Cell migration involves complex mechanical interactions between cells or between cells and the underlying substrate. Using a newly developed technique, "traction force microscopy", I have been able to visualize the dynamic characteristics of mechanical forces exerted by migrating fibroblasts such as magnitude, direction, and shear. For NIH 3T3 fibroblasts, I found that the lamellipodium provides nearly all of the force necessary for cell migration. A high shear zone separates the lamellipodium from the remainder of the cell body, suggesting that they are mechanically distinct entities. The timing of the tractions at the leading edge, as well as the spatial distribution, bears no apparent relationship to concurrent local protrusive activities, yet changes in traction force patterns often precede changes in migration direction. In H-ras transformed cells I found isolated regions of weak, transient traction forces in pseudopods all along the cell that appeared to act against one another. The resulting shear pattern suggested that there were multiple disorganized mechanical domains. These results support a frontal towing model for cell migration where the dynamic traction forces at the leading edge served to actively pull the cell body forward. In H-ras transformed cells, the weak poorly coordinated traction forces coupled with weak cell substrate-adhesions were likely responsible for the abnormal motile behavior of these cells. To probe the mechanical interactions beneath various regions of migrating fibroblasts, a cell substrate inhibitor (GRGDTP peptide) was locally applied while imaging stress distribution on the substrate utilizing traction force microscopy. I found that both spontaneous and GRGDTP induced detachment of the trailing edge resulted in extensive cell shortening with no change in overall traction force magnitude or cell migration. Conversely, leading edge disruption resulted in a dramatic global loss of traction forces pnor to any significant cell shortening. These results suggested that fibroblasts transmit their contractile forces to the substrate through two distinct types of adhesions. Leading edge adhesions were unique in their ability to transmit active propulsive forces whereas trailing end adhesions created passive resistance during cell migration and readily redistributed their loads upon detachment. I have also investigated how fibroblasts regulate traction forces based on mechanical input. My results showed that stretching forces applied through the flexible substrate induced increases in both intracellular calcium concentration and traction forces in fibroblasts. Treatment with gadolinium, a well known stretch-activated ion channel inhibitor, was found to inhibit both traction forces and cell migration without inhibiting cellular spread morphology or protrusive activities. Gadolinium treatment also caused a pronounced decrease in vinculin and phosphotyrosine concentrations from focal adhesions. Local application of gadolinium to the trailing region had no detectable effect on overall traction forces or cell migration, whereas local application to the leading edge caused a global inhibition of traction forces and an inhibition of migration. These observations suggest that stretch activated entry of calcium ions in the frontal region serves to regulate the organization of focal adhesions and the output of mechanical forces. Together my experiments elucidate how fibroblasts exert mechanical forces to propel their movements, and how fibroblasts utilize mechanical input to regulate their movements.

Orientador: Fernanda da Cruz Landim / Resumo: As pesquisas envolvendo a biologia das células-tronco abordam um amplo espectro de fenômenos, que vão desde o nível tecidual e celular, até o seu uso em terapias celulares. Esta crescente atenção sugere que é necessário estudar conceitos básicos da biologia das células-tronco para compreender completamente os processos de diferenciação funcional. Desta forma, o instrumento da reprogramação celular por meio da manipulação gênica fornece subsídios para melhor compreender os processos de renovação e diferenciação que constituem as características fundamentais das células-tronco. A obtenção dessas células em medicina veterinária visa validar diversos modelos experimentais domésticos, como o equino, na busca de novos fármacos e terapias alternativas para reabilitação. Uma série de estudos, porém, ainda são necessários para que tais aplicações sejam viáveis, uma vez que os mecanismos fundamentais das técnicas empregadas ainda não estão totalmente elucidados. Embora a reprogramação celular por meio de vetores virais tenha sido relatada com sucesso em diversas espécies animais, outras técnicas também podem ser empregadas, como o uso de transposons, sequências de DNAs capazes de se movimentar de uma região para outra no genoma de uma célula. Não se tem conhecimento de qual o melhor tipo celular a ser utilizado, e nem tão pouco qual a metodologia de reprogramação mais eficiente. Sabe-se que o cordão umbilical possui uma reserva rica em células-tronco mesenquimais, as quais por serem mu... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Researches on the biology of stem cells cover a broad spectrum of phenomena, ranging from tissue and cellular level, to their use in cell therapy. This growing attention suggests that is necessary to study basic concepts of stem cells organization in order to fully understand the functional differentiation processes. Thus, the cell reprogramming through gene manipulation provides grants to better understand the processes of renewal and differentiation which are the essential characteristics of stem cells. Obtaining these cells in veterinary medicine aims to validate various household experimental models, such as horses, on the search for new drugs and alternative therapies for rehabilitation. However, a number of studies is still necessary for such applications to be feasible, since the fundamental mechanisms of techniques employed are not fully elucidated yet. Although cell reprogramming using viral plasmid has been reported with success in several animal species, other techniques may also be employed, such use transposons, this is, DNAs sequences capable of moving from one region to another in the cell genome. The unawereness of what the best cell type to be used, and nor what is the most efficient reprogramming methodology. It is known that the cord has rich reserves mesenchymal stem cells, which are multipotent and can improve the efficiency of obtaining the induced Pluripotent Stem Cells (iPS) compared to the use of fibroblast, inefficient to be reprogrammed. The aim of this study was to obtain iPS through viral transfection and nonviral adult fibroblasts and equine cord cells, aiming to observe which transfection and cell type is more efficient for cell reprogramming. Both cell types was infected with viral vectors and transposons containing the genes OCT-4, SOX-2, c-MYC, and KLF-4; transformed cells were evaluated for morphology, immunocytochemistry... (Complete abstract click electronic access below) / Doutor

Der humanen dermalen Wundheilung liegt ein komplexes Zusammenspiel verschiedener Faktoren zugrunde. Die Bedeutung dieses fein regulierten Gleichgewichts wird deutlich, wenn es durch Fehlregulationen oder Störungen zu chronischen Wundheilungsstörungen oder lokaler Fibrose mit überschießender Narbenbildung kommt. Eine der möglichen Methoden zur Prävention und Behandlung ist die Deckung der Wunde mit einem Hautersatz. Dabei werden zunehmend sogenannte Biomaterialien aus natürlichen Substanzen mit hoher Biokompatibilität und der Möglichkeit zur Interaktion mit dem nativen Gewebe verwendet. In Studien wurde gezeigt, dass vor allem sulfatierte Glykosaminoglykan-Derivate durch die Interaktion ihrer negativ geladenen Sulfatgruppen mit Zytokinen, Wachstumsfaktoren und dermalen Zellen einen positiven Einfluss auf den Wundheilungsprozess haben können. In der vorliegenden Arbeit wurden daher kollagenbasierte artifizielle extrazelluläre Matrizes mit unsulfatierter oder sulfatierter Hyaluronsäure hinsichtlich ihres Einflusses auf humane dermale Fibroblasten als Komponenten der Wundheilung untersucht. Dermale Fibroblasten spielen im Ablauf der Wundheilung eine tragende Rolle und interagieren eng mit der umgebenden Matrix. Anhand ihrer Proteinexpression lassen sich Rückschlüsse auf wichtige Funktionen wie Adhäsion, Proliferation, Differenzierung und Matrixsynthese ziehen. In den durchgeführten Experimenten zeigte sich, dass sulfatierte Matrix in der Kultur mit dermalen Fibroblasten kein entzündliches Milieu förderte. Die Proliferation, Differenzierung und Migration der Fibroblasten schienen gesteigert, während sich die Matrix-Synthese und ihr Remodeling weder pathologisch gehemmt noch überschießend zeigten. Daher wäre die weitere Untersuchung dieses Biomaterials ein vielversprechender Ansatz, um langfristig dem Risiko von Wundheilungsstörungen wie chronischen Wunden oder fibroproliferativen Wundheilungsstörungen effektiv entgegenzuwirken.

Our study contributes to the tissue engineering, mainly to the construction of appropriate scaffolds for regeneration of damaged skin. Simultaneously, it brings valuable insights for basic research in the field of molecular mechanisms of adhesion, proliferation and phenotypic maturation of cells and the control of the cell behavior through the cell extracellular matrix (ECM), represented by synthetic nanofibrous material. Nanofibrous polylactic-co-glycolic acid (PLGA) membranes were prepared by needle-less electrospinning technology. These membranes were further modified with cell adhesion-mediating biomolecules, e.g. collagen, fibronectin and fibrin in order to increase their affinity to colonizing cells. Adhesion, growth and differentiation of keratinocytes (HaCaT) and fibroblasts, i.e. major cell types of epidermis and dermis, were evaluated on these nanofibrous membranes. The results show that the membrane modification using fibrin structures improved adhesion and proliferation of human dermal fibroblasts. The collagen structure on the surface of membranes improved the adhesion and proliferation of human HaCaT keratinocytes. Furthermore, fibrin structure stimulated fibroblasts to produce collagen, which is a major component of ECM in the natural skin dermis. Fibronectin enhanced cell attachment...

There is an increasing interest for the use of nanostructures as potential tools in areas that include biology and medicine, for applications spanning from cell separation to treatments of diseases. Magnetic nanoparticles (MNPs) have been the most widely studied and utilized nanostructures in biomedical applications. Despite their popularity, the regular shape of MNPs limits their potential for certain applications. Studies have shown that magnetic nanowires (MNWs), due to their high-­-aspect ratio and specific magnetic properties, might provide improved performance for some biomedical applications. As a consequence, MNWs have received increasing attention from researchers in the last years. However, as with any other nanostructure intended for biomedical applications, rigorous studies must be carried out to determine their potential toxicity and adverse effects before they can be successfully incorporated in clinical applications. This work attempts to elucidate the cytotoxic effects of nickel NWs (Ni NWs) in human fibroblasts by measuring cell viability under different parameters. Ni NWs of three different lengths (0.86 ± 0.02 μm, 1.1 ± 0.1 μm and 6.1 ± 0.6 μm) were fabricated by electrodeposition using porous aluminum oxide (PAO) membranes as templates. Energy dispersive X-­-Ray analysis (EDAX) and X-­-Ray diffraction (XRD) were used for the chemical characterization of the Ni NWs. Their physical characterization was done using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) imaging. MTT assays were performed to assess cell viability of human fibroblasts in the presence of Ni NWs. NW length, NW/cell ratio and exposure time were changed throughout the experiments to elucidate their effects on cell viability. The results showed that NWs length has a strong effect on internalization and cytotoxicity. Smaller NWs showed higher toxicity levels at earlier times while longer NWs had stronger effects on cell viability at later times. NW/cell ratio did not seem to have a very strong effect at low concentrations. However, at high concentration (1000 NW/cell) significant loss of cell viability was observed, with the effects becoming stronger at later times. Other factors such as cell surface area, presence of oxide layer on NWs, and the cytotoxicity of Ni salts, were also studied and found to affect cell viability. For our knowledge, this is the first systematic study done in human fibroblasts wi-­-38 using ferromagnetic NWs; where the toxic effects of equivalent amounts of Ni in its salt and in its NW form are compared. It is also the first study to provide insights of the interaction between wi-­-38 cells and Ni NWs. The results of this study complement and enrich previous cytotoxicity studies of Ni NWs. This work aims at providing a more comprehensive understanding of the interaction between NWs and biological systems. Despite the advancements, further studies will be required to fully understand the factors affecting NW cytotoxicity. Only when we understand the underlying mechanisms, will we be able to design suitable nanostructures for biomedical applications.

iii Abstract Glycobiology represents a very progressive subject of cell biology. Protein-saccharide interactions play not only supporting and cell organization role, but they also represent medium for information storage and its decoding. Galectins, group of animal lectins (saccharide-binding proteins), which have selective affinity to ß-galactosides, are multifactorial molecules. They participate in cell-cell and cell-matrix interaction, transmembrane signaling, apoptosis, pre- mRNA splicing and are also present in various types of carcinomas. High expression of galectin-1 has been detected in cancer stroma originated from squamous cell epithelium. In the previous study we established that the fibroblasts - myofibroblasts transition, apart from the known TGF- beta, is also induced by galectin-1. We compared relationship between galectin-1 expression, presence of myofibroblasts and gene expression in tissue samples from patients with head and neck squamous cell carcinoma. Cancer stroma with myofibroblasts was rich in galectin-1 expression in comparison with stroma without myofibroblasts. Moreover, we used microarray analysis (ILLUMINA) to compare the whole genome transcriptome from samples with and without presence of galectin-1. High expression of galectin-1 in tissue samples corresponded with expression...

Human esophageal squamous cell carcinoma (hESCC) is a very aggressive form of cancer due to its ability to easily metastasize into proximal lymph nodes and adjacent organs. The role of the extracellular matrix (ECM) and its stromal cells in metastasis remains unclear. To better understand the effect of the ECM and fibroblast cells on esophagus cancer cell migration and invasion, we propose a biomimetic human esophagus model cultured with hESCC and human primary fibroblast cells (fibroblast). To mimic the extracellular matrix of human esophagus we use decellularized porcine esophagus matrix (DEM) to culture with hESCC and fibroblasts in static conditions. This DEM can recapitulate the human esophagus tumor microenvironment with relevant cues. This model will provide valuable information regarding esophagus cancer cell migration with respect to the heterogeneous extracellular matrix and stromal fibroblast cells. We expect to discover the mechanisms by which extracellular matrix and stromal cells affect cancer migration and invasion in vitro. Characterizing this process will provide vital insight towards the effects of fibroblasts cells on facilitating migration and invasion of esophageal cancer cells. This esophagus cancer model also provides promising potential to study drug screening and develop new strategies against esophagus metastasis. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2020. / FAU Electronic Theses and Dissertations Collection

PART I The basic helix-loop-helix (bHLH) family of transcription factors are involved in a variety of developmental processes. MyoR is the mouse homologue of the human transcription factor ABF-1 . MyoR is classified as a class II basic-helix-loop-helix transcription factor. In order to better understand the relationship between MyoR and muscle cell differentiation, we analyzed the temporal expression at both the mRNA and protein level. Unlike previous studies, we have utilized reverse transcriptase quantitative PCR to analyze mRNA expression. This allows quantitative analysis of MyoR mRNA levels during muscle cell differentiation. We have also analyzed MyoR expression at the protein level. Our studies suggest that the temporal expression of MyoR at the mRNA level is similar to the expression profile seen at the protein level. To ascertain differences in the MyoR DNA-binding activity during myogenesis we performed EMSA. Results suggest that changes in MyoR expression fail to account for differences in the DNAbinding complexes to an E-box site. Part II Members of the basic helix-loop-helix (bHLH) family are required for a number of different developmental pathways, including lymphopoiesis, myogenesis, neurogenesis and sex determination. Screening a eDNA library prepared from silk-producing glands of the black widow spider, we have identified a new bHLH transcription factor named BW6. Within the bHLH region, BW6 shows considerable conservation with other HLH proteins, including Drosophila melanogaster achaete and scute, as well as three HLH proteins identified by gene prediction programs. The expression pattern of bw6 is restricted to a subset of silk producing glands, which includes the tubuliform and major ampullate glands. BW6 is capable of binding an E-box element as a heterodimer with E2A, but was unable to bind this motif as a homodimer. BW6 is also capable of inhibiting the transactivation of rE47 in mammalian cells. BW6 represents the first example of a silk-gland-restricted bHLH protein, and its expression pattern suggests that BW6 may play a role in regulating differentiation of cells in the spider that control silk gland formation or egg case silk gene expression.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20084号 / 医博第4177号 / 新制||医||1018(附属図書館) / 33200 / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 山田 亮, 教授 椛島 健治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20230号 / 医博第4189号 / 新制||医||1019(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 野田 亮, 教授 武田 俊一, 教授 杉田 昌彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM