP, 20/104 (19,2%) com TE e 49/104 (47,1%) com outras doenças hemtológicas. Foi feita a avaliação da prevalência de mutações somáticas, seja por sequenciamento ou análise de fragmentos, nos genes JAK2 (exon 12 e Y931C), MPL, IDH1, IDH2, CALR, TET2 e ASXL1. PCR-RFLP foi realizada para identificação de mutações em JAK2V617F. Resultados: A mutação JAK2V617F foi observada em 30 (28,8%) pacientes (12 PV, 11 TE e 7 MFP), a mutação JAK2 exon 12 foi observada em apenas um (0,96%) paciente com PV, mutação JAK2Y931C em 4 (3,8%) pacientes (1 PV, 2 TE e 1 MFP) e 8 (7,7%) pacientes apresentaram mutações em CALR (3 TE e 5 MFP). Mutações nos genes epigenéticos como IDH1 foram observadas em 9 (8,7%) pacientes (2 TE, 2 MFP, 1 SMD, e 4 pacientes com suspeita de NMP), mutações em IDH2 estão presentes em 5 (4,8%) pacientes (2 TE, 1 SMD/leucemia e 4 pacientes com suspeita de NMP), mutações em ASXL1 foram identificadas em 13 (12,5%) pacientes (1 PV, 3 TE, 2 MFP, 3 SMD/leucemias e 4 com suspeita de NMP) e finalmente, mutações em TET2 foram encontradas em 33 (31,7%) pacientes (3 PV, 5 TE, 4 MFP, 8 SMD/leucemias e 13 pacientes com suspeita de NMP). Além disso, no caso da PV, os pacientes que apresentam mutações em JAK2V617F apresentam valores aumentados de plaquetas (mediana de 5,41 x 105/mm3 plaquetas) em relação aos pacientes sem a mutação (mediana de 2,06 x 105/mm3 plaquetas), com diferença estatística (p=0,031). Pacientes do mesmo grupo que apresentam mutações em TET2 apresentam, opostamente aos com mutações em JAK2V617F, menores valores de plaquetas (mediana de 1,75 x105/mm3 plaquetas) em relação aos pacientes sem mutações no gene TET2 (mediana de 5,41 x 105/mm3 plaquetas), com diferença estatística (p=0,048). No caso da MFP, os pacientes que apresentam mutações em JAK2V617F apresentam valores maiores de leucócitos (mediana de 1,09 x104/mm3 leucócitos) do que os pacientes que não apresentam a mutação (mediana de 6,99 x103/mm3 leucócitos) com diferença estatística (p=0,046), já os pacientes que apresentam mutações no gene ASXL1 apresentam valores menores de hemácias (mediana de 2,43 x106/mm3 hemácias) em relação aos pacientes que não apresentam mutação (mediana de 3,71 x106/mm3 hemácias) com diferença estatística (p=0,042). Conclusão: O trabalho permitiu fornecer um perfil genético dos pacientes com NMP estudados. Além disso, é possível observar que algumas mutações epigenéticas podem influenciar em diferenças clínicas / Myeloproliferative neoplasms (MPNs) Philadelphia (Ph) chromosome negative, such as polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) are clonal disorders of hematopoietic stem cell characterized by increased proliferation of differentiated myeloid cells. This phenomenon occurs due somatic mutation (JAK2V617F) that constitutively stimulates the JAK-STAT signaling pathway. This mutation is more frequent in PV, around 95%, and between 50% in ET and PMF. Other genetic aberration can be observed in the thrombopoietin (TPO) receptor MPL. Mutations in MPL can be in the germline line or somatic and less than 10% of patients with TE or PMF would harbor this genetic alteration. Otherwise, patients with TE or PMF without JAK2V617F or MPL mutation could present somatic mutations in calreticulin (CALR). In addition to somatic mutations that cause myeloproliferation, other genetic alterations that function as epigenetic regulators were identified in genes as TET2, IDH1, IDH2 e ASLX1 in MPN. Objective: Establish genetic profile in patients with diagnosis of PV, ET, and PMF through genetic alterations in the following genes: JAK2, MPL, CALR, IDH1, IDH2, TET2 e ASXL1, and correlate those alterations with demographic characteristic of the study population. Casuistic and Methods: Peripheral blood samples from 104 patients referred to the Tumor Biology Laboratory of the Department of Hematology of Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo for diagnostic investigation were analyzed. Fifteen out 104 samples were from PV patients (14.4%), 20/104 (19.2%) in PMF, 20/104 (19.2%) in ET and 49/104 (47.1%) with other hematologic diseases. Identification of somatic mutations was made, either by direct sequencing or fragment analysis in JAK2 (exon 12 and Y931C), MPL, IDH1, IDH2, CALR, TET2 and ASXL1. PCR-RFLP was performed to identify JAK2V617F mutation. Results: JAK2V617F mutation was observed in 30 (28.8%) patients (12 PV, 11 ET and 7 PMF), JAK2 exon 12 in only one (0.96%) patient with PV, JAK2Y931C in 4 (3.8%) patients (1 PV, 2 ET and 1 PMF), and 8 patients (7.7%) presented CALR mutation (3 ET and 5 PMF). Mutations in the epigenetic genes as IDH1 were observed in 9 (8.7%) patients (2 ET, 2 PMF, 1 MDS and 4 patients with suspected MPN), IDH2 mutations were present in 5 (4.8%) patients (2 ET, 1 MDS/leukemia, and 4 patients with suspected MPN), ASLX1 mutations were identified in 13 (12.5%) patients (1 PV, 3 ET, 2 PMF, 3 MDS/leukemia and 4 with suspected MPN) and finally, TET2 mutations were present in 33 (31.7%) patients (3 PV, 5 ET, 4 PMF, 8 MDS/leukemia, and 13 with suspected MPN). In addition, patients with PV who harbor JAK2V617F have increased platelet counts (median 5.41 x 105/mm3 platelets) compared to those without the mutation (median 2.06 x 105/mm3 platelets, p=0.031). Patients in the same group with TET2 mutation, as opposed to those with JAK2V617F, presented low platelets counts (median of 1.75 x 105/mm3 platelets) compared to those without TET2 mutation (median 5.41 x 105/mm3 platelets, p=0.048). Presence of JAK2V617F in patients diagnosed with PMF have a greater number of leukocytes (median 1.09 x104/mm3 leukocytes) when compared to patients without the mutation (median 6.99 x 103/mm3 leukocytes, p=0.046). Patients with PMF who presented mutations in ASXL1 gene have a lower number of red blood cells (median of 2.43 x 106/mm3) compared to patients without mutations in the same gene (median 3.71 x 106/mm3, p=0.042). Conclusion: The present study allows us to provide a genetic profile of patients with MPN. Furthermore, it is possible to observe that some epigenetic mutations could influence in some clinical differences

Une étude théorique couplée à une partie expérimentale a été entreprise sur la dynamique de l'échange de ligand sur des surfaces de nanoclusters (GNC) dans le but de montrer qu'il était possible de contrôler les propriétés structurales et optiques de GNC à travers la composition de la couronne de ligand. Nos études de calcul ont été effectuées par la théorie fonctionnelle de la densité en chimie quantique (approche Kohn - Sham). Nous avons analysé les principales caractéristiques UV - Visible des spectres calculés par TD - DFT / niveau de CAMB3LYP pour les clusters métalliques Au13, Au25 et Au28 protégées par des ligands thiolate, chlorure, et phosphine. Nos résultats montrent qu'il est possible de régler l'énergie de la bande d'absorption la plus basse des clusters d'or par une répartition spécifique des ligands qui contrôle de fait la répartition des charges entre la couronne de ligand et le noyau métallique.En parallèle, nous avons synthétisé une série de clusters de composition Au25 (ATP)x (TP) 18 - x avec 4ATP (4 - aminothiophénol) et TP (thiophénol) par synthèse directe et par échange de ligands. Les mesures de spectroscopie de masse ESI - MS montrent que la nucléarité Au25 est préservée pour tous ces différents clusters. En revanche, l'échange de ligands TP par le DDT (1 - dodécanethiol) dans le mélange conduit à la formation de nanoparticules. Les mesures de spectroscopies IR confirment la présence de deux ligands différents sur la surface de l'or et les analyses SAXS montrent que nous avons une bonne corrélation entre la distance entre deux cœurs métalliques et la longueur du ligand de surface. / We performed a joint computational – experimental investigation of the dynamics of ligand exchange on gold nanoclusters (GNC) surface with the aim to understand how to control the structural and optical properties of GNC through the design of their ligand shell. Our computational studies were carried out in the framework of the Kohn – Sham implementation of density functional theory in quantum chemistry. We analyzed the main features of UV – Vis spectra computed at the TD – DFT / CAMB3LYP level for the Au13, Au25, and Au28 metallic cores protected by thiolate, chloride, and phosphine ligands. Our results show that it is possible to tune the energy of the lowest absorption band of gold clusters by ligand shell engineering in order to control the charge redistribution between ligand shell and metallic core.In parallel we synthesized a set of Au25(ATP)x(TP)18 – x clusters with different ATP/TP ratios using an adapted Demessence protocol by combining 4ATP (4 – aminothiophenol) and TP (thiophenol) ligands. ESI – MS measurements evidence that for these mixed ligand shells the Au25 nuclearity is preserved. However, the addition of the DDT (1 – dodecanethiol) ligand in the mixture leads to nanoparticle formation. FT – IR spectroscopy confirms the absorption of two different ligands on the gold surface and SAXS shows that we have a good correlation between the distance between two clusters and the length of the ligand protecting them.Finally, we carried out a comparison of the mode of binding and the structural and optical properties of the fully ligated PH3 and NHC GNC with metallic cores of different nuclearities.

Dans cette thèse nous avons étudié la mise en forme de matériaux carbonés par des méthodes électrochimiques pour des applications dans les domaines des capteurs et de l’énergie. Dans la première partie, l’électrochimie bipolaire, qui permet de réaliser des réactions électrochimiques sur un objet conducteur présent dans une solution et soumise à un champ électrique, a été utilisée pour générer des objets de type Janus. Ces objets asymétriques ont été modifiés à une extrémité par du poly(N-isopropylacrylamide (pNIPAM), un hydrogel sensible à la température, et par une peinture électrophorétique à l’autre extrémité. En contrôlant l’intensité du champ électrique ainsi que son temps d’application il a été possible de varier la longueur ainsi que l’épaisseur de l’hydrogel. Ces objets sensibles à la température, émettant de la lumière, ont des applications potentielles dans le domaine des capteurs ou dans le milieu médical.Dans la seconde partie, la mise en forme de carbone poreux pour des applications électrochimiques a été étudiée. La carbonisation de polymères contenant du zinc a été utilisé pour synthétiser du carbone micro/mésoporeux possédant ainsi une grande surface spécifique. Les polymères contenant du zinc ont été préparés à partir de différents types de ligands d’acide dicarboxylique par une méthode solvothermique. Ils ont ensuite été carbonisés pour obtenir des matériaux poreux avec des caractéristiques et des propriétés particulières. Ils ont été utilisés comme matériaux d’électrode pour des supercondensateurs, montrant des capacités élevées. De plus ils possèdent également une activité électrocatalytique à la réaction de réduction de l’oxygène. / In this thesis, the design of advanced carbon materials via electrochemical techniques and for electrochemical applications have been studied. In the first part, the concept of bipolar electrochemistry, which allows carrying out electrochemical reactions on a free-standing conductive object in an electric field, was employed to generate Janus-type objects. These objects are modified with a thermoresponsive hydrogel of poly(N-isopropylacrylamide) (pNIPAM) on one side and an electrophoretic deposition paint (EDP) on the other side. The results show that the length and the thickness of the hydrogel can be controlled by varying the electric field and the time of the experiment. The concept can be further generalized to other micro- and nanometer-sized objects, thus opening up perspectives for various applications.In the second part, the design of porous carbon structures for electrochemical applications was studied. The direct carbonization of non-porous zinc containing polymers was used to synthesize micro/mesoporous carbons with high surface area, pore volume. Non-porous zinc containing polymers with various types of dicarboxylic acid ligands prepared by solvothermal method were used as templates and starting materials. After carbonization porous carbons with various characteristics and properties were obtained. The synthesized porous carbon samples showed good electrochemical performance with high capacitance values. In addition, the derived materials exhibit excellent electrocatalytic activity with respect to the oxygen reduction reaction (ORR).

The main focus of the research work presented in the thesis is the understanding of structural and conformational reorganizations in hyperbranched and linear polymers. The thesis includes three different investigations: a) the design, synthesis, conformational reorganizations and self-assembly of hyperbranched polymers (HBPs), b) the Raman spectroscopic studies of the melting of polyethylene glycol (PEG), and c) the preparation of functionalized porous polymer films. HBPs are structurally imperfect analogues of the defect-free branched polymers called dendrimers. Dendrimers prepared using a stepwise polymerization methodology will carry all the unreacted B groups at the periphery and therefore modification of these peripheral units with suitable linear segments (e.g. PEG) would naturally generate a core-shell structure. On the other hand, in the case of HBPs, that contain a large number of linear defects, the B groups are distributed throughout the highly branched backbone, and hence modification of these units may not generate a core-shell configuration. However, our earlier studies have demonstrated the unimolecular micelle type behavior (core-shell) of PEG-ylated aromatic HB polyethers in solution. Hence, even though the PEG segments are randomly placed throughout the HBP backbone, the polymer achieves a core-shell configuration through conformational reorganizations; this suggests that the core of HBPs may be flexible and adaptable. In order to further examine this issue, we have investigated the self-assembly of these PEG-ylated HBPs. These polymers were found to organize into uniform nano-aggregates (Figure 1). More interestingly, these aggregates were found to restructure on mica surface when exposed to solvent vapor. This study confirmed the formation of core-shell domains in the PEG-ylated HBPs and the ability of the HB core to reconfigure. Figure 1. Restructuring of nano-aggregates formed from PEG-ylated HBPs on mica surface. As mentioned earlier, the precise control of the placement of functional groups would be impossible in the case of hyperbranched polymers (HBPs); most approaches would result in random placement of the functional entities as the growth of the polymer occurs in a statistically governed manner. Hence, creating Janus hyperbranched structures appears a daunting task. Janus is the name given for facially amphiphilic systems, such as a sphere made by joining a hemisphere with a hydrophobic surface and one with a hydrophilic surface. Since we have demonstrated that the HBP backbone is adaptable, it would be interesting to test the possibility of generating a Janus configuration through reorganization of peripheral segments. Peripherally “clickable” HB polyesters were synthesized using the transesterification polymerization methodology. The peripheral groups were modified with two dissimilar segments, PEG and docosyl (C22 alkyl, DOCO) chains, by azide-yne click reaction using the corresponding azides. The immiscibility of the chains along with the tendency of alkyl chain to crystallize was expected to cause self-segregation of segments at the periphery aided by the reconfiguration of HB core. Melting transition associated with alkyl segments was evident in the DSC thermogram. In addition, the melting enthalpies, normalized with respect to the weight of alkyl segments, were close to that of the C22 alkyl chain for three HBPs with different compositions of DOCO and PEG. This suggests complete phase segregation of segments at the periphery. The behavior of these HBPs at the air-water interface was also investigated. In this experiment a single layer of molecules, deposited at the air-water interface, is compressed and decompressed at a predetermined rate. The surface pressure variation during the compression-decompression cycle reveals the molecular organization at the surface. Stable and reversible isotherms were obtained for these HBPs; which suggested their Janus nature with PEG chains solvated in water and alkyl chains projecting out into air. A change in slope was observed in the isotherm and we have ascribed this to the crystallization of docosyl segments. In order to verify the assignment, quasi-static compression (QSC) experiments were performed. In QSC experiment, the monolayer was rapidly compressed to a required surface area and the drop in surface pressure, as a function of time at this fixed surface area, was recorded. During the rapid compression the molecules may not have adequate time to organize optimally and therefore, holding the area constant often leads to a drop in surface pressure with time. The total pressure-drop after the compression reflects the extent of molecular reorganizations; this, for example, could be due to the crystallization of alkyl segments. In the present study, the extent of pressure drop scaled with the composition of alkyl chains; this proves the assignment and supports the self-segregation of the peripheral segments to generate a Janus configuration (Figure 2). AFM images of the transferred monolayer allowed the estimation of monolayer height, which was roughly in agreement with the expected single molecule dimension. Small angle X-ray scattering experiments (SAXS) on HBPs demonstrated existence of a lamellar morphology in the solid state. Janus structures may be expected to exist as bilayers in the solid state. As expected, the lamellar dimensions estimated were twice the monolayer height observed in AFM images. This confirms the bilayer structure and the Janus configuration of HBPs. We have also investigated the self-assembly of these Janus HBPs in solution and it was found that disk-like aggregates were formed in methanol, whereas vesicles were formed in water by systems that had the appropriate balance of hydrophilic and hydrophobic segments. Figure 2. a) The structure of Janus HBP. b) A cartoon depiction of reconfiguration of randomly functionalized HBP to Janus. c) DSC thermograms showing clear melting and crystallization peaks. d) Langmuir isotherms for Janus HBPs. The change in slope is indicated by orange arrow. e) Quasi-static compression isotherms. The extent of pressure drop for different HBPs (inset). f) The SAXS pattern suggesting lamellar structure in the solid state and a proposed model (inset). It was clear from the study that the incompatibility between segments is the primary reason for the reconfiguration. In order to test the generality of the approach, we have prepared two other types of HBPs: one with PEG and fluorocarbon (FC) and the other with DOCO and FC. These segments were chosen because of their mutual incompatibility. In both these systems, the formation of Janus configuration was similarly confirmed by DSC, SAXS and Langmuir isotherm studies. We have extended these studies to HBPs functionalized with three different immiscible segments (tripod HBPs) namely, PEG, DOCO and FC; in the case of PEG two different molecular weights were used: PEG350 and PEG1000; the latter exhibits a strong tendency to crystallize. Melting transition associated with each of the segments was observed in the DSC experiments. The normalized melting enthalpy of the docosyl domain was found to vary substantially depending on the composition and configuration of HBPs (Janus or tripod). Considering the variation of these values it was concluded that the presence of an immiscible amorphous segment, like PEG350, enhances the phase separation and crystallization of DOCO by providing more flexibility to the core. The presence of one or more immiscible but crystallizable segments (e.g. FC or PEG1000), on the other hand, lead to less effective crystallization. Interfacial behavior of the tripod HBPs, that carry the hydrophilic PEG units, were also studied using Langmuir trough. The tripod HBPs formed clear monolayer at the air-water interface at higher surface pressures (40 mN/m); where the hydrophobic segments, FC and DOCO, oriented away from water into air and PEG segments remained solvated in water. Due to the incompatibility between FC and DOCO, it may be anticipated that these segments also form phase segregated domains. While the AFM images allowed the estimation of monolayer thickness, these domains could not be resolved in the images. Interestingly, the transferred monolayer appeared smoother after thermal annealing. To investigate the reason, contact angle (CA) measurements were performed. It is widely accepted that the contact angle is very sensitive to the structure and chemistry of a few angstrom thick region of the top surface of a monolayer. The water CA on the monolayer before thermal annealing was 84˚ while it increased to 104˚ after annealing. This behavior was attributed to the effective phase segregation and crystallization of FC and DOCO domains. The crystallization causes CF3 end groups to orient normal to the surface and hence increases the water contact angle. The receding contact angle, on the other hand, was found to be substantially smaller (45˚); this large contact angle hysteresis could be indicative of chemical heterogeneity on the surface, and it has been suggested that the presence of small domains of low surface free energy could indeed result in such large contact angle hysteresis. Interestingly, when the film was aged under ambient conditions for 12 h, the contact angle dropped to the initial value of 84˚; re-annealing the film at 75˚C for 12 h again raised the contact angle back to 104˚. This reversible annealing-aging behavior reveals dynamic nature of the monolayer that permits gradual restructuring at the surface (Figure 3). Self-assembly of these tripod hybramers resulted in the formation of unique aggregated structures. Morphology of these aggregates, observed in AFM images, were different from those formed by Janus hybramers. These results provide a clear evidence for the segregation of all the three segments into individual domains -the hypothesized tripod hybramer configuration. Figure 3. AFM images of transferred monolayer of tripod HBP before and after thermal annealing. Contact angle (CA) measured on the corresponding monolayers are shown in the inset. A model proposed for explaining the variation in the appearance of the monolayer (image) and the CA is also provided in the figure. Effective phase segregation of FC and DOCO is proposed the reason for the change. Another interesting question we have investigated as a part of the thesis is the molecular mechanism of melting of PEG. Crystallization of polymer is hypothesized as polymer chains moving down the free energy landscape though sequence of conformational reorganizations into an ordered crystalline (lamellar) state. Such conformational reorganizations also occur during melting. We have attempted to probe conformational dynamics in PEG during its melting using Raman spectroscopy, polarized optical microscopy, DSC and DFT calculations. Single crystal X-ray and vibrational spectroscopic studies have demonstrated helical configuration of PEG chains in the solid state with a gauche O-C-C-O, trans C-C-O-C and trans C-O-C-C conformations. There are three ways in which the PEG-helix can change during heating; one where C-O single bond rotates, second where the C-C rotates and third where both rotate, but the barriers to these rotations are different, as was established by earlier NMR studies. The intensity ratio of two Raman bands, I2880/2850, was found to be sensitive to the configuration about O-C-C-O units along the polymer backbone. An invariant I2880/2850 ratio of ~0.78 was ascribed to the consistent C-C dihedral angle (gauche configuration) during melting. This agrees with the well-known “gauche effect” in ethylene glycol. Prominent spectral changes were also observed in the methylene rocking region (~800 cm-1). In order to gain insight into the new conformers formed during melting, we carried out Gaussian calculations using model conformers. Among helical models considered the heptamer and tetradecamer helix models were found to be most suitable for the solid state helical structure of PEG. The Gaussian calculations using different model conformers revealed that the Raman band at 810 cm-1 corresponds to CH2 rocking vibrations of the gauche C-C-O-C units along the polymer chain; while the band at ~1500 cm-1 region was characteristic of O-C-C-O trans conformer. This agrees with the earlier assignment of 1500 cm-1 band to CH2 scissoring vibrations of PEG in an all-trans configuration. Interestingly, no peak was observed in the 1500 cm-1 region of the Raman spectrum during melting of PEG, but new peaks appeared in the 810 cm-1 region (Figure 4). Hence, it was concluded that C-C bond rotation does not occur during melting of PEG. Thus, our study confirmed that C-O single bond rotation is the molecular mechanism of PEG melting. Figure 4. a) Raman spectra of PEG at different temperatures. b) helical model considered for the study. c) Comparison of the Raman spectrum of molten PEG (2) with calculated spectra of two different models. The dynamics of a system, where the successive changes occur as a function of external perturbation, can be studied using 2D correlation spectroscopy (2D-CoS). 2D-Raman Correlation spectroscopy was employed to study the molecular structural dynamics during the melting of PEG. Conventional rules of 2D-CoS were used to retrieve the order in which the vibrational bands respond to temperature. Vibration at 934 cm-1 corresponding to the amorphous domain of PEG solid state was found to respond to temperature first. As the temperature of the system rises, CH2 rocking (1280 cm-1), CH2 wagging (1472 cm-1) and CH2 scissoring (1124 cm-1) vibrations becomes active and this provides flexibility to the chain. As the polymer chain gains adequate energy (at particular temperature) bond rotation takes place resulting in the transformation of a few TGT segments to GGG segments. On increasing the temperature further more C-O bond rotations occur, leading to destruction of lamellar domains and eventually PEG melts. The final study presented in this thesis deals with the generation of functionalized porous polymer films. Condensation of water droplets during solvent evaporation from a polymer solution, under humid conditions, is known to generate uniformly porous polymer films. We have investigated the possibility of pore formation through water phase separation strategy (Figure 5). In the presence of added surfactants (SDS and CTAB in the present study), the interface of phase separated water droplets and the polymer would naturally become lined with the surfactants and consequently the internal walls of the pores generated, upon removal of the water, could become decorated with the hydrophilic head groups of the surfactant molecules. The size of the pores and their distribution were examined using AFM and IR imaging methods. We have demonstrated that the ATR-IR imaging is an efficient method for analyzing a few nanometer thick surface section of the polymer film (Figure 5e). It was observed that the presence of surfactant is important for the pore formation and irregular pore formation resulted in the absence of surfactant. In addition, both the surfactant concentration and the relative volume fraction of the surfactant solution were found to govern the size of the pores formed. Cloud point measurements suggested that the occurrence of surfactant facilitated the phase separation of water. Although IR imaging possessed inadequate resolution to confirm the presence of surfactants at the inner surface of pores, exchange of the inorganic counter-ion, such as the sodium-ion of sodium dodecyl sulphate (SDS), with suitable ionic organic dyes permitted the unequivocal demonstration of the presence of the surfactants at the interface with confocal fluorescence microscopy (Figure 5f). Figure 5. Schematic depiction of a homogeneous polymer solution (sky blue) in THF–water; yellow lines with red dots depicts dissolved surfactant molecules. (b) Initial stages of water droplet formation that are stabilized by surfactants. At this stage some polymer precipitation may also occur at the interface (depicted by the dark ring around the droplet). (c) Some droplet coalescence and continued formation of new water droplets leads to a slightly broad size distribution (coalescence and redistribution could be retarded due to the high solution viscosity). (d) Complete removal of THF followed by water generates the internally functionalized porous structures. e) IR spectral Image of the porous film. f) confocal fluorescence image of the porous film where the inner surface was functionalized with Rhodamine B. (For figures pl refer the pdf file)

The exploitation of colloidal building blocks with morphological and functional anisotropy facilitates the generation of complex structures with unique properties, which are not exhibited by isotropic particle assemblies. Herein, we demonstrate an easy and scalable bottom-up approach for the programmed assembly of hairy oppositely charged homogeneously decorated and Janus particles based on electrostatic interactions mediated by polyelectrolytes grafted onto their surface. Two different assembly routes are proposed depending on the target structures: raspberry-like/half-raspberry-like or dumbbell-like micro-clusters. Ultimately, stable symmetric and asymmetric microstructures could be obtained in a well-controlled manner for the homogeneous–homogeneous and homogeneous–Janus particle assemblies, respectively. The spatially separated functionalities of the asymmetric Janus particle-based micro-clusters allow their further assembly into complex hierarchical constructs, which may potentially lead to the design of materials with tailored plasmonics and optical properties.

De nombreux systèmes nanoparticulaires ont été développés pour modifier la délivrance de molécules par la voie cutanée. Dans ce travail de thèse, nous nous sommes intéressés aux nanoparticules lipidiques type Janus (JNP), une forme galénique innovante caractérisée par la combinaison de deux compartiments, de polarité chimique opposée, un compartiment aqueux accolé à un compartiment lipidique. L’objectif principal a été la caractérisation des JNP. La spectroscopie ATR-FTIR a permis de mettre au point un descripteur IR permettant de suivre la stabilité physique des JNP à l’air libre et en fonction du temps. Le même descripteur a permis de suivre leur devenir à la surface de la peau, et de constater une pénétration significative à partir de 3 heures d’application. Nous avons prouvé que l’AFM-IR est une technique prometteuse pour étudier la nanostructure de la peau. De plus, elle a permis de montrer qu’après 24 heures d’application, les JNP se sont accumulées dans les premières couches du SC avec un gradient dans les couches plus profondes du SC. En revanche, il n’a pas été possible de déterminer si elles ont pénétré à l’état intact ou dégradé. Les JNP semblent avoir une influence sur la pénétration cutanée de l’acide hyaluronique, elles ont permis une augmentation significative de son flux de pénétration. La caractérisation de la phase lipophile des JNP par différentes techniques (LC-MS, DLS, Cryo-TEM, diffraction des rayons X…) a permis de mieux comprendre leur instabilité aux températures élevées (32°C - 43°C). / Many nanocarriers have been developed to improve the delivery of molecules into the skin. In this PhD thesis, we are interested in lipid-based Janus nanoparticles (JNP), an innovative galenic form characterized by the combination of two compartments of opposite chemical polarity, an aqueous compartment associated to a lipid compartment. The main aim was the characterization of JNP. ATR-FTIR spectroscopy allowed to identify an infrared descriptor to follow the physical stability of JNP in open air and over time. The same descriptor allowed to follow their behavior on the surface of the skin, and to note a significant penetration from 3 hours of application. AFM-IR has been shown to be a promising technique for studying the nanostructure of the human skin. In addition, it has shown that after 24 hours of application, JNP were accumulated in the first layers of the SC with a gradient in the deeper layers of the SC. However, it was not possible to conclude if they have penetrated in the intact or degraded form. JNP seem to have an influence on the cutaneous penetration of the hyaluronic acid, they allowed a significant increase of its penetration flux. The characterization of the lipophilic phase of JNP by different techniques (LC-MS, DLS, Cryo-TEM, X-ray diffraction...) allowed to better understand their instability at high temperatures (32°C - 43°C).

The main subject of this thesis is to examine thermo-osmotic flows, which occur on interfaces of non-uniform temperature. Such thermo-osmotic flows are purely non-thermal equilibrium phenomena. Along the non-isothermal interface, specific interaction of a liquid and its solutes with a boundary vary in strength across the interface, according to the local temperature. This boundary can be a solid, a membrane or a phase boundary. The flow is thereby continuously pumping fluid across the interface in direction of the local temperature gradient, resulting in an extended flow pattern in the bulk due to mass conservation. In a system containing particles and heat sources in a liquid under spatial confinement, the thermo-osmotic flow may drive particles in a directed manner, or can lead to collective phenomena. To approach this broad topic of (self-)thermophoresis and collective motion of active particles and quantify the role of the thermo-osmotic flow upon the latter effects, different experiments have been performed: The first experiments aim to quantify the thermo-osmotic flow at a non-isothermal liquid/solid interface for two fundamentally different substrate properties. Further, the bulk flow was investigated for two different systems. The form and spatial extension of this bulk flow pattern depends sensitively on the form of the container and the interface, as well as on the thermo-osmotic flow. The first system is a liquid film confined between two planar glass cover slips. The second case is a Janus particle immobilized on one of the glass slips. In the first case, the non-uniform temperature profile is generated by optical heating of a nanometer sized gold colloid, and in the second case, the heat source is the Janus particle. The bulk flow pattern consists, for the second case, of the flow pattern created by the glass cover slips and the one created by the Janus particle. The following experiments are focusing on the dynamics of mobile self-thermophoretic Janus particles. In particular, their dynamics and the contributions of the thermo-osmotic flow to the interaction of multiple active particles are investigated. To investigate those particles under controlled conditions and examine their interactions at low concentrations for an effectively unlimited amount of time, a real-time feedback algorithm was co-developed to gain control of the motion of multiple active particles simultaneously, called ”photon nudging”. With the help of this method, first experiments have been performed to quantify the dynamics of a Janus particle located close to a heat source.

Die Fähigkeit künstlicher Mikroschwimmer, auf äußere Reize zu reagieren und deren mechanistische Ursprünge, gehören zu den umstrittensten Fragen der interdisziplinären Wissenschaft. Die Erzeugung chemischer Gradienten ist dabei eine technische Herausforderung, da sie aufgrund von Diffusion schnell abflachen. Inspiriert von ‘Stop-flow’ Experimenten aus der chemischen Kinetik zeigen wir, dass die Erzeugung eines mikrofluidischen Gradienten durch Kombination mit einer Druckrückkopplungsschleife zur präzisen Kontrolle des Stoppens erfolgen kann. Das ermöglicht es uns, die mechanistischen Details der Chemotaxis von künstlichen katalytischen Janus-Mikromotoren zu untersuchen. Wir stellen fest, dass diese Kupfer-Janus-Partikel eine chemotaktische Bewegung entlang des Konzentrationsgradienten sowohl in positiver als auch in negativer Richtung zeigen, und wir demonstrieren die mechanische Reaktion der Partikel auf unausgewogene Widerstandskräfte, die dieses Verhalten erklären.