Dissertationen zum Thema „Cell interaction“

Understanding surface-cell interactions is essential to fabricating a successful biomaterial. In vivo, cells interact with asymmetric features on the micro- and nanoscale. Some of these features, described as valleys, ridges, and spheres, are random, but methodically placed. There are many techniques used to duplicate the topographical features that cells encounter, many of which rely on precision and are labor intensive. Alternatively, the synthetic poly(2-methoxystyrene) (P2MS) homopolymer selfassembled into desirable features, was easily processed and produced the random surface preferred by cells. The features achieved with P2MS were the result of secondary and tertiary conformations confirmed by circular dichroism. The features were also a consequence of the optical activity revealed by polarimetry. Advanced microscopy verified that the features were indeed biomimetic and measured between 150—600 nm in depth and height. Polymers were synthesized using free radical and anionic techniques; some involved the use of a chiral initiator. Spin-casting and solvent annealing were employed to create polymer films for substrate-cell studies. Reaction conditions and molecular weight were varied to achieve different topographical features and thermal profiles. In showing that the films were able to be sterilized, the films were further subjected to cytotoxicity studies involving both Escherichia coli and Bacillus cereus. The results of turbidity measurements and colony counting revealed increased cell viability. The gram positive bacteria, B. cereus, showed increased adhesion through hydrophobic interactions, the same type of interactions proteins rely on for deposition prior to cell adhesion. The cell adhesion study used the human epithelial carcinoma (HeLa) cell line, and showed increased adhesion on chiral initiated P2MS. As a result, this work verified that topographical features can influence cell behavior without the presence of biochemical cues and that P2MS may provide a viable option for tissue engineering applications.

Bennett and O'Brien [Biochemistry 1995 34, 3102] showed that the ultraviolet light exposure of two-component large unilamellar liposomes (LUV) composed of a 3:1 molar mixture of dioleoylphosphatidylethanolamine (DOPE) and 1,2-bis[10-(2'-hexadienoyloxy)-decanoyl]-sn-glycero-3-phosphatidylcholine (bis-SorbPC) facilitated liposome fusion. The rate and extent of fusion was dependent on the extent of photopolymerization, the temperature, and the pH. Here, the effect of the molar lipid ratio of DOPE/bis-SorbPC liposomes on the temperature for the onset of fusion, was characterized by changing the relative amounts of unreactive polymorphic lipid, and reactive lamellar lipid. The cellular uptake of liposomes is mediated by nonspecific adsorption of liposomes onto the cell surface and subsequent endocytosis. This research compared the effect of liposome surface charge on liposomal binding and endocytosis by a human cancer cell line, HeLa, and a murine macrophage cell line, J774. LUV were composed of dioleolylphosphatidylcholine with and without either a cationic lipid, dioleoyldimethylammonium propanediol, or an anionic lipid, dioleolylphosphatidylserine. HeLa cells endocytosed cationic liposomes to a greater extent than either neutral or anionic liposomes and with PEG- LUV, a neutral PEG-lipid over the anionic PEG-PE2000. In contrast, the extent of liposome endocytosis by J774 cells was quite similar for both cationic and anionic liposomes, both greater than neutral liposomes. Incorporation of a neutral PEG lipid may minimize interactions with cells of the RES, yet strongly interact with proliferative cells. Clapp et al., [Macromolecules 1997 30, 32] demonstrated that certain amphiphilic cyanine dyes are capable of sensitizing lipid polymerization to visible light. The individual effects of pH, light intensity, temperature, and the requirement for oxygen suggested that the polymerization process is initiated by electron transfer from the dye excited state to oxygen, to yield superoxide anion, which in aqueous media combines to form hydrogen peroxide. Here, irradiation of cell-associated visible light sensitive liposomes sensitized with either the cationic dye, N, N' -dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine, DiIC(18)3, or a sulfonated derivative, DiI-DS, caused cell membrane damage and cytoplasmic delivery of liposomal contents could not be confirmed.

Thesis (M.S. in Chemistry)--Washington State University, August 2009.
Title from PDF title page (viewed on Sept. 21, 2009). "Department of Chemistry." Includes bibliographical references (p. 60-66).

We investigated the role of Fas ligand in the development of anti-tumor immunity. The LSA tumor specific cytotoxic T lymphocyte (CTL) clone, PE-9, expressed both Fas and Fas ligand. This CTL clone upregulated Fas and Fas ligand expression upon activation through the T-cell receptor and induced apoptosis in Fas+, LSA tumor cells using the FasL-based pathway. However, LSA and EL-4 tumor cells constitutively expressed Fas ligand and killed Fas+ PE-9 CTLs and Fas+, but not Fas-negative (Fas-) activated T cells and thymocytes. These data suggested that T cells and cancer cells can kill each other and that cancer cells may use Fas ligand to evade the action of the immune T cells. In addition to the expression of membrane-bound form, FasL+ LSA and EL-4 tumor cells produced a soluble form of Fas ligand when they grew in vivo and in vitro. Serum from EL-4 or LSA-bearing wild type mice contained significant levels of Fas ligand. The soluble FasL induced apoptosis in liver and thymus of C57BL/6 wild type (Fas+) mice, but not C57BL/6 lpr/lpr (Fas-) mice. The detection of apoptosis in the liver of C57BL/6 gld/gld (FasL-defective) mice suggested that the source of Fas ligand found in the sera of EL-4 or LSA-bearing mice was from the tumor cells rather than the host cells. CTL or NK cells used FasL-based apoptosis to kill the target cells when activated. To this end, we tested whether constitutive expression of Fas on tumor cells generate enhanced anti-tumor immunity. IL-2 or poly-I-C induced/ activated NK/LAK cells displayed higher cytotoxicity against L1210 Fas+, but not L1210 Fas- tumor cells. Furthermore, growth of L1210 Fas+, but not Fas- tumor, in vivo, generated Fas-specific cytotoxic T lymphocytes. Therefore, mice bearing L1210 Fas+ tumor cells survived for a longer time than mice bearing L1210 Fas- tumor cells. To determine the role of the Fas, FasL, and perforin in the initiation of tumor, C57BL/6 +/+ (FasL+, Fas+), C57BL/6 lpr/lpr (Fas-), C57BL/6 gld/gld (FasL-), and perforin knock-out (PKO) (FasL+, Fas+, but perforin-deficient) mice were injected with methylcholanthrane (MCA). Tumor development in lpr or gld mice was faster and uncontrollable, compared to C57BL/6 (wild-type) and PKO mice. However, wild-type and PKO mice showed delayed tumor appearence and were able to suppress tumor growth. In addition to the deficiency of Fas or FasL, high levels of TGF-b and IL-10 expression detected in lpr and gld mice were also responsible for the early tumor development. Together these data suggested that interactions between Fas and Fas ligand, expressed on immune cells and tumor cells, play an important role in the generation of anti-tumor immunity. Tumor cells use FasL to evade the action of the immune system, and upregulation of FasL makes T cells more cytolytic. Tumor growth may depend on the number of cancer cells vs. the number of cancer specific T cells.
Ph. D.

Thesis (Ph. D.)--Biomedical Engineering, Georgia Institute of Technology, 2008.
Committee Chair: Meredith, Carson; Committee Co-Chair: Galis, Zorina; Committee Co-Chair: McIntire, Larry; Committee Member: García, Andrés; Committee Member: Prausnitz, Mark.

"March 2003" Bibliography: leaves 206-233. The experimental data presented in this thesis provide evidence that PTP-Pez is an active phosphatase that interacts with and dephosphorylates the adherens junction protein ℓ-catenin. PTP-Pez also associates with proteins that form part of the tight junction complex, the scaffolding protein ZO-1 and the transmembrane protein occludin.

Tissue engineering involves the use of cells, growth factors/cytokines, and scaffolds to regenerate damaged tissue. The choice of cells, scaffold and their delivery is crucial to the successful outcome of the treatment and this is particularly the case in bone and cartilage where the repair tissue has to recreate a structural hierarchy to restore long lasting function. One approach to deliver high numbers of cells to a defect site is as cell aggregates or spheroids. Experiments are described here that aim to understand how mesenchymal stem cells (MSCs) and osteoblastic cells behave in a cell aggregate and how this could be refined by the inclusion a self -assembling hydrogel to influence cellcell and cell-extracellular matrix (ECM) interactions. Forming cellular spheroids in vitro can be achieved using various methods, including hanging drop, static suspension culture, encapsulation/entrapment, and low adherence multi-well. Extensive analysis identified that the most efficient and reproducible method for the formation of spheroids using rat osteoblasts and human MSCs was through their culture at a specific concentration in polyHEMA coated plates. Both viability and ability of cells to differentiate was investigated. The MTT assay was used to assess cells viability while their ability to differentiate was assessed by measuring alkaline phosphatase activity as well as measuring gene osteogenic markers expression via qRT-PCR. Analysis of cell differentiation under these conditions revealed that alkaline phosphatase activity appeared more elevated in 2D cultures compared to 3D. However, it was noted that there were contrasting results between the two types of cells with expression of osteogenic genes higher when MSCs were grown in osteogenic media while with calvarial significant expression was also observed when grown in normal media. Because of the distinct regulatory cues given by cell-cell contact in the spheroid, analysis was performed for connexin (Cx)-43, a gap junction protein and members of the ephrin/Eph family. Cx-43 iv was immunolocalised to gap junction structures in cells after osteogenic treatment on a flat substrate but this was more difficult to assess in the 3D spheroids. Analysis of transcript patterns reflected the increased abundance of Cx-43 in cells treated with osteogenic supplements and parallel changes in expression of Ephrin B1 and Ephrin B2. Experiments were also performed including a Puramatrix hydrogel nanofibers scaffold that could encase the cells in an ECM-like environment, provide mechanical support and protect them and manipulate cell-cell interactions. The results obtained in this study concluded that calvaria cells viability and hence proliferation increased when grown embedded within 0.25% Puramtrix while mesenchymal stem cells increased when embedded in 0.5% Puramatrix. Similarly, alkaline phosphatase activity was higher in cells embedded within 0.25% Puramatrix while mesenchymal stem cells favoured 0.5%. On the other hand, osteogenic gene expression of both cells was enhanced with the use of Puramatrix scaffold.

This thesis presents the development of a non-intrusive method for monitoring fuel cells. The instrumentation demands that only the readily accessible AC output terminals of the fuel cell be monitored, making it easy to install on an existing fuel cell. The monitoring hardware senses the current and voltage output of the fuel cell and converts the measurements into the digital domain. The original algorithm to process the data was the Non-Intrusive Load Monitor (NILM) system which detects specific events, classifies and stores them. The software that is described in this thesis is a data specific compression algorithm that is used to store a continuous stream of measurements so that it can be processed off line. This gives the user the ability to search for critical interactions between the fuel cell and loads in the system after the fact, rather than demanding that load and interaction dynamics be known before the monitor is installed.

Pancreatic ductal adenocarcinoma (PDAC) is a disease with very poor prognosis amongst all pancreatico-biliary cancers. PDAC is characterised by a pronounced desmoplastic stroma which upon depletion has been associated with immune cell mediated tumour clearance. In situ analyses of various immune cell markers in the stromal compartments may provide a lucid picture of immune cell migration to the tumour epithelia. Automated, unbiased, high throughput imaging and analysis of specifically designed tissue microarrays, from surgically resected tissue samples of PDAC, advanced PDAC, and other pancreatico-biliary diseases; stained for distinct immune cell markers was carried out in the juxtatumoural stroma and the panstromal compartments. Prognostic significance was determined with X-Tile software. In vitro and in vivo assays were undertaken to outline the possible mechanisms. Immune cell infiltration to PDAC was higher than infiltration to other pancreatico-biliary diseases with the exception of CD8+ T cells. While CD4+, CD68+ and myeloperoxidase+ cells could infiltrate the juxtatumoural stroma of PDAC; CD3+, CD8+, Foxp3+ and CD20+ cells could not in the early stage PDAC patients tissue analysed and also in an independent validation cohort of advanced stage PDAC patients. Survival analyses demonstrated pro-survival effects of having high CD8+ densities. CD8+ T cells could only infiltrate the juxtatumoural compartment of KPC mice after stromal collapse resulting from targeting stellate cells with All-trans Retinoic Acid. 17 In vitro migration assays demonstrated increased CD8+ T cell migration towards activated pancreatic stellate cells compared to quiescent pancreatic stellate cells and appeared to be dependent on CXCL12. T cells are hindered from migrating to the juxtatumoural compartment by activated pancreatic stellate cells as a result of an increase in CXCL12 secretion. Rendering activated pancreatic stellate cells quiescent results in a reduction of CXCL12 secretion which may allow CD8+ T cells to migrate to the tumours and perform cytotoxic functions.

An association between painful lumbar intervertebral discs (IVDs) and innervation deep with the nucleus pulposus (NP) of the degenerate IVD has been highlighted by a number of studies. However the mechanisms responsible for driving this innervation and hence the pathogenesis of symptomatic IVD degeneration is complex. The extracellular matrix (ECM) component aggrecan has been shown to inhibit neurite outgrowth and a role for cell mteractions via the e production of soluble mediators from degenerate disc cells, hypothesised to be neurotrophic factors, has been proposed. This study was undertaken to investigate NP cell/neural cell interactions in relation to degeneration of the IVD.

Améliorer la connaissance des interactions cellules-matériau est un enjeu crucial pour la biologie cellulaire et l’ingénierie tissulaire ainsi que pour la fabrication de dispositifs biomédicaux élaborés. Ce travail de thèse s’est basé sur le développement de technologies de micro- et nanofabrications avancées et de l’imagerie cellulaire de haute résolution, afin d’obtenir une vue plus claire sur les phénomènes d’élongation, alignement, extention, et déformation locale de membrane cellulaire sur des motifs topographiques. La culture cellulaire sur des substrats comportant des trous et des sillons à dimensions micro et nanométriques a été étudiée, ce qui a permis de démontrer une forte dépendance de la géométrie des motifs et de la décoration de surface. En général, les membranes cellulaires sont localement déformées à cause de la présence des trous ou des sillons micrométriques, qui aident, entre autre, l’attachement et l’adhésion cellulaire. Nous avons aussi noté que les cellules cancereuses sont plus agressives pour exploiter les motifs topographiques. Enfin, nos études ont été élargies à des motifs de géometrie plus complexe tels que des réseaux de microsillons croisés avec des différentes périodicités et profondeurs de gravure.

Thesis (MSc)--Stellenbosch University, 2015.
ENGLISH ABSTRACT: The use of non-Saccharomyces yeasts together with the yeast S. cerevisiae in multistarter wine fermentations has emerged as a useful tool to modulate wine aroma and/or to decrease the concentration of undesirable compounds. However, upon inoculation, these yeast species do not co-exist passively, but interact in various ways. While competition for nutrients and the excretion of killer toxins in an antagonistic relationship are obvious and well established types of interactions, some studies have suggested the existence of other forms of cellular or molecular interactions. One of these includes physical cell-cell contact and to our knowledge, only one previous study has confirmed its existence in wine yeasts. Yeast interactions are also influenced by other factors, such as ethanol concentration, however some studies have highlighted the role that dissolved oxygen plays on the survival of non-Saccharomyces yeasts and their ability to compete for space with S. cerevisiae and little research has focused on this. This study aimed to investigate the occurrence of a physical cell-cell and/or metabolic interaction between S. cerevisiae and L. thermotolerans in mixed culture fermentations of synthetic grape must. For this purpose, fermentations in a Double Compartment Bioreactor (DCB) which separates yeast population through the use of a membrane were compared to mixed fermentations in the absence of the membrane, using the same reactor. Furthermore, the impact of oxygen supply on yeast behaviour was also assessed. Following mixed culture fermentations in a DCB, it was observed that the presence of S. cerevisiae led to a significant decline in viability in L. thermotolerans. This decline was significantly less prominent in mixed cultures where the cells were in indirect contact. Together, the data provided evidence for both cell-cell and metabolic interactions whereby S. cerevisiae had a strong negative influence on the growth of L. thermotolerans. However, it was also observed that L. thermotolerans had some negative impact on the growth of S. cerevisiae, leading to a reduction in biomass (when in indirect contact) and a reduced maximum CFU/mL compared to pure cultures. The data also suggest that direct physical contact may increase the production of glycerol and propanol, but this needs further investigation. By decreasing the frequency at which oxygen pulses were provided, a reduction in biomass and increase in fermentation duration was observed for all fermentations. However, this effect was somewhat reduced in mixed cultures. Here, no impact on fermentation duration was observed and the decrease in biomass was less compared to pure cultures. The impact of these oxygen pulses was also greater on L. thermotolerans. In the latter yeast’s pure culture a slight increase in glycerol was observed when less oxygen was provided and in general there appeared to be no impact on acetic acid production. Furthermore, there was little or no impact on volatile production, however, more repeats might reveal different results and therefore more research is needed to confirm these results. To our knowledge, this is the first study of its kind to confirm a physical cell-cell interaction between the yeast pair S. cerevisiae and L. thermotolerans.
AFRIKAANSE OPSOMMING: Die gebruik van nie-Saccharomyces gis saam met die gis S. cerevisiae in multi-inokuleringskulture het die afgelope paar jaar as n goeie hulpmiddel na vore gekom om wyn aroma te moduleer en/of om die konsentrasie van ongewensde verbindings te verminder. Sodra inokulasie plaasgevind het, het hierdie gis die potensiaal om op verskeie maniere teenoor mekaar te reageer. Kompetisie vir nutriente en die afskeiding van toksiese verbindings in n antagonistiese verhouding is alreeds goed beskryf in die literatuur. Somige studies het, alhoewel, die bestaan van ander vorme van sellulêre of molekulêre interaksies voorgestel. Een van hierdie sluit in n fisiese sell-sell interaksie en so ver as wat ons kennis strek, het nog net een studie van tevore so ‘n interaksie bevestig tussen wyn giste. Gis interaksies word ook beïnvloed deur ander faktore, soos byvoorbeeld etanol konsentrasie. Terwyl sommige studies die rol wat opgelosde suurstof speel in die oorlewing van nie-Saccharomyces gis en hulle vermoë om te kompeteer vir spasie met S. cerevisiae alreeds beklemtoon, het min navorsing al hierop gefokus. Hierdie studie het gestreef om die voorkoms van n fisiese sell-sell en/of metaboliese interaksie tussen S. cerevisie en L. thermotolerans in gemengde kultuur fermentasies van sintetiese druiwe sap te ondersoek. Vir hierdie doeleinde was fermentasies uitgevoer met behulp van ‘n Dubbel Kompartement Bioreaktor (DKB) wat gis populasies skei deur middel van ‘n membraan en hierdie was vergelyk met gemengde kultuur fermentasies sonder die membraan in dieselfde reaktor sisteem. Verder was die impak van suurstof toevoer op gis gedrag ook geassesseer. Na afloop van gemengde kultuur fermentasies in ‘n DKB, was daar waargeneem dat die teenwoordigheid van S. cerevisiae gelei het tot ‘n betekenisvolle afname in lewensvatbaarheid in L. thermotolerans. Hierdie afname was aansienlik minder in gemengde kulture waar die gis in indirekte kontak was. Saam verskaf hierdie data bewyse vir n sell-sell asook metaboliese interaksie waardeur S. cerevisiae ‘n sterk, negatiewe invloed op die groei van L. thermotolerans gehad het. Daar was egter ook waargeneem dat L. thermotolerans tot ‘n mindere mate ‘n negatiewe impak op die groei van S. cerevisiae gehad het en dat dit gelei het tot ‘n verlaging in biomassa (toe die gis in indirekte kontak was) en ‘n verlaagde maksimum CFU/mL in vergelyking met suiwer kulture. Die data dui ook aan dat fisiese kontak kon gelei het tot ‘n verhoging in gliserol en propanol produksie, maar hierdie kort verdere ondersoek. Deur die frekwensie te verminder waardeur suurstof pulse aan die fermentasies verskaf was, was ‘n verlaging in biomassa produksie en ‘n verlenging in fermentasie tydperk waargeneem. Hierdie tendense was waargeneem in almal, behalwe die gemengde kultuur fermentasies. Die effek van suurstof puls verlaging was minder op hierdie fermentasies aangesien daar geen impak op fermentasie tydperk was nie en die verlaging in biomassa minder was. Die impak van hierdie suurstof pulse was ook groter op L. thermotolerans. ‘n Klein toename in gliserol produksie was waargeneem in laasgenoemde gis se suiwer kultuur toe minder suurstof beskikbaar was en oor die algemeen was asynsuur onveranderd. Verder was daar ‘n klein of geen impak op vlugtige verbindings nie, alhoewel, meer herhalings mag verskillende resultate lewer en daarom is meer navorsing nodig om hierde resultate te bevestig. So ver as wat ons kennis strek is hierdie die eerste studie van sy soort om ‘n fisiese sell-sell interaksie tussen die gispaar S. cerevisiae en L. thermotolerans te bevestig.

Thesis (Ph. D.) -- University of Maryland, College Park, 2006.
Thesis research directed by: Food Science. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

Functionalized nano materials have an expansive range of potential uses in bio medical applications. Functionalized synthetic (biocompatible and/or biodegradable) polymers that control or monitor cell signaling can be effective antagonists and promising drug candidates. We have developed a series of biodegradable functional polymer systems (with dimensions in the nanoscale) for creating allergy-effective drugs, using RBL mast cells and anti-2,4 dinitrophenyl (DNP) IgE antibodies that sensitize these cells by binding to high affinity IgE receptors(FceRI); creating polymers which are effective inhibitors of degranulation of mast cells stimulated by a potent allergen. The inhibition is possible because of the specific interaction of the functional polymers with the proteins (IgE) on the mast cell surfaces to control cell-signaling i.e. intelligent design of functional materials to manipulate cellular functions. The functional polymer system is based on biodegradable poly(lactides) carrying two, three or four 2,4-dinitrophenyl (DNP) groups. Although these DNP-functionalized ligands do not stimulate the granule exocytosis response, they have the potential to inhibit the robust response stimulated by other multivalent DNP ligands. These polymeric ligands are effective inhibitors of degranulation of mast cells stimulated by a potent allergen and thus are a potential model drug system. Additionally these lactide based polymers bind and achieve steady state binding with solution IgE within a few seconds at low concentrations; achieving about 75% of the binding below 0.2 uM. The preparation, characterization, processing and effectiveness of the functional polymers to control material-cell interactions will be discussed

[EN] This thesis deals with the development of a living biointerface between synthetic substrates and living cells to engineer cell-material interactions for tissue engineering purposes. This living biointerface is made of Lactococcus lactis, a non-pathogenic lactic bacteria widely used as starter in the dairy industry and, recently, in the expression of heterologous proteins in applications such as oral vaccine delivery or membrane-bound expression of proteins. L. lactis has been engineered to display the III 7-10 fragment of the fibronectin fused to GFP as reporter protein. Fibronectin is a ubiquitous protein present in the extracellular matrix, a complex mesh of structural and adhesive proteins which serve as mechanical support and development niche for cells of a wide variety of tissues. This fragment contains two important sequences, RGD and PHSRN. RGD is an adhesive sequence that interacts with a wide range of integrins, membrane-bound receptors that play a role in cellular processes such as adhesion, migration, proliferation and differentiation. On the other hand, PHSRN binds synergistically with RGD to some integrins such as alpha-5-beta-1 and others, increasing the specificity of this interaction. Genetically engineered L. lactis has been thoroughly characterized to test its capabilities as a living interface. This strain was found to express the FNIII 7-10-GFP fragment covalently linked to the cell wall and biological activity and expression levels of this fragment was assessed with techniques such as Western blot, ELISA and immunofluorescence. Moreover, this strain still holds the ability to develop biofilms, communities of sessile, attached bacteria to abiotic surfaces which helps greatly in the generation of a stable monolayer of bacteria between synthetic substrates and mammalian cells. Mammalian cell behaviour in response to the expressed fibronectin fragment on L. lactis membrane was also assessed. Several cell lines were tested, such as Fn-/Fn- and NIH3T3 fibroblasts, C2C12 myoblasts and human bone-marrow derived mesenchymal cells. This living biointerface was found to trigger cell adhesion and FAK phosphorylation, a marker for intracellular integrin-mediated signalling in all of the tested cell lines. It also triggered myoblast-to-myotube differentiation on C2C12 cells. In hMSCs, the cell-wall exposed fibronectin fragment was found to enhance the phosphorylation of ERK1/2, a kinase involved in the MAPK pathway, which is deeply involved in a multitude of cellular processes related to differentiation, proliferation and migration. Nevertheless, this thesis is a proof of concept that this novel system can be further exploited to express almost any desired protein or small molecule to help in the development of new tissues from progenitor cells. These molecules can be either secreted in the medium or displayed in the membrane, and can also be constitutively expressed or in-demand, due to the great flexibility of L. lactis and the wide variety of expression systems available. This interface based on living bacteria establishes a new paradigm in surface functionalization for biomedical engineering applications.
[ES] Esta tesis aborda el desarrollo de una biointerfase viviente entre materiales sintéticos y células vivas con el objetivo de dirigir la interacción célula-material en aplicaciones de ingeniería tisular. Esta biointerfase está compuesta de Lactococcus lactis, una bacteria láctica no patógena, ampliamente usada en la industria láctea como inóculo, y, recientemente, en la expresión heteróloga de proteínas para su uso como vacunas de administración oral o su expresión en membrana. L. lactis ha sido genéticamente modificado para expresar el fragmento III 7-10 de la fibronectina, unida a GFP como reporter. La fibronectina es una proteína presente de forma ubicua en la matriz extracelular, una compleja red de proteínas adhesivas y estructurales cuyo propósito es servir como soporte estructural y como nicho de desarrollo para diversos tejidos. Este fragmento contiene dos secuencias importantes, RGD y PHSRN. RGD es una secuencia adhesiva de unión que interacciona con una amplia variedad de integrinas, receptores de membrana que juegan muchos e importantes papeles en diferentes procesos celulares, como adhesión, proliferación, migración o diferenciación. Por otra parte, PHSRN se une a las integrinas de forma sinérgica con RGD facilitando aún más estos procesos y aumentando la especificidad de esta interacción. Esta cepa de L. lactis modificada ha sido ampliamente caracterizada para estudiar su idoneidad como interfaz funcional viviente. Se ha demostrado que L. lactis es capaz de expresar el fragmento FNIII7-10-GFP covalentemente anclado a la pared celular bacteriana, habiéndose caracterizado también su actividad biológica con técnicas como Western blot, ELISA e inmunofluorescencia. Esta cepa mantiene la capacidad de desarrollo de biofilms presente en la gran mayoría de microorganismos. Los biofilms son comunidades de bacterias sésiles adheridas a un sustrato que pueden ser usadas como interfase física entre células de mamífero y sustratos abióticos. También se ha estudiado la respuesta celular a la fibronectina expuesta en la membrana de L. lactis. Se estudiaron varias líneas celulares, como fibroblastos Fn-/Fn- y NIH3T3, mioblastos C2C12 y células mesenquimales humanas derivadas de médula ósea. Esta interfase viviente fue capaz de provocar respuesta celular en forma de adhesión en todas las líneas estudiadas, además de inducir diferenciación de mioblastos a miotubos en C2C12 y de provocar la fosforilación de FAK, un marcador de señalización celular mediada por integrinas. En células mesenquimales humanas se demostró la capacidad del fragmento de fibronectina expuesto para fosforilar ERK1/2, una kinasa perteneciente a la ruta de señalización MAPK, ruta que forma parte de muchos procesos celulares importantes como diferenciación, proliferación y migración. Pese a todo, esta tesis es sólo una prueba de concepto de un sistema que puede ser utilizado para expresar casi cualquier proteína o molécula pequeña deseada, que puede ser muy útil en el desarrollo de nuevos tejidos a partir de sus células progenitoras. Estas moléculas pueden ser secretadas en el medio o ancladas en la pared celular, de forma constitutiva o bajo demanda, debido a la flexibilidad y amplia variedad de sistemas de expresión disponibles para L. lactis. Esta biointerfase basada en bacterias vivas establece un nuevo paradigma en el campo de la funcionalización de superficies para aplicaciones de ingeniería biomédica.
[CAT] Aquesta tesi aborda el desenvolupament d'una interfase viva entre materials sintètics i cèl·lules vives amb l'objectiu de dirigir la interacció cèl·lula-material, per al seu ús en aplicacions d'enginyeria tissular. Aquesta interfase està composta de Lactococcus lactis, un bacteri làctic, no patogènic i àmpliament utilitzat en l'industria làctica com a inòcul, i, recentment, en l'expressió heteròloga de proteïnes per al seu ús com vacunes d'administració oral o per a la seva expressió en membrana. L. lactis ha sigut genèticament modificada per a expressar el fragment III7-10 de la fibronectina, unida a GFP com a reporter. La fibronectina és una proteïna present de forma ubiqua en la matriu extracel·lular, una complexa xarxa de proteïnes adhesives i estructurals que s'utilitzen com a suport estructural i com a nínxol de desenvolupament per a diversos teixits. Aquest fragment conté dos seqüències importants, RGD i PHSRN. RGD és una seqüència adhesiva d'unió a integrines, receptors de membrana que juguen molts i molt importants papers en diferents processos cel·lulars, com poden ser adhesió, proliferació, migració o diferenciació. Per altra banda, PHSRN s'uneix a les integrines de forma sinèrgica amb RGD facilitant encara més aquests processos i augmentant l'especificitat d'aquesta interacció. Aquesta modificació genètica de L. lactis ha estat àmpliament caracteritzada per provar les seves característiques com a interfase funcional vivent. S'ha demostrat que L. lactis és capaç d'expressar el fragment FNIII 7-10-GFP covalentment ancorat a la paret cel·lular bacteriana, havent-se caracteritzat també la seva activitat biològica amb tècniques com Western blot, ELISA i immunofluorescència. A més, aquest cep manté la capacitat de desenvolupament de biofilms, comunitats de bacteris sèssils adherits a un substrat que poden ser utilitzades com a interfase física entre cèl·lules de mamífer i substrats abiòtics. També s'ha estudiat la resposta cel·lular a la fibronectina expressada en la paret cel·lular de L. lactis. El estudi es va fer utilitzant diverses línies cel·lulars, com fibroblasts Fn-/Fn- i NIH3T3, mioblasts C2C12 i cèl·lules mesenquimals humanes derivades de medul·la òssia. Aquesta interfase vivent va ser capaç de provocar resposta cel·lular en forma d'adhesió a totes les línies estudiades, a més d'induir diferenciació de mioblasts a miotubs en C2C12 i de provocar la fosforilació de FAK, un marcador de senyalització cel·lular mediat per integrines, en les línies assajades. En cèl·lules mesenquimals humanes es va demostrar la capacitat del fragment de fibronectina exposat per fosforilar ERK1/2, una kinasa pertanyent a la ruta de senyalització MAPK, ruta que forma part de molts processos cel·lulars importants com diferenciació, proliferació i migració. Malgrat tot, aquesta tesi mostra només una prova de concepte d'un sistema que pot ser utilitzat per expressar gairebé qualsevol proteïna o molècula petita desitjada, que pot ser molt útil en el desenvolupament de nous teixits a partir de les seves cèl·lules progenitores. Aquestes molècules poden ser secretades en el medi o ancorades a la paret cel·lular, de manera constitutiva o sota demanda, a causa de la flexibilitat i àmplia varietat de sistemes d'expressió disponibles per L. lactis Aquesta biointerfase basada en bacteris vius estableix un nou paradigma en el camp de la funcionalització de superfícies per a aplicacions d'enginyería biomèdica.
Rodrigo Navarro, A. (2015). Functional living biointerfaces to direct cell-material interaction [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/51461
TESIS

Pulsed Electric Fields (PEF) allow non-thermal pasteurization and sterilization of liquids, involving the application of high intensity electric field pulses (20-80 kV/cm) of short duration (1-10 microseconds). The electric field interacts with microorganisms at the level of plasma membrane, through the mechanism of electroporation, but, although many theories have been proposed to describe this phenomenon, a satisfactory explanation has not been found yet. An extensive description of the state of the art of the knowledge on this field is given, along with the current research needs and the main obstacles to a wide diffusion of PEF applications. The time course of cell transmembrane voltage is studied, developing an analytical expression for planar, spherical, cylindrical and prolate spheroidal membranes, with the aim to evaluate the charging time constants and the steady state intensity upon variation of treatment conditions. The results of this approach are used to investigate the impact of the electric field in rod-like bacteria. The electric field distribution in test chamber is studied by means of computer simulations for various electrode geometries, optimizing the shape for intensity and uniformity of electric field. The impact on PEF treatment of a normal distribution of cell dimensions in a microbial population and the rotational movement of bacteria inside treatment chambers are also investigated. Computer simulations are used to obtain a possible explanation to the deviations from first order inactivation kinetics, and to the intrinsic variability of microbial laboratory results. PEF inactivation experiments of Escherichia Coli are carried out in a test system under different conditions of treatment duration and microbial concentration. Inactivation kinetics is compared to theories of electroporation and computer simulations previously carried out. Experimental evidence of a variation in the effectiveness of PEF as a function of bacterial concentration is discovered and a possible explanation proposed. The application of high permittivity ceramics materials to PEF is also studied, with the aim of increasing the volumes of treatment chambers, improving the duration of electrodes, and allowing the use of the most energy efficient square wave pulses to large volumes of liquid. These materials can also be used to test the possibility of a relation between energy deposited in treatment chambers and microbial inactivation, in order to acquire more insight on the interaction between Pulsed Electric Fields and cell membranes. A novel chamber is prepared with the use of a high permittivity non-toxic material (Sodium Potassium Niobate) and it is ready to be tested in PEF applications.

[Truncated abstract] Melanoma cell adhesion molecule (MCAM) is highly expressed in more than 70% of metastatic melanoma and is correlated with invasive potential. However, the specific contribution MCAM makes to invasion and metastasis in melanoma is not clear. In this study, I have demonstrated that transfection of MCAM into MCAM-negative melanoma and CHO cells leads to changes in cell shape, and the modulation of cell-to-cell and cell-matrix interactions. MCAM positive cells were slower to spread on collagen type I, collagen type IV and laminin 1 than MCAM negative cells, although these differences were not apparent on vitronectin, fibronectin and laminin 10. In contrast, MCAM expression had little effect on cell adhesion to any of the matrices tested. MCAM positive (compared to negative) cells also showed morphological changes and a rearrangement of the actin cytoskeleton when plated on a matrix containing laminin 5. Taken together, these data suggest that MCAM expression modulates β1-integrinmediated spreading on matrix, but has little effect on αvβ3-mediated cell-matrix interactions. As this study provided little evidence to suggest that MCAM transfection altered β1 integrin expression levels on melanoma cells, it is proposed that a competitive interaction between the cytoplasmic domains of MCAM and β1 integrin may affect mature focal adhesion assembly. MCAM expression in melanoma cells was also associated with decreased cell movement over matrix into a scratch-wound site and an increased tendency to form cell cords on Matrigel. These two assays gauge the propensity of a cell to engage in cell-cell versus cell-matrix interactions, and suggest that MCAM positive cells favour cell-cell adhesion. Interestingly, MCAM transfection was also associated with an increased ability of melanoma cells to migrate through a basement membrane towards a chemoattractant. ... Analysis of the intracellular domain of MCAM revealed the presence of tyrosine and dileucine endocytosis signals. Interestingly, disruption of these two motifs did not seem to impair the internalization of MCAM from the cell surface. The di-leucine motif, however, was necessary for the recycling of MCAM back to the surface following endocytosis. Lastly, MCAM was found to exists as dimers within the cell membrane in the absence of ligand, although the exact location of the dimerization motif is not yet clearly defined. Collectively, findings from my study suggest: MCAM expression in melanoma cells facilitates cell-cell interactions, whilst concomitantly modulating cell-matrix interactions. MCAM transfection also leads to enhanced migration of melanoma cells through a basement membrane. Thus, MCAM expression may increase the ability of melanoma cells to migrate as a collective, a feature of highly invasive cancer. The intracellular domain of MCAM interacts with ApxL2, a novel member of the Shroom family of actin-binding proteins. It is likely that ApxL2 links a proportion of MCAM within the cell to the actin cytoskeleton, contributing to cell shape determination and other processes, such as migration. MCAM exists as dimers on the cell surface and is internalized at least partially by a clathrin-mediated mechanism.

The scope of this dissertation is to give a contribution to the understanding of the mathematical description of cell and cellular aggregate mechanics, focusing on remodelling and growth processes which occur inside living structures and on the interactions among cells and the extracellular environment, during the process of cell migration.

A complex system is a collection of parts, that can be identical or different, that interact with each other and environment, and exhibit emergent behaviour. Here, I consider the formation of vascular structures in the body as a complex system consisting of an emergent pattern in interacting endothelial cells. A cancer tumour is a different but related complex system that contains various types of cells, some of which having cancer-inducing mutations. To understand the formation of a vascular structure or a cancer tumour, it is important to understand both the single cells and cell-cell interactions. To study the physical interaction among cells in vascular formation or cancer cell growth, in this thesis an agent-based model is built based on the physical properties of cells which includes the size, shape, direction, and position of cells. In this way the mathematical equations in the model can show the physical variation among modelled cells. The 3-dimensional shape of cells is modelled, and so while I start with cell interactions in petri-dish the model can be easily extended to describe motility of cells in a 3-dimensional system in the future. The physical model is implemented and then simulated with in silico experiments, and then the spatial distribution of cells in in vitro experiments is analysed and used to calibrate the model. In vitro experiments with and without a drug in normal and hypoxic conditions are carried out. Also the patterns formed by cells with different treatment are analysed to produce different parameter combinations in the model. This physical model is shown to be able to predict vessel formation and be reused to predict the spatial distribution of cancer cells in in vitro growth experiments. With biological data such as cell size, cell shape, etc. this model is able to predict behaviours of various cell types, and can also be used to predict more complex phenomena, such as mixed type of cancer cells growing in 3-dimensions with vascular structures.

Includes bibliographical references (leaves 199-253).
This study attempted to characterize at a transcriptional level, the defence responses of Arabidopsis thaliana after infection by Botrytis cinerea, using microarrays. The first microarray experiment focused on profiling Arabidopsis genes induced by B. cinerea over time (temporal) while the second investigated spatial expression of Arabidopsis genes from the point of inoculation. A number of genes were up- and down-regulated specifically at 12 hrs, others at 24 hrs while others were up- and down-regulated at both time points. Similarly, some genes were specifically induced very close to the lesion while others in more distal tissue.

The termina1 enzyme in the linoleoyl-CoA desaturase enzyme complex, delta-6-desaturase was implied in the control of cell proliferation in cancer cells. One of the aims of this study was to isolate the terminal enzyme. It was decided that in order to isolate this enzyme it was first necessary to isolate the entire complex and then to enzymatically solubilise the first two components of the complex i e cytochrome b5 reductase and cytochrome b5 from the complex resulting in a pure delta-6-desaturase . The first two components were isolated and purified using simplified and easily reproducible methodologies which could be utilised in the final purification of delta-6- desaturase. The entire enzyme complex, linoleoyl-CoA desaturase was also isolated in a pure form and this pure complex was used to attempt to isolate delta-6-desaturase. The terminal enzyme was isolated with some cytochrome b5 still bound to it. The methods used had proven to be successful and with some modifications should yield a pure enzyme. Zinc and GLA were known to play a role in the inhibition of cancer cell proliferation and zinc was hypothesised to inhibit cell growth by stimulating the activity of the linoleoyl-CoA desaturase enzyme complex which is involved in the regulation of cell proliferation. GLA is the product of the reaction that this enzyme complex catalyses and GLA has been shown to inhibit cancer ce ll growth. The effect of GLA on cell growth and linoleoyl-CoA desaturase activity was thus investigated. Results showed that both zinc and GLA inhibited cell growth and that the combined addition of zinc and GLA generally resulted in the inhibition of cell growth and the activation of linoleoyl-CoA desaturase activity in the BL-6 cells while having a less pronounced effect on the LLCMK cells. The results of this study support the hypothesis that zinc may be a cofactor of linoleoyl-CoA desaturase.

Bibliography: leaves 152-223. Experiments were designed to identify and purify human bone marrow stromal precursor cells by positive immunoselection, based on the cell surface expression of the VCAM-1 and STRO-1 antigens. The data presented demonstrates a hierarchy of bone cell development in vitro.

In initial experiments, lines of transgenic parasites were generated in which the MHC II-dependent T cell epitope Moth Cytochrome C (MCC) was expressed within a number of different fusion proteins, to provide a model in which the fate of parasite-derived antigens could be followed in infected DC. MHC II-MCC complex formation in CD and MΦ complex-specific T cell line. Although the fusion proteins were clearly demonstrated to be secreted at high levels, no cell surface staining could be detected with D4 and neither infected DC nor infected MΦ could stimulate T cell proliferation. Leishmania-infected DC were however, shown to efficiently process and present exogenous antigen to T cells in vitro. Alternative strategies were therefore developed to probe the DC:Leishmania interaction in more detail. Investigation into the effect of uptake of EGFP-expressing L. mexicana parasites by different DC cultures in vitro demonstrated that, in the absence of exogenous factors, uptake of L. mexicana amastigotes did not cause activation of DC. Uptake of L. mexicana promastigotes resulted in activation of a small proportion of DC indicating that promastigotes do encode an activation signal, but that this is not sufficient to activate the entire DC population. However, neither L. major promastigotes nor L. mexicana promastigote mutants lacking surface lipophosphoglycan (LPG) activated DC in vitro. Therefore these data suggest that L. mexicana promastigotes encode an activation signal, but that this is not sufficient to stimulate all DC. As the promastigotes which did not activate DC either lacked, or expressed a modified version of, LPG, we propose that LPG is a L. mexicana pathogen-associated molecular pattern (PAMP). The work presented in this thesis demonstrates that infected DC are capable of initiating that anti-Leishmania response in vivo, as they efficiently present antigen to T cells. However, infection per se is not sufficient to activate all DC. These data therefore suggest that during the initiation of an anti-Leishmania T cell response DC are likely to be activated by factors produced in response to injection of parasites by the insect vector, such as pro-inflammatory cytokines.

Les peptides pénétrateurs de cellule (CPP) sont des oligopeptides cationiques faisant parti des vecteurs les plus étudiés dans le cadre du développement du transport ciblé de médicament à l’intérieur de l’organisme. Les applications principales sont par exemple le traitement des cancers ou la thérapie génique. Néanmoins, certaines caractéristiques des CPPs rendent leur utilisation médicale compliquée, tels que leur manque de spécificité à l’égard des cellules cibles ou la perte de leurs propriétés pénétrantes lorsqu’un cargo moléculaire leur est greffé. L’une des solutions envisagées pour résoudre ces problèmes est le greffage sur des polypeptides di-blocs auto-assemblés basés sur de l’élastine (ELPBC), des systèmes développés par l’équipe d’Ashutosh Chilkoti à l’Université de Duke (USA). Des travaux précédents ont montré que ces macromolécules, que l’on appelle CPP-ELPBC, retrouvaient les propriétés pénétrantes du CPP même en présence d’un cargo et permettaient également d’induire une spécificité à l’encontre des cellules cancéreuses. En revanche, le mécanisme de pénétration de ces systèmes restait inconnu.Dans cette thèse, je me suis concentré sur l’étude du mécanisme de pénétration des CPP et des CPP-ELPBC au travers de membranes lipidiques modèles, et en particulier sur l’adsorption de ces molécules à la surface de vésicules unilamellaires géantes (GUV). Le développement d’une nouvelle méthode de quantification de la fluorescence en microscopie confocale m’a permis de réaliser des mesures simples de comptage de peptides à la surface des vésicules, ce qui m’a permis par la suite de procéder à des mesures thermodynamiques de l’adsorption des peptides
Cell-penetrating peptides (CPP) are cationic oligopeptides currently investigated as potential vectors for targeted drug delivery design, for applications in cancer treatment and/or gene therapy. Nevertheless, some drawbacks make the CPP complex for medical applications, such as their lack of specificity toward target cells or the loss of their penetrating properties once they have been grafted with a molecular cargo. One of the solutions studied to overcome these issues is the binding of the CPP unit on a self-assembling elastin-like diblock polypeptide (ELPBC), a macromolecular system designed by the team of Ashutosh Chilkoti from Duke University (USA). While it has already been proven that these molecules, named CPP-ELPBC, recover the penetrating properties of the CPP despite the presence of a cargo and also induce a selectivity toward tumorous cells, the exact mechanism of translocation is still under debate.In this PhD thesis, I focused on the investigation of the translocation mechanism of the CPP and CPP-ELPBC using model lipid membranes, and specifically the adsorption of these molecules at the surface of giant unilamellar vesicles (GUV). The development of a new quantification method of fluorescence in confocal microscopy allowed me to directly count the peptides adsorbed on the surface of the GUVs, which I used to perform thermodynamic measurements on the peptide adsorption

Exposure to spores and hyphae of Alternaria alternata, an airborne ubiquitous fungus, is clinically associated with allergic airway disorders including allergic rhinitis, asthma, and chronic rhinosinusitis. Dendritic cells are known as the type of antigen presenting cells most often associated with allergic inflammation. In this study, we used mouse bone marrow-derived dendritic cells (BMDCs) as a model to study the ability of A. alternata spores and different components of the spore cell wall to stimulate innate immune responses. We found that BMDCs were highly sensitive to A. alternata spores, chitin and the major allergen Alt a 1. Following stimulation with these molecules, the expression of MHC II and other co-stimulators, like CD40, CD86, and OX40L, were highly up regulated. In order to determine how different cell wall components affect the T cells, we conducted co-culture experiments of BMDCs and lymphocytes in this study. Both spores and Alt a1 did not induce IL-4 in mixed lymphocytes reactions. Interestingly, we found that Alt a 1 induced the switching of the CD4+ T cell population to the Th17 type, with a major increase in IL-17A secretion. This study reveals that A. alternata components may balance the innate immune responses between Th2 and Th17 pathways, and/or contributes to the development and exacerbation of more serve neutrophilic forms of asthma.
Master of Science

Particle-laden flow in a microchannel resulting in aggregation of microparticles was investigated to determine the dependence of the cluster growth rate on the following parameters: suspension void fraction, shear strain rate, and channel-height to particle-diameter ratio. The growth rate of an average cluster was found to increase linearly with suspension void fraction, and to obey a power-law relationships with shear strain rate as S^0.9 and channel-height to particle-diameter ratio as (h/d)^-3.5. Ceramic liposomal nanoparticles and silica microparticles were functionalized with antibodies that act as targeting ligands. The bio-functionality and physical integrity of the cerasomes were characterized. Surface functionalization allows cerasomes to deliver drugs with selectivity and specificity that is not possible using standard liposomes. The functionalized particle-target cell binding process was characterized using BT-20 breast cancer cells. Two microfluidic systems were used; one with both species in suspension, the other with cells immobilized inside a microchannel and particle suspension as the mobile phase. Effects of incubation time, particle concentration, and shear strain rate on particle-cell binding were investigated. With both species in suspension, the particle-cell binding process was found to be reasonably well-described by a first-order model. Particle desorption and cellular loss of binding affinity in time were found to be negligible; cell-particle-cell interaction was identified as the limiting mechanism in particle-cell binding. Findings suggest that separation of a bound particle from a cell may be detrimental to cellular binding affinity. Cell-particle-cell interactions were prevented by immobilizing cells inside a microchannel. The initial stage of particle-cell binding was investigated and was again found to be reasonably well-described by a first-order model. For both systems, the time constant was found to be inversely proportional to particle concentration. The second system revealed the time constant to obey a power-law relationship with shear strain rate as τ∝S^.37±.06. Under appropriate scaling, the behavior displayed in both systems is well-described by the same exponential curve. Identification of the appropriate scaling parameters allows for extrapolation and requires only two empirical values. This could provide a major head-start in any dosage optimization studies.

Fibrillins are large structural glycoproteins in the extracellular matrix that multimerize to form microfibril suprastructures in connective tissues. Fibrillin-containing microfibrils confer mechanical stability to tissues and regulate bioavailability of the transforming growth factor beta (TGF-β) superfamily. Mutations in fibrillin-1 lead to a number of fibrillinopathies, most notably, Marfan syndrome (MFS), an autosomal dominant connective tissue disorder affecting the cardiovascular, ocular and skeletal system. It has been shown in animal models recapitulating MFS that enhanced levels of active TGF-β1 trigger the typical MFS phenotype.We report that recombinant fibrillin-1 fragments expressed in HEK293 cells and purified by metal affinity chromatography contained active TGF-β1 and its latent form, LAP-TGF-β1. Further analysis of the purification scheme demonstrated that TGF-β1 secreted by HEK293 cells non specifically co purified with fibrillin-1 fragments, and was present in all protein preparations. We demonstrate that non specific co purification of active TGF-β1 and LAP-TGF-β1 was eliminated by gel filtration chromatography, under high salt conditions, for fibrillin 1 fragments spanning the N-terminal and C-terminal half of the protein. However, gel filtration chromatography of the central fibrillin-1 fragment, rF20, did not completely dissociate LAP-TGF-β1 associated with this fragment, suggesting an interaction between rF20 and LAP-TGF-β1.
Les fibrillines sont de larges glycoprotéines structurales dans la matrice extracellulaire qui multimérisent pour former des suprastructures microfibrillaires dans les tissus conjonctifs. Les microfibrilles contenant les fibrillines confèrent la stabilité tissulaire et régulent la biodisponibilité de la superfamille des facteurs de croissance transformant beta (TGF-β). Les mutations dans la fibrilline-1 entraînent des fibrillinopathies, dont la plus commune est le syndrome de Marfan (MFS). Le MFS est une maladie génétique à transmission autosomique dominante des tissus conjonctifs affectant les systèmes cardiovasculaire, oculaire et squelettique. À partir de modèles animaux, il a été démontré qu'une augmentation des niveaux de TGF-β actifs est observée dans les animaux ayant le phénotype de MFS par rapport aux animaux contrôle. Dans cette étude, nous démontrons que des fragments de la fibrilline-1 recombinante exprimés par des cellules HEK293 et purifiés par chromatographie d'affinité métallique contiennent du TGF-β1 sous forme active et sous forme latente (LAP- TGF-β1). Des analyses subséquentes du schéma de la purification démontrent que le TGF-β1 sécrétés par les cellules HEK293 sont co-purifiées de manière non spécifiques avec les fragments de la fibrilline-1, et que ce facteur de croissance était présent dans toutes les préparations protéiques. Nous avons aussi démontré que la co-purification non-spécifique de la TGF-β1 sous forme active ou la LAP-TGF-β1 était éliminée par chromatographie d'exclusion en présence de concentration saline élevée pour les fragments représentant les moitiés de la fibrilline-1 en C-terminal et N-terminal. Cependant, la chromatographie d'exclusion du fragment rF20, représentant la partie centrale de la fibrilline-1, n'a pas complètement dissocié la LAP-TGF-β1 associée avec ce fragment, suggérant une interaction entre ce fragment et la LAP-TGF-β1.

Thesis (Ph. D.)--Illinois State University, 1995.
Title from title page screen, viewed May 8, 2006. Dissertation Committee: Hou Tak Cheung (chair), David W. Borst, Herman E. Brockman, Alan J. Katz, Anthony J. Otsuka. Includes bibliographical references (leaves 98-110) and abstract. Also available in print.