Biomolecular Shuttles under Dielectrophoretic Forces

Motor proteins and filaments are essential elements in living cells. They are employed in skeletal muscles to generate forces, they transport cargos such as organelles to specific locations in the cells or they reorganize themselves to change a cell\u27s structure. Moreover, motor proteins and filaments use hydrolysis of adenosine triphosphate (ATP) as chemical fuel to generate mechanical movement in their interaction. Understanding the behavior of these enticed nano-sized machines and their properties, yet to be mimicked and synthesized by humans is very important to the future development of transport in nanoscale. Thus far, researchers succeeded in demonstrating the interaction of motor proteins and filaments in in vitro environment and controlling their random movement by various methods such as with the influence of DC electric field, driven flow field and engineered tracks by photolithographic method. In this thesis, dielectrophoretic forces, which are generated under nonuniform electric field by AC, are explored as a candidate to control the direction of biomolecular shuttles, actin filaments which glide on heavy meromyosin coated surface. Under dielectrophoretic forces, actin filaments showed bidirectional movement between embedded electrodes. The orientation and velocity of actin filaments were measured under various AC voltages, frequencies and distances between electrodes. Additionally, the effect of temperature on myosin-actin motility was further investigated and loading cargo on actin filaments was demonstrated by using a streptavidin-biotin binding system

The Interaction Between Elk-1 And Microtubules In Neurons

Tez (Yüksek Lisans) -- İstanbul Teknik Üniversitesi, Fen Bilimleri Enstitüsü, 2008Thesis (M.Sc.) -- İstanbul Technical University, Institute of Science and Technology, 2008Elk-1, ETS grubu transkripsiyon faktörüdür ve anti-apoptotik ve hücreyi bölünmeye götüren genlerin ekspresyonlarını kontrol etmektedir. Ancak Elk-1 in bölünme özellikğini kaybetmiş olan sinir hücrelerinde bulunması ve sinirlerin akson ve dendrit bölgelerinde gözülmesi bu proteinin başka görevlerinin de olabileceğini göstermektedir. Bu çalışmada, Elk-1 proteininin hücrenin iskeletini oluşturan mikrotübül yapısıyla olan etkileşimi sinir hücrelerinde moleküler ve biyokimyasal yöntemler uygulanarak araştırılmıştır. Öncelikle bu iki proteinin, konfokal mikroskobu tekniği kullanılarak hücrenin içinde yerleri belirlenmiş ve ayni bölgelerde lokalize oldukları görülmüştür. Sonrasında yapılan in vitro ve in vivo bağlanma deneylerinde ise Elk-1 ve mikrotübül arasında direkt interaksiyon olduğu tespit edilmiştir. Daha sonra ise Elk-1 proteininin mikrotübüle bağlanma bölgesinin belirlenmesi amacıyla delesyen mutantları kullanılarak çökertme deneyleri tasarlanmış ve sonuç olarak Elk-1’in değişik bölgelerinin mikrötübüle bağlanma yatkınlığının olduğu bulunmuştur. Elde ettiğimiz bulgular mikrotübüllerin Elk-1 ile interaksiyona geçebildiğini göstermektedir.Elk-1 is an ETS-domain transcription factor which is known to control the expression of immediate early genes such as c-fos and egr-1. Because the expressions of these genes are induced upon external proliferation or survival stimuli, Elk-1 is expected to be present in dividing cells and mostly around or in the nucleus.. However, in the previous studies, Elk-1 has been shown to localize in the axonal and dendritic regions of neurons which are non-dividing cells. For this reason, we wanted to explore whether Elk-1 can associate with microtubules in neuroblastoma cells and primary hippocampal neurons. Our results indicate that Elk-1 co-localizes with microtubules in both cell types, mostly in the cytoplasm and proximal axons. In addition to this, bacterially expressed and purified GST-Elk proteins containing different regions of the whole protein, can pull-down microtubules both from the mouse brain lysate or in the purified form. The interaction between Elk-1 and microtubules were also confirmed by in vitro binding and microtubule sedimentation assays. Finally, microtubules were co-immunoprecipitated with Elk-1 protein in neuroblastoma cells. These data altogether show that Elk-1 can associate with microtubules in neuronsYüksek LisansM.Sc

Multiple trap optical tweezers for cell force measurements

Adherent cells establish transient adhesion sites that serve as stable anchors of the cell cytoskeleton to the substrate and concomitantly allow for force transduction and cell motility. We established a multiple trap optical tweezers system for non-invasive cell force measurements at individual adhesion sites. A force study was conducted to analyze the impact of bead functionalization, adhesion site area, location, spacing, and orientation as well as the role of vinculin on adhesion formation

Advanced Computer Simulations Of Nanomaterials And Stochastic Biological Processes

This dissertation consists of several parts. The first two chapters are devoted to of study of dynamic processes in cellular organelles called filopodia. A stochastic kinetics approach is used to describe non-equilibrium evolution of the filopodial system from nano- to micro scales. Dynamic coupling between chemistry and mechanics is also taken into account in order to investigate the influence of focal adhesions on cell motility. The second chapter explores the possibilities and effects of motor enhanced delivery of actin monomers to the polymerizing tips of filopodia, and how the steady-state filopodial length can exceed the limit set by pure diffusion. Finally, we also challenge the currently existing view of active transport and propose a new theoretical model that accurately describes the motor dynamics and concentration profiles seen in experiments in a physically meaningful way. The third chapter is a result of collaboration between three laboratories, as a part of Energy Frontier Research Center at the University of North Carolina at Chapel Hill. The work presented here unified the fields of synthetic chemistry, photochemistry, and computational physical chemistry in order to investigate a novel bio-synthetic compound and its energy transfer capabilities. This particular peptide-based design has never been studied via Molecular Dynamics with high precision, and it is the first attempt known to us to simulate the whole chromophore-peptide complex in solution in order to gain detailed information about its structural and dynamic features. The fourth chapter deals with the non-equilibrium relaxation induced transport of water molecules in a microemulsion. This problem required a different set of methodologies and a more detailed, all-atomistic treatment of the system. We found interesting water clustering effects and elucidated the most probable mechanism of water transfer through oil under the condition of saturated Langmuir monolayers. Together these computational and theoretical studies compose a powerful and diverse set of physical approaches and both analytical and numerical methodologies, that can be successfully applied in the fields of biology, chemistry and biophysics.Doctor of Philosoph

Development of natural-based hydrogel particles using a biomimetic methodology

Tese de doutoramento em BioengenhariaSuperhydrophobic (SH) surfaces have been greatly explored in the biomedical field. Such surfaces are inspired by the repellent properties of natural structures. The most well known example is the lotus leaf, which has the capacity to repeal the water droplets due to the presence of micro/nano topographical features and the low surface energy. One particular application of artificial SH surfaces is their employment as supports where liquid droplets of aqueous-based polymeric solutions are dispensed, acquiring an almost spherical shape. Through application of one or multiple hardening steps solid particles are obtained in a fast way. Using this methodology, a variety of functional natural-based hydrogel spherical systems encapsulating cells and/or drugs are proposed in this thesis. The strategy of the first proposed system is the incorporation of cyclodextrins (CDs) in the dextranmethacrylate (Dex-MA) microgels networks in order to improve their loading efficiency for hydrophobic drugs. The formation of inclusion complexes between CDs and dexamethasone, when the CDs were copolymerized, increased significantly the amount of drug in the Dex-MA particles, when compared with formulations without or with freely dispersed CDs. Chitosan (Chi), unless it has been modified, is soluble in acidic media, which turn it incompatible with encapsulation of cells or pH sensitive molecules. The second system proposed envisaged a mild Chi-based system with two sequential hardening steps, where dexamethasone or fibroblast-like cells were successfully entrapped. SH surfaces methodology was also used to co-encapsulate cells and proteins into hydrogel particles without compromise the cell viability and protein activity. Collagen combined with platelet lysates were used to obtain easy-to-handle spherical formulations being capable to act as in situ growth factors release system as well as reservoirs of human adipose derived stem cells, for applications in skin regeneration. Mesenchymal stem cells derived from bone marrow and fibronectin were also encapsulated inside alginate spheres and the system was studied for bone regeneration. The control of the release of bioactive agents may be achieved by adjusting the chemistry and physical parameters such as particles architecture. Multicompartmentalized systems have emerged and are envisioned to be the next area of development due to the possibility to confines various bioactive agents exhibiting a variety of release kinetics. Taking advantage of the simplicity of the SH surfaces methodology, core/shell and multilayered particles composed by Dex-MA and alginate were efficiently prepared, with cells or drugs encapsulated into individual compartments. The developed work shows that a wide variety of particles useful for biomedical application, ranging from homogeneous spherical matrices to compartmentalized systems, could be obtained under mild conditions and in a fast way, using SH surfaces.Superfícies superhidrofóbicas (SH) têm vindo a ser exploradas no campo biomédico. Estas superfícies são inspiradas nas propriedades repelentes de estruturas naturais. O exemplo mais conhecido é a folha de lótus, com a sua capacidade de repelir gotas de água devido à presença de micro/nano estruturas e baixa energia de superfície. Uma aplicação particular das superfícies SH artificiais é a sua utilização como suportes onde gotas de soluções poliméricas de base aquosa são dispensadas, adquirindo uma forma quase esférica. Através da aplicação de um ou múltiplos passos de solidificação, partículas sólidas são obtidas de uma forma rápida. Usando esta metodologia, uma variedade de hidrogéis funcionais de base natural, encapsulando células e/ou fármacos, são propostos nesta tese. A estratégia do primeiro sistema proposto é a incorporação de ciclodextrinas (CDs) em microgéis de dextrano-metacrilatado (Dex-MA), com o intuito de melhorar a sua capacidade de carga para fármacos hidrofóbicos. A formação de complexos de inclusão entre CDs e dexametasona, quando as CDS estavam co-polimerizadas, aumentou significativamente a quantidade de fármaco no interior das partículas, quando comparadas com formulações sem, ou com CDs livres. O quitosano, a não ser que esteja modificado, é solúvel em meios ácidos, o que o torna incompatível com o encapsulamento de células ou moléculas sensíveis ao pH. O segundo sistema proposto teve como objectivo a produção de sistemas não agressivos, através de dois passos sequenciais de gelificação do quitosano, onde dexametasona e/ou fibroblastos foram encapsuladas com sucesso. A metodologia das superfícies SH foi também usada para o co-encapsulamento de células e proteínas em hidrogéis sem comprometer a viabilidade das células nem a atividade das proteínas. Colagénio combinado com lisados de plaquetas foram usados para obter formulações fáceis de manusear, sendo estas capazes de atuar como sistema de libertação de factores de crescimento in situ e também como reservatório de células estaminais derivadas do tecido adiposo, para a regeneração de pele. Em outro sistema apresentado, células estaminais mesenquimais e fibronectina foram encapsulados em esferas de alginato e o sistema foi estudado para regeneração óssea. O controlo da libertação de agentes bioativos pode ser conseguido através do ajuste da química e de parâmetros físicos tal como a arquitetura das partículas. O potencial dos sistemas multicompartimentalizados tem emergido devido à possibilidade de encapsulamento de vários agentes bioativos exibindo diferentes cinéticas de libertação. Aproveitando a simplicidade da metodologia das superfícies SH, partículas com uma ou mais camadas, compostas por Dex-MA e alginato foram eficientemente preparadas, encapsulando células e fármacos em compartimentos individuais. Os trabalhos desenvolvidos mostram que uma grande variedade de partículas úteis para aplicações biomédicas, desde matrizes esféricas homogéneas até sistemas compartimentalizados, podem ser obtidos em condições não agressivas e de uma forma rápida, usando superfícies SH.Fundação para a Ciência e Tecnologoa (FCT) SFRH/BD/71395/2010 e PTDC/CTM-BIO/1814/2012

Mechanisms for directed transport and organization at subcellular scales

The timely and faithful segregation of genetic material is an essential cellular function that relies on the transport and stable positioning of subcellular components despite the disruptive influence of thermal fluctuations. In prokaryotes, a two-protein system (known as ParABS) has been identified as being responsible for the positioning of low-copy number plasmids and chromosomes prior to cell division. Multiple experimental observations, in vitro reconstitutions and computational modelling efforts support the idea that this system is powered by the ‘burnt-bridge’ Brownian ratchet mechanism. In this thesis we provide computational models that complement these studies to understand how this mechanism generates and sustains directional transport through the transduction of chemical energy into mechanical motion. In particular we study the effects of chemical kinetics, inter-protein interaction strength, system size and availability of proteins that drive this mechanism with an application to the rich protein dynamics observed in vivo. Finally, we simulate a coarse-grained model for a highly polyvalent ‘burnt-bridges’ Brownian ratchet capable of translocating either by rotation or translation and detail the system parameters that govern the transitions between these two distinct modes of motion. The models presented in this thesis provide key insights and make experimentally testable predictions which can be used for the engineering of novel synthetic motor systems