Dissertations / Theses on the topic 'Biomoleculaire'

En el presente trabajo de tesis doctoral se aborda la problemática de la dinámica del proceso de domesticación animal y las prácticas ganaderas iniciales en el extremo más occidental del área mediterránea a lo largo del intervalo temporal que abarca entre el 5.500-4.700 cal ANE. El objetivo principal de la tesis se aborda a partir del estudio de una muestra significativa de yacimientos neolíticos situados en el noreste de la Península Ibérica. Se ha evaluado con esta tesis doctoral los principales modelos explicativos planteados en torno a los orígenes del neolítico en la Península Ibérica, prestando especial atención a las hipótesis y explicaciones formuladas sobre el proceso de domesticación animal y practicas ganaderas iniciales. A partir de los nuevos datos aportados con el estudio de una muestra significativa de yacimientos neolíticos del área del noreste peninsular se ha analizado la información existente bajo el prisma de la tafonomía, demostrando la importancia de considerar no solo la composición de los conjuntos de restos de fauna, sino su grado de representatividad histórica. La contextualización de los nuevos datos obtenidos a nivel peninsular y europeo, con un énfasis especial en el área mediterránea, ha aportado documentos significativos sobre las implicaciones que tuvieron la adopción y cría de rebaños de las cuatro principales especies domésticas. Los análisis efectuados han permitido constatar la explotación polivalente de los diferentes tipos de producciones animales, la plena integración de los ciclos agrícola y ganaderos, la práctica de estrategias ganaderas tanto intensivas como extensivas y la existencia de explotaciones ganaderas permanentes en zonas elevadas del Pirineo durante el Neolítico Inicial, documentos todo ellos que obligan a replantear algunas de las asunciones que han guiado hasta la actualidad el debate sobre los orígenes del Neolítico. A nivel metodológico, el método seguido contempla como novedad de manera integrada los procesos analíticos desarrollados en el marco de la disciplina arqueológica, arqueotafonómica, arqueozoológica y biomolecular. Esta aproximación ha permitido documentar, caracterizar y evaluar los procesos de trabajo vinculados a la explotación y gestión de los animales domésticos durante el Neolítico Inicial. La integración de los análisis de isotopos estables ha supuesto una aportación significativa al estudio de la gestión ganadera, y ha permitido caracterizar las estrategias implementadas en la alimentación de los primeros animales domésticos. Los resultados ponen en evidencia la rápida adaptación de los animales domésticos a los ambientes peninsulares, documentándose la practica una estrategia ganadera plenamente consolidada caracterizada por la posibilidad de modelar la estructura demográfica de los rebaños, la productividad de las especies y las capacidades de adaptación alimentaria de los animales dependiendo de características fisiológicas y etológicas de cada especie. La interpretación de los resultados muestra que la adopción de las técnicas ganaderas no es un proceso lineal ni homogéneo a inicios del Neolítico en la Península Ibérica. La documentación de modalidades regionales pone en relieve la importancia y magnitud del estudio de la domesticación animal y practicas ganaderas iniciales en el marco del proceso de neolitización en esta área geográfica.
In the present doctoral PhD thesis the problematic of the dynamics of the process of animal domestication and the initial husbandry practices in the most western part of the Mediterranean area throughout the temporal interval that covers between 5,500-4,700 cal ANE is addressed. The main objective of this research is approached from the study of a significant sample of Neolithic sites located in the northeast of the Iberian Peninsula. The main explanatory models proposed about the origins of the Neolithic in the Iberian Peninsula have been evaluated with this doctoral thesis, focusing on the hypotheses and explanations formulated about the process of animal domestication and initial livestock practices. Based on the new data provided by the study of a significant sample of Neolithic sites in the northeastern peninsular area, the existing information has been analyzed from a taphonomical perspective, pointing out the importance of considering not only the composition of the faunal remains, but also its degree of historical representativeness. The contextualisation of the new data obtained at peninsular and European level, with a special emphasis on the Mediterranean area, has provided significant documents of the implications of adoption and herding of the four main domestic species. The analyzes carried out have shown polyvalent exploitation of the different types of animal production, full integration of the agricultural and livestock cycles, practice of intensive and extensive livestock strategies and existence of permanent livestock farms in the high areas of the Pyrenees during the Early Neolithic. All these documents force us to rethink some of the assumptions that have guided the debate on the origins of agriculture and pastoralism. At a methodological level, it is to be note as a novelty that the method implemented integrated several analytical processes developed within the framework of archaeological, archaeo-taphonomical, archaeozoological and biomolecular disciplines. This approach has allowed to document, characterize and evaluate the work processes linked to the exploitation and management of domestic animals during the Early Neolithic period. The integration of stable isotope analysis has involved contributing in a significant manner to the study of livestock management, allowing to characterize the strategies implemented in the feeding of the first domestic animals. Results show the rapid adaptation of domestic animals to peninsular environments. Indeed, the practice of a fully consolidated livestock strategy characterized by the possibility of modeling the demographic structure of the herds, the productivity of the species and the food adaptation capacities of the animals depending on physiological and ethological characteristics of each species, has been documented. Therefore, results allow to interpret that the adoption of livestock techniques was a non-linear, non-homogenous process at the beginning of the Neolithic in the Iberian Peninsula. The documentation of regional modalities highlights the importance and magnitude of the study of animal domestication and initial livestock practices in the framework of the neolithization process in this geographical area.

Le cancer du sein est le plus fréquent chez la femme en France, représentant 33% des cas, suivi par le cancer colorectal et celui du poumon. Il est également le plus meurtrier, étant responsable de 18% des décès féminin par cancer.Mes travaux de thèse visent à améliorer la prise en charge des patientes atteintes d'un cancer du sein. Pour cela, il est essentiel de prendre en compte leur qualité de vie ainsi que les caractéristiques biomoléculaires de leur tumeur. J'ai ainsi structuré mes travaux de thèse autour de ces deux axes.Concernant la qualité de vie des patientes, je suis en charge de l'analyse finale et de la valorisation de l'étude MENOCOR qui étudie l'impact de la ménopause chimio-induite (MCI) chez les femmes jeunes en âge de procréer. Dans ce manuscrit, je vous présente les premiers résultats de l'étude (impact de la MCI sur le score fonctionnel du QLQ-C30 ainsi que sur les hormones). Les patientes sont réparties en 2 groupes à la fin de l'étude selon leur statut ménopausique : ménopausées et non ménopausées. Les résultats montrent une tendance indiquant que l'évolution du score dans le temps entre les 2 groupes est différente : les patientes ménopausées voient leur qualité de vie diminuer dans le temps contrairement aux patientes non ménopausées (p=0.058). Les 2 groupes diffèrent selon l'âge (p<0,001) et les récepteurs aux œstrogènes (p=0,03). L'hormone anti-müllérienne (AMH), dont la différence entre les 2 groupes est significative à l'inclusion (p<0,001), se révèle être un marqueur utile dans la définition du statut ménopausique des patientes. Les résultats finaux permettront d'avoir des données concernant la ménopause (incidence, facteurs de risque) ainsi que son impact sur la qualité de vie afin de permettre, dans l'avenir, d'adapter la prise en charge des patientes jeunes et ménopausées de manière précoce.Je me suis également consacrée à la conception du projet AR-GBS visant à mettre au point une technique de réalité augmentée pour le repérage préopératoire des lésions infracliniques du cancer du sein. La technique de réalité augmentée qui sera développée à l'issue de cette étude permettra aux patientes de ne pas avoir recours aux techniques de repérages actuelles invasives et donc d'améliorer leur prise en charge chirurgicale et par conséquent leur qualité de vie.La caractérisation biomoléculaire des tumeurs est investiguée dans l'étude PERCEPTION qui s'intéresse à la corrélation entre les éléments figurés du sang et l'infiltration lymphocytaire tumorale chez les femmes atteintes d'un cancer du sein triple négatif. Je me suis occupée de la gestion de l'étude, de l'analyse intermédiaire ainsi que de sa valorisation. Les résultats obtenus n'ont pas permis de mettre en évidence de corrélation entre le Neutrophil-to-Lymphocyte Ratio (NLR) et le pourcentage de lymphocytes infiltrant la tumeur (TILs) (rs = -0.19, IC 95% [-0.49 ; 0.16], p = 0.3). Contrairement aux attentes basées sur la littérature existante, les résultats montrent une tendance à une corrélation positive entre le NLR et le ratio des TILs CD8/FoxP3 (rs = 0.36, IC 95% [0.03 ; 0.64], p = 0.043). En raison de la fiable taille de l'échantillon et du manque de puissance statistique, l'absence de différence observée doit être interprétée avec prudence. Il est donc nécessaire d'attendre les résultats de l'analyse finale pour conclure. Cette analyse intermédiaire a également permis d'identifier certains éléments à optimiser pour l'analyse finale telle que la méthode utilisée pour déterminer le nombre de TILs de chaque sous-population qui n'est pas suffisamment représentative de l'infiltration réelle.Ces travaux offrent ainsi des perspectives prometteuses pour améliorer la prise en charge et la qualité de vie des patientes atteintes d'un cancer du sein. La poursuite de ces études permettra non seulement de confirmer les tendances observées mais aussi d'affiner les stratégies thérapeutiques adaptées aux besoins spécifiques des patientes
Breast cancer is the most common cancer among women in France, accounting for 33% of cases, followed by colorectal and lung cancers. It is also the deadliest, responsible for 18% of female cancer deaths.My doctoral research aims to improve the management of patients with breast cancer. To achieve this, it is crucial to consider both their quality of life and the biomolecular characteristics of their tumors. I have structured my thesis work around these two themes.Regarding the quality of life of patients, I am responsible for the final analysis and publication of the MENOCOR study, which examines the impact of chemotherapy-induced menopause (CIM) in young women of childbearing age. In this manuscript, I present the initial results of the study (the impact of CIM on the functional score of the QLQ-C30 and hormone levels). At the end of the study, the patients are divided into two groups based on their menopausal status: menopausal and non-menopausal. The results indicate a trend suggesting that the evolution of the score over time differs between the two groups: menopausal patients experience a decrease in quality of life over time, unlike non-menopausal patients (p = 0.058). The two groups differ in terms of age (p < 0.001) and estrogen receptors (p = 0.03). The Anti-Müllerian Hormone (AMH), which shows a significant difference between the two groups at baseline (p < 0.001), proves to be a useful marker in defining the menopausal status of patients. The final results will provide data on menopause (incidence, risk factors) and its impact on quality of life, enabling the adaptation of early management for young and menopausal patients in the future.I also took part of the AR-GBS project conception, which aims to develop an augmented reality technique for the preoperative localization of subclinical breast cancer lesions. The augmented reality technique developed as a result of this study will allow patients to avoid current invasive localization techniques, thereby improving their surgical management and, consequently, their quality of life.The biomolecular characterization of tumors is investigated in the PERCEPTION study, which explores the correlation between blood components and tumor infiltration lymphocytes in women with triple-negative breast cancer. I was responsible for managing the study, conducting the interim analysis, and its publication. The results did not demonstrate a correlation between the Neutrophil-to-Lymphocyte Ratio (NLR) and the percentage of tumor-infiltrating lymphocytes (TILs) (rs = -0.19, 95% CI [-0.49; 0.16], p = 0.3). Contrary to expectations based on existing literature, the results show a trend toward a positive correlation between the NLR and the CD8/FoxP3 TIL ratio (rs = 0.36, 95% CI [0.03; 0.64], p = 0.043). Due to the small sample size and lack of statistical power, the absence of an observed difference should be interpreted with caution. Therefore, it is necessary to await the final analysis results to draw conclusions. This interim analysis also identified areas for optimization in the final analysis, such as the method used to determine the number of TILs in each subpopulation, which is not sufficiently representative of the actual infiltration.These works thus offer promising perspectives for improving the management and quality of life of breast cancer patients. Continuing these studies will not only confirm the observed trends but also refine therapeutic strategies tailored to the specific needs of patients

Este trabalho apresenta o estudo e caracterização de dois complexos moleculares, hRXRálfadeltaAB e hemocianina de Acanthoscurria gomesiana, através de técnicas estruturais e biofísicas. O uso da técnica de crio-microscopia eletrônica para o estudo da hemocianina de Acanthoscurria gomesiana, resultou em um modelo estrutural com resolução de 14 angstron- pelo métodode Fourier Shell Correlation com critério de 1/2 bit. Neste limite de resolução, já é possível observar detalhes estruturais que o mostram como sendo comptível com outros modelos de hemocianinas. Com relação ao estudo de hRXRalfadeltaAB, mostrou-se, através das técnicas de cromatografia analítica de exclusão por tamanho, eletroforese de gel de poliacrilamida e SAXS, que a proteína pode se apresentar no estado dimérico em solução, mesmo na ausência do seu ligante, 9-cis-RA. Também foi estudado a associação de hRXRalfadeltaAB a elementos responsivos: DR1, DR4, F2 e PAL. Suas constantes de dissociação foram calculadas através da técnica de espectroscopia por anisotropia de fluorescência. Os resultados obtidos mostram maior afinidade por DR1 e DR2 e indicam uma origem entrópica para o processo de associação
This work describes characterization of two biomolecular complexes: hRXR deltaAB and a hemocyanin from Acanthoscurria gomesiana using structural and biophysical techniques. Application of cryo-electron microscopy to studies of a hemocyanin from Acanthoscurria gomesiana resulted in its structural model to 14Å resolution, which was calculated by Fourier Shell Correlation with cut-off of 1/2 bit. At this resolution limit one can observe structural details of the complex which are compatible with other hemocyanin models. With respect to hRXR deltaAB, we showed using analytic size exclusion chromatography, SDS PAGE and SAXS, that the protein is dimeric in solution even at the absence of its ligand, 9-cis-RA. hRXR deltaAB binding to the responsive elements of DNA, DR1, DR4, F2 and PAL was investigated and the binding constants to these responsive elements have been determined using fluorescence anisotropy technique. Our results show higher affinity of the receptor to DR1 and DR4 and indicate entropic mechanism of DNA binding

As interações moleculares, em especial as de caráter não-covalente, são processos-chave em vários aspectos da biologia celular e molecular, desde a comunicação entre as células ou da velocidade e especificidade das reações enzimáticas. Portanto, há a necessidade de estudar e criar métodos preditivos para calcular a afinidade entre moléculas nos processos de interação, os quais encontram uma gama de aplicações, incluindo a descoberta de novos fármacos. No geral, entre esses valores de afinidade, o mais importante é a energia livre de ligação, que normalmente é determinada por modos computacionalmente rápidos, porém sem uma forte base teórica, ou por cálculos muito complexos, utilizando dinâmica molecular, onde mesmo com um grande poder de determinação da afinidade, é muito custoso computacionalmente. O objetivo deste trabalho é avaliar um modelo menos custoso computacionalmente e que promova um aprofundamento na avaliação de resultados obtidos a partir de simulações de docking molecular. Para esta finalidade, o método de Monte Carlo é empregado para a amostragem de orientações e conformações do ligante do sítio ativo macromolecular. A avaliação desta metodologia demonstrou que é possível calcular grandezas entrópicas e entálpicas e analisar a capacidade interativa entre complexos proteína-ligante de forma satisfatória para o complexo lisozima do bacteriófago T4.
The molecular interactions, especially the ones with a non-covalent nature, are key processes in general aspects of cellular and molecular biology, including cellular communication and velocity and specificity of enzymatic reactions. So, there is a strong need for studies and development of methods for the calculation of the affinity on interaction processes, since these have a wide range of applications like rational drug design. The free energy of binding is the most important measure among the affinity measurements. It can be calculated by quick computational means, but lacking on strong theoretical basis or by complex calculations using molecular dynamics, where one can compute accurate results but at the price of an increased computer power. The aim of this project is to evaluate a computationally inexpensive model which can improve the results from molecular docking simulations. For this end, the Monte Carlo method is implemented to sample different ligand configurations inside the macromolecular binding site. The evaluation of this methodology showed that is possible to calculate entropy and enthalpy, along analyzing the interactive capacity between receptor-ligands complexes in a satisfactory way for the bacteriophage T4.

O trabalho descrito nesta tese mostra a aplicação de um sistema de detecção condutométrica sem contato acoplado capacitivamente (C4D) para monitorar interações biomoleculares em microssistemas analíticos. Inicialmente, o desempenho analítico de microssistemas fabricados em vidro, poli(dimetilsiloxano) (PDMS) e poliéster-toner (PT) foi avaliado de modo a escolher o melhor material (em termos de facilidades de fabricação, custo e repetibilidade) para os ensaios biomoleculares. Dentre os materiais estudados, os dispositivos fabricados em PT mostraram-se mais adequados para testes rápidos, onde a repetibilidade analítica não é o parâmetro mais importante. Os dispositivos fabricados em PDMS e selados contra uma placa de vidro apresentaram os melhores resultados em termos de repetibilidade e o desempenho analítico foi similar aos dispositivos de vidro. Dessa maneira, os dispositivos fabricados em PDMS/vidro foram escolhidos para a demonstração dos objetivos da tese. Por outro lado, os dispositivos fabricados em PT foram explorados para estudar a configuração geométrica do sistema de C4D. A instrumentação para monitoramento dos ensaios de ligação foi composta basicamente de dois sistemas de C4D, um software escrito em LabVIEW e um sistema de bombeamento das soluções. De modo a encontrar a configuração ideal da cela de detecção, geometrias contendo três, quatro e cinco eletrodos foram avaliadas em dispositivos de PT. A configuração ótima foi composta de três eletrodos, espaçados simetricamente. Nesta geometria, um eletrodo é utilizado para aplicar o sinal senoidal de excitação e os outros dois são utilizados para capturar o sinal resultante. As dimensões dos eletrodos (largura e espaçamento entre eles) foram otimizados usando ferramentas quimométricas. O complexo avidina-biotina foi utilizado como modelo de ligação para mostrar a aplicabilidade do sistema proposto. Para os microssistemas biomoleculares, os eletrodos (com geometria otimizada) foram fabricados sobre a superfície de uma placa de vidro por fotolitografia, sputtering e lift-off. Os eletrodos de detecção foram isolados com uma camada de óxido de silício com espessura de 50 nm, depositada pelo processo de deposição química em fase de vapor assistida por plasma. A camada de SiO2 foi modificada quimicamente com solução de 3-amino-propil-trietóxi-silano em etanol. Para imobilização covalente de biotina, uma alíquota de 10 ?L de fotobiotina dissolvida em água (0,1 mg/mL) foi adicionada à superfície e exposta a radiação ultravioleta (365 nm, 10 mW/cm2) durante 15 min. A detecção foi realizada aplicando um sinal senoidal, a partir de um gerador de funções, ao eletrodo de excitação registrando o sinal resultante nos dois eletrodos receptores. Para minimizar a captura de ruído elétrico, os experimentos foram realizados em uma gaiola de Faraday. O controle e a aquisição de dados foi feito mediante um software escrito em LabVIEW monitorando os sensorgramas de condutividade em tempo real. Os canais microfluídicos foram fabricados em PDMS por litografia suave e selados irrevesivelmente contra a placa de vidro contendo os eletrodos isolados e modificados quimicamente. As soluções (tampão e amostra) foram manuseadas com auxílio de uma bomba peristáltica ou duas bombas seringas. Soluções contendo tampão e avidina foram introduzidas nos microcanais e as mudanças de conductividade foram monitoradas em função do tempo. As soluções contendo avidina permaneceram em contato com a superfície modificada até o sinal de condutividade atingir um patamar de equilíbrio. Depois disso, solução tampão foi introduzida no microcanal para remover os analitos adsorvidos à superfície. Duas válvulas solenóides foram utilizadas para permitir um controle automático da distribuição das soluções nos microcanais. O limite de detecção obtido para a interação entre avidina e biotina foi de 75 nmol L-1.
The study reported in this thesis shows the application of a capacitively coupled contactless conductivity detection (C4D) for monitoring biomolecular interactions on analytical microsystems. Initially, the analytical performance of the microsystems fabricated in glass, poly(dimethylsiloxane) (PDMS) and polyester-toner (PT) was investigated in order to choose the best material (in terms of fabrication facilities, costs and repeatability) for the biomolecular assays. Among all substrate materials studied, devices fabricated in PT showed suitability for quick experiments, in which the analytical repeatability is not the most important parameter. The devices fabricated in PDMS and sealed against a glass plate presented the best results in terms of repeatability and the analytical performance was similar to that one of glass devices. For this reason, PDMS/glass devices were chosen for showing the goals of this thesis. On the other hand, PT devices were employed to study the geometrical design of the C4D system. The instrumentation for monitoring binding assays was basically composed of two C4D systems, a software written in LabVIEW and a solution pumping system. In order to find the suitable detection cell configuration for this dual-C4D system, designs containing three, four and five electrodes were evaluated on PT devices. The optimal design was composed of three electrodes symmetrically spaced. In this configuration, one electrode is used for applying an excitation sinusoidal wave and the other two for picking up the resulting signal. The dimensions of the electrodes (width and gap) were optimized by chemometric tools. The avidin-biotin complex was used as a binding model for showing the feasibility of the proposed system. For the biomolecular microsystems, electrodes were fabricated on glass surface using photolithographic, sputtering and lift-off processes. Detection electrodes were insulated with a 50-nm silicon oxide layer deposited by plasmaenhanced chemical vapor deposition. The SiO2 layer was functionalized by immersing the cleaned surface in a 3-aminopropyltriethoxy-silane solution in ethanol for 3 h. For biotinylation of the amino-silane layer, 10 ?L of photobiotin dissolved in deionized water (0.1 mg/mL) was dropped on the modified glass surface and exposed to a 365 nm UV radiation at intensity of 10 mW/cm2 for 15 min. Detection was carried out by passing a sinusoidal excitation signal from the function signal generator to the first electrode and picking up the resulting signal at the two receiver electrodes. To reduce electrical noise pickup, all measurements were carried out in a Faraday cage. The data acquisition was obtained in a software written in LabVIEW and the conductivity sensorgrams were recorded in real-time. The microfluidic network was fabricated in PDMS by soft lithography and irreversibly sealed against the electrodes plate. Solutions were handled into microfluidic channels using a peristaltic pump or two syringe pumps. Buffer and avidin-containing solution was injected into the microchannels and conductivity changes were monitored over time. Avidin solutions were allowed to remain in contact with the surface until a stable conductivity had reached equilibrium. Avidin-free buffer solutions were then injected to rinse off non-specifically bound analytes. Two solenoid valves were used to allow an automatic dispensing of the sample/buffer solution into microchannels. The limit of detection found for avidin-biotin system was 75 nmol L-1.

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Físicas e Matemáticas. Programa de Pós-graduação em Química
Made available in DSpace on 2012-10-22T04:09:00Z (GMT). No. of bitstreams: 0
O presente trabalho analisou materiais provenientes de sítios arqueológicos da população pré-colonial Jê (ca. 1000 AD), localizados na faixa costeira de Santa Catarina. O objetivo principal do trabalho foi identificar a origem dos resíduos orgânicos preservados nos fragmentos de cerâmica escavados dos sítios pré-coloniais Jê, através das seguintes técnicas hifenadas: GC-MS, HT GC-MS e GC-C-MS IR. Foram analisados 118 fragmentos de cerâmica, sendo que 53 % dos fragmentos continham lipídios. A maioria classificada como gordura degradada de origem animal, sendo, embora em menor número, detectado resíduos de origem vegetal. A interpretação destes resíduos de origem animal foi realizado a partir da comparação com amostras de referencia arqueológicas (valores de d13C para colágenos de ossos) e modernas (distribuição dos ácidos graxos saturados em gordura de capivara, mamíferos aquáticos, bivalves e peixes,), pela identificação de novos biomarcadores (ácidos 4,8,12-trimetil-tridecanóico, pristânico, fitânico, C16, C18 e C20 w-(o-alquil-fenil)alcanóicos e di-hidroxiácidos) e através dos valores de d13C dos ácidos graxos Ac14:0, Ac16:0 e Ac18:0. Através destes foi possível comprovar que grande parte dos lipídios tinha sua origem em gorduras marinhas. Já a identificação dos resíduos de origem vegetal foi evidenciada através de triterpenos e ésteres de ceras epicuticulares.

Glycosylation is a posttranslational modification affecting the fast majority of cellular surfaces through glycolipids and glycoproteins. It has been estimated that over 80% of all proteins are glycosylated and that the glycans decorating the periphery of these molecules are fundamental for the biological function of the entity. Many of these functions have yet to be unraveled. As the modification is a secondary-gene event, it depends on the availability and activity of a set of glycosyltransferases, the availability of both donor and acceptor substrate (both in time and place), as well as many other factors that may include temperature, pH, etc. As a consequence a single glycosylation site may express a certain number of different glycans, i.e., structural microheterogeneity. The importance of glycosylation as primary mediators of cellular communication, protein-protein cross-talk, and even cell-pathogen interaction has been attributed precisely to this phenomenon. In the past few years substantial advancements have been made in the understanding of the function of particular glycosidic epitopes, such as the most relevant blood group antigens ABO, and Sda, (sialyl)Lewisx,y,a,b, the HNK-1 antigen, etc., but many more structures have been identified without an attributed functionality, either as a stand-alone epitope or in conjunction with the underlying peptide structure. Similarly, a serious lack of knowledge still exists on the carbohydrate recognizing molecules, i.e. lectins and their function; even so they have been recognized over the last decades as decisive players in numerous biological processes, ranging from cell-cell communication, fertilization, pathogen-cell adhesion to metastasis. Both deficiencies are directly related to the absence of proper tools to elaborate well-defined carbohydrate epitopes for the study of their interaction characteristics and their employment in the discovery of new complementary molecules. Consequently, there is an increasing interest in finding powerful and nanosized tools to screen for these molecules and to study their carbohydrate interactions in detail. In this Thesis, two complementary approaches are described to characterize lectin-carbohydrate interactions with high sensitivity, low sample consumption, and without the need for sample labeling: SPR and CREDEX-MS. In SPR, we have developed an approach where the sugar is immobilized onto a sensor surface through a tailor-made peptide module that allows (1) to capture the lectin, (2) to characterize the interaction through kinetic and thermodynamic parameters, and (3) to identify the interacted protein by mass spectrometry. In CREDEX-MS, based on proteolytic excision of protein-carbohydrate complexes and mass spectrometric analysis, the peptides conforming the carbohydrate binding domain are identified. After completing a preliminary phase where the above mentioned methodologies are optimized and used for the study of carbohydrate-protein interactions using purified well-known lectins; the established platforms are employed to analyze carbohydrate-driven interactions in more complex systems. Specifically, SPR and CREDEX-MS techniques are applied in a research project on molecular aspects of reproduction with the main objective of disclosing the molecular elements that participate in the first steps of fertilization in bovine species, namely: formation of the sperm reservoir in the oviductal epithelium and gamete recognition (oocyte (ZP)-sperm interaction).
La glicosilació, el procés enzimàtic que uneix sacàrids per produir glicans que s'adhereixen a proteïnes, lípids o altres molècules biològiques, és una modificació co-traduccional i post-traduccional present a la pràctica totalitat de components cel•lulars, on trobem glicoproteïnes, glicolípids i altres glicoderivats. Pel que fa específicament a les proteïnes, s’estima que més d’un 80% estan glicosilades i que aquests glicans són fonamentals en processos biològics com la senyalització cel•lular, el cicle infecciós de certs patògens, les respostes inflamatòria i immune, la fertilització, etc. En els últims anys s’ha avançat substancialment en el coneixement bàsic de la funció de determinats epítops o cadenes glicosídiques concretes. No obstant, es desconeixen les funcions de moltes altres estructures glicosídiques. D’altra banda, també existeix un cert desconeixement sobre les molècules que reconeixen els carbohidrats, les lectines “lectores del codi glicòmic”. Aquestes proteïnes es caracteritzen per reconèixer i unir-se de forma reversible i específica a certs monosacàrids o epítops oligosacàrids donant lloc a interaccions similars a les reaccions antigen-anticòs o enzim-substrat. El paper de les lectines en processos com l'aglutinació i la definició de grups sanguinis, la inflamació (selectines), la mielinització del teixit nerviós, la progressió tumoral, etc., demostra la ubiqüitat i diversitat d'activitats en què es veuen implicades aquestes proteïnes. Per això, disposar d'una eina ràpida i fiable per al seu aïllament i identificació, previ a l'estudi de les seves interaccions amb polisacàrids, constitueix un objectiu de màxim interès en l'actual investigació biomèdica. En la present Tesi Doctoral, es descriuen dues aproximacions complementàries mitjançant les quals es poden caracteritzar les interaccions lectina-carbohidrat amb gran sensibilitat, poca mostra i sense la necessitat de cap marcatge. En la tècnica de ressonància de plasmó superficial (SPR), el sucre és immobilitzat sobre una superfície a través d'un mòdul peptídic, la qual cosa permet (1) capturar la lectina, (2) caracteritzar la seva interacció mitjançant paràmetres cinètics i termodinàmics i (3) identificar posteriorment la proteïna mitjançant espectrometria de masses. Complementàriament, la tècnica CREDEX-MS, basada en l'excisió proteolítica del complex proteïna-sucre i posterior anàlisi per espectrometria de masses, ens permet identificar els pèptids que formen part del domini d'unió al sucre.

This thesis deals with fragmentation of complex molecular ions, especially biomolecules, in gas phase collision experiments. The aim is to investigate the relations between energy deposition and fragmentation and to shed light on the mechanisms behind energy and charge transfer processes in collisions involving the building blocks of life. Further, the question how a solvent environment influences the dissociation behavior is elucidated. In the first part of the thesis, results from different collision experiments with biomolecular ions are presented, focusing on electron capture induced dissociation of hydrated nucleotides and small peptides. The investigated processes may be relevant for the understanding of radiation damage and the optimization of sequencing methods used in protein research. Our results clearly demonstrate that effects due to surrounding solvent molecules are substantial. While the dissipation of internal energy by evaporation of the loosely bound solvent molecules may protect the biomolecule, the influence which this environment has on the electronic structure may lead to an enhancement or suppression of certain dissociation channels. The second part of the thesis focuses on recent instrumental developments. Here, the aim was to optimize and complement the techniques used in the experiments above and to have versatile tools available for different kinds of gas phase collision studies involving complex molecular ions. Therefore, we have constructed an electrospray ion source platform for the preparation of intense beams, with options of accumulation and cooling of mass selected ions, allowing for a large variety of experiments. This device is also intended to serve as an ion source for the new storage ring facility DESIREE (DoubleElectroStatic Ion Ring ExpEriment), which is currently under construction at Stockholm University. In these unique storage rings, oppositely charged ions may interact at very low relative velocities in a cryogenically cooled and ultrahigh vacuum environment.

This thesis describes experimental studies of ultrafast charge transfer processes in molecules which are the building blocks of proteins and DNA; the amino acid phenylalanine and the nucleoside adenosine respectively. This was achieved by using femtosecond (10-15 and attosecond (10-18) laser pulses with a range of wavelengths to create a positive charge in these molecules through ionisation and to probe the subsequent electron motion. This work has been performed using gas phase molecular targets which were liberated from solid samples using a technique known as laser induced acoustic desorption (LlAD). An experimental investigation was performed supported by simulations undertaken using a radiation transport code, HYADES, to optimise and understand this technique. The results presented for the adenosine and the nucleobase adenine suggest that fragmentation of the sugar group following ionisation (which corresponds to strand breakages in DNA) is mostly via relatively slow dissociation processes (nano or microsecond timescales). In vivo such a slow statistical process is likely to be quenched by cooling of the vibrational energy in the molecule by the surrounding environment. This shows that, to a degree, cells can be protected from the direct action of ionising radiation in DNA. In phenylalanine two different ultrafast processes were observed. The first of these was charge transfer to the phenyl group with a time constant of 80 fs, attributed to transfer between electronic states. A second process lasting 30 fs was observed, which is consistent with a number of theoretical predictions of a purely electronic process, termed charge migration. This work provides the first experimental evidence for charge migration in a complex molecule, and is one of the fastest processes ever measured in a biological system. It could ultimately pave the way for control of electron transport in natural or synthetic nanostructures.

Sistemas biológicos podem ser representados por redes que armazenam não apenas informações de conectividade, mas também informações de características de seus nós. No contexto biomolecular, esses nós podem representar proteínas, metabólitos, entre outros tipos de moléculas. Cada molécula possui características anotadas e armazenadas em bases de dados como o Gene Ontology. A comparação visual dessas redes depende de ferramentas que permitam o usuário identificar diferenças e semelhanças entre as anotações feitas sobre as moléculas (atributos) e também sobre as interações conhecidas (conexões). Neste trabalho de mestrado, buscou-se desenvolver técnicas que facilitem a comparação desses atributos sobre as moléculas, tentando manter no processo a visualização das redes em que essas moléculas estão inseridas. Como resultado, obteve-se a ferramenta VisPipeline-MultiNetwork, que permite comparar até seis redes, utilizando operações de conjuntos sobre as redes e sobre seus atributos. Dessa forma, diferentemente da maioria das ferramentas conhecidas para a visualização de redes biológicas, o VisPipeline-MultiNetwork permite a criação de redes cujos atributos são derivados das redes originais por meio de operações de união, intersecção e valores exclusivos. A comparação visual das redes é feita pela visualização do resultado dessas operações de conjuntos sobre as redes, por meio de um método de comparação lado-a-lado. Já a comparação dos atributos armazenados nos nós das redes é feita por meio de diagramas de Venn. Para auxiliar este tipo de comparação, a técnica InteractiVenn foi desenvolvida, em que o usuário pode interagir com um diagrama de Venn efetuando operações de união entre conjuntos. Essas operações de união aplicadas sobre os conjuntos são também aplicadas sobre as respectivas formas no diagrama. Esta característica da técnica a diferencia das outras ferramentas de criação de diagramas de Venn. Integrando essas funcionalidades, o usuário é capaz de comparar redes sob diversas perspectivas. Para exemplificar a utilização do VisPipeline-MultiNetwork, dois casos no contexto biomolecular foram estudados. Adicionalmente, uma ferramenta web para a comparação de listas de cadeias de caracteres por meio de diagramas de Venn foi desenvolvida. Ela também implementa a técnica InteractiVenn e foi denominada InteractiVenn website.
Biological systems can be represented by networks that store not only connectivity information, but also node feature information. In the context of molecular biology, these nodes may represent proteins, metabolites, and other types of molecules. Each molecule has features annotated and stored in databases such as Gene Ontology. A visual comparison of networks requires tools that allow the user to identify differences and similarities between nodes attributes as well as known interactions between nodes (connections). In this dissertation, we sought to develop a technique that would facilitate the comparison of these biological networks, striving to maintain in the process the visualization of the network connectivities. As a result, we have developed the VisPipeline-MultiNetwork tool, which allows comparison of up to six networks, using sets of operations on networks and on their attributes. Unlike most known tools for visualizing biological networks, VisPipeline-MultiNetwork allows the creation of networks whose attributes are derived from the original networks through operations of union, intersection and unique values. A visual comparison of the networks is achieved by visualizing the outcome of such joint operations through a all-in-one comparison method. The comparison of nodes attributes is performed using Venn diagrams. To assist this type of comparison, the InteractiVenn technique was developed, in which the user can interact with a Venn diagram, performing union operations between sets and their corresponding diagrams. This diagram union feature differs from other tools available for creating Venn diagrams. With these tools, users manage to compare networks from different perspectives. To exemplify the use of VisPipeline-MultiNetwork, two case studies were carried out in the biomolecular context. Additionally, a web tool for comparing lists of strings by means of Venn diagrams was made available. It also implements the InteractiVenn technique and its site has been named InteractiVenn.

Dissertação para obtenção do Grau de Doutor em Biotecnologia
The main objective of this thesis was to study the fluorescence modulation induced by gold nanoparticles (AuNPs) on fluorophores nearby and/or bonded to the AuNPs’ surface through nuclei acid molecules. The understanding of the effect of distance in the spectral properties of fluorophores would allow the development of a biosensor for the characterisation of DNA and/or RNA sequences. To study the photophysics involved in the fluorescence modulation by AuNPs it was necessary to develop an experimental approach that removed the effect of the optical interference caused by the presence of AuNPs. By comparing the samples with controlled reference solutions it was possible determine the fluorescence quantum yield and fluorescence decay time of the fluorophores in the vicinity of AuNPs. During the characterisation several non-photophysical phenomena involving nanoparticles were unveiled, such as a local pH effect, coupling of the plasmonic oscillator with transition moments of the fluorophore or AuNP-induced fluorophore aggregation. The developed experimental method was applied to the study of the effect of distance in the modulation of fluorescence caused by AuNPs. Using DNA molecules as spacer, the photophysical properties of fluorophores at different distances of the surface of the AuNPs showed a distance-dependence fitting into a 1/r6 dependence. The knowledge gathered on AuNP-DNA-fluorophore systems allowed for a successful semi-quantitative detection of RNA in solution. The same system showed to be useful for the simultaneous quantification and control RNA synthesis in vitro. In situ detection and gene silencing was demonstrated by targeting EGFP mRNA as proof-of-concept. A similar approach was successfully achieved in siRNA and endogenous miRNA targets. The application of this system to micro-deletions and RNA isoforms analysis was also demonstrated in synthetic targets.
Fundação para a Ciência e Tecnologia - (SFRH/BD/43320/2008)

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Mechanical Engineering, 2020
Cataloged from student-submitted PDF of thesis.
Includes bibliographical references (pages 194-206).
The ability of cells to sense and respond to their environment is encoded in biomolecular reaction networks, in which information travels through processes such as production, modification, and removal of biomolecules. These reaction networks can be modeled as input-output systems, where the input, state and output variables are concentrations of the biomolecules involved in these reactions. Tools from non-linear dynamics and control theory can be leveraged to analyze and control these systems. In this thesis, we study two key biomolecular networks. In part 1 of this thesis, we study the input-output behavior of signaling systems, which are responsible for the transmission of information both from outside and from within the cells, and are ubiquitous, playing a role in cell cycle progression, survival, growth, differentiation and apoptosis. A signaling pathway transmits information from an upstream system to downstream systems, ideally in a unidirectional fashion.
A key obstacle to unidirectional transmission is retroactivity, the additional reaction flux that affects a system once its species interact with those of downstream systems. In this work, we identify signaling architectures that can overcome retroactivity, allowing unidirectional transmission of signals. These findings can be used to decompose natural signal transduction networks into modules, and at the same time, they establish a library of devices that can be used in synthetic biology to facilitate modular circuit design. In part 2 of this thesis, we design inputs to trigger a transition of cell-fate from one cell type to another. The process of cell-fate decision-making is often modeled by means of multistable gene regulatory networks, where different stable steady states represent distinct cell phenotypes. In this thesis, we provide theoretical results that guide the selection of inputs that trigger a transition, i.e., reprogram the network, to a desired stable steady state.
Our results depend uniquely on the structure of the network and are independent of specific parameter values. We demonstrate these results by means of several examples, including models of the extended network controlling stem-cell maintenance and differentiation.
by Rushina Shah.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Mechanical Engineering

Recently, research processes in Life sciences have evolved at a rapid pace. This evolution, mainly due to technological advances, offers more powerful equipment and generalizes the digital format of research data. In the data deluge context, we need to overcome the current tsunami of data and prepare for the future. The current model, consisting to regularly add hardware resources into centralized core facilities without global coordination, is no longer sustainable. Scientific data management and analysis should be enhanced in order to offer services and developments corresponding to the new e-Science uses, and infrastructures are the vehicles to achieve so. We propose and implement research support infrastructures in line with new science directives, adapting them to the scenarios presented by the divergent use cases. Three different domain-specific infrastructures framed in three different scientific projects are assembly and introduced in this dissertation. The first case is framed in the clinical data management field, and focuses on the data platforms build around two epidemiologic case studies on Immune Mediated Inflammatory diseases (IMIDs), IMID-clinica and IMID-longitudinal. Making the leap to infrastructures more oriented to analysis process support, the transPLANT infrastructure represents a first intrusion into the topical cloud computing model. It is focused on plant genomics and its design became the seed for a more integrative cloud-based solution, this time developed for the non-programmer’s members of the 3D/4D genomics community. MuGVRE is the front cover of the resulting platform. Becoming obvious the transversal potential of cloud-based computational infrastructures as virtual research environments, openVRE is implemented as an abstraction of MuGVRE. It offers a vanilla platform encompassing computation, data and administration services ready to be adopted and customized by other scientific communities. They all represent an opportunity to establish better research processes through enhanced collaboration, data management, analysis practices and resources optimization.

This thesis describes three readout formats for molecular analyses. A common feature in all works is probing techniques that upon specific target recognition ideally results in equimolar amounts of DNA circles. These are then specifically amplified and detected using any of the techniques presented herein. The first paper presents a method that enables homogeneous digital detection and enumeration of biomolecules, represented as fluorescence-labelled DNA macromolecules. This method offers precise measurements to be performed with a wide linear dynamic range. As an application, two closely related bacterial species were selectively detected. The second paper further investigates and optimizes the properties of the technique presented in paper one. The third paper demonstrates a platform that enables simultaneous quantitative analysis of large numbers of biomolecules. The array format and decoding scheme together propose a digital strategy for decoding of biomolecules. The array and the decoding procedure were characterized and evaluated for gene copy-number measurements. The fourth paper examines a new strategy for non-optical measurements of biomolecules. Characteristics of this technique are investigated, and compared to its optical equivalent, fluorescence polarization.

Abstract Electrostatic interactions are very important in biomolecular systems. Electrostatic forces have received a great deal of attention due to their long-range nature and the trade-off between desolvation and interaction effects. It remains a challenging task to study and to predict the effects of electrostatic interactions in biomolecular systems. Computer simulation techniques that account for such interactions are an important tool for the study of biomolecular electrostatics. This study is largely concerned with the role of electrostatic interactions in biomolecular systems and with developing novel models to estimate the strength of such interactions. First, a novel formulation based upon continuum electrostatics to compute the electrostatic potential in and around two biomolecules in a solvent with ionic strength is presented. Many, if not all, current methods rely on the (non)linear Poisson-Boltzmann equation to include ionic strength. The present formulation, however, describes ionic strength through the inclusion of explicit ions, which considerably extends its applicability and validity range. The method relies on the boundary element method (BEM) and results in two very similar coupled integral equations valid on the dielectric boundaries of two molecules, respectively. This method can be employed to estimate the total electrostatic energy of two protein molecules at a given distance and orientation in an electrolyte solution with zero to moderately high ionic strength. Secondly, to be able to study interactions between biomolecules and membranes, an alternative model partly based upon the analytical continuum electrostatics (ACE) method has been also formulated. It is desirable to develop a method for calculating the total solvation free energy that includes both electrostatic and non-polar energies. The difference between this model and other continuum methods is that instead of determining the electrostatic potential, the total electrostatic energy of the system is calculated by integrating the energy density of the electrostatic field. This novel approach is employed for the calculation of the total solvation free energy of a system consisting of two solutes, one of which could be an infinite slab representing a membrane surface.

The amalgamation of nanotechnology and biology has led to the development ofnew types of hybrid materials that are expected to produce major advances in areas such as materials science, therapeutics and diagnostics. One of the most promising developments is the use ofnanoparticles (NPs) as labels for the detection of analytes in biological assays. The aim of this research project was to prepare gold nanoparticle (GNP) labels for use in such assays. In chapter 1, the optical properties and the use of GNPs in homogeneous and heterogeneous colorimetric assays are reviewed. In chapter 2 a simple conjugation method is introduced that not only allows almost any biological molecule or hapten to be attached to GNPs but also allows the user to control or vary the mean number of molecules per particle. In this method a high molecular weight aminodextran polymer is functionalized with the molecule of choice and chemical attachment groups that are used to covalently anchor the polymer to the GNPs. This method was used to conjugate biotin and 1125 functionalized dextrans to GNPs. These functionalized dextrans were then used to investigate the conjugation procedure in more_detail. Results from GNP titrations and microbead assays demonstrate that the minimum amount of functionalized dextran required to prevent salt-induced flocculation ofthe GNPs (equivalence point) is the amount required to coat all of the GNPs and at this point there is no free functionalized dextran in solution. In chapter 3 the described method was used to conjugate different numbers DNP haptens to GNPs and then these labels were used in non-traditional reagent-limited lateral flow immunoassays. The number of molecules per GNP is varied by simply adjusting the stoichiometry of reagents in the dextran functionalization reaction. Controlling the number of molecules per particle can have important consequences on the sensitivity of a biological assay. Results showed that when the number of DNP molecules per particle decreased, there was an increase in the sensitivity of the assay. Furthermore when the results from these immunoassays were compared to those obtained from traditional reagent-limited lateral flow immunoassays, the nontraditional format proved to be over 50 % more sensitive. In chapter 4 the conjugation method was used to attach oligonucleotides to GNPs for use in a nucleic acids lateral flow (NALF) device. Although NALF devices are available commercially, detection is usually achieved with the use of antibodies or haptens which can be both problematic and expensive. In addition, many of these devices have issues with sensitivity and are often interfaced with complicated target amplification / purification protocols. In chapter 4 an antibody / hapten independent NALF device is described that can be used to detect the un-purified products from a simple polymerase chain reaction (PCR) amplification protocol. Using the developed NALF device it was possible to detect specific amplification products corresponding to ~1 attomole oftemplate molecules with the unaided eye.

Thesis (Ph. D.)--University of California, San Diego, 2007.
Title from first page of PDF file (viewed June 21, 2007). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 142-157).

Thesis (Ph. D.)--University of Missouri-Columbia, 2007.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on February 14, 2008) Vita. Includes bibliographical references.

Microfluidics is the science and technology of manipulating fluids at small scales ranging from microlitres (10$^{-6}$) to picolitres (10$^{-12}$). The fundamental physics is distinct from fluid behaviour on bulk scales and laminar flow is the key characteristic on this scale. Microfluidic systems have a wide range of applications in many disciplines from engineering to physics, chemistry and biotechnology. In this thesis, I explore different strategies exploiting the capabilities of microfluidic devices for manipulating and analysing biomolecules. A particular focus of the work is on the study of amyloid fibrils. These species are protein aggregates related to a wide range of human diseases and functional materials. In chapter 3 I demonstrate an efficient way to separate particles in different sizes based on a microfluidic diffusion method. This method enables us to explore the properties of amyloid fibrils, such as their growth kinetics and interaction with small molecules. Rapid binding information could be obtained with only microlitres of sample in tens of seconds time scale. A further manipulation method for charged particles is introduced in chapter 4, based on the integration of microfluidics and free flow electrophoresis. I present a very effective and simple way to overcome one of the most critical problem in this situation. High electric filed can be applied through two streams of conductive solutions, with all the electrolysis by products, e.g., gas bubbles and other deposits, removed simultaneously without interfering with the system. In addition to microfluidic devices made by soft lithography in PDMS, I also set up a hot embossing fabrication process with the Teflon material (chapter 5). Teflon has many advantages compared with PDMS, such as lower protein adsorption, higher mechanical strength and better chemical compatibility. With different materials and structures, microfluidic devices can be expanded to more applications.

Thesis (Ph. D.)--University of California, San Diego, 2009.
Title from first page of PDF file (viewed June 16, 2009). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references.

Thesis (M.S.)--West Virginia University, 2008.
Title from document title page. Document formatted into pages; contains ix, 115 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 103-105).

Thesis (M.S.)--University of Cincinnati, 2010.
Advisor: Carla Purdy. Title from electronic thesis title page (viewed Apr. 19, 2010). Includes abstract. Keywords: Biological pathways; weighted gate; BMDL; pyrimidine. Includes bibliographical references.

Aquesta tesi ha estat realitzada al Centre Nacional de Microelectrònica, Institut de Microelectrònica de Barcelona (CNM-IMB) que és un institut d'investigació que forma part del Consell Superior d'Investigacions Científiques (CSIC). La memòria és un recull de la feina realitzada per en Luis Guillermo Villanueva Torrijo sota la direcció del Professor d'Investigació Joan Bausells Roigé al període comprès entre setembre de 2002 i octubre de 2006. El treball queda dividit en tres apartats, tots tres relacionats amb el disseny i la fabricació de bigues de mida micromètrica (micro-cantilevers en anglès) per a diferents aplicacions. Al segon capítol es descriu la feina realitzada amb bigues piezoresistives. L'objectiu fonamental d'aquesta part del treball consistia en la fabricació d'un element sensor capaç de detectar forces dins del rang de 10 a 100 pN. Per això, en primer lloc, es va realitzar una anàlisi teòrica del comportament d'aquestes estructures mecàniques quan se les hi aplica una força al seu extrem lliure. També es va estudiar el soroll (tant electrònic com mecànic) que presentaven. D'aquesta manera, es van establir uns criteris per a la maximització de la sensibilitat i la resolució del sensor. Els resultats analítics es van comparar amb els resultats de simulacions per elements finits, obtenint divergències molt baixes. Això va ser interpretat com una validació dels resultats analítics. Es van dissenyar i fabricar unes bigues piezoresistives de polisilici amb forma de "U". Les dimensions i la resta de paràmetres es van determinar mitjançant els criteris obtinguts per l'optimització del comportament de les bigues. Aquestes es van fabricar a la Sala Blanca del CNM i també fent servir una tecnologia CMOS comercial (0.8 m de AustriaMicroSystems). Els processos de fabricació dins de la Sala Blanca del CNM es van optimitzar per augmentar el rendiment de les oblies. Així, finalment, es va arribar a un rendiment que estava a prop del 95% (aproximadament 95 de cada 100 dispositius es van obtenir correctament). Es va optimitzar el post procés dels xips CMOS al CNM per obtenir un alt rendiment. En aquest cas no només es va considerar la supervivència de les estructures, sinó també la dels circuits CMOS integrats al costat de les bigues. Aquests circuits, dissenyats al ETH de Zürich, consisteixen en un filtre i un amplificador per a millorar la resolució del sensor. Una vegada fabricats, els dispositius es van caracteritzar. La part principal d'aquesta caracterització recollia dos aspectes: la mesura del soroll del senyal de sortida del circuit i la determinació de la sensibilitat dels dispositius. Considerant tots dos resultats es va calcular la resolució dels sensors. Els millors resultats obtinguts van ser aproximadament 30 nN per a les bigues fabricades al CNM i 30 pN per les bigues fetes amb tecnologia CMOS. Aquesta diferència de tres ordres de magnitud a la resolució és deguda als circuits amplificadors i ens permetria mesurar forces al rang requerit.
Per altra banda, amb l'objectiu de realitzar mesures de conducció en un ambient líquid, es van fabricar unes bigues conductores però aïllades. La capa conductora en aquestes bigues (una capa d'or) ha d'estar aïllada del exterior per una capa dielèctrica (nitrur de silici) per disminuir d'aquesta manera les capacitats paràsites. Al extrem lliure, s'ha de situar una punta de polisilici afilada per poder escanejar superfícies. La punta ha d'estar coberta per or i, sobre l'or, tenir nitrur a tot arreu menys al vèrtex. Per obtenir aquests dispositius, es va optimitzar el gravat de puntes de polisilici obtenint finalment puntes amb un diàmetre de vèrtex més petit que 20 nm (fent servir un atac sec en un equip DRIE seguit d'unes oxidacions per esmolar). A més, es va realitzar un estudi dels esforços interns per intentar obtenir bigues planes. A l'última part del treball, es va dur a terme la fabricació de sondes per AFM (bigues amb una punta esmolada al seu extrem lliure). Aquests dispositius es fan servir moltíssim actualment per caracteritzar superfícies i realitzar experiments que requereixen molta precisió i/o resolució. L'objectiu fonamental d'aquesta feina era el possibilitar la fabricació de sondes per AFM al nostre centre de manera que els dissenys poguessin ser triats pels investigadors d'acord amb les necessitats de cadascú d'ells. Per això, es van considerar diferents materials i processos de fabricació de puntes. La millor opció va ser el gravat sec amb un equip DRIE d'unes puntes "tipus coet" amb una part superior afilada, situada al cim d'una columna cilíndrica. Els processos de gravat es van optimitzar per així obtenir una alta uniformitat arreu de l'oblia, així com uns perfils de puntes apropiats per poder fer-les servir en un AFM. A continuació, es van fabricar sondes completes. Per comprovar com de bona era la tecnologia de fabricació que havíem dissenyat, es van fabricar dispositius de dos tipus diferents: per fer-les servir en mode contacte (constant elàstica baixa) i per fer-les servir en mode dinàmic (constant elàstica alta). Aquests dispositius es van utilitzar per escanejar unes mostres d'alumini i es van comparar amb els resultats obtinguts amb sondes comercials, obtenint resultats similars en ambdós casos.
Finalment, es van fabricar sondes per a aplicacions específiques: sondes amb puntes amb la part superior plana per l'estudi de la elasticitat de polímers i materials biològics (molt baix mòdul de Young) i sondes amb bigues d'una geometria especial per a que les freqüències de ressonància del mode fonamental i del primer harmònic transversal estiguessin més juntes, per així millorar la detecció del potencial de superfície en la tècnica KPFM. Amb la fabricació d'aquestes puntes, es va demostrar que el disposar d'una tecnologia que permetés la fabricació de sondes pot ser molt útil per al desenvolupament de noves aplicacions de l'AFM.
Este trabajo queda dividido en tres apartados, todos ellos relacionados con el diseño y fabricación de vigas en voladizo de tamaño micrométrico (micro-cantilevers en inglés) para diferentes aplicaciones. En el segundo capítulo se describe el trabajo realizado con vigas piezorresistivas. El objetivo fundamental de esa parte del trabajo consistía en la consecución de un elemento sensor capaz de detectar fuerzas en el rango de 10 a 100 pN. Para ello, en primer lugar, se realizó un detallado análisis teórico del comportamiento de estas estructuras mecánicas cuando se les aplica una fuerza en su extremo libre. Se estudió asimismo el ruido (tanto eléctrico como mecánico) presente en ellas. De esta manera se establecieron unos criterios para la maximización de la sensibilidad y la resolución del sensor. Los resultados analíticos se compararon con los resultados de simulaciones por elementos finitos, obteniendo divergencias muy bajas, lo cual fue interpretado como una validación de los primeros. Se diseñaron y fabricaron unas vigas piezorresistivas de polisilicio con forma de U. Las dimensiones y demás parámetros se fijaron mediante los criterios obtenidos para la optimización del comportamiento de las vigas. Las vigas se fabricaron tanto en la Sala Blanca del CNM como usando una tecnología CMOS comercial (0.8 m de AustriaMicroSystems). Los procesos de fabricación dentro de la Sala Blanca del CNM se optimizaron para aumentar el rendimiento de las obleas. De esta forma, finalmente, se alcanzó un rendimiento cercano al 95% (aproximadamente 95 de cada 100 dispositivos se obtuvieron correctamente). Se optimizó asimismo el post proceso de los chips CMOS en el CNM para obtener un alto rendimiento. En este caso, se consideró la supervivencia de las estructuras mecánicas así como de la circuitería CMOS integrada junto con las vigas. Esta circuitería, diseñada en el ETH de Zürich, consistía en un filtro y un amplificador para mejorar la resolución del sensor. Una vez fabricados, los dispositivos se caracterizaron. La parte central de esta caracterización englobó dos aspectos: la medida del ruido de la señal de salida del circuito y la determinación de la sensibilidad de los dispositivos. Teniendo en cuenta ambos resultados se calculó la resolución de nuestros sensores. Los mejores resultados obtenidos fueron de unos 30 nN para las vigas fabricadas en el CNM y de unos 30 pN para las provenientes de la tecnología CMOS. Esta diferencia de tres órdenes de magnitud en la resolución es debida a la circuitería adjunta a los dispositivos transductores (vigas) y nos permitiría medir fuerzas del orden de magnitud requerido.
Por otro lado, con el objetivo de realizar medidas de conducción en medio líquido, se fabricaron unas vigas conductoras pero aisladas. La capa conductora en dichas vigas (capa de oro) ha de estar aislada del exterior por medio de una capa dieléctrica (nitruro de silicio) para así disminuir las capacidades parásitas. En el extremo libre, se ha de situar una punta de polisilicio afilada para poder escanear superficies. Dicha punta ha de estar cubierta por oro y, sobre el oro, tener nitruro en todas partes salvo en el vértice. Para obtener estos dispositivos, se optimizó el grabado de puntas de polisilicio, obteniendo finalmente puntas con un diámetro de vértice menor que 20 nm (usando un ataque en un equipo DRIE seguido por unas oxidaciones para afilar). Además, se realizó un estudio de los esfuerzos internos para intentar obtener vigas lo más planas posible. En la última parte del trabajo, se llevó a cabo la fabricación de sondas para AFM (vigas con una punta afilada en su extremo libre). Estos dispositivos son ampliamente usados en la actualidad para caracterizar muestras y para realizar experimentos en los que se requiere una alta precisión y/o resolución. El objetivo fundamental de este trabajo era el posibilitar la fabricación de sondas para AFM en nuestro centro de manera que los diseños pudieran ser elegidos a voluntad y acordes con las necesidades de cada
investigador. Para ello se consideraron diferentes materiales y procesos de fabricación de puntas. La mejor opción fue la definición por medio de un equipo DRIE de puntas "tipo cohete" con una parte superior afilada, situada sobre una columna cilíndrica. Los procesos de grabado se optimizaron para así obtener una alta uniformidad a lo largo y ancho de la oblea así como unos perfiles de puntas apropiados para poder ser usadas después en un AFM. A continuación, se fabricaron sondas completas. Para comprobar cómo de buena era la tecnología de fabricación que habíamos diseñado, se fabricaron puntas de dos tipos diferentes: para ser usadas en modo contacto (constante elástica baja) y para ser usadas en modo dinámico (constante elástica alta). Dichos dispositivos se usaron para escanear algunas muestras y se compararon con algunos disponibles comercialmente, obteniendo resultados similares tanto para modo contacto como para dinámico.
Finalmente, se fabricaron sondas para aplicaciones específicas: sondas con puntas con la parte superior plana para el estudio de la elasticidad de polímeros y materiales biológicos (con bajo módulo de Young) y sondas con vigas de una geometría especial para que las frecuencias de resonancia del modo fundamental y del primer harmónico transversal estuvieran más juntas, para así mejorar la detección del potencial de superficie en la técnica KPFM. Con la fabricación de estas puntas, se demostró que el disponer de una tecnología que permita la consecución de puntas puede ser muy útil para el desarrollo de nuevas aplicaciones del AFM.
The main objective of this thesis has been the research in the design and fabrication of micro-cantilevers that are one of the most used mechanical transducers because of their versatility. The use of polysilicon piezoresistive cantilevers has been explored in order to detect binding forces between biomolecules. Force resolution under 100 pN was required. A detailed analytical study has been performed in order to calculate sensitivity and resolution when applying a force at their free end. The results obtained with this analysis have been confirmed by the use of FEM simulations and hence used to determine the optimum design of the piezoresistive sensor. U-shaped polysilicon cantilevers have been fabricated at CNM clean room facilities using a novel and dedicated technology. Designs were made following the criteria imposed by the previously obtained analytical results. The high force resolution required implied the fabrication of some cantilevers among the softest piezoresistive cantilevers reported up to date (elastic constants down to 0.5 mN/m). With the final optimized fabrication process, a yield of 95% has been achieved. Using a commercial CMOS technology (0.8 m from AustriaMicroSystems), polysilicon piezoresistive cantilevers have been designed and fabricated following again the criteria imposed by the theoretical analysis and, in this case, also design rules from the CMOS technology. Cantilevers were integrated with a filtering and amplifying circuitry to reduce noise. The softest piezoresistive CMOS integrated cantilevers have been obtained with a high yield and with an undamaged circuitry. In order to determine the actual sensitivity of such soft sensors and their gauge factor, a characterization method (consisting in AFM actuation) has been developed. Gauge factor for polysilicon deposited at CNM and at AustriaMicroSystems was -12 and -9 respectively. The maximum force sensitivity and force resolution obtained for CNM fabricated sensors have been 11 V/nN and 28 nN respectively. The maximum force sensitivity and force resolution obtained for CMOS fabricated sensors have been 11 V/pN and 27 pN respectively. In both cases, resolution is limited by the noise in the circuit, whose main contributions are Hooge noise (or 1/f) and Johnson noise (or thermoelectric). Conductive, but isolated, nitride cantilevers (with a wrapped gold layer) with a sharp tip (that has an opened contact) have been designed and fabricated to be used in conductive measurements in liquid environments. Polysilicon tips definition has been optimized to improve the whole probes fabrication process, achieving apex radii smaller than 20 nm using a dry etching by means of a DRIE equipment followed by sharpening oxidation.
A complete and novel technological process has been developed for the fabrication of AFM cantilevers. Different tip materials and machining processes have been analyzed, obtaining the best results for crystalline silicon tips defined using a DRIE equipment to machine rocket tips. Isotropic processes with low cross-wafer dispersion and anisotropic processes with low cross-wafer dispersion and low scalloping have been achieved. After a sharpening oxidation, apex radii smaller than 5 nm have been achieved. Complete AFM probes have been fabricated. In order to test the developed technology, probes with similar characteristics to commercial ones were fabricated and used to raster scan some samples (in contact and non-contact mode) yielding results similar to those obtained with commercial probes. In addition, some special probes have been fabricated for nanoindentation over polymers and also to improve Kelvin Probe Force Microscopy (KPFM) performance. Thus, the availability of a technology that allows
the fabrication of customized cantilevers is very useful for the development of new SPM applications.

Neuerliche Fortschritte im Verständnis biomolekularer Motoren rücken ihre Anwendung als Nanomaschinen in den Bereich des Möglichen. So könnten sie zum Beispiel als Nanoroboter arbeiten, um in molekularen Fabriken kleine – aber dennoch komplizierte – Strukturen auf winzigen Förderbändern herzustellen, um Netzwerke molekularer Nanodrähte und Transistoren für elektronische Anwendungen zu assemblieren oder sie könnten in adaptiven Materialien patrouillieren und diese, wenn nötig, reparieren. In diesem Sinne besitzen biomolekulare Motoren das Potenzial, die Basis für die Konstruktion, Strukturierung und Wartung nanoskaliger Materialien zu bilden
Recent advances in understanding how biomolecular motors work have raised the possibility that they might find applications as nanomachines. For example, they could be used as molecule- sized robots that work in molecular factories where small, but intricate structures are made on tiny assembly lines, that construct networks of molecular conductors and transistors for use as electrical circuits, or that continually patrol inside “adaptive” materials and repair them when necessary. Thus biomolecular motors could form the basis of bottom-up approaches for constructing, active structuring and maintenance at the nanometer scale

Islet transplantation has emerged as a promising cell-based therapy for the treatment of diabetes, but its clinical efficacy remains limited by deleterious host responses that underlie islet destruction. In this dissertation, we describe the assembly of cell surface-supported thin films that confer molecular-level control over the composition and biophysicochemical properties of the islet surface with implications for improving islet engraftment. Specifically, the process of layer-by-layer (LbL) polymer self assembly was employed to generate nanothin films of diverse architecture with tunable properties directly on the extracellular surface of individual islets. Importantly, these studies are the first to report in vivo survival and function of nanoencapsulated cells, and have helped establish a conceptual framework for translating the diverse applications of LbL films to cellular interfaces. Additionally, through proper design of film constituents, coatings displaying ligands and bioorthogonally reactive handles may be generated, providing a modular strategy for incorporating exogenously derived regulators of host responses alongside native constituents of the islet surface. Towards this end, a strategy was developed to tether thrombomodulin to the islet surface in a site-specific manner, thereby facilitating local generation of the powerful anti-inflammatory agent, activated protein C. Collectively, this work offers novel biomolecular strategies for cell surface engineering with broad biomedical and biotechnological applications in cell-based therapeutics and beyond.

Flexor tendon injuries in zone II of the hand (i.e. between the distal volar crease and the distal interphalangeal joint) can be costly for both the afflicted individual and society because of the high cost of a long rehabilitation period, complicated by tendon ruptures or scarring with adhesion formation, causing impaired range of motion. The aim of the present thesis was to characterize more fully the deep flexor tendon, the tendon sheath and their response to injury in a rabbit model in order to find potential targets to improve the outcome of repair. The intrasynovial rabbit deep flexor tendon differed from the extrasynovial peroneus tendon in the expression of collagens and transforming growth factor-β1 gene expression. Differences were also found in collagen III and proteoglycans between regions of the flexor tendon subjected to either compressive or tensile load. After laceration and subsequent repair of the flexor tendon, a shift in collagen gene expression from type I to type III occurred. Proteoglycans were generally increased with the notable exception of decorin, a potential inhibitor of the profibrotic transforming growth factor-β1 which was markedly increased during the first two weeks after repair in tendon tissue but remained unaltered in the sheaths. Both vascular endothelial growth factor and basic fibroblast growth factor mRNA levels remained essentially unaltered, whereas insulin-like growth factor-1 increased later in the healing process, suggesting potential beneficial effects of exogenous addition, increasing tendon strength through stimulating tenocyte proliferation and collagen synthesis. Matrix metalloproteinase-13 mRNA levels increased and remained high in both tendon and sheath, whereas there was only a transient increase of matrix metalloproteinase-3 mRNA in tendon. We could also demonstrate a significant increase of the proportion of myofibroblasts, mast cells and neuropeptide containing nerve fibers in the healing tendon tissue, all components of the profibrotic myofibroblast-mast cell-neuropeptide pathway.
Biomolecular aspects of flexor tendon healing

Biomolecular interactions, including protein-protein, protein-DNA, and protein-ligand interactions, are of special importance in all biological systems. These interactions may occer during the loading of biomolecules to interfaces, the translocation of biomolecules through transmembrane protein pores, and the movement of biomolecules in a crowded intracellular environment. The molecular interaction of a protein with its binding partners is crucial in fundamental biological processes such as electron transfer, intracellular signal transmission and regulation, neuroprotective mechanisms, and regulation of DNA topology. In this dissertation, a customized surface plasmon resonance (SPR) has been optimized and new theoretical and label free experimental methods with related analytical calculations have been developed for the analysis of biomolecular interactions. Human neuroglobin (hNgb) and cytochrome c from equine heart (Cyt c) proteins have been used to optimize the customized SPR instrument. The obtained Kd value (~13 µM), from SPR results, for Cyt c-hNgb molecular interactions is in general agreement with a previously published result. The SPR results also confirmed no significant impact of the internal disulfide bridge between Cys 46 and Cys 55 on hNgb binding to Cyt c. Using SPR, E. coli topoisomerase I enzyme turnover during plasmid DNA relaxation was found to be enhanced in the presence of Mg2+. In addition, a new theoretical approach of analyzing biphasic SPR data has been introduced based on analytical solutions of the biphasic rate equations. In order to develop a new label free method to quantitatively study protein-protein interactions, quartz nanopipettes were chemically modified. The derived Kd (~20 µM) value for the Cyt c-hNgb complex formations matched very well with SPR measurements (Kd ~16 µM). The finite element numerical simulation results were similar to the nanopipette experimental results. These results demonstrate that nanopipettes can potentially be used as a new class of a label-free analytical method to quantitatively characterize protein-protein interactions in attoliter sensing volumes, based on a charge sensing mechanism. Moreover, the molecule-based selective nature of hydrophobic and nanometer sized carbon nanotube (CNT) pores was observed. This result might be helpful to understand the selective nature of cellular transport through transmembrane protein pores.

The modulation of biological interactions with artificial surfaces is a vital aspect of biomaterials research. Protein adsorption is established as an early biological response to implanted materials that influences biocompatibility, hence an understanding of how to direct specific protein and cellular responses is critical for the development of future biomaterials. The effects of protein adsorption and subsequent cellular interactions on a variety of surfaces are investigated. Acrylic-based hydrogels are used as a model system in which to investigate both tear and serum protein adsorption from simple and complex solutions. The effect of surface topography, created by colloidal silica, on serum protein adsorption and conformation as well as cell adhesion is also investigated. Tantalum (Ta) and oxidised polystyrene (PSox) are investigated for their ability to support cell adhesion when precoated with various serum proteins. Protein interactions are examined using a combination of quartz crystal microbalance with dissipation (QCM-D), surface plasmon resonance (SPR), dual polarisation interferometry (DPI) and enzyme-linked immunosorbent assay (ELISA) while cellular interactions are analysed using QCM-D, microscopy and adhesion assays. The QCM-D technique was evaluated for its ability to provide new insight into cell-surface interactions. Most tear and serum proteins were found to adsorb onto the acrylic hydrogels, however, lysozyme was found to absorb into the hydrogel matrix and decrease the hydration, which may lead to an adverse biological response. Fibronectin adsorbed onto nanotextured colloidal silica surfaces was found to be conformationally changed compared to flat controls which is likely to correlate with the reduced endothelial cell adhesion observed on these textured surfaces. Ta and PSox precoated with either serum or fibronectin were shown to support cell adhesion and spreading, while surfaces precoated with albumin were not. QCM-D responses varied between underlying surfaces, protein precoating, ECM deposition, cytoskeletal activity and length of exposure indicating that alterations in cell-material responses are reflected in QCM-D measurements. QCM-D parameters were found to correlate with adhered cell numbers, cell contact area and cytoskeletal activity. The results highlight that characterisation of interfacial interactions with a wide range of analytical techniques is necessary to gain insight into cell-protein-material interactions which can then be utilised in the development of new generations of biomaterials with improved properties designed for specific applications.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2005.
Includes bibliographical references (leaves 115-124).
Microfabricated transducers enable the label-free detection of biological molecules in nanoliter sized samples. Integrating microfluidic detection and sample-preparation can greatly leverage experimental efforts in systems biology and pharmaceutical research by increasing analysis throughput while dramatically reducing reagent cost. Microfabricated resonant mass sensors are among the most sensitive devices for chemical detection, but degradation of the sensitivity in liquid has so far hindered their successful application in biology. This thesis introduces a type of resonant transducer that overcomes this limitation by a new device design: Adsorption of molecules to the inside walls of a suspended microfluidic channel is detected by measuring the change in mechanical resonance frequency of the channel. In contrast to resonant mass sensors submersed in water, the sensitivity and frequency resolution of the suspended microchannel resonator is not degraded by the presence of the fluid. Our device differs from a vibrating tube densitometer in that the channel is very thin, and only molecules that bind to the walls can build up enough mass to be detected; this provides a path to specificity via molecular recognition by immobilized receptors.
(cont.) Suspended silicon nitride channels have been fabricated through a sacrificial polysilicon process and bulk micromachining, and the packaging and microfluidic interfacing of the resonant sensors has been addressed. Device characterization at 30 mTorr ambient pressure reveals a quality factor of more than 10,000 for water filled resonators; this is two orders of magnitude higher than previously demonstrated Q-values of resonant mass sensors for biological measurements. Calculation of the noise and the sensitivity of suspended microchannel resonators indicate a physical limit for mass resolution of approximately 0.01 ng/cm2 (1 Hz bandwidth). A resolution of -0.1 ng/cm2 has been experimentally demonstrated in this work. This resolution constitutes a tenfold improvement over commercial quartz crystal microbalance based instruments. The ability to detect adsorbing biomolecules by resonance frequency has been validated through binding experiments with avidin and various biotinylated proteins.
by Thomas P. Burg.
Ph.D.

Electrochemistry is a powerful technique that offers multiple possibilities and which is in constant evolution. Simple modifications of the electrode surface can result in an improvement of the selectivity and sensitivity of the method. However some situations require more complex modifications such as the incorporation of an external agent to the electrode surface, or within the actual electrode. This thesis describes the development and characterization of a range of novel electrochemical sensors for multiple applications covering agri-food, biomedical and environmental contexts. The foundations of the approach rest upon the development of carbon-loaded polycarbonate composite films. Their fabrication is described and the ease with which they can be modified and physically adapted is highlighted and critically evaluated. The response of the resulting sensors have been validated against conventional techniques. An overview of the technologies employing carbon electrodes is presented in Chapter 1 and serves to set the context of the subsequent research. The various methodologies employed are outlined in Chapter 2. Preliminary modifications of the analytical process has evolved from the ex situ functionalisation of the conventional carbon electrodes with copper (Chapter 3) through to the examination of the versatility and complexities of sample pre-treatment (Chapter 4). The pre-treatment of the sample using naphthoquinones as labeling agents has been developed and this work was extended to examine a wholly new derivatisation agent which could have analytical and clinical/veterinary diagnostic merit. A new direction was sought to overcome the limitations of the conventional analytical approach and composite systems were envisaged as providing an accessible yet flexible method of developing electrochemical sensors for discrete probe and flow systems. The basic procedure has been characterization and optimized for a range of analytes such as neurotransmitters (Chapter 5), anti-oxidants (Chapter 6), purine metabolites (Chapter 8) and phosphate (Chapter 9). Each chapter highlights a different aspect and applicability of the composite and go from simple physical surface modification (Chapter 5) to the incorporation of chemical agents (Chapter 6) and more complex systems such as enzymes (Chapters 8 and 9).

This thesis explores the use of cell theory calculations to characterise hydration thermodynamics in small molecules (cations, ions, hydrophobic molecules), proteins and protein-ligand complexes. Cell theory uses the average energies, forces and torques of a water molecule measured in its molecular frame of reference to parameterise a harmonic potential. From this harmonic potential analytical expressions for entropies and enthalpies are derived. In order to spatially resolve these thermodynamic quantities grid points are used to store the forces, torques, and energies of nearby waters which giving rise to the new grid cell theory (GCT) model. GCT allows one to monitor hydration thermodynamics at heterogeneous environments such as that of a protein surface. Through an understanding of the hydration thermodynamics around the protein and particularly around binding sites, robust protein-ligand scoring functions are created to estimate and rank protein-ligand binding affinities. GCT was then able to retrospectively rationalise the structure activity relationships made during lead optimisation of various ligand-protein systems including Hsp90, FXa, scytalone dehydratase among others. As well as this it was also used to analyse water behaviour in various protein environments with a dataset of 17 proteins. The grid cell theory implementation provides a theoretical framework which can aid the iterative design of ligands during the drug discovery and lead optimisation processes, and can provide insight into the effect of protein environment to hydration thermodynamics in general.

Thesis (Ph. D.)--University of Oregon, 2008.
Typescript. Includes vita and abstract. Includes bibliographical references (leaves 164-171). Also available online in Scholars' Bank; and in ProQuest, free to University of Oregon users.

xiv, 171 p. ; ill. (some col.) A print copy of this title is available through the UO Libraries. Search the library catalog for the location and call number.
Biological molecular motors, which use chemical energy from ATP hydrolysis to generate mechanical force, are involved in a variety of important mechanical processes in eukaryotic cells, such as intracellular transport, cell division and muscle contraction. These motors, which produce motion on the nanoscale, operate in the presence of substantial thermal noise. In this dissertation, two approaches are used to model the physics of nanoscale motors: (1) A theoretically established type of Brownian motor called the "flashing ratchet" is studied. This motor transports diffusive particles in a preferred direction. (2) A coarse-grained mechanical model for the biological molecular motor myosin-V is developed, and used to study the role of Brownian diffusion, and the interaction between chemical and mechanical degrees of freedom, in the transport mechanism of this motor. In chapter III, Brownian dynamics simulations and analytical calculations demonstrate that the average velocity of rigid chains of particles in a flashing ratchet reverses direction in response to changing the size of the chain or the temperature of the heat bath. Recent studies have introduced policies for "closed-loop" control of a flashing ratchet, in which the system is controlled based on information about its internal state (such as the positional distribution of particles). In chapter IV, the effect of time delay on the implementation of closed-loop control of a flashing ratchet is investigated. For a large ensemble, a well-chosen delay time improves the ratchet performance (increasing the velocity) by synchronizing into a quasi-stable mode that takes advantage of the semi-deterministic nature of the time development of average quantities for a large ensemble. I n chapter V, a coarse-grained mechanical model is presented for the transport mechanism of myosin-V, which walks along intracellular filaments. The model is well constrained by experimental data on the mechanical properties of myosin V and on the kinetic cycle. An experimentally motivated model for the intramolecular coordination of the motor's steps is proposed and tested.
Adviser: Heiner Linke