The 1980's heralded the discovery and identification of extra-pituitary sources of the neurohypophysial hormone oxytocin in non-neural tissues of several animal species. The presence, location and biosynthesis of significant amounts of oxytocin in the ovarian corpus luteum was followed by the immunocytochemical demonstration of an oxytocin-like peptide in the testicular interstitial cells. Leydig cells, which comprise up to 80% of the testicular intertubular cell population, are known to synthesize testosterone in situ. Indirect evidence indicated that an oxytocin-like peptide was also present in Leydig cells. The question arose whether this peptide was synthesized de novo by Leydig cells or was taken up and stored by the cells following biosynthesis at some other intra- and/or extra-gonadal source(s). Since luteinizing hormone (LH) and ascorbate are known to augment the production of oxytocin in ovarian granulosa cells, varying concentrations of these two stimulants were used to monitor the biosynthesis of oxytocin from isolated Leydig cells in culture. / Ph. D.

Esta tesis aborda la actividad biológica de la fibronectina (FN) como proteína de interfase en la interacción célula-material. La tesis investiga la respuesta de la proteína, en términos de cantidad adsorbida y conformación, ante diferentes propiedades físico-químicas del material. Además, se correlaciona la respuesta celular temprana y la funcionalidad celular con el estado de la proteína adsorbida sobre el material. Para ello se prepararon diferentes series de materiales con propiedades físico-químicas controladas. La distribución de FN sobre las diferentes superficies se caracterizó mediante el uso de la microscopía de fuerza atómica (AFM) y la densidad superficial adsorbida fue cuantificada mediante técnicas de marcado radioactivo y western blot. La respuesta celular se evaluó en términos de la adhesión inicial a las superficies, así como los procesos posteriores de diferenciación, proliferación, reorganización y producción de matriz extracelular. Se investigó el efecto de la nanotopografía en la adsorción de la FN y el comportamiento celular sobre una serie de topografías controladas en la escala nanométrica, obtenidas mediante el spin casting de soluciones de ácido poli(L-láctico)/poliestireno (PLLA/PS) de distintas concentraciones. La migración del PLLA hacia la superficie del film durante el proceso de spin coating proporciona superficies de PLLA con nanopicos de diferentes tamaños (14, 29 y 45 nm). El tamaño de la nanoestrutura afecta a la densidad de FN adsorbida, siendo mayor en la superficie de menor nanotopografía. En cuanto a la respuesta celular inicial, se observan adhesiones focales más desarrolladas y mejor reorganización celular de la capa de FN adsorbida en las superficies de mayor topografía (29 and 45 nm), lo que resulta en una mayor producción y organización de nueva matriz. Por otra parte se empleó una familia de materiales con sutiles variaciones en la composición química: polímeros acrílicos (polimetil, etil y butil acrilato -PMA, PEA y P / González García, C. (2012). BIOLOGICAL ACTIVITY OF FIBRONECTIN AT THE CELL-MATERIAL INTERFACE [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/17701

Abstract While most of the in vitro cultures are carried out on bi-dimensional (2D) substrates, most of the in vivo extracellular matrices are threedimensional (3D). Consequently cells behave differently on 2D substrates as a way to self-adaptation to a non-physiological environment. This fact has encouraged the development of more relevant culture conditions seeking to provide more representative models for biomedicine (e.g. cancer, drug discovery and tissue engineering) and further insights into any dimension-dependent biological mechanism. Different 3D culture systems have been established though their variability and complexity hinder their standardisation in common cell culture procedures. So, this thesis deals with the dimensionality issue in cell/material interactions and introduces sandwich-like microenvironments as a versatile tool to study cell behaviour. Cells cultured within this system use both dorsal and ventral receptors to adhere and spread, undergoing important changes with respect to the 2D cultures and approaching to 3D conditions. Stimulation of dorsal receptors has been previously addressed by overlaying a protein gel on cells already attached on a 2D surface. Here we propose a sandwich-like system that consists of two 2D surfaces so that wider spectra of conditions can be investigated by changing the nature of the substrate (material, topography…) and the protein coatings of both ventral and dorsal sides. Since sandwich culture provides an altered cellular adhesion compared to the traditional 2D substrates by the excitation of the dorsal receptors, changes in the intracellular signalling are expected, which might alter important processes such as proliferation, morphology, migration and differentiation. Hence this thesis evaluates the effect of different sandwich culture parameters in cell behaviour. First, cell fate upon adhesion was evaluated in terms of morphology, proliferation and adhesion. Different conditions were studied such as materials with different properties or protein coatings (dorsal and ventral substrates), as well as the effect of sandwiching cells just after seeding or after been allowed to adhere to the ventral substrate. Interesting results were obtained such as the relationship between the ability of cells to reorganise the ECM with cell morphology, proliferation and adhesion, similarly as observed in 3D hydrogels (degradable vs nondegradable systems). Then, cell migration within sandwich culture was studied by live imaging of a wound healing assay. Results revealed the key effect of both ventral and dorsal substrates in determining the migration rate as well as the migration mode used by cells. Moreover cells within the sandwich culture migrating in the wound healing assay adopted an elongated cell morphology that resembled cells migrating in other 3D systems. Beyond differences in cell morphology and migration, dorsal stimulation promoted cell remodelling of the extra-cellular matrix (ECM) over simple ventral receptor activation in traditional 2D cultures. Finally the effect of sandwich culture on cell differentiation was evaluated. First we showed an increase in C2C12 myogenic differentiation when cultured within the sandwich system. This enhancement was shown to be dorsal stimulation dependent and related to an alteration of the signalling pathway and the growth factor release. To determine if sandwich culture leads only to myogenic differentiation or whether it allows differentiation to other lineages, 4 different human mesenchymal stem cells (hMSCs) lines were cultured under the same conditions. Results showed the same sandwich environment triggered different cell differentiation. This points out the importance of the microenvironment cell niche in vivo, which highly influence cell fate, and thus the need of mimicking it properly in vitro. Overall, sandwich-like microenvironments switch cell behaviour towards 3D-like patterns, demonstrating the importance of this versatile, simple and robust approach to mimic cell microenvironments in vivo. / Ballester Beltrán, J. (2014). Sandwich-like systems to engineer the cellular microenvironment [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48166

Amoebae are commonly studied to understand embryogenesis, and the best-characterized amoebozoan species is Dictyostelium discoideum (Dd). Dd has a very simple life cycle with a range of developmental stages, among which we are most interested in the stage of a migrating slug. It has been observed that different sizes of Dd slugs maintain a proportional distribution of prestalk cells and prespore cells: prestalk cells occupy the anterior 20% of the slug, while prespore cells occupy the posterior 80%. However, it remains unknown how the migrating slug forms and preserves this anterior-posterior proportional pattern under so many different dynamics including cell movement, signaling, and cell differentiation. Therefore, we constructed a mathematical model to simulate the cell movement and chemical distribution during slug migration, and we conducted numerical experiments to explore possible factors for this pattern. In particular, we divided the problem of interest into the following three parts to be investigated. (1) differential motion: the ability of prestalk cells to move through all the prespore cells and stay in the anterior region of the slug; (2) signaling: how cells of different types produce, receive, and respond to the signals in the environment; (3) cell differentiation: how prestalk and prespore cells differentiate into each other under the regulation of signaling. We finally combined and balanced these mechanisms appropriately to achieve the desired patterns observed in migrating slugs.

A developmentally regulated cyclic AMP-dependent protein kinase has been recently reported in Dictyostelium discoideum. This report describes some of the physical and kinetic properties of the cAMP-dependent holoenzyme and its subunits. Gel filtration data suggests a holoenzyme Mr of 170,000-190,000, and catalytic and regulatory subunit Mrs of 40,000 and 49,000, respectively. These molecular weight determinations are compatible with an R₂C₂ subunit arrangement of the holoenzyme. Kinase activity required the presence of Mg²⁺ but cAMP binding to the enzyme was not dependent on divalent metal ions. The pH optimum for kinase activity was 7.5; the cAMP binding activity was not affected over a pH range of 5.0-10.0. The holoenzyme and isolated regulatory subunit had identical cAMP Kds of 28 nM. Cyclic AMP was able to dissociate the subunits when analyzed by density gradient centrifugation. Histone VII-S activated the subunits in the absence of cAMP but did not produce their dissociation. In contrast to the gel filtration data, sedimentation values indicated a dimeric holoenzyme structure. Reassociation of the subunits in the absence of cAMP occurred rapidly and was not dependent upon a preincubation with MgATP. High NaCl and low pH depressed both the total kinase activity and the ability of the subunits to reassociate as determined by activity ratio. MgATP did not decrease the ability of the holoenzyme to bind cAMP, neither did the holoenzyme possess a high affinity MgATP binding site. By the use of microdissection techniques holoenzyme levels were determined in individuals at each stage of development and in each cell type during development. Kinase activity was low and non-cAMP dependent in early aggregates but increased and became cAMP-dependent in later aggregates. Maximum activity and cAMP-dependency occurred during the slug and culmination stages. The only differential distribution of the kinase within a single-stage occurred during culmination when the activity in the stalks was approximately one-fourth that in the prespore mass. Preliminary evidence indicates that this difference is not due to an inhibitor. / Ph. D. / incomplete_metadata

Glycogen phosphorylase 2 (gp-2) is a key enzyme during the development of Dictyostelium discoideum. The gp-2 enzyme breaks down glycogen into glucose monomers that are subsequently used to synthesize the terminal end products of cellular differentiation. This gene is an ideal candidate for studying the process of selective gene expression because its product figures so prominently in the development of this organism, implying a dependable control mechanism responsible for its developmentally regulated expression. I present in this thesis the identification of several putative cis-acting elements of gp-2 as revealed through footprint analysis. Due to the extreme AT-bias characteristic of Dictyostelium promoters, footprinting conditions required intensive optimization with respect to template, nonspecific competitor, source of protein extract and DNase I digestion. Using an endlabeled fragment containing seven repeated sequences (3 TA boxes [TAATTATA], 2 TAG boxes [TAAAAATGGT] and 2 C boxes [ACCCACT]), purified replication protein A and several developmental nuclear extracts were tested for DNA binding activity. Small footprints were observed on the TAG and C boxes of the promoter for both protein sources. However, using a more sensitive footprinting strategy involving multiple rounds of primer extension, larger footprints spanning the same promoter regions were detected. In both cases, the appearance of the footprints coincided with the documented transcriptional activity of the gene. It can be concluded from the data obtained that the TAG and C boxes are very likely cis-acting elements involved in the regulation of gp-2 expression. / Master of Science

Bone marrow mesenchymal stromal cells (BM MSCs) represent a heterogeneous population of progenitors with potential for generation of skeletal tissues. However the identity of BM MSC subpopulations is poorly defined mainly due to the absence of specific markers allowing in situ localization of those cells and isolation of pure cell types. Here, we aimed at characterization of surface markers in mouse BM MSCs and in their subsets with distinct differentiation potential. Using conditionally immortalized BM MSCs we performed a screening with 176 antibodies and high-throughput flow cytometry, and found 33 markers expressed in MSCs, and among them 3 were novel for MSCs and 13 have not been reported for MSCs from mice. Furthermore, we obtained clonally derived MSC subpopulations and identified bipotential progenitors capable for osteo- and adipogenic differentiation, as well as monopotential osteogenic and adipogenic clones, and thus confirmed heterogeneity of MSCs. We found that expression of CD200 was characteristic for the clones with osteogenic potential, whereas SSEA4 marked adipogenic progenitors lacking osteogenic capacity, and CD140a was expressed in adipogenic cells independently of their efficiency for osteogenesis. We confirmed our observations in cell sorting experiments and further investigated the expression of those markers during the course of differentiation. Thus, our findings provide to our knowledge the most comprehensive characterization of surface antigens expression in mouse BM MSCs to date, and suggest CD200, SSEA4 and CD140a as markers differentially expressed in distinct types of MSC progenitors.

Résumé: Le muscle squelettique possède une excellente capacité à se regénérer notamment grâce à ses cellules progénitrices stromales (mrSC) et myogéniques (CPM). À la suite de certains traumas et pour des raisons encore méconnues, la qualité de sa régénération est compromise ce qui mène à l’apparition de structures aberrantes tel l’os mature, aussi appelée ossification hétérotopique (OH) post-traumatique. Notre laboratoire a montré dans un modèle murin que les mrSC sont pleinement impliquées dans cette pathologie. De plus, un facteur fortement ostéoinducteur, BMP9, ne cause l’OH que si, et seulement si, le muscle est endommagé. Ce modèle d’étude est unique puisqu’il présente les particularités physiopathologiques de l’OH post-traumatique, un dommage du muscle étant essentiel à la formation d’os. De plus, ce modèle a permis de mettre en évidence le rôle prédominant du microenvironnement des cellules progénitrices dans le développement de cette pathologie. Nous avons donc émis l’HYPOTHÈSE selon laquelle le microenvironnement du muscle endommagé contient des facteurs qui peuvent influencer le phénotype de ses cellules progénitrices stromales et myogéniques favorisant ainsi le développement de l’OH. Nos résultats montrent que l’état hypoxique d’un muscle sévèrement endommagé augmente la prolifération et la différenciation ostéogénique des mrSC. De plus, l’hypoxie induit spécifiquement l’expression de BMP9 par les mrSC. L’impact de BMP9 a également été évalué sur la différenciation des CPM. Les résultats montrent qu’à des concentrations physiologiques, BMP9 inhibe le potentiel myogénique des CPM en faveur d’une différenciation ostéogénique, et cela tant dans la lignée myoblastique murine C2C12 que chez les CPM primaires humaines. En résumé, le muscle endommagé développant l’OH possède un microenvironement spécifique responsable du débalancement de la capacité régénérative de ses progéniteurs. Nos travaux montrent que ce microenvironnement cause un retard de la myogenèse et une ostéogenèse où participeront non seulement les mrSC mais également les CPM. L’identification et la compréhension des mécanismes régulant ces facteurs s’avèrent clé pour offrir aux cliniciens des outils de diagnostic mais également des alternatives ou des approches complémentaires aux traitements prophylaxiques actuels. / Abstract: Skeletal muscle has an extraordinary ability to regenerate due to its resident stromal cells (mrSCs) and myogenic progenitor cells (MPCs). Following certain traumas, the quality of the regeneration of skeletal muscle can be compromised for unknown reasons, leading to the appearance of aberrant structures such as mature bone, a process called posttraumatic heterotopic ossification (HO). Our laboratory developed a mouse model to show that mrSCs are fully involved in this pathology. We also showed that BMP9, a highly osteoinductive factor, causes HO if and only if the muscle is damaged. This model is unique in that it recapitulates the pathophysiological features of post-traumatic HO in which muscle damage is essential for bone formation. The model was also used to show that the progenitor cell microenvironment plays a predominant role in the development of this pathology. Based on these results, we HYPOTHESIZED that the microenvironment of the damaged muscle contains factors that can influence the phenotype of its progenitor cell populations, thus promoting the development of HO. Our results showed that the hypoxic state of a severely damaged muscle increases the proliferation and osteogenic differentiation of mrSCs and also specifically induces the expression of BMP9 by mrSCs. The impact of BMP9 on the differentiation of MPCs was also evaluated. At physiological concentrations, BMP9 inhibited the myogenic differentiation potential of murine myoblast C2C12 cells and primary human MPCs, and triggered their differentiation into an osteogenic lineage. In summary, we showed that damaged muscle that develops HO has a specific microenvironment that is responsible for the loss of the regenerative capacity of progenitor cells, leading to a delay in myogenesis, and that mrSCs and MPCs are both involved in osteogenesis. The identification and understanding of the mechanisms regulating these key factors could provide clinicians with valuable diagnostic tools as well as alternative and/or complementary approaches to current prophylactic treatments.

BACKGROUND: Bone remodeling relies on the tightly regulated interplay between bone forming osteoblasts and bone digesting osteoclasts. Several studies have now described the molecular mechanisms by which osteoblasts control osteoclastogenesis and bone degradation. It is currently unclear whether osteoclasts can influence bone rebuilding. METHODOLOGY/PRINCIPAL FINDINGS: Using in vitro cell systems, we show here that mature osteoclasts, but not their precursors, secrete chemotactic factors recognized by both mature osteoblasts and their precursors. Several growth factors whose expression is upregulated during osteoclastogenesis were identified by DNA microarrays as candidates mediating osteoblast chemotaxis. Our subsequent functional analyses demonstrate that mature osteoclasts, whose platelet-derived growth factor bb (PDGF-bb) expression is reduced by siRNAs, exhibit a reduced capability of attracting osteoblasts. Conversely, osteoblasts whose platelet-derived growth factor receptor beta (PDGFR-beta) expression is reduced by siRNAs exhibit a lower capability of responding to chemotactic factors secreted by osteoclasts. CONCLUSIONS/SIGNIFICANCE: We conclude that, in vitro mature osteoclasts control osteoblast chemotaxis via PDGF-bb/PDGFR-beta signaling. This may provide one key mechanism by which osteoclasts control bone formation in vivo.

The eukaryotic nuclear envelope (NE), separates the nucleoplasm from cytoplasm and is made up of two concentric lipid membranes, the outer and the inner nuclear membranes (ONM and INM), the nuclear pore complexes (NPCs) and an underlying filamentous nuclear lamina. The INM contains hundreds of unique transmembrane proteins of which only a handful have been characterized. In this thesis, I aimed to understand the functional organization of proteins in the nuclear envelope and I focused on investigating the functions of a recently identified INM transmembrane protein, Samp1. We have developed a novel and robust approach, MCLIP, to identify specific protein-protein interactions taking place in live cells. Using MCLIP, we have shown that Samp1 interacts with proteins of the LINC complex, the nuclear lamina and components of the mitotic spindle. Samp1's specific interactions with a variety of binding partners, suggest that Samp1 plays important roles both in interphase and in mitosis.  We have also shown that Samp1 can provide a binding site at the INM for the GTPase Ran, a master regulator of protein interactions in interphase and in mitosis. Furthermore, we have also investigated the role of Samp1 in cell differentiation using two independent model systems. In human iPSCs, ectopic expression of Samp1 promoted differentiation despite pluripotent culture conditions. In C2C12 myoblast, depletion of Samp1 completely blocked differentiation into myotubes. The two studies complement each other and suggest that Samp1 has a strong differentiation promoting activity. Taken together, the findings in this thesis, give insights on the unexpected and unforeseen roles played by a transmembrane protein in different fundamental cellular process. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript. Paper 5: Manuscript.</p>