Rozprawy doktorskie na temat „LMWG”

Ce travail consiste en une étude théorique de la formation et de la structure d’organogels supramoléculaires. Ces gels sont obtenus par dispersion dans un solvant organique de molécules de bas poids moléculaire (Low Molecular Weight Gelators, LMWGs). Ces molécules LMWG s'auto-assemblent de manière non covalente, par exemple par liaison hydrogène, empilement π, interactions de Van der Waals, etc. et forment finalement des réseaux fibrillaires. Ces réseaux enchevêtrés piègent mécaniquement le liquide, principalement par tension superficielle, générant un gel. La description précise des phénomènes de gélation de solvants organiques reste encore partielle, laissant des questions ouvertes qui empêchent de prédire si un candidat LMWG donné sera capable de gélifier un certain liquide d'intérêt. Si, par la modélisation, des règles de conception pouvaient être établies entre la structure chimique d'un LMWG et ses propriétés de gélification, il serait possible de concevoir des LMWG pour des liquides spécifiques, tout en améliorant la compréhension de la formation des organogels. Pour répondre à ces objectifs, ce travail étudie des familles de LMWG chimiquement différentes, dans le but de corréler leur structure chimique avec leur comportement de gélification. L'approche suivie dans cette thèse consiste à modéliser l'auto-assemblage de différentes séries de LMWG (d’une part des composés bisamide-cyclohexane, d’autre part des composés de thiazole, tous porteurs de chaînes alkyles de différentes longueurs), dans le but de comprendre la formation des fibres de gel et de déterminer leur structure. La plupart des LMWG que nous avons étudiés cristallisent pour former des gels, et pour de tels systèmes cristallins, notre méthodologie de modélisation débute par la prédiction de la structure cristalline des fibres de gel, en combinant la génération de cellules cristallines et des si-mulations des diagrammes de diffraction des rayons X sur poudres. Ensuite, nous déterminons la morphologie des cristaux en utilisant les principes de cinétique de croissance. Enfin, nous caractérisons la capacité de gélification des fibres cristallines en utilisant des paramètres d'énergie de surface. Il est important de souligner que nos activités de modélisation ont été menées en interaction très étroite avec les efforts expérimentaux correspondants entrepris dans les groupes du Prof. Laurent Bouteiller (Sorbonne Université) et du Prof. Pierre-Antoine Albouy (Université Paris-Sud). Leurs résultats d'expériences de gélification, de diffraction des rayons X sur poudres et de caractérisation SEM ont été comparés à nos données de modélisation
This work deals with supramolecular organogels. These gels are obtained by dispersing in the organic solvent low molecular weight molecules (Low Molecular Weight Gelators, LMWGs), which are not soluble at room temperature and form a suspension. This suspension is heated, achieving solution, and cooled down back to room temperature where LMWG molecules self-assemble in non-covalently bonded Self-Assembled Fibrillar Networks (SAFiNs), e.g., by hydrogen-bonding, π-stacking, Van der Waals interactions, etc. This entangled network traps mechanically the liquid, principally by surface tension, trigger-ing a gel state. A precise description of the phenomena remains partially unknown, leaving open questions that still impede to predict beforehand whether a given LMWG candidate will be able to gelate a certain liquid of interest. If design rules could be established between the chemical structure of a LMWG and its gelation properties, it could be possible to design LMWGs for specific liquids of interest while providing insight about organogel formation. Thus, this work investigates sets of chemically diverse LMWG families, with the aim of correlating their chemical structure with their corresponding gelation behavior. The approach followed in this thesis consists in modelling the self-assembly of different series of LMWGs, bisamide-cyclohexane compounds and thiazole compounds with alkyl chains of different lengths, with the aim of understanding the formation of the gel fibers and determining their structure. Most of the LMWGs that we have studied crystallize to form gels, and for such crystalline systems, our methodology starts with a Crystal Structure Prediction (CSP) of the gel fibers, combining crystal cell generation and powder X-ray diffraction simulations. Then, we determine their crystal morphology using growth kinetics principles, to finally characterize the gelation ability of the gel fibers using surface energy parameters. Our modelling activities have been carried out in very close interaction with corresponding experimental efforts undertaken in the groups of Prof. Laurent Bouteiller (Sorbonne Université) and Prof. Pierre-Antoine Albouy (Université Paris-Sud). Their results of gelation experiments, powder X-ray diffraction and SEM characterization were compared with our modelling data

La résistance aux antibiotiques est une réalité à laquelle nous devons faire face. La résistance bactérienne aux antibiotiques peut être conférée par plusieurs mécanismes, dont la surexpression de pompes à efflux, certaines appartenant à la superfamille des transporteurs ABC (“ATP-binding cassette”). Les transporteurs ABC sont des protéines omniprésentes qui utilisent l'hydrolyse de l'ATP pour pomper une large gamme de substrats. Ils sont également responsables du développement des phénotypes de résistance à de multiple drogues dans les cellules cancéreuses et les microorganismes pathogènes.L'exportateur bactérien ABC BmrA (“Bacillus multidrug resistance ATP”), est homologue à ABCB1, un transporteur humain impliqué dans les phénotypes de résistance dans les cellules cancéreuses. Avec une connaissance approfondie de sa surexpression et de sa purification, BmrA est un archétype utile pour obtenir des informations sur le fonctionnement des transporteurs ABC de multiples drogues. Notre objectif est de déchiffrer les changements conformationnels associés au transport des médicaments.Nous avons montré que BmrA existe dans au moins deux conformations différentes, dans des micelles de détergent ou reconstitué dans des nanodisques. En l'absence de ligand (forme apo), différentes partie de BmrA fixe rapidement du deutérium comme le montre l'échange hydrogène deutérium couplé à la spectrométrie de masse (HDX-MS). La forme piégée par l'ADP induite par le vanadate montre une grande protection globale contre l'incorporation de deutérium. De plus, il a été observé que BmrA dans les nanodisques présente un profil de deutération différent en présence de médicament, indicatif d'une nouvelle conformation intermédiaire. De plus, en utilisant deux mutants affectés dans différentes étapes du cycle catalytique, il a été montré comment BmrA change de conformations au cours du cycle d'export des médicaments. Les résultats obtenus à partir de la diffusion de neutrons aux petits angles (SANS), brossent un tableau similaire et renforcent les résultats obtenus sur le cycle catalytique de BmrA.Ces résultats conduisent à une meilleure compréhension des changements de conformation de BmrA qui s’opèrent pour permettre le phénotype de résistance aux médicaments
Antibiotic resistance is not the story of the future but a reality today. Bacterial resistance to antibiotics can be conferred by several mechanisms, including the overexpression of dedicated efflux pumps, some of them belonging to the ABC (“ATP-binding cassette”) transporters superfamily. ABC transporters are ubiquitous proteins that use ATP hydrolysis to pump a wide range of substrates. They are also responsible for the development of MDR (“MultiDrug Resistance”) phenotypes in cancer cells and pathogenic microorganisms.The bacterial ABC exporter BmrA (“Bacillus multidrug resistance ATP”), is structurally and functionally close to ABCB1, a human transporter involved in MDR phenotypes in cancer cells. Together with extensive knowledge in its overexpression and purification, BmrA is a useful archetypical transporter to gain information on the functioning of multidrug ABC transporters. Our goal is to decipher the conformational changes associated with drug transport.We showed that BmrA exists in at least two different conformations, in detergent micelles or when reconstituted in nanodiscs. In the absence of ligand (apo form), BmrA gets quickly exchanged with deuterium as shown by Hydrogen Deuterium Exchange Coupled to Mass Spectrometry (HDX-MS). The vanadate-induced ADP trapped form shows a large overall protection against deuterium incorporation. Moreover, it was observed that BmrA in nanodiscs shows a different deuteration profile in the presence of drug, indicative of a new intermediate conformation. In addition, using two different catalytic mutants of BmrA, that are trapped in two opposite conformations of the catalytic cycle, it was shown how BmrA changes conformations during the drug export cycle. The results obtained from Small Angle Neutron Scattering (SANS), on WT BmrA and the mutants, paint a similar picture and strengthen the results obtained on the catalytic cycle of BmrA.These results could potentially lead to a better understanding of the structural basis of MDR

Mortality from cardiovascular disease in patients on chronic hemodialysis (HD) is 10 to 20 times greater than in the general population. One major risk factor is renal dyslipidemia, characterised by an impaired catabolism of triglyceride (TG)-rich lipoproteins with accumulation of atherogenic remnant particles. A contributing factor may be derangement of the lipoprotein lipase (LPL) system, the major lipase in the catabolism of TG-rich lipoproteins. The functional pool of LPL is located at vascular surfaces, and is released by heparin into the circulating blood and extracted and degraded by the liver. Unfractionated heparin (UFH) is commonly used during dialysis to avoid clotting in the extracorporeal devices, but is increasingly replaced by various low molecular weight heparin (LMWH) preparations. Plasma LPL activity is usually lower after injection of LMWH which is therefore said to release less LPL and cause less disturbance of lipoprotein metabolism than UFH. However, animal studies have revealed that LMWH is as efficient as UFH in releasing LPL but is less efficient in retarding hepatic uptake. The aim of this study was to explore the effects of UFH and a LMWH (dalteparin) on LPL activity and TG concentrations in HD-patients compared with healthy controls, matched for age and gender. A disturbed LPL system might contribute to an impaired lipoprotein metabolism, and hence, an aggravated cardiovascular condition. An 8-hour primed infusion of UFH to controls gave rise to an initial peak of LPL activity within 30 minutes. The activity then dropped by almost 80% over the next two hours and levelled off to a plateau that corresponded to 15% of the peak level. When UFH was infused to HD-patients the curve for LPL activity resembled that for controls, but was reduced by 50% during the peak, while the plateau activities were comparable. The interpretation was that the functional pool, represented by the initial peak, was impaired in HD-patients, while the production of lipase molecules, reflected by the plateau, was only marginally reduced. During the peak of LPL activity TG decreased in both groups, but less in HD-patients, as was expected from the lower circulating lipase activity. During the plateau phase with low lipase activity, TG increased towards and beyond baseline values. When dalteparin was infused, the same pattern of plasma LPL activity was observed, although remarkably reduced. In controls the peak was only 30% and the subsequent plateau 40% compared with the activities during the UFH infusion. A bolus of UFH given when the LPL activity had levelled off to a plateau brought out about the same amount of activity, regardless of whether dalteparin or UFH had been infused. The conclusion was that both heparin preparations had reduced endothelial LPL to a similar extent, but that dalteparin less efficiently retarded the hepatic uptake of the enzyme. As a consequence to this, TG tended to reach higher levels after the dalteparin infusion. The LPL activities were further reduced in HD-patients during infusion with dalteparin, the peak was only 27% and the plateau 35% compared with the activities when UFH was infused. There was no decrease in TG, but rather a continuous increase, suggesting a profound depletion of functional LPL. In another study in HD-patients, two anticoagulation regimes based on present clinical practice were compared, and the doses were adjusted to the respective manufacturers recommendation. UFH was administered as a primed infusion, whereas dalteparin was given only as a single bolus pre-dialysis, not followed by an infusion. The results were in line with those in the experimental studies and indicate that also in the clinical setting LMWH interferes with the LPL system as least as much as an infusion of UFH does, and temporarily impairs lipolysis of TG. This interference might, in consequence, contribute to an aggravated cardiovascular condition in HD-patients.

Protein tyrosine phosphorylation in eukaryotes is a key mechanism for cellular control, since it is involved in several processes, such as cellular metabolism, proliferation, differentiation and oncogenic transformation. A fine balancing of cellular protein tyrosine phosphorylation levels is determined by regulating the activities of protein-tyrosine kinases and/or protein-tyrosine phosphatases (PTPs) (Alonso et al., 2004). The PTP superfamily comprises almost 70 enzymes that, despite very limited sequence similarity, share a common CX5R active-site motif and an identical catalytic mechanism. LMW-PTPs are a group of cytosolic enzymes of 18 kDa that are widely expressed in different tissues. They are represented by two most abundant isoforms (arised from mutually exclusive alternative splicing), named fast and slow, according to their electrophoretic mobility (Tabernero et al., 2008; Wo et al., 1992; Dissing et al., 1991; Xing et al., 2007). LMW-PTP interacts with several receptor tyrosine kinases and docking proteins that may be involved in cancer progression, but the identification of functionally relevant interactors is not yet conclusive. Several works have shown how LMW-PTP overexpression is associated with human tumorigenesis, especially in breast, colon and kidney. In fact, results obtained with a wide array of human carcinomas indicate a significant increase in the expression of LMW-PTP in tumor tissue and a correlation between higher expression of LMW-PTP on one hand and worse prognosis and reduced survival on the other (Malentacchi et al., 2005). LMW-PTP acts also as a positive regulator of tumor onset and growth in in vivo animal models. (Chiarugi et al., 2004). Moreover there are many findings that LMW-PTP plays a key role in chemoresistance: Ferreira et al., demonstrated how the phosphatase overexpression induce resistance towards vincristine and imatinib, in a leukemia cell lines (Ferreira et al., 2012). Our studies were conducted on a Melanoma cell line, A375, to study in depth the role of LMW-PTP in skin tumor onset, in order to elucidate the molecular mechanism and pathways in which this enzyme is involved and also in the view of identifying new possible therapeutic targets. Using transient silencing technique, we tested the apoptotic response of A375 cells: citofluorimetric analysis showed that upon phosphatase silencing, treatment with 5FU increase cell mortality up to 50%. In agreement with these results, western blot analysis of apoptotic markers (Caspase3, Bcl2 and Bim) confirmed that LMW-PTP silencing sensitize cancer cells towards chemotherapic drugs. Looking for a link between LMW-PTP and apoptosis, we speculate that the phosphatase exerts its action through Cav1-Bcl2 pathways. In fact the phosphorylation on Tyr14 of Cav-1 leads to Bcl2 degradation, increasing apoptosis. Our data demonstrate how LMW-PTP knocking-down lead to Cav-1 Tyr phosphorylation, and Bcl-2 down-regulation. Several studies have demonstrated how tumor cells can develop chemoresistance increasing the activity or the expression level of membrane transporters that actively expel chemotherapy drugs from inside. Our experiments suggest that LMW-PTP may confer resistance against anti-tumoral drugs, increasing the activity of some of these membrane transporters, thus limiting the toxic effect of chemotherapy. One of the most important treatment for Melanoma is Radiotherapy. Using therapeutic doses of radiation (2Gy), we demonstrated that silenced cells were more responsive to this treatment, with respect to control samples, confirming that LMW-PTP plays a key-role not only in chemio- but also in radio-resistance. Furthermore resistant cancer cells, usually, show common phenotypes: one of this features is self-renewal capability. Colony formation assay demonstrates that melanoma cells are able to reform new colonies, even when cells are exposed to a damage, such as 5FU treatment or 2Gy irradiation. When LMW-PTP is knocked-down and cells treated with 5FU or radiated, we observed no colony formation: we can assume that LMW-PTP silencing leads to the loss of some characteristics of self- renewal, fundamental for metastasis. LMW-PTP exerts its action also through some molecules implicated in cell migration and adhesion. Adhesion and Detachment assay showed that LMW-PTP silencing lead cells to be more adherent to a substrate. Accordingly Wound Healing Assay demonstrated that melanoma cells are able to migrate and refill the wound very quickly. We tested also the Invasiveness of A375 cells: silenced cells showed a decreased ability to invade, respect to untreated cells: in fact LMW-PTP down-regulation lead to a MMP-9 reduction. Considering the importance of LMW-PTP in tumor onset we investigate the possibility to inhibit this enzyme as a new therapeutic approach.. Previous work demonstrated that a natural compound, Morin, is an enzymatic inhibitor of LMW-PTP. Morin is not toxic for Melanoma cells, but its combination with 5FU, causes a strong increase of apoptotic cells. Interestingly, this sensitization is not reproducible in non tumoral cell line, such as C2C12 myoblast: this co-treatment could be specific for cancer cells. Furthermore our studies demonstrate that Morin doesn’t act only as an inhibitor of LMW-PTP: western blot analysis showed that the flavonol lead to phosphatase degradation, in a dose and time-dependent manner. This down-regulation may be due to different mechanism, but since this effect start 2h after Morin incubation, we hypothesized that a proteasome activation may be involved. Incubation with Morin together with a proteasome inhibitor (MG132), confirmed our hypothesis. The enhancement of chemotherapic action, obtained with Morin is reproducible even with Radiation therapy. A375 cells pre-treated with Morin and then radiated with 2Gy, decrease dramatically their viability. Moreover irradiation exposition didn’t influence self-renewal capability of Melanoma cells: on the contrary, Morin treatment before irradiation is able to affect the formation of new colonies after the treatment. LMW-PTP is involved in cell-adhesion, migration and invasion: in fact Morin can affect this markers. After Morin treatment Melanoma cells are less able to migrate and invade; furthermore cells increase the number of focal adhesions. To better understand the role of LMW-PTP in tumor onset, we looked for new substrates. To analyze this aspect we studied the phospho-proteomic profile of silenced Melanoma cells. Through these analysis we identified different proteins showing an higher levels of Tyr phosphorylation, upon LMW-PTP silencing. One of the new substrates identified with this experimental approach is Annexin-A1, a protein involved in apoptosis: this finding suggest a further possible link between LMW-PTP and apoptosis resistance, a link that will be further investigated. More interestingly, four of the proteins identified with the proteomic analysisbelong to glycolysis pathway, PKM2, α-Enolase, GAPDH and TIM. To confirm this results we performed immune and co-immunoprecipitations: these analyses confirmed that the mentioned enzymes get in contact with LMW-PTP and, presumably, are direct substrates of the phosphatase. It is well known that the “Warburg effect” causes alteration in cancer cell energetic metabolism, leading cells to consume large quantity of glucose, metabolizing it predominantly through glycolysis, and producing high level of lactate. Considering that four of these substrates are involved in glycolysis pathway, we tested some metabolic parameters. When LMW-PTP is silenced A375 cells consume less O2, and consequently their glucose up-take is higher, producing more lactate respect to controls. Pyruvate kinase controls the final and rate-limiting reaction of glycolysis: PKM2 undergoes conformational conversion between a tetrameric/full active and a dimeric/less active state. The conversion to a less active state, induced by Tyr phosphorylation, confers to PKM2 “non-metabolic” abilities. Indeed, PKM2 translocates into the nucleus and acts as a transcriptional co-activator of β-catenin and hypoxia-inducible factor 1α (HIF-1α), cooperating to control cell proliferation and glucose catabolism, respectively. Western Blot Analysis of the Glucose transporter GLUT-1 and Hesokinase II, confirmed our previous data. When LMW-PTP is down-regulated both proteins had an increased expression level, explaining, at least in part, the glycolytic metabolism showed by silenced cells. Moreover LMW-PTP influences not only the Tyr phosphorylation state of PKM2, but also its expression level: in fact when the phosphatase is down-regulated PKM2 protein level is higher. In conclusion, our results demonstrated that LMW-PTP plays a key role in chemo and radio-resistance acquisition of Melanoma cells: in fact when the phosphatase is knocked-down cells are more responsive to therapy. Considering that gene silencing cannot be used in patients, the discovery that Morin is able to reproduce the same phenotype, open new possibilities for therapies. Moreover LMW-PTP seems to influence metabolism of Melanoma cells, a parameter often deregulated in cancer cells. Further studies will be conducted to better characterize the role of this enzyme, and its role in the regulation of tumor metabolism.

In this work, the influence of different cooling rates (5, 20, 50 and 100 °C/s) on the microstructure and mechanical properties of Laser Metal Wire Deposited (LMwD) Ti-6Al-4V was investigated, this was done using a thermal-mechanical physical simulation system (Gleeble 3800, DSI). Two different soak times above β transus (held at 1100 °C), 5 and 40 s, were used and after cooling to 150 °C, the samples were tensile tested. The samples were characterized with optical microscopy (OM) and scanning electron microscopy (SEM) and hardness testing. The results were then compared, both with each other and with two reference samples, that were only heated to 150 °C and then tensile tested. It was found that for the lowest cooling rate, 5 °C/s, the microstructure had transformed from a basketweave α microstructure to a colony α microstructure in the center of the specimen waist where heating was most efficient. Ultimate tensile strength (UTS) was found to be in the range of 858 – 977 MPa, with the highest average being recorded for the reference samples, similar results were noted for the strain, with a range of  ⁓5 – 14 %, where the highest recorded average was for the reference samples. However, the extensometer used was not optimized for this kind of test, therefore percent reduction of area (RA) measurements were performed. The RA measurements produced a significantly different result than that obtained from the testing, a large scatter in the ductility was found, possibly due to thermal instability that occurred during testing. Overall, the microstructure appears to be relatively stable over the cooling range of 20 - 100 °C/s, no major differences were observed, the microstructure consisted of a homogeneous basketweave α microstructure, with little to no change in the measured average α lath thickness.

Tumor progression is not only due to the aggressiveness of cancer cells but also to the support given by tumour reactive stroma; reason why the study of stromal cell involvement in tumor microenvironment has become extremely important in the last decades. Tumor mass is a complex network of cancer and stromal cells, and fibroblasts are the main component. Under the influence of tumor cells, fibroblasts engage a transdifferentiation program converting them into their active form (myofibroblast), the so called cancer associated fibroblasts (CAFs), that, in turn, are able to enhance tumor cells growth, migration and invasion. This crosstalk is mediated by soluble factors, cell–cell contacts and extracellular vesicles (EVs) trafficking. Our work is focused in particular on cellular interaction based on EVs trafficking. Two types of Evs has been described, ectosomes (with a diameter from 100 nm to 1 μm) and exosomes (from 30 to 100 nm) that show differences in size, biogenesis and composition. It has been discovered by my research group that a transfer of proteins and lipids between CAFs and cancer cells mediated by ectosomes exists and that this is, essentially unidirectional from CAFs to cancer cells. We have identified about two hundreds proteins that are specifically transferred to cancer cells by this type of cargo. One of the most interesting proteins, considering its role in cancer progression is Galectin-1 We have found that Galectin-1 silencing in CAFs reduce the migration of cancer cells, revealing a novel mechanism by which tumor stroma contribute to cancer progression. These results are important because Galectin-1 has been highlighted as a good target in both cancer and fibroblast cells, increasing the possibilities to counteract cancer aggressiveness by reducing Galectin- 1 action through specific inhibitors. In the second part of my thesis the role of low molecular weight protein tyrosine phosphatase (LMW-PTP) in fibroblasts during their activation has been investigated for the first time. It is known that LMW-PTP expression in cancer increases with the staging of tumor and that it is implicated in several biological processes. Our findings show that the activation induces in CAFs an increase of LMW-PTP expression that is associated to cytoskeletal rearrangement. As a consequence CAFs show a more invasive phenotype that is reversed when LMW-PTP is silenced. Additionally our results suggest the LMW-PTP involvement in cell metabolism. The increase of LMW- PTP induces a more gycolytic metabolism and its silencing causes the induction of a more OXPHOS behaviour. We hypothesize that LMW-PTP could drive fibroblast infiltration and migration during tumor progression. These findings, taken together, contribute to highlight the role of CAFs within tumor microenvironment in sustaining tumors.

This thesis improves on the design of the leaky-mode spatial light modulator, LMW-SLM, presented by Dr. Smalley[1]. Improvements include: input coupling gratings, a pulsed laser input, output coupling gratings, and a 3D printed adjustable module for the stabilization of critical alignments. First, input coupling gratings reduce the cost of the LMW-SLM from $500 to around $2, a drop in cost of over two orders of magnitude. This enables multiple modulators to be used in a single display and allows for an inexpensive modular design to be created. Second, a pulsed laser input allows for image creation without the use of a polygon for derotation. Removal of the polygon allows for direct viewing of the LMW-SLM output enabling near-eye and flat panel displays. Third, output coupling gratings allow for bottom exit devices that are essential for thin substrates and flat panel displays. Fourth, the 3D printed module allows for the critical alignments of the LMW-SLM to become permanent. This in turns allows for transportation of the created displays without a trained technician by abstracting away the complexities of the device. The resulting changes simplify hardware, reduce cost, and enable the LMW-SLM to be modularized and the resulting 3D displays to be transportable. These improvements are made possible by the addition of a one new mask step during fabrication, a simple circuit design, and a 3D printed module designed in SOLIDWORKS. Included in this thesis as attachments are the MATLab, Eagle, and SOLIDWORKS files used to create the improved LMW-SLM.

Statement of the problem The primary objective of this thesis was to determine the best study design to evaluate the safety and effectiveness of an intermediate dose of low molecular weight heparin for secondary prevention of pregnancy associated VTE (PAVTE). An RCT was deemed unfeasible,so the use of a single arm study with prior evaluation of feasibility with a pilot study is proposed. // Methods - A systematic review was conducted to evaluate the efficacy of current strategies used for secondary prevention of PAVTE.A survey was used to elicit the non-inferiority margin. // Results - The pooled proportion of recurrent VTE in patients treated with full dose LMWH was 0.012(95% CI 0.006 to 0.02) and the rate of major bleeding was 0.025(95% CI=0.01 to 0.041). The non-inferiority margin was elicited at 2.5%. // Conclusions - Although a randomized controlled trial should be conducted whenever possible, in certain scenarios they are unfeasible. Therefore, an alternative study design should perhaps be used to evaluate the safety and efficacy of therapeutic strategies.

Orientadores: Ricardo Aparicio, Munir Salomão Skaf
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química
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Resumo: O câncer é uma doença cuja incidência e prevalência atinge proporções alarmantes, estabelecendo-se, hoje, como um problema mundial de saúde pública. A fosforilação de proteínas é um evento dinâmico e reversível, governado pela atividade oposta de proteínas tirosina quinases e proteínas tirosina fosfatases. Níveis elevados das fosfatases LMW-PTP e CDC25B foram observados em uma ampla variedade de tumores e, assim, estas foram selecionadas como alvo para o desenvolvimento de novos inibidores. Utilizando métodos in silico, uma coleção, contendo aproximadamente 500 mil fragmentos, foi montada a partir de um banco de compostos comerciais. Para cada enzima, esses fragmentos foram submetidos a distintos protocolos de docagem molecular, através dos quais 19 pequenas moléculas foram selecionadas e adquiridas comercialmente. Os resultados computacionais foram validados por ensaios de inibição enzimática, tendo sido identificados novos esqueletos moleculares capazes de inibir mais do que 50% da atividade enzimática, obtendo-se valores de eficiência do ligante de até 0,33 kcal mol-1 por átomo diferente de hidrogênio. Paralelamente, uma série de compostos derivados do ácido benzenofosfônico foi ensaiada frente à LMW-PTP após estudos de docagem, seguindo-se estudos cristalográficos que levaram à obtenção de duas estruturas inéditas: uma com a proteína na forma apo e outra de um complexo LMW-PTP:inibidor. Além do sítio ativo já conhecido, observou-se um segundo sítio cristalográfico cuja potencial função biológica, se confirmada, poderia abrir novas possibilidades para modular a atividade da LMW-PTP, perspectiva que demanda investigação
Abstract: Cancer is a disease whose incidence and prevalence have reached alarming proportions, emerging today as a major public health problem. Protein phosphorylation is a dynamic and reversible event, governed by the opposite activities of protein tyrosine kinases and protein tyrosine phosphatases. High levels of the phosphatases LMW-PTP and CDC25B have been observed in a wide variety of tumors and, for this reason, they have been selected as targets for inhibitor development. Using in silico methods, a collection of approximately 500,000 fragments was assembled from a database of commercial compounds. For each enzyme, these fragments were subjected to different molecular docking protocols, through which 19 small molecules have been selected and purchased. The computational results were validated by enzyme inhibition assays, with the identification of new molecular scaffolds capable of inhibiting in more than 50% the enzyme activity, resulting in ligand efficiency values up to 0.33 kcal mol-1 per non-H atom. Similarly, a number of compounds derived from benzenophosphonic acid was tested against the LMW-PTP after docking studies, followed by crystallographic studies which resulted in two new structures: one of the apo protein and another of a complex LMW-PTP:inhibitor. In addition to the previously described active site, a second crystallographic site was identified, whose potential biological function, if confirmed, might open new possibilities to modulate LMW-PTP activity, in a perspective which demands further investigation.
Doutorado
Físico-Química
Doutora em Ciências

Ce travail de thèse avait pour objectif d’étudier différents vieillissements des copolymères d’éthylène et de norbornène (ENC), utilisés comme conditionnement de produits pharmaceutiques. Grâce à la stratégie analytique adoptée qui a fait appel à différentes techniques de caractérisation, telles que les techniques séparatives comme la chromatographie d’exclusion stérique, la chromatographie liquide haute performance à polarité de phases inversée, les techniques spectrales dont la spectroscopie infra rouge à transformée de Fourier et la spectroscopie UV, les techniques d’analyse thermique à travers l’analyse thermogravimétrie et la calorimétrie différentielle à balayage, puis d’une étude de toxicité des produits de dégradations, nous avons pu mettre en évidence différents types de modifications dans le volume du matériau après vieillissement. La modification principale dans la masse du matériau, observée à la dose réglementaire de stérilisation (25 kGy), est la scission des chaînes du polymère qui s’accompagne de la création de composés de basses masses molaires, donc de migrants potentiels risquant d’influencer la sécurité d’emploi des ENC. Puis pour des doses élevées de rayonnement (150 kGy) et pendant 500h d’exposition UV, on a la réticulation des chaînes.La présence de l’additif (l’antioxydant phénolique l’Irganox 1010®) empêche la création des CBMM après vieillissements. Cependant, en absence d’additif, les vieillissements génèrent de nouveaux CBMM.Toutefois, l’étude de toxicité montre une certaine toxicité à 150 kGy du grade ENC
The aim of this thesis work was to study different ages of copolymers of ethylene and norbornene (ENC), used as packaging of pharmaceutical products. Thanks to the analytical strategy adopted using different characterization techniques, such as separation techniques such as size exclusion chromatography, reverse phase high performance liquid chromatography, spectral techniques including infrared spectroscopy transforming of Fourier and UV spectroscopy, thermal analysis techniques through thermogravimetric analysis and differential scanning calorimetry, and then a toxicity study of degradation products, we were able to highlight different types of modifications in the volume of the material after aging. The main modification in the bulk of the material, observed at the prescribed sterilization dose (25 kGy), is the cleavage of the polymer chains, which is accompanied by the creation of compounds with low molar masses, and therefore potential migrants, which are likely to influence ENC job security. Then for high doses of radiation (150 kGy) and for 500h UV exposure, there is the crosslinking of the chains. The presence of the additive (the phenolic antioxidant Irganox 1010®) prevents the creation of MBMM after aging. However, in the absence of an additive, aging generates new CBMMs. However, the toxicity study shows some toxicity at 150 kGy of the ENC

In human erythrocytes, Syk kinase is a key enzyme that triggers membrane protein band 3 Tyr-phosphorylation (Tyr-P). This enzyme, also called p72Syk, undergoes significant proteolysis in the absence of protease inhibitors, giving rise to p36Syk formation, which induces much greater band 3 Tyr-P then p72Syk. Besides, proteolysed Syk is capable to prepare membranes for further phosphorylation by p72Syk: when membranes are pre-phosphorylated by p36Syk, subsequent p72Syk-catalysed phosphorylation is much higher than that obtained with non-pretreated membranes. Since proteolytic enzymes are present in isolated membranes and p72Syk binds closely to the cytoskeleton, probably to the spanning domain of the anchored band 3, this binding could allow the activation of the enzyme by proteolysis. In human erythrocytes, the P-Tyr level of proteins, mainly transmembrane band 3, is closely controlled by the antithetic activity of Tyr-protein kinases and phosphatases, resulting in a dephosphorylated state. Only after particular stimuli, as with oxidizing agents, diamide or pervanadate, or thiol alkylating compound, N-ethyl maleimide (NEM), Tyr-phosphorylation of band 3 can be triggered, due to the inhibition of Tyr-phosphatase action and the reorganisation of erythrocyte membrane. SHP-1 is a SH2-domain containing protein Tyr-phosphatase expressed in hematopoietic cell lines. We demonstrate that, in human erythrocytes, SHP-1 is present in membranes from resting cells, but in 5% of the protein amount. This amount increases up to three fold following NEM treatment of intact cells, whereas diamide and pervanadate do not alter the normal protein location. In addition, SHP-1 translocation from cytosol to membrane is not affected by band 3 P-Tyr level and localizes into the cytoskeletal compartment. Band 3 is the target of SHP-1, which dephosphorylates Tyr residues 8, 21 and 904. From the cytosol of the red blood cells, through a DEAE-Sepharose ion-exchange chromatography followed by a Sephadex G-75 column, we purified an enzyme with phosphatasic activity on pNPP. Its characterization revealed that the enzyme is an acid phosphatase with a low molecular weight, and western blotting followed by immunostaining with the appropriate antibody confirmed that the enzyme was the Low Mr PTPase. The purified enzyme was able to dephosphorylate the four Tyr residues of band 3: 8, 21, 359 and 904, thus inducing for the first time the total dephosphorylaion of this protein. Dapsone is a drug used in the treatment of leprosy, malaria or AIDS-related Pneumocystis pneumonia. N-hydroxylation of dapsone (DDS) leads to the formation of the toxic hydroxylamines responsible for the clinical methaemoglobinaemia associated with DDS therapy. In addition, the drug has been associated with shortening of the erythrocyte lifespan, with resulting potential clinical consequences such as anaemia and morbidity in areas where DDS is used to treat malaria. We studied how DDS and/or its hydroxylamine (DDS-NHOH) induce erythrocyte membrane alterations leading to premature cell removal. Results indicate that the hydroxylamine, but not dapsone, is able to trigger Tyr-phosphorylation of membrane proteins, mainly of band 3. DDS-NHOH-induced band 3 Tyr-P peaked in 30’ (at 0.3 mM) and was already completely reversed after 45’ of incubation. However, when analysed for the enzymes involved in this process, Syk and SHP-2, membranes revealed dose- and time-dependent recruitment of both enzymes. Band 3 Tyr-phosphorylation is not due to an imbalance between enzymatic activities, since both Tyr-kinase and phosphatase activities were promptly inhibited by DDS-NHOH in both dose- and time-dependent manners, but more probably by a favoured substrate-kinase interaction. The band 3 Tyr-phosphorylation process is very useful in detecting early DDS-NHOH-induced alterations. The membrane modifications continue, with the increase in DDS-NHOH incubation time, leading to aggregation of band 3. Band 3 high molecular weight aggregates (HMWA) location in the first 30’ of incubation was in the Triton-soluble fraction of the RBCs’membranes, while prolonging the incubation time, not only the content of band 3 HMWA further increased, but complete membrane reorganisation also occurred, the cytoskeleton containing almost all the band 3 HMWA complexes formed. When we analysed membranes from erythrocytes subjected to dose- and time-dependent DDS-NHOH treatments in the presence of autologous plasma, immunostaining with anti-human IgG revealed net enhancement of the autologous antibodies content. Since removal of human erythrocytes is mediated by antibody recognition, this can explain the shortening of the erythrocyte lifespan seen when dapsone is used clinically.
Negli eritrociti umani la tirosin chinasi Syk è un enzima chiave nella tirosin fosforilazione della proteina di membrana banda 3. Questo enzima, chiamato anche p72Syk, va incontro ad una significante proteolisi in assenza di inibitori proteasici, portando alla formazione di p36Syk, capace di indurre con maggiore efficienza la tirosin fosforilazione della banda 3 rispetto al p72Syk. Inoltre, la fosforilazione innescata dal p36Syk prepara le membrane per la successiva fosforilazione da parte dell’oloenzima p72Syk. Infatti quando le membrane sono pre-fosforilate con p36Syk, la successiva fosforilazione catalizzata dal p72Syk è maggiore rispetto a quella ottenuta in assenza della pre-fosforilazione. Poiché gli enzimi proteolitici sono presenti nelle membrane isolate e il p72Syk si lega al citoscheletro della cellula, probabilmente al dominio transmembrana della banda 3 ancorata a questa frazione della membrana, questo legame potrebbe permettere l’attivazione dell’enzima p72Syk attraverso la sua proteolisi. Negli eritrociti umani il livello di tirosin fosforilazione delle proteine, soprattutto della proteina transmembrana banda 3, è strettamente controllato dall’attività antitetica delle protein tirosin chinasi e fosfatasi, risultando in condizioni basali in uno stato di quasi completa defosforilazione. Solo stimoli particolari, come agenti ossidanti, diamide o pervanadato, o composti alchilanti, N-etilen maleimide (NEM), possono innescare la tirosin fosforilazione della banda 3, dovuta l’inibizione delle tirosin fosfatasi. SHP-1 è una protein tirosin fosfatasi contenente due domini SH2 espressa nelle cellule ematopoietiche. Noi dimostriamo che, negli eritrociti umani, SHP-1 è presente nelle membrane delle cellule in condizioni basali, ma solo il 5% della quantità totale dell’enzima. Questa percentuale incrementa fino a tre volte dopo il trattamento della cellula con NEM, mentre diamide e pervanadato, pur inducendo la tirosin fosforilazione della banda 3, non alterano la localizzazione basale dell’enzima. Ciò indica che la traslocazione della SHP-1 dal citoplasma alle membrane non dipende dalla tirosin fosforilazione della banda 3. Abbiamo dimostrato che la banda 3 è un substrato per l’enzima e che esso defosforila i residui tirosinici 8, 21 e 904 della proteina. Dal citoplasma del globulo rosso abbiamo purificato un’enzima con attività fosfatasica su para-nitro fenilfosfato (pNPP) attraverso una cromatografia a scambio ionico su DEAE-Sepharose, seguita da una gel filtrazione su una colonna G-75 Sephadex. La caratterizzazione dell’enzima purificato ha portato alla sua identificazione con la fosfatasi acida a basso peso molecolare. Tale identificazione è stata confermata dal western blotting, seguito dalla immunorivelazione con l’anticorpo appropriato. La fosfatasi acida a basso peso molecolare purificata è in grado di defosforilare i quattro siti fosfo tirosinici della banda 3: 8, 21, 359 e 904. In questo modo si è ottenuta per la prima volta la totale defosforilazione della proteina. Dapsone è un farmaco utilizzato nel trattamento della lebbra, malaria o pneumonia dovuta a Pneumocystis associata all’ AIDS. La sua N-idrossilazione porta alla formazione di idrossilamine tossiche responsabili della metaemoglobinemia associata al trattamento con dapsone. Inoltre, il farmaco è stato correlato alla riduzione della vita media dell’eritrocita, ottenendo come risultato conseguenze cliniche come anemia e morbidità nelle aree dove il dapsone è utilizzato per trattare la malaria. Noi abbiamo studiato il processo attraverso il quale il dapsone ed il suo metabolita idrossilamindapsome (DDS-NHOH) inducono alterazioni a livello della membrana eritrocitaria che portano alla rimozione prematura dell’eritrocita. I risultati indicano che DDS-NHOH, ma non il dapsone, è capace di innescare la tirosin fosforilazione delle proteine di membrana, soprattutto della banda 3. L’indotta tirosin fosforilazione raggiunge un massimo in 30’ di trattamento (con 0.3 mM) poi essa comincia a calare e dopo 45’ dall’inizio del processo sparisce completamente. Il reclutamento alla membrana degli enzimi coinvolti in questo processo, Syk e SHP-2, avviene in maniera dose e tempo dipendente, per entrambi gli enzimi. Inoltre la tirosin fosforilazione della banda 3 non è dovuta ad uno sbilanciamento tra le attività enzimatiche, dal momento che sia l’attività tirosin chinasica che quella tirosin fosfatasica sono inibite dal DDS-NHOH in maniera dose e tempo dipendente, ma più probabilmente è dovuta ad una favorevole interazione substrato-chinasi. Le modifiche che avvengono nella membrana indotte da DDS-NHOH continuano anche dopo che la la fosforilazione è annulata (dopo 45’) e portano all’aggregazione della banda 3. La localizzazione degli aggregati ad alto peso molecolare di banda 3 (HMWA) nei primi 30’ di incubazione è prevalentemente nella frazione solubile in Triton X-100. Con il prolungamento del tempo d’incubazione si ha non solo l’ulteriore incremento della quantità di aggregati, ma anche un loro definitivo spostamento nel citoscheletro della membrana eritrocitaria. Questo è un indizio di una completa riorganizzazione della membrana. L’analisi delle membrane ottenute da eritrociti incubati con concentrazioni crescenti di DDS-NHOH a vari tempi d’incubazione in presenza del plasma autologo ha rivelato un netto aumento nel contenuto di anticorpi autologhi. Poiché la rimozione dell’eritrocita è mediata dal riconoscimento da parte dei macrofagi degli anticorpi autologhi, la presenza di quest’ultimi sulla membrana del globulo rosso trattato con DDS-NHOH spiega la riduzione della vita media degli eritrociti nei pazienti trattati con dapsone.

The aim of this study was to validate the two presumptive blood tests LMG, LCV and the three visualising blood methods Bluestar Forensics, Lumiscene and the Ruhoff method. The methods’ sensitivity, durability, matrices effects, false positive results and the methods effect on subsequent DNA analysis were studied. DNA analyses were also performed to assess the detection limit of the forensic DNA analysis. Drops of diluted blood were applied on different absorptive matrices and the sensitivity was investigated. The solutions were also placed under different conditions to investigate the durability of the solutions. The solutions were applied upon panels using different chemicals and materials and the false positive results were studied. The DNA analyses were performed by diluting the blood with Bluestar Forensics, the hydrogen peroxide method, the Ruhoff method and deionised water. The study showed that the LMG with a 3 % H2O2 concentration performs the best and it is suited for practical casework. The positive results of LMG was easier to interpret than those of LCV, this is probably due to the fixative agent of the used LCV solution. Bluestar Forensics and Lumiscene did perform similar on the different matrices tested, but the Lumiscene solution had a slightly higher durability. The results strongly indicate that the Ruhoff method can be used without luminol, hence only as a hydrogen peroxide solution (the hydrogen peroxide method). All three visualising blood methods decreases chances of retrieving a positive DNA profile, however the visualising blood methods could be used if the blood cannot be found in any other way. A DNA profile was obtained from the one blood sample analysed at dilution of 1:256 in deionized water.

Oxidative processes play important role in cell physiology and pathology as well. Balance of these processes is supplied by cooperating antioxidative systems; function of antioxidant defense systems depens on high levels of antioxidants in organism. Presented work is focused on developement and optimization of methods for analysis of important enzyme and non-enzyme antioxidants as well as total antioxidant capacity of selected types of biological material. Extractions and analyses of vitamin E, carotenoids, superoxide dismutase, catalase, peroxidase and lipoxygenase in barley and malt were optimized. RP-HPLC and HPLC/ESI-MS were used for analysis of vitamin E, phenolic and carotenoid content, spectrophotometry was used for enzymes activity analysis. A new methods for catalase and lipoxygenase activities were developed and compared with direct UV methods. Superoxide dismutase activity was determined by commercial diagnostic kit. A colorimetric method was used for peroxidase activity determination. Some kinetic parameters of enzymes were provided too. Optimized methods were used in the analyses of antioxidants in plant material - in barley and malt - in sets of samples of 6 varieties cultivated in four different locations for two years. Content of individual antioxidants differed depending on the variety, but usually were not found significant differences in the levels, depending on growing location. Perhaps climatic conditions have the greatest influence on levels of low molecular weight and enzymatic antioxidants at the specific location; oxidation processes are influenced both the quantity of moisture, both by sunlight, which induces oxidative processes in cultivated plants. The activity of antioxidants in barley caryopses is rapidly increasing during the malting process; an elevated temperature and moistness first induces activation the enzyme systems including antioxidant. In caryopsis is metabolic activity increased during which we can expect an increased production of radicals; for this purpose can antioxidant systems be activated that protect cells from damage by oxidative stress. In the second part of work optimized methods were applied in two clinical trials focused on study of the influence of exogenous antioxidants intake on metabolic and antioxidant status in human organism. In the first clinical study influence of food supplement containing polyunsaturated fatty acids and vitamin E on metabolism of hyperlipidaemics was evaluated. After 3-month supplemenation a lipid profile was improved and serum antioxidant levels increased. The second experiment was focused on enzyme and non-enzyme antioxidant levels in healthy subjects after temporarily intake of specific foods rich in antioxidants. After two-month intake plasma phenolic substances were slightly increased. Total antioxidant capacity and activities of enzyme antioxidants were not affected. Results of both clinical exeriments showed that supplying of antioxidants in natural form or in the form of food supplements does not markedly affect metabolism of healthy subjects, while in patients with chronic diseases antioxidant supplementation can positively influence metabolic status. Results of this work showed that optimized methods are suitable for analyses of antioxidant status parameters and also for monitoring of exogenous antioxidant intake.

Background . Dent's disease is an X-linked recessive renal proximal tubular disorder. It is usualy characterized by low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, nephrocalcinosis and progressive renal failure. Aminoaciduria, phosphaturia, glycosuria, uricosuria and an acquired impairment of urinary acidification may also occur. At the present time two kinds of Dent's disease are known: Dent's disease-1(OMIN No 300009) and Dent's disease-2 (OMIN No 300555). The first associated with mutations in CLCN5 that encodes the renal chloride channel CIC-5. the progression of the nephropaty into uraemia would occur between the third and fifth decade of life. Object of the study. The aim of this study is to carry out a metabolic- instrumental survey of the renal calculosis in a family where some members have mutations in the CLCN5 gene. Material and methods. A patient from clinical pediatrics was admitted to our centre due to a suspected Dent's disease so a molecular analysis was performed and a CLCN5 gene mutation was detected. We created a family tree over four generations, the molecular analysis was performed on 26 out of the generations, the molecular analysis was performed on 26 out of the 52 members of the family. The nephrological clinic followed up a group of 9 patients, from the second and third generation, and carried out a metabolic-instrumental survey of the nephrolitiasis. For each patient we evaluated the creatinine, uric acid, Ca, P, K, Cl, Mg both at the plasmatic level and urinary level, pH hematic (venous). Furthemore we tested PTH, osteocalcina, vt.D3, bony ALP at the plasmatic level. The 24h urine excretion, oxalate (Ox) and citrate were tested. All patients were studied with renal ultrasonography. Results. Besides the proband, the mutation was detected also in 7 males out 11 and 11 heterozygotic females out of 15. Six patients out of 9 shoved hyperphosphaturia, hypersodiuria and hyperuricuria was associated with hyperuricemia. In 5/9 patients hypercalciuria and hyperossaluria were detected; in 2/9 hypocitraturia was detected. None of the 9 patients showed hypomagnesiuria or hypomagnesaemia. All patients had a normal level of PTH, osteocalcina and bony ALP, the same was for the clearance of the creatinine. The statistical analysis carried out with t Student's tests between mutaded and non-mutaded patients did not reveal differences as for UCa e UPO4. The linear regression test revealed the following correlations: TmPO4/VFG vs UPO4 (r=-0.82 p=0.006); EFNa/UNa /r=0.77 p=0.013); UNa/UPO4(r=0.73 p=0.023), UCa vs UPO4 (r=0.74 p=0.02), UNa vs UCa (r =0.72 p=0.026). According to the renal ultrasonography 8/9 patients showed bilateral microlithiasis. Conclusions. None of the members of the 3rd generation had renal failure including the 3 mutated patients. Moreover these patients showed hyperuricemia that didn't seen related to renal involvement. In all the subjects we studied, there was the presence and the bilaterality of the microlithiasis. Although in the 3 subjects there were the distinctive signs of the proximal tubulopathy, from the metabolic survey we detected a picture similar to the typical bilateral relapsing nephrolithiasis.

Over the last 20 years "composite" insulators have been increasingly used in high voltage applications as an alternative traditional materials. More recently, polydimethylsiloxane (PDMS) have been used as weather sheds on these composite insulators. The main attraction with PDMS is that the surface hydrophobicity can be recovered following pollution or surface discharges. Among the possible mechanisms for recovery the most likely is the migration of low molecular weight silicone oil (LMWS) from the bulk to the surface encapsulating pollutant particles. Although it is widely recognised that the migration of LMWS is the cause of this recovery of hydrophobicity, the mechanism of what actually occurs is not well understood. It is also not known for how long this process will continue. The main objective of this study program was to gain improved understanding of the surface hydrophobic recovery process that is unique to polydimethlysiloxane high-voltage insulators. Fundamental knowledge of this mechanism has been increased through the development of the Contact Angle DRIFT Electrostatic Deposition (CADED) novel analytical technique. This technique enabled study of the degradation of silicone elastomers subjected to high voltage environments by closely following LMWS migration from the bulk material to the surface and linking it to the contact angle measurements. The migration rate data showed that the aged material recovered faster that the virgin material. Differences in the rate and maximum surface levels of silicone were seen between materials from different manufacturers. This has significant implications for the life-time of these materials A model system has been developed to examine LMWS diffusion through the bulk material and into the interface of surface and pollutant. This was achieved by examining theoretical and empirically derived equations and using existing experimental data to better understand the mechanism of recovery. This diffusion was Fickian in the initial stages of recovery. X-ray photoelectron spectroscopy (XPS) and contact angle measurements were used to substantiate the degree of degradation in in-field silicone insulators by quantifying the levels of the major degradation products: silica and silica-like material and alumina.

Over the last 20 years "composite" insulators have been increasingly used in high voltage applications as an alternative traditional materials. More recently, polydimethylsiloxane (PDMS) have been used as weather sheds on these composite insulators. The main attraction with PDMS is that the surface hydrophobicity can be recovered following pollution or surface discharges. Among the possible mechanisms for recovery the most likely is the migration of low molecular weight silicone oil (LMWS) from the bulk to the surface encapsulating pollutant particles. Although it is widely recognised that the migration of LMWS is the cause of this recovery of hydrophobicity, the mechanism of what actually occurs is not well understood. It is also not known for how long this process will continue. The main objective of this study program was to gain improved understanding of the surface hydrophobic recovery process that is unique to polydimethlysiloxane high-voltage insulators. Fundamental knowledge of this mechanism has been increased through the development of the Contact Angle DRIFT Electrostatic Deposition (CADED) novel analytical technique. This technique enabled study of the degradation of silicone elastomers subjected to high voltage environments by closely following LMWS migration from the bulk material to the surface and linking it to the contact angle measurements. The migration rate data showed that the aged material recovered faster that the virgin material. Differences in the rate and maximum surface levels of silicone were seen between materials from different manufacturers. This has significant implications for the life-time of these materials A model system has been developed to examine LMWS diffusion through the bulk material and into the interface of surface and pollutant. This was achieved by examining theoretical and empirically derived equations and using existing experimental data to better understand the mechanism of recovery. This diffusion was Fickian in the initial stages of recovery. X-ray photoelectron spectroscopy (XPS) and contact angle measurements were used to substantiate the degree of degradation in in-field silicone insulators by quantifying the levels of the major degradation products: silica and silica-like material and alumina.

It is widely known that early estimates about the binding properties of drug candidates are important in the drug discovery process. Surface plasmon resonance (SPR) biosensors have become a standard tool for characterizing interactions between a great variety of biomolecules and it offers a unique opportunity to study binding activity. The aim of this project was to develop a SPR based assay for pre-screening of low molecular weight (LMW) drug compounds, to enable filtering away disturbing compounds when interacting with drugs. The interaction between 47 LMW compounds and biological ligands were investigated using the instrument BiacoreTM, which is based on SPR-technology.

Partie I: Modèles de spin collectif On utilise le formalisme des états cohérents de spin pour étudier des modèles de spin collectif, qui ont plusieurs champs d'application en physique. Le modèle de Lipkin-Meshkov-Glick (LMG) a en particulier été analysé à la limite thermodynamique. La méthode développée au cours de ce travail peut être utilisée, en principe, pour des Hamiltoniens plus généraux, s'écrivant en fonction des générateurs de l'algèbre su(2). Nous avons pu dériver exactement la densité d'états intégrée du modèle. La nature des singularités de la densité d'états a été mise en évidence. Les premières corrections de taille finie ont également été calculées. Les valeurs moyennes d'observables ont été étudiées. Près des singularités, la quantification de Bohr-Sommerfeld, adaptée aux spins, n'est pas valable. Pour traiter ces cas, nous avons développé une nouvelle approche, permettant alors de décrire le spectre au voisinage des points critiques. Partie II : Calcul quantique adiabatique Nous avons construit un modèle simple permettant de mettre en évidence la relation entre les transitions de phase quantiques et le calcul (quantique) adiabatique. Ce modèle met en évidence l'importance du choix du Hamiltonien initial et du chemin adiabatique considéré dans l'espace des paramètres, et peut servir comme un cas d'école pour des modèles plus réalistes. Nous avons enfin étudié la dynamique des populations des états à travers une transition de phase, pour le cas du modèle LMG abordé dans la première partie. Une analyse numérique nous a montré que ces changements de population sont très sensibles à la présence des points exceptionnels dans le spectre, ce qu'un modèle simplifié de l'évolution quantique permettait de suggérer.

碩士
國立陽明大學
生化暨分子生物研究所
104
Alzheimer’s disease (AD) is the most common cause of dementia in elderly people. It is a chronic neurodegenerative disease. Currently, the hypothesis for the pathogenic mechanism of AD is amyloid cascade hypothesis. It states that the aggregation of β-amyloid (Aβ) is the primary cause of AD. Aβ contains 39-42 residues amino acid. It was a proteolytic product derived fromβ-amyloid precursor protein (βAPP). The aggregation process of Aβ involves conformational changes and self-assembly, indicating that the structural property plays an important role in Aβ aggregation. Previous studies also reported that the aggregation of Aβ was linked to its interaction with cell membrane-like enviroment. The interaction between Aβ and cell membrane might alter the structural property and conformational stability of Aβ. Some studies suppoted the view that cell membrane environment might inhibit the aggregation of Aβ, whereas others thought that cell membrane enviroments would promote the aggregation of Aβ. In this study, we used negatively charged LMPG micelles and familial Alzheimer’s disease-linked Iowa-type (Aβ40(D23N)) as model systems for investigating the Aβ-cell membrane interaction mechanism, and applied nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD) spectroscopy, thioflavine-T (Th-T) fluorometric assay and transmission electron microscopy (TEM) to characterize the interaction between Aβ and membrane lipid. By compairing the structure and aggregation behavior of Aβ40(D23N) in aqueous solution and LMPG micelles environment, we obtained that LMPG micelles would interact with Aβ40(D23N), leading to an increase of the α-helical propensity of Aβ40(D23N) and an inhibition of Aβ40(D23N) aggregation. Structural analysis showed that Aβ40(D23N) interacted with LMPG micelles through two short α-helical regions, L17VFFAENVGS26 and K28GAIIGLM35. These results may provide the information about the mechanism of Aβ-cell membrane interaction.

Leung, Ching Yee = LMG模型中的保真度對數和絶熱要求 / 梁靜儀.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2010.
Includes bibliographical references (leaves 53-58).
Abstracts in English and Chinese.
Leung, Ching Yee = LMG mo xing zhong de bao zhen du dui shu he jue re yao qiu / Liang Jingyi.
Chapter 1 --- Quantum phase transition and fidelity --- p.1
Chapter 1.1 --- What is a quantum phase transition --- p.1
Chapter 1.2 --- Use of fidelity in describing QPT --- p.3
Chapter 1.3 --- Quantum fidelity versus classical fidelity --- p.5
Chapter 1.4 --- Motivation of the project --- p.8
Chapter 2 --- Introduction to LMG model --- p.11
Chapter 2.1 --- The LMG model --- p.11
Chapter 2.2 --- General ground-state solution of LMG model --- p.13
Chapter 2.3 --- Analytical solution of ground-state fidelity of LMG model --- p.16
Chapter 2.4 --- Numerical diagonalization of the Hamiltonian --- p.23
Chapter 3 --- Scaling dependence of logarithmic fidelity in the LMG model --- p.26
Chapter 3.1 --- Symmetry-broken phase --- p.26
Chapter 3.2 --- Polarized phase --- p.29
Chapter 3.3 --- Scaling behavior of logarithmic fidelity around the critical point --- p.30
Chapter 4 --- Quench dynamics --- p.35
Chapter 4.1 --- Introduction to quench dynamics --- p.35
Chapter 4.2 --- Quantum adiabatic theorem --- p.35
Chapter 4.3 --- Ground-state quench dynamics --- p.37
Chapter 4.4 --- Motivation --- p.38
Chapter 4.5 --- "Adiabaticity, residue energy and fidelity" --- p.39
Chapter 4.6 --- Adiabatic requirement --- p.40
Chapter 5 --- LMG model in quench dynamics --- p.42
Chapter 5.1 --- Numerical analysis method --- p.42
Chapter 5.2 --- Loss of adiabaticity --- p.44
Chapter 5.3 --- The adiabatic requirement in the symmetry-broken phase --- p.45
Chapter 5.4 --- The adiabatic requirement in the polarized phase --- p.46
Chapter 5.5 --- In the critical region --- p.47
Chapter 6 --- Summary --- p.50
Chapter 6.1 --- Scaling dependence of logarithmic fidelity --- p.50
Chapter 6.2 --- Scaling dependence of duration time in quench dynamics --- p.52
Bibliography --- p.53

Next generation of sequencing (NGS) technologies have taken life science research into a new era. With the rapid advances in these technologies and the associated reduction in overall costs, the sequencing and assembly of genomes have come within reach of most laboratories. Studies related to the evolution, ecology and biology of an organism now rely heavily on genomic data and obtaining a genome sequence has become an essential resource for the rapid progress and success of these studies. Pantoea ananatis is recognised as an emerging but rather unconventional pathogen capable of infecting a wide range of different hosts. Numerous plants of agricultural and economic importance including maize, rice, onion, pineapple, melon, sudan grass and Eucalyptus trees have been affected. With the outbreak of P. ananatis in a South African Eucalyptus nursery in 1998, it was realised that very little is known about this pathogen. A better understanding of the pathogenicity, metabolism and ecology of the bacterium is required to develop strategies for the control of the disease. During this study, the genome sequence of P. ananatis strain LMG 20103 was obtained using the Roche 454 technology. To aid in the assembly of this Eucalyptus pathogen’s genome sequence, the type strain of P. ananatis LMG 2665 was also sequenced using Illinima’s Genome Analyzer (GA). A draft assembly of P. ananatis LMG 20103, consisting of 117 contigs, was generated after optimization of the Newbler assembly parameters and comparison with other genome assemblies and genomes. This study demonstrated that the assembly could be completed using both in-vitro, and in-silico approaches such as contig scaffolding, gap closure with conventional PCR reactions and sequencing, manual curation and automated genome annotation. The final complete genome consisted of a 4 386 227 bp chromosome and a 317 146 bp mega-plasmid. With the complete genome sequence available, the reconstruction of metabolic network of P. ananatis LMG 20103 was attempted using two pathways reconstruction pipelines namely, Pathway Tools and Model SEED. It was found that missing metabolic reactions and incomplete pathways in the draft metabolic networks were mainly caused by incorrect gene annotations or bioinformatic errors during the automated network reconstruction. These two pipelines differed substantially in the way network reconstruction is undertaken. Performing a comparison between the two proposed networks, annotation errors could be detected and corrected. Although some improvement could be made to the predicted network further experimental data is still required to improve the accuracy of the draft metabolic network. Despite the amount of effort and cost, it is believed that the complete genome and a draft metabolic network of P. ananatis LMG 20103 will be a valuable resource for many subsequent studies to investigate the evolution and biology of this emerging plant pathogen. This information will be essential for the development of strategies to predict and control future disease outbreaks associated with this pathogen.
Dissertation (MSc)--University of Pretoria, 2012.
Microbiology and Plant Pathology
unrestricted

碩士
國立陽明大學
生命科學系暨基因體科學研究所
104
Alzheimer's disease (AD) a chronic neurodegenerative disease. It is the main cause of dementia in the elderly people. β-amyloid peptide (Aβ) is the main component of neuritic plaques which are a pathological hallmark of AD. β-amyloid peptide (Aβ) contains 39~42 amino acid residues. It was a proteolytic product derived from β-amyloid precursor protein (βAPP). Currently, the leading theory for explaining the etiology and pathogenesis of AD is the “Amyloid cascade hypothesis” which stated that Aβ aggregation resulted in brain cell death and dementia. Studies have shown that the aggregation of Aβ was linked to its interaction with cellular membranes. However, the underlying mechanism remains unclear. From a structural perspective, the interaction might induce conformational changes of Aβ resulting in an alteration of it’s aggregation behavior. To understand the role of cellular membranes in Aβ aggregation, the effects of lipids on the structure and aggregation behavior of Aβ were characterized by using thioflavine-T (Th-T) fluorometric assay, nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopies. An anionic lipid, lyso-myristoylphosphatidylglycerol (LMPG) and a genetic Aβ mutant, Dutch-type Aβ40 (Aβ40(E22Q)), were used in the present study. Structural studies indicated that LMPG micelles increased the α-helical content of Dutch-type Aβ40. The results of aggregation kinetics showed that LMPG micelles inhibited the aggregation of Dutch-type Aβ40. These results suggested that the interaction of Dutch-type Aβ40 with anionic lipids would increase its conformational stability leading to a reduction of its aggregation rate. These findings may help us gain an insight into the possible role of Aβ-lipid interaction in the process of Aβ aggregation.

Tese de mestrado, Microbiologia Aplicada, Universidade de Lisboa, Faculdade de Ciências, 2020
Lactic acid bacteria (LAB) have long had a prominent role in human society, where many of its individuals are used to produce various fermented food products and a few others are clinicallyrelevant pathogens. Since the late twenty-first century, many of these bacteria have also demonstrated their potential as biotechnological organisms, thanks to their proven safety for human health. Lactococcus lactis is one of such LAB, with a long history in the production of dairy products and a recently well established role in biotechnology. There, L. lactis strains have been used as microbial cell factories for the production of recombinant protein, as vectors for mucosal vaccination, and more. L. lactis subsp. lactis LMG 19460 is a strain whose genome was recently sequenced and whose lack of intrinsic plasmids makes it an ideal candidate for biotechnological applications. For the better understanding and use of such biotechnological organisms, genome-scale metabolic models, or genome-scale models (GEMs), can be a great tool. However, developing robust GEMs for organisms which have no available experimental data can be a difficult task, since they cannot be validated through comparison with published phenotypes. As such, strategies different from traditional methodologies are necessary. Here, two GEMs were developed, one for the well characterised, reference strain L. lactis subsp. lactis IL1403 and another for L. lactis LMG 19460. The GEM for L. lactis IL1403 accounts for 575 genes, 921 reactions and 639 metabolites. It was reconstructed through comparative genomic approaches, where metabolic functions in strain IL1403 were inferred from high-quality published GEMs. The assembled model was then refined and validated through comparison with the comprehensive published data available for the organism. The model demonstrates good capabilities in predicting experimentally determined phenotypes of strain IL1403. Using this validated and working model, a GEM was then developed for the lesser-known strain LMG 19460. The metabolic model for L. lactis LMG 19460 accounts for 570 genes, 916 reactions and 638 metabolites. It is a functional model, capable of performing in silico predictions using data available for other L. lactis strains. However, it still requires true validation through comparison with experimentally determined, strainspecific phenotypes. As a first step in the experimental characterisation of L. lactis LMG 19460, a chemically defined medium was here developed and optimised, supporting clear growth and considerable biomass production when compared with published media (final OD600 = 2.02). The GEM for L. lactis LMG 19460 is capable of simulating unconstrained growth in this medium. In future applications, both metabolic reconstructions here assembled should be further refined and validated, in order to fully develop them into high-quality GEMs. Then, these models will be of significant use for further studying the metabolism of their respective strains, where they can be usedto map high-throughput data and drive experimental design. Furthermore, by testing synthetic biology hypothesis and predicting the effects of metabolic engineering, these models will be invaluable tools for the applications of their respective strains in biotechnology.
As bacterias do acido lactico (BAL), correspondentes a ordem taxonomica Lactobacillales, sao um grupo de bacterias Gram-positivas com baixo conteudo G+C, caracterizadas por produzirem acido lactico como principal produto do metabolismo. Sao um grupo altamente diversificado, quer nas suas caracteristicas fisionomicas, quer no leque de habitats que ocupam, os quais vao desde numerosos produtos alimentares fermentados, a ambientes vegetais, superficies animais e vias gastrointestinais. Embora alguns membros desta ordem sejam agentes patogenicos, a maioria destas especies e reconhecida como segura para a saude humana. Estes microrganismos estao tradicionalmente associados a industria alimentar, onde servem para a producao de variados produtos fermentados. Mais ainda, desde o final do seculo vinte, tem vindo a adquirir um papel cada vez mais relevante na area da biotecnologia. Uma das especies mais bem caracterizadas no grupo de BAL e Lactococcus lactis. Esta bacteria partilha ja uma longa historia com o ser humano, em grande parte devido ao seu uso na producao de inumeros lacticinios. Recentemente, adquiriu tambem um papel distinto na biotecnologia, onde a sua seguranca para a saude humana lhe confere inumeras vantagens sobre os organismos tradicionalmente utilizados nesta area. Um exemplo destas aplicacoes e a sua utilizacao como fabrica celular microbiana para a producao de inumeros compostos e enzimas de relevancia industrial. Outro exemplo, e a sua aplicacao nas areas da terapeutica e imunologia, onde L. lactis tem sido utilizada para a producao e administracao in vivo de compostos terapeuticos e para a vacinacao atraves de mucosas. A estirpe L. lactis spp. lactis LMG 19460, cujo genoma foi recentemente sequenciado, e uma boa candidata para estas varias aplicacoes biotecnologicas. Isto deve-se, em particular, ao facto de nao ter plasmideos intrinsecos, o que reduz os seus custos metabolicos e permite, em principio, maior rendimento na producao de proteina recombinante. Qualquer aplicacao biotecnologica de um organismo beneficia de um conhecimento integral e abrangente das suas funcoes celulares, nomeadamente do seu metabolismo. Para alcancar essa compreensao holistica, existem na area da biologia de sistemas inumeras ferramentas, dais quais se destacam os modelos metabolicos a escala genomica (MMEG). Estes modelos sao representações matematicas e, consequentemente, computacionais de todas funcoes metabolicas de um dado organismo, permitindo, assim, simular estados fisiologicos e prever fenotipos em variadas condições ambientais. Surgiram no final dos anos 1990, logo apos a sequenciacao dos primeiros genomas, e tem desde entao sido desenvolvidos para cada vez mais organismos, cobrindo agora todos os dominios da vida celular. Quando estes modelos sao gerados de um modo cuidado e compreensivo, resultam em MMEG de alta qualidade, que podem, entao, ser aplicados para inumeros fins. Destes, destacam-se particularmente o mapeamento de dados omicos, permitindo, assim, a melhor interpretacao dos mesmos, e, reciprocamente, a melhoria e o refinamento do modelo. Destacam-se, tambem, as aplicacoes na area da biotecnologia, designadamente na biologia sintetica e engenharia metabolica. Ai, MMEG simulam, entre outras coisas, a insercao de plasmideos e manipulacoes geneticas, permitindo, assim, testar hipoteses antes da sua aplicacao experimental. A construcao de um MMEG e um processo trabalhoso e minucioso que, de modo geral, segue quatro passos essenciais. No primeiro, e gerada uma reconstrucao esboco da rede metabolica do organismo em questao. No segundo passo, o esboco obtido e revisto e refinado, de modo a conceder maior qualidade e realismo a reconstrucao metabolica, mas tambem para lhe dar a estrutura necessária aos passos seguintes. O terceiro passo e a conversao da rede metabolica para um formato matemático e, consequentemente, computacional. A lista das reacoes metabolicas de uma reconstrucao pode ser representada numa matriz, denominada matriz estequiometrica (ou matriz S), onde as colunas correspondem a cada reacao, as linhas a metabolitos e as entradas aos seus respetivos coeficientes estequiometricos. E esta abstracao da rede metabolica numa matriz matematica que permite a computacao de estados fisiologicos e previsao de fenotipos. O ultimo passo para o desenvolvimento de um MMEG e a sua avaliacao e validacao. Em primeiro, sao analisados e corrigidos possiveis erros na rede metabolica, e, de seguida, as capacidades do modelo sao validadas pela comparacao com dados experimentais. Como tal, para desenvolver o modelo de um dado organismo, e essencial que exista boa e variada literatura experimental para o mesmo. Alternativamente, estes dados experimentais podem ser obtidos em paralelo ao desenvolvimento do modelo. Para organismos cuja literatura metabolica e bastante limitada ou inexistente, sao, entao, necessarias estrategias alternativas para a construcao e validacao do seu MMEG. Neste trabalho foram desenvolvidos dois MMEG, um para a estirpe de referencia L. lactis spp. lactis IL1403 e outro para a estirpe L. lactis LMG 19460. O MMEG para a estirpe IL1403 contabiliza 575 genes, 921 reacoes e 639 metabolitos. Para a sua construcao, foram aplicadas abordagens de genomica comparativa que permitiram inferir as funcoes metabolicas de L. lactis IL1403. Nomeadamente, foram detetadas homologias bidirecionais entre a estripe e uma serie de organismosalvo para os quais estao publicados MMEG de alta qualidade. De seguida, foram corrigidos erros na rede metabolica e o modelo foi validado pela comparacao das suas capacidades com dados disponíveis na extensa literatura de L. lactis IL1403. O MMEG resultante demonstra boas capacidades de simular fenotipos determinados experimentalmente, tais como, requisitos nutritivos, capacidade de utilizar diferentes fontes de carbono, crescimento em meios quimicamente definidos e crescimento respeitante de taxas especificas de consumo de nutrientes e producao de metabolitos. Apos a construcao de um MMEG para L. lactis IL1403 validado e funcional, o mesmo foi utilizado como base para inferir as funcoes metabolicas da estirpe LMG 19460, ainda não caracterizada ao nivel do seu metabolismo. O MMEG aqui desenvolvido para L. lactis LMG 19460 contabiliza 570 genes, 916 reacoes e 638 metabolitos. E funcional e capaz de simular crescimento e diferentes fenotipos quando utilizados dados publicados para outras estirpes de L. lactis. De qualquer forma, para a sua correta validacao e melhoria da especificidade, as capacidades deste modelo precisam de ser comparadas com dados experimentais especificos a estirpe, ainda a obter. De modo a iniciar o processo da caracterizacao metabolica de L. lactis LMG 19460, foi aqui desenvolvido um meio quimicamente definido capaz de suster crescimento da estirpe. Este meio e constituido por uma fonte de carbono, todos os aminoacidos e uma serie de vitaminas, minerais e outros micronutrientes. Pela sua otimizacao, nomeadamente no que diz respeito a concentracao da fonte de carbono e tampao, foi possivel obter um meio capaz de suster uma producao consideravel de biomassa de L. lactis LMG 19460 (densidade otica final de 2,02, a 600 nm). De modo a iniciar, tambem, o processo de validacao do MMEG desenvolvido para este organismo, o meio aqui construido foi aplicado como condicoes ambientais in silico. Depois, o modelo foi avaliado quanto a sua capacidade de reproduzir a ocorrencia de crescimento verificada in vitro; teste para o qual foi positivo. De qualquer forma, sao ainda necessarios muitos mais dados experimentais para corretamente validar o modelo para L. lactis LMG 19460. No futuro serao necessarios ainda mais esforcos de refinamento e validacao dos dois modelos aqui construidos, de modo a eventualmente torna-los em MMEG de alta qualidade. Para o modelo de L. lactis IL1403, isto significa continuar o trabalho de revisao de todas as reacoes incluidas, quer pela continuacao da pesquisa de funcoes metabolicas na respetiva literatura publicada, quer pela investigacao mais detalhada das homologias aqui detetadas entre a estirpe e os organismos-alvo utilizados. Mais ainda, o processo de validacao pode ser melhorado e continuado pela obtencao de dados experimentais de maior qualidade e dados ainda nao disponiveis para a estirpe, tais como a determinacao dos seus genes letais. Quanto ao modelo de L. lactis LMG 19460, tudo o que foi referido para o anterior modelo aplica-se tambem a este, com o acrescimo de ser necessaria a obtencao de ainda mais dados experimentais especificos a estirpe. Estes dados serao, tais como, a determinacao das suas auxotrofias e requisitos nutritivos, a sua capacidade de utilizar diferentes fontes de carbono e as suas taxas de consumo de nutrientes e producao de metabolitos. So quando determinados estes fenotipos da estirpe e que sera possivel a devida validacao do MMEG. Para muitos destes fins, pode ser aplicado o meio sintetico aqui desenvolvido. Este deve tambem continuar a ser desenvolvido e otimizado para o crescimento de L. lactis LMG 19460. Quando atingido o ponto da alta qualidade, os MMEG aqui desenvolvidos para as duas estirpes de L. lactis poderao entao ser utilizados para fins mais aplicados, tais como o estudo detalhado dos seus processos metabolicos, a previsao realistica de fenotipos resultantes de manipulações geneticas e a participacao no desenho e otimizacao destas estirpes como fabricas celulares microbianas.

碩士
臺灣大學
生化科學研究所
96
Liver abscess with metastatic complications caused by Klebsiella pneumoniae (K. pneumoniae) is an emerging infectious disease in Taiwan in the recent years. The human bacterial pathogen was wrapped in a physical barrier of exopolysaccharides. The sugar coated structure, termed capsular polysaccharides (CPS), is an important virulence factor which protects the pathogen from attack by the host immune system. It has been documented that a cassette of genes involved in CPS development are gathered at the capsule biosynthesis locus (cps). The Wzb gene is localized within such gene cluster which determines synthesis and assembly of CPS. It has been suggested that wzb is involved in the regulation of CPS biosynthesis, since the capsule is eliminated in the wzb knockout strain. Furthermore, Wzb is also considered as a kind of low molecular weight protein tyrosine phosphatases (LMW-PTP) based on the protein sequence alignment. In order to confirm the role of Wzb in regulating bacterial CPS biosynthesis through protein tyrosine phosphorylation, we first cloned the Wzb from the liver abscess strain K. pneumoniae NTUH-K2044 and tagged the protein with both histidines (His) and haemagglutinin epitop (HA) at the C-terminus. Meanwhile, substrate-trapping mutants, C9S, D115A and C9S/D115A, were also generated based on the information of the active site. The maximal enzymatic activity of Wzb was achieved at a pH of 5.5 and the corresponding kinetic constants Km, Vmax, Kcat and Kcat/Km, measured at 26.8°C, were 1.35 mM, 34.8 μmole min-1 mg-1, 641.1 min-1and 475 mM-1 min-1, respectively. The inhibition assay revealed that Wzb is not inhibited by NaF and EDTA, even at a high concentration (10 mM). On the contrary, pre-incubation with 10 mM H2O2 or 1 μM vanadate or 1 mM indoacetamide caused the phosphatase to lose its activity completely. These results confirmed that Wzb of K. pneumoniae NTUH-K2044 can be classified as a LMW-PTP. In vitro dephosphorylation assay indicated that several endogenous tyrosine phosphorylated proteins can be dephosphorylated by Wzb, suggesting that these unknown proteins are potential targets of Wzb and governed by it. On the other word, the cellular activity regulated by tyrosine phosphorylation system in bacteria can be clarified by substrate identification. In order to uncover the endogenous substrates of Wzb, we perform substrate-trapping experiments to pull them out where the LMW-PTP is first adopted. Against all expectations, substrate-trapping analysis falls to identify any endogenous tyrosine phosphorylated proteins, however the tyrosine-autokinase, Wzc, can serve as a substrate of Wzb that was verified by a straightforward manner. There are still several obstacles to overcome in the recognition of natural substrates. For further understanding of the precise roles of tyrosine phosphorylation system in bacterial CPS biosynthesis or other associated cellular responses, more precise experimental methods must be exploited in the future to answer those questions. Our investigation on the substrate identification is still ongoing.

Consumers are currently concerned about improving their health, and therefore demand foods that are beneficial to overall health. This has caused the rising interest in probiotics, which are live microorganisms which when ingested in sufficient amounts, restore balance in the gastrointestinal tract and consequently improve health. Probiotic bacteria have been incorporated into various food products which are now referred to as functional foods, and represent about 65% of the world‟s food market. Probiotics are sensitive to various environmental factors such as oxygen, moisture, pH and temperature. It is of great importance that probiotics remain viable and alive throughout the stages of processing, storage in food products and during gastrointestinal transit in order for them to confer health benefits. The use of prebiotics and microencapsulation to protect and ensure viability of probiotics has been used in food industries. Challenges faced when using most microencapsulation techniques include the need for a food grade encapsulating material, stability of the probiotic cells during encapsulation processes and storage, the need to minimize negative effects they might have on the organoleptic properties of foods into which they are incorporated. The freeze drying technique, which is known to be suitable for the preservation of probiotic cells, avoids heat induced injuries to cells and also slows down detrimental chemical reactions, was used in the current study to prepare microparticles encapsulating probiotic bifidobacteria. Due to limited reports on the use of lipid based food grade encapsulating materials for the microencapsulation of probiotics, this study explored the use of such materials and developed a lipid based synbiotic material which is expected to protect and improve probiotic viability. A lipid based excipient Vegetal BM 297 ATO and various concentrations of the prebiotic inulin were used to prepare different formulations, followed by an investigation to determine which concentration of inulin resulted in better protection and survival of Bifidobacterium longum LMG 13197 during the freeze drying process. Bifidobacterium longum LMG 13197 was successfully encapsulated in Vegetal using freeze drying method. It was observed that the formulation prepared with 2% (w/v) inulin resulted in better protection of B. longum LMG 13197 during the encapsulation process. Characterization of the microparticles revealed that they contained high numbers of bacterial cells resulting from relatively high encapsulation efficiency. The presence of inulin resulted in microparticles with an acceptable size which is desirable for food applications. These results led to further investigation of the potential of Vegetal-inulin matrix to protect bifidobacteria in simulated gastrointestinal fluids and improve shelf life under different storage conditions. This study demonstrates that the Vegetal-inulin matrix protected B. longum LMG 13197 during transit in the simulated gastric fluid (SGF) and subsequently released the cells in the simulated intestinal fluid (SIF). In comparison with the unencapsulated cells, the number of cells released in SIF was higher, which suggests that the Vegetal-inulin matrix has the potential to release probiotics in the colon for health benefits to be exerted. The shelf life of encapsulated B. longum LMG 13197 powders stored in glass bottles was investigated under two different storage temperatures for 6 weeks. The study demonstrates that although there was a high loss of viable probiotic cells during storage at 25°C, Vegetal-inulin matrix improved survival of probiotics for 3 weeks as opposed to the unencapsulated cells. On the other hand, encapsulation with Vegetal did not offer improved survival of bacteria when compared to the unencapsulated cells at 4°C, but the addition of inulin offered better protection for up to 5 weeks. Therefore, better shelf life of Vegetal-inulin microparticles containing B. longum LMG 13197 can be achieved at 4°C than at 25°C.
Dissertation (MSc)--University of Pretoria, 2015.
tm2015
Microbiology and Plant Pathology
MSc
Unrestricted

碩士
國立中興大學
化學系所
100
CaroS2K, which contains ribonuclease activity, is an 85kDa protein of bacteriocin protein, which produced by Pectobacterium carotovorum subsp. carotovorum (Pcc) strain 3F-3. From our previous studies, the immunity protein, caroS2I (Mw=10kDa), can associate with the CaroS2K to form a complex, and inhibit the antibiotic function. By homologous analysis from FASTA protein database, the results suggest that CaroS2K has an organization of functional domains as follows: an N-terminal part is present as a transmembrane domain; a central domain is the domain for target cell surface receptor binding; and a C-terminal domain encoding the antibiotic function. We have reported that the target for ribonuclease activity of caroS2K is bacterial ribosomal RNA. Here, to determine the exact C-terminal region of caroS2K needed for rRNA cleavage, we constructed a series of N-terminally truncated carocin S2 by Site-directed, Ligase-Independent Mutagenesis (SLIM). The deleted derivatives starting at residue Gln400, Ile450, Arg600, Gly677 and Lys691 all show the hydrolysis of RNA in vitro, while at Lys692 does not. These results suggest that the minimal ribonuclease region of caroS2K extent from C-terminal residue and locate between Lys691 and Arg783. By alanine-scanning mutagenesis experiment, we have figured out several amino acid residues that were essential for cytotoxicity of CaroS2K. In this study, we take truncated caroS2K derivative caroS2TKD677 as template, substitute its residues mentioned above with alanine. All the point mutants lose the in vitro RNase activity except Y734A and W764A. The results indicate these residues are involved in RNA hydrolysis activity, corresponding to the prediction of computational approach analysis. In order to protect carocin-producing cell itself, there is coordinate synthesis of immunity protein, CaroS2I, which associates with CaroS2K to neutralize its cytotoxicity. By co-immunoprecipitation assays, we identify that there is a interaction-site between caroS2I and C-terminus of caroS2K. In addition, the region within the uncharacterized Domain III of caroS2K resists the association between N-terminally truncated CaroS2K and CaroS2I.

Triticale belongs to the amphiploid cereals and was derived by crossing wheat and rye. Its baking quality is substantially worse than in wheat. The baking quality is determined by a composition and a content of the storage proteins. Both high and low molecular glutenin subunits have a major effect on the final quality of dough. The secalins of rye belong amongst the storage proteins of triticale, which negatively influence the baking quality. These are the reasons why lineages having translocated chromosome 1R and containing subunit Glu-D1d, which positively influences the baking quality, were created. The thesis is focused on identification of the allelic composition of loci of high molecular glutenin subunits (HMW-GS), low molecular glutenin subunits (LMW-GS), loci Pina a Pinb and null alleles of Waxy genes. 23 selected genotypes of triticale were analysed by using DNA markers based on polymerase chain reaction (PCR). Allelic composition of loci HMW-GS (Glu-A1, Glu-B1, Glu-D1), LMW-GS (Glu-A3) and Pina-D1, Pinb-D1 was described and null alleles were detected in the loci Wx-A1 and Wx-B1.

Mestrado de dupla diplomação com a UTFPR - Universidade Tecnológica Federal do Paraná
Os cogumelos apresentam uma grande diversidade na sua composição química, quer de compostos de alto peso molecular, quer compostos de baixo peso molecular (LMW-“Low Molecular Weight”). Devido a sua composição química os cogumelos apresentam inúmeras bioactividades, incluindo: atividade antioxidante, antitumoral, antimicrobiana entre outras. A atividade antitumoral de cogumelos tem sido associada a presença de polissacarídeos nos cogumelos, no entanto ha uma crescente base de conhecimento que mostra que compostos LMW também tem um papel essencial na atividade antitumoral de diferentes espécies de cogumelos. Neste trabalho começou-se por realizar uma pesquisa bibliográfica, de forma a selecionar compostos LMW, presentes em diferentes espécies de cogumelos e associados de alguma forma a uma atividade antitumoral. No total selecionaram-se 115 compostos que formaram a nova biblioteca LMW 2.0, que foi cuidadosamente preparada para estudos in silico. Em uma segunda parte do trabalho foram realizados estudos de screening virtual da biblioteca LMW 2.0 utilizando o software de docking molecular AutoDock 4.0, de forma a tentar estimar quais os compostos da biblioteca que poderão ser os melhores inibidores de 3 proteínas da família Bcl-2: a Bcl-2 (linfoma de célula B 2), a Bcl-XL (linfoma de células B extra grandes) e a MCL-1 (leucemia mieloide-1). Estas proteínas foram escolhidas pois estão envolvidas nas vias de sinalização da apoptose promovendo a sua inibição, sendo atualmente conhecidos alvos terapêuticos em processo tumorais. Assim este trabalho teve como objetivo tentar identificar compostos LMW, presentes em cogumelos, que possam potenciar a apoptose tumoral, interagindo com a família Bcl-2 de proteínas anti-apoptóticas. No geral a proteína MCL-1 parece ser mais sensível aos compostos da biblioteca LMW 2.0, com valores de Ki (constante de inibição) estimados mais baixos, variando entre 17,1 nM e 64,7 nM para os dez melhores compostos. De seguida os melhores resultados foram obtidos para a Bcl-XL com valores entre os 51,5 e 185,6 nM e finalmente os resultados menos interessantes foram da Bcl-2 com valores de Ki entre 140,7 nM e 281 nM. Os compostos glicosilados apresentaram no geral uma melhor capacidade inibidora estimada. A visualização em detalhe das conformações de interação previstas mostra que a melhor capacidade inibidora dos compostos glicolisados fica provavelmente a dever-se à interação da glucose com alguns aminoácidos polares que formam a orla exterior do centro ativo das proteínas em estudo.
Mushrooms exhibit great diversity in their chemical composition, both in high molecular weight compounds and in low molecular weight (LMW) compounds. Due to their chemical composition mushrooms have numerous bioactivities, including: antioxidant, antitumor, antimicrobial and others. The antitumor activity of mushrooms has been associated with the presence of polysaccharides in mushrooms, however a growing knowledge base as emerged showing that LMW compounds also play an essential role in the antitumor activity of different species of mushrooms. In this work, a bibliographical research was performed in order to select LMW compounds, present in different species of mushrooms, and associated in some way with an antitumor activity. In total, 115 compounds were selected establishing the new LMW 2.0 library. These compounds were carefully prepared for in silico studies and will be made available to the scientific community. In a second part of the work, virtual screening studies of the LMW 2.0 library were performed using AutoDock 4.0 molecular docking software, in an attempt to estimate which library compounds may be the best inhibitors of Bcl-2 family proteins: a Bcl-2 (B-cell lymphoma 2), Bcl-XL (extra-large B-cell lymphoma) and MCL-1 (myeloid leukemia-1). These proteins were chosen because they are involved in the apoptosis signalling pathways opromoting apoptosis inhibition, and are currently known therapeutic targets in several tumor types. Thus, this work aims to identify LMW compounds, present in mushrooms, which could potentially promote tumoral apoptosis by interacting with the Bcl-2 family of anti-apoptotic proteins. Overall the MCL-1 protein appears to be more sensitive to LMW 2.0 library compounds with lower estimated Ki (inhibition constant) values, ranging from 17.1 nM to 64.7 nM for the top ten compounds. The best results were then obtained for Bcl-XL with values between 51.5 and 185.6 nM and finally the less interesting results were Bcl-2 with Ki values between 140.7 nM and 281 nM. Glycosylated compounds generally presented better estimated inhibitory capacity. The detailed visualization of predicted interaction conformations shows that the best inhibitory capacity of the glycosylated compounds is probably due to the interaction of glucose with several polar amino acids that form the outer edge of the active centre of the proteins under study.

The present thesis entitled “Design and Synthesis of Novel Soft Composites from Physical Gels and Nanomaterials” deals with soft materials derived from low molecular weight gels and nanomaterials. Chapter 1 gives a general introduction and overview of the low molecular weight gel (LMOG) which forms the basis of the work. It delves with the history of research in physical gel field, design of different types of gelator molecules, their interesting self-assembly patterns, potential applications of these gelator molecules as well as challenges to design new gelator molecules. It also encompasses the relatively recent area of two component gel system to conveniently bypass the cumbersome synthetic protocol. The aspect of liquid crystallinity in the gel phase is also discussed to throw light on the pattern of assembly and potential uses of these materials. Towards the end there is a comprehensive discussion on the smart nanocomposites derived from LMOGs and nanomaterials. The design, synthesis and numerous applications of inorganic-organic hybrid composites are discussed. Chapter 2A describes the synthesis and characterization of a variety of fatty acid amides of different naturally occurring L-amino acids whose molecular structures are shown in Chart 2A.1. Some of them were found to form gels with various hydrocarbons. The gelation properties of these compounds were studied by a number of physical methods including FT-IR spectroscopy, X-ray diffraction, scanning electron microscopy (SEM), differential scanning calorimetry, rheology and it was found that gelation was critically dependent on the fatty acid chain length and nature of the amino acid. Among them, L-alanine based gelators were found to be the most efficient and versatile as they self-assemble into a layered structure to form the gel network. Mechanisms for the assembly and formation of gels from these molecules are discussed. (Structural formula) Chart 2A.1. Molecular structures of various fatty acid amides of different amino acids. Chapter 2B describes efficient gelation of both aliphatic and aromatic hydrocarbon solvents by a fatty acid amide, n-lauroyl-L-alanine (Chapter 2B.1). In addition, this compound was found to gelate the binary solvent mixtures comprised of aromatic hydrocarbon e.g. toluene and aliphatic hydrocarbon e.g. n-heptane. SEM and AFM showed that the fiber thickness of the gel assembly increases progressively in the binary mixture of n-heptane and toluene with increasing percentage of toluene. The self- Chart 2B.1. Molecular structure of the gelator. assembly patterns of the gels in individual solvents, n-heptane and toluene are however, different. The toluene gel consists of predominantly one type of morphological species while n-heptane gel has more than one species leading to polymorphic nature of the gel. The n-heptane gel is thermally more stable than the toluene gel as evident from the measurement using differential scanning calorimetry. The thermal stability of the gels prepared in the binary mixture of n-heptane and toluene is dependent on the composition of solvent mixture. Rheology of the gels shows that they are shear-thinning material and show characteristic behavior of soft viscoelastic solids. For the gels prepared from binary solvent mixture of toluene and n-heptane, with incorporation of more toluene in the binary mixture, the gel becomes a more viscoelastic solid. The time sweep rheology experiment demonstrates that the gel made in n-heptane has faster gel formation kinetics than that prepared in toluene. Chapter 2C describes lyotropic mesophase formation by organogels of different fatty acid amides of L-alanine in aromatic solvents. The helical assembly, characteristic of the cholesteric mesophase was found to exhibit reflection bands in circular dichroism spectra. The reflection bands corresponded to the pitch of the helical arrangement of the gelator molecules in the aromatic solvent. Transmission Electron Microscopy (TEM) showed presence of twist in the gel fibres. Polarising optical microscopy of the organogel exhibited weak birefringence confirming lyotropic nature of the assembly. Chapter 3 deals with synthesis and characterization of a new class of molecules with molecular structures shown in Chart 3.1. Among a variety of amino acid based molecules only alanine and serine based molecules were found to form translucent gels in aliphatic hydrocarbons such as n-heptane. TEM showed presence of fiber like structures for alanine whereas serine based gelator produces unique network like structures. SEM of the dried gels exhibited presence of three dimensional fibrous networks to spongy globular cauliflower like structures depending on the molecular structure of the gelators. Rheological studies of the organogels showed that they behave like typical LMOG gels. The oscillatory rheological studies demonstrated that the L-serine based gelator, 5 formed more viscoelastic solid like gel than that of L-alanine based gelator, 1 in n-heptane. Chart 3.1. Molecular structures of different amino acid derivatives from 3,4,5-tri-dodecyloxybenzoic acid scaffold. Chapter 4A presents design and properties of new nanocomposites from LMOG and metal nanoparticles (Chart 4A.1). The profound influence of nanoparticle (NP) incorporation into physical gels was evident from various microscopic and bulk properties. The interaction of nanoparticles with the gelator assembly was found to depend critically on the capping agent coating the nanoparticles. TEM showed long range Chart 4A.1. Molecular structures of the gelator and various AuNPs synthesized. directional assembly of the certain AuNPs along the gel fibers. SEM of the dried gels and nanocomposites indicated that the morphological transformation in the composite microstructures depended profoundly on the capping agent of the nanoparticle. Differential Scanning Calorimetry showed that gel formation from sol postponed to lower temperature with incorporation of AuNPs having capping agents which were able to interact with the gel fibers. Rheological studies indicated that the gel-nanoparticle composites exhibit greater rigidity as compared to the naked gel only when the capping agents were able to interdigitate into the gelator assembly. Also, very low percentage of the AuNPs incorporation could switch the cholesteric mesophase of gel assembly, as evident from circular dichroism. We have been able to define a relationship between materials and molecular properties via manipulation of the molecular structures of NP capping agents. Chapter 4B discusses the design and preparation of novel organogel-carbon nanotube composites by incorporation of single-walled carbon nanotubes (SWNT) into physical gels formed by an L-alanine based Low Molecular Mass Organogelator (Chart 4B.1). The gelation process and the properties of the resulting nanocomposites were found to depend on the kind of SWNTs incorporated in the gels. With pristine SWNTs, only a limited amount could be dispersed in the organogels. Attempted incorporation of higher amounts of pristine SWNTs led to precipitation from the gel. To improve their solubility in the gel matrix, a variety of SWNTs functionalized with different aliphatic and aromatic chains were synthesized (Chart 4B.1). Scanning electron microscope images of the nanocomposites showed that the texture and organization of the gel aggregates were altered upon incorporation of SWNTs. The microstructures of nanocomposites were found to depend on the kind of SWNTs used. Incorporation of functionalized SWNTs into the organogels depressed the sol to gel transition temperature, with the n-hexadecyl chain functionalized SWNTs being more effective than the n-dodecyl chain functionalized counterpart. Rheological investigations of pristine SWNT containing gels indicated that the flow of nanocomposites became resistant to applied stress at a very low wt-% of SWNT incorporation. Again, more effective control of flow behavior was achieved with functionalized SWNTs possessing longer hydrocarbon chains. This happens presumably via effective interdigitation of the pendant chains with the fatty acid amides of L-alanine in the gel assembly. Also, the helical cholesteric mesophase formed by the toluene gel could be switched to a layer stacked assembly by doping functional SWNT. Remarkably, by using a near IR laser irradiation at 1064 nm for a short duration (1 min) at room temperature, it was possible to selectively induce a gel-to-sol phase transition of the nanocomposites, while prolonged irradiation (30 min) of the organogel under identical conditions did not cause gel melting. Chart 4B.1. Molecular structures of the gelator and different functionalized SWNT synthesized. Chapter 5A presents design of two component hydrogels and their potential utilization as a template for metal nanoparticle synthesis. Among a variety of acids and amines (Chart 5A.1) only stearic acid or eicosanoic acid when mixed with di- or oligomeric amines in specific molar ratios form stable gels in water. The formation of such hydrogels depends on the hydrophobicity of the fatty acid, and also on the type of amine used. The gelation properties of these two component systems were investigated using electron microscopy, FTIR, 1H NMR spectroscopy, differential scanning calorimetry (DSC) and both single crystal and cast film X-ray diffraction. FTIR spectral analysis suggests salt formation during gelation. 1H NMR of the gels indicates that the fatty acid chains are immobilized in the gel state and when the gel is melted, these chains regain their mobility. Analysis of DSC data indicates that increase in spacer length in the di-/oligomeric amine lowers the gel melting temperature. Two of these gelator salts developed into crystals and structural details of such systems could be secured by single-crystal X-ray diffraction analysis. The structural information of the salts thus obtained was compared with the XRD data of the self-supporting films of those gels. Such analyses provided pertinent structural insight on the supramolecular interactions that prevail within these gelator assemblies. From the crystal structure it is confirmed that the multilayered lamellar aggregates exist in the gel and it also showed that only one plane of symmetry is present in the gel state. Finally, the hydrogel was used as a medium for the synthesis of silver nanoparticles. The nanoparticles were found to position themselves on the fibers and produce a long ordered assembly of gel-nanoparticle composite (Figure 5A.1). Chart 5A.1. Structures and abbreviations of different acids and amines checked for gelation. Figure 5A.1. TEM images of gel-Ag-NP composite. (a) Ag-NP synthesized in hydrogel of SA-IBPA (1:3.5), (b) Magnified images of Ag-NP preferentially residing on gel fibers. Chapter 5B demonstrates the aptitude of supramolecular hydrogel formation using simple bile acids e.g. lithocholic acid (LCA) in aqueous solution containing di- or oligomeric amines (Chart 5B.1). By variation of the choice of the amines in such mixture the hydrogelation properties could be modulated. However, replacement of LCA by cholic acid or deoxycholic acid resulted in no hydrogelation. FT-IR studies show that the carboxylate and ammonium residues of the two components are primarily involved in salt formation. This promotes further assembly of the components reinforced by continuous Chart 5B.1. Structures and abbreviations of different bile acids and amines checked for gelation. hydrogen bonded network leading to gelation. Electron microscopy shows that the morphology of the gels of two component systems which also depends strongly on the amine part. Variation of amine component from the simple ethanediamine (EDA) to oligomeric amine with lithocholic acid changes the morphology of the assembly from long one dimensional nanotubes to three dimensional complex structures. Single crystal X-ray diffraction analysis with one of the amine-LCA complexes suggested the motif of fiber formation where the amines participate with the carboxylate and hydroxyl moiety through H-bonding and electrostatic forces. The rheological properties of this class of two component system provide clear evidence that this system is a shear-sensitive hydrogel and the flow behavior can be modulated varying the acid-amine ratio. From small angle neutron scattering study, it becomes clear that loose gel from LCA-EDA shows scattering oscillation due to the presence of non interacting nanotubules while for gels of LCA with oligomeric amine the individual fibers come together to form complex three dimensional structures of higher length scale.(For structural formula pl refer the pdf file)

The present thesis entitled “Design and Synthesis of Novel Soft Composites from Physical Gels and Nanomaterials” deals with soft materials derived from low molecular weight gels and nanomaterials. Chapter 1 gives a general introduction and overview of the low molecular weight gel (LMOG) which forms the basis of the work. It delves with the history of research in physical gel field, design of different types of gelator molecules, their interesting self-assembly patterns, potential applications of these gelator molecules as well as challenges to design new gelator molecules. It also encompasses the relatively recent area of two component gel system to conveniently bypass the cumbersome synthetic protocol. The aspect of liquid crystallinity in the gel phase is also discussed to throw light on the pattern of assembly and potential uses of these materials. Towards the end there is a comprehensive discussion on the smart nanocomposites derived from LMOGs and nanomaterials. The design, synthesis and numerous applications of inorganic-organic hybrid composites are discussed. Chapter 2A describes the synthesis and characterization of a variety of fatty acid amides of different naturally occurring L-amino acids whose molecular structures are shown in Chart 2A.1. Some of them were found to form gels with various hydrocarbons. The gelation properties of these compounds were studied by a number of physical methods including FT-IR spectroscopy, X-ray diffraction, scanning electron microscopy (SEM), differential scanning calorimetry, rheology and it was found that gelation was critically dependent on the fatty acid chain length and nature of the amino acid. Among them, L-alanine based gelators were found to be the most efficient and versatile as they self-assemble into a layered structure to form the gel network. Mechanisms for the assembly and formation of gels from these molecules are discussed. (Structural formula) Chart 2A.1. Molecular structures of various fatty acid amides of different amino acids. Chapter 2B describes efficient gelation of both aliphatic and aromatic hydrocarbon solvents by a fatty acid amide, n-lauroyl-L-alanine (Chapter 2B.1). In addition, this compound was found to gelate the binary solvent mixtures comprised of aromatic hydrocarbon e.g. toluene and aliphatic hydrocarbon e.g. n-heptane. SEM and AFM showed that the fiber thickness of the gel assembly increases progressively in the binary mixture of n-heptane and toluene with increasing percentage of toluene. The self- Chart 2B.1. Molecular structure of the gelator. assembly patterns of the gels in individual solvents, n-heptane and toluene are however, different. The toluene gel consists of predominantly one type of morphological species while n-heptane gel has more than one species leading to polymorphic nature of the gel. The n-heptane gel is thermally more stable than the toluene gel as evident from the measurement using differential scanning calorimetry. The thermal stability of the gels prepared in the binary mixture of n-heptane and toluene is dependent on the composition of solvent mixture. Rheology of the gels shows that they are shear-thinning material and show characteristic behavior of soft viscoelastic solids. For the gels prepared from binary solvent mixture of toluene and n-heptane, with incorporation of more toluene in the binary mixture, the gel becomes a more viscoelastic solid. The time sweep rheology experiment demonstrates that the gel made in n-heptane has faster gel formation kinetics than that prepared in toluene. Chapter 2C describes lyotropic mesophase formation by organogels of different fatty acid amides of L-alanine in aromatic solvents. The helical assembly, characteristic of the cholesteric mesophase was found to exhibit reflection bands in circular dichroism spectra. The reflection bands corresponded to the pitch of the helical arrangement of the gelator molecules in the aromatic solvent. Transmission Electron Microscopy (TEM) showed presence of twist in the gel fibres. Polarising optical microscopy of the organogel exhibited weak birefringence confirming lyotropic nature of the assembly. Chapter 3 deals with synthesis and characterization of a new class of molecules with molecular structures shown in Chart 3.1. Among a variety of amino acid based molecules only alanine and serine based molecules were found to form translucent gels in aliphatic hydrocarbons such as n-heptane. TEM showed presence of fiber like structures for alanine whereas serine based gelator produces unique network like structures. SEM of the dried gels exhibited presence of three dimensional fibrous networks to spongy globular cauliflower like structures depending on the molecular structure of the gelators. Rheological studies of the organogels showed that they behave like typical LMOG gels. The oscillatory rheological studies demonstrated that the L-serine based gelator, 5 formed more viscoelastic solid like gel than that of L-alanine based gelator, 1 in n-heptane. Chart 3.1. Molecular structures of different amino acid derivatives from 3,4,5-tri-dodecyloxybenzoic acid scaffold. Chapter 4A presents design and properties of new nanocomposites from LMOG and metal nanoparticles (Chart 4A.1). The profound influence of nanoparticle (NP) incorporation into physical gels was evident from various microscopic and bulk properties. The interaction of nanoparticles with the gelator assembly was found to depend critically on the capping agent coating the nanoparticles. TEM showed long range Chart 4A.1. Molecular structures of the gelator and various AuNPs synthesized. directional assembly of the certain AuNPs along the gel fibers. SEM of the dried gels and nanocomposites indicated that the morphological transformation in the composite microstructures depended profoundly on the capping agent of the nanoparticle. Differential Scanning Calorimetry showed that gel formation from sol postponed to lower temperature with incorporation of AuNPs having capping agents which were able to interact with the gel fibers. Rheological studies indicated that the gel-nanoparticle composites exhibit greater rigidity as compared to the naked gel only when the capping agents were able to interdigitate into the gelator assembly. Also, very low percentage of the AuNPs incorporation could switch the cholesteric mesophase of gel assembly, as evident from circular dichroism. We have been able to define a relationship between materials and molecular properties via manipulation of the molecular structures of NP capping agents. Chapter 4B discusses the design and preparation of novel organogel-carbon nanotube composites by incorporation of single-walled carbon nanotubes (SWNT) into physical gels formed by an L-alanine based Low Molecular Mass Organogelator (Chart 4B.1). The gelation process and the properties of the resulting nanocomposites were found to depend on the kind of SWNTs incorporated in the gels. With pristine SWNTs, only a limited amount could be dispersed in the organogels. Attempted incorporation of higher amounts of pristine SWNTs led to precipitation from the gel. To improve their solubility in the gel matrix, a variety of SWNTs functionalized with different aliphatic and aromatic chains were synthesized (Chart 4B.1). Scanning electron microscope images of the nanocomposites showed that the texture and organization of the gel aggregates were altered upon incorporation of SWNTs. The microstructures of nanocomposites were found to depend on the kind of SWNTs used. Incorporation of functionalized SWNTs into the organogels depressed the sol to gel transition temperature, with the n-hexadecyl chain functionalized SWNTs being more effective than the n-dodecyl chain functionalized counterpart. Rheological investigations of pristine SWNT containing gels indicated that the flow of nanocomposites became resistant to applied stress at a very low wt-% of SWNT incorporation. Again, more effective control of flow behavior was achieved with functionalized SWNTs possessing longer hydrocarbon chains. This happens presumably via effective interdigitation of the pendant chains with the fatty acid amides of L-alanine in the gel assembly. Also, the helical cholesteric mesophase formed by the toluene gel could be switched to a layer stacked assembly by doping functional SWNT. Remarkably, by using a near IR laser irradiation at 1064 nm for a short duration (1 min) at room temperature, it was possible to selectively induce a gel-to-sol phase transition of the nanocomposites, while prolonged irradiation (30 min) of the organogel under identical conditions did not cause gel melting. Chart 4B.1. Molecular structures of the gelator and different functionalized SWNT synthesized. Chapter 5A presents design of two component hydrogels and their potential utilization as a template for metal nanoparticle synthesis. Among a variety of acids and amines (Chart 5A.1) only stearic acid or eicosanoic acid when mixed with di- or oligomeric amines in specific molar ratios form stable gels in water. The formation of such hydrogels depends on the hydrophobicity of the fatty acid, and also on the type of amine used. The gelation properties of these two component systems were investigated using electron microscopy, FTIR, 1H NMR spectroscopy, differential scanning calorimetry (DSC) and both single crystal and cast film X-ray diffraction. FTIR spectral analysis suggests salt formation during gelation. 1H NMR of the gels indicates that the fatty acid chains are immobilized in the gel state and when the gel is melted, these chains regain their mobility. Analysis of DSC data indicates that increase in spacer length in the di-/oligomeric amine lowers the gel melting temperature. Two of these gelator salts developed into crystals and structural details of such systems could be secured by single-crystal X-ray diffraction analysis. The structural information of the salts thus obtained was compared with the XRD data of the self-supporting films of those gels. Such analyses provided pertinent structural insight on the supramolecular interactions that prevail within these gelator assemblies. From the crystal structure it is confirmed that the multilayered lamellar aggregates exist in the gel and it also showed that only one plane of symmetry is present in the gel state. Finally, the hydrogel was used as a medium for the synthesis of silver nanoparticles. The nanoparticles were found to position themselves on the fibers and produce a long ordered assembly of gel-nanoparticle composite (Figure 5A.1). Chart 5A.1. Structures and abbreviations of different acids and amines checked for gelation. Figure 5A.1. TEM images of gel-Ag-NP composite. (a) Ag-NP synthesized in hydrogel of SA-IBPA (1:3.5), (b) Magnified images of Ag-NP preferentially residing on gel fibers. Chapter 5B demonstrates the aptitude of supramolecular hydrogel formation using simple bile acids e.g. lithocholic acid (LCA) in aqueous solution containing di- or oligomeric amines (Chart 5B.1). By variation of the choice of the amines in such mixture the hydrogelation properties could be modulated. However, replacement of LCA by cholic acid or deoxycholic acid resulted in no hydrogelation. FT-IR studies show that the carboxylate and ammonium residues of the two components are primarily involved in salt formation. This promotes further assembly of the components reinforced by continuous Chart 5B.1. Structures and abbreviations of different bile acids and amines checked for gelation. hydrogen bonded network leading to gelation. Electron microscopy shows that the morphology of the gels of two component systems which also depends strongly on the amine part. Variation of amine component from the simple ethanediamine (EDA) to oligomeric amine with lithocholic acid changes the morphology of the assembly from long one dimensional nanotubes to three dimensional complex structures. Single crystal X-ray diffraction analysis with one of the amine-LCA complexes suggested the motif of fiber formation where the amines participate with the carboxylate and hydroxyl moiety through H-bonding and electrostatic forces. The rheological properties of this class of two component system provide clear evidence that this system is a shear-sensitive hydrogel and the flow behavior can be modulated varying the acid-amine ratio. From small angle neutron scattering study, it becomes clear that loose gel from LCA-EDA shows scattering oscillation due to the presence of non interacting nanotubules while for gels of LCA with oligomeric amine the individual fibers come together to form complex three dimensional structures of higher length scale.(For structural formula pl refer the pdf file)