Orientador: Rui Seabra Ferreira / Resumo: Desenvolvido em 1990 por um grupo de pesquisadores do Centro de Estudo de Venenos e Animais Peçonhentos (CEVAP), no Estado de São Paulo, Brasil, o Biopolímero de Fibrina (BPF) possuía o principal objetivo de ser um adesivo à base de fibrina sem o uso de sangue humano, a fim de evitar a transmissão de doenças infecciosas por meio deste insumo. Após diversas pesquisas com o BPF, comprovou-se não somente sua capacidade adesiva, como também sua ação coagulante, sua ação como auxiliar no reparo ósseo e cartilaginoso e sua função como arcabouço para células-tronco mesenquimais (CTMs), devido ao fato de que o BPF possui uma estrutura tridimensional adequada. Em estudos recentes e ao exercer essa função, tal material não afetou o microambiente biológico das células, ou seja, permitiu a adesão, proliferação e diferenciação celular, e aderência e crescimento destas. Tais características, apresentadas pelo BPF, são desejáveis na maioria dos biopolímeros utilizáveis, o que ressalta a importância do aprofundamento das pesquisas com BPF e suas interações em experimentos in vivo. Assim, no capítulo 1 realizamos uma ampla revisão na literatura sobre biopolímeros de fibrina, células-tronco e reparação de tecido ósseo. No capítulo 2 é apresentado o artigo científico “Arcabouço de fibrina para células-tronco mesenquimais como potencial osteogênico”. / Abstract: Developed in 1990 by a group of researchers from the Center for the Study of Venomous and Poisonous Animals (CEVAP) in the State of São Paulo, Brazil, the Fibrin Biopolymer (GMP) had the main objective of being a fibrin-based adhesive without the use of human blood in order to avoid the transmission of infectious diseases by means of this input. After several investigations with BPF, it was verified not only its adhesive capacity, but also its coagulant action, its action as an aid in bone and cartilage repair and its function as a framework for mesenchymal stem cells (MSCs), due to the fact that the BPF has an adequate three-dimensional structure. In recent studies and in carrying out this function, such material did not affect the biological microenvironment of the cells, that is, it allowed cell adhesion, proliferation and differentiation, and adhesion and growth of these cells. These characteristics, presented by BPF, are desirable in most usable biopolymers, which underscores the importance of deepening GMP research and its interactions in in vivo experiments. Thus, in Chapter 1 we conducted a broad review in the literature on biopolymers of fibrin, stem cells and repair of bone tissue. In chapter 2 the scientific paper "Fibrin scaffold for mesenchymal stem cells as osteogenic potential" is presented. / Doutor

Scaffold proteins facilitate many aspects of intracellular signaling. These proteins can regulate two or more proteins in the same pathway, or coordinate signaling from multiple pathways. Scaffold proteins are therefore key control points for the flux of signaling and play essential roles in biological systems. There are four possible mechanisms by which scaffold proteins achieve activation and propagate signaling: 1) rigid protein binding between two or more proteins to co-localize binding partners, 2) ligand-induced activation such as may result from a conformational change, 3) disorder-to-order transition where the scaffold protein folds as a result of a protein-protein interaction, and 4) dynamic processes such as phosphorylation. The scaffold protein NF-κB essential modulator (NEMO) functions via ligand-induced activation and serves as the key control point for canonical NF-κB signaling. The work described in this thesis investigates the role of a previously uncharacterized domain within NEMO that is required for function, which we term the Intervening Domain (IVD). Bioinformatic analysis reveals a high level of sequence conservation across species within this domain. Conformational changes following ligand binding are observed for NEMO and these changes require conserved sequences in the IVD. Additionally, a functional IVD is shown to increase the binding affinity of NEMO for IKKβ, enhance the thermal stability of NEMO, and is required to propagate NF-κB signaling in cells. A fluorescence-based assay is also developed to characterize the formation of a complex composed of NEMO, a zinc ion, and IκBα. A separate fluorescence-based assay is developed to measure IKK activity and is used to determine that NEMO alone or in the presence of linear tetraubiquitin does not enhance the rate of IKKβ phosphorylation of an IκBα-derived peptide. Furthermore, a number of organic small molecules and macrocycles are screened against the NEMO-IKKβ interaction. One small molecule was validated as an inhibitor and its biophysical properties and inhibition kinetics are described in this thesis. These analyses represent the first characterization of a highly conserved domain required for the function of the key control point in NF-κB signaling. The IVD domain of NEMO could be targeted for development of an allosteric effector for therapeutic discovery.

<p>Validerat; 20130918 (global_studentproject_submitter)</p>

Diabetes is a chronic metabolic disorder whereby the body loses its ability to maintain normal glucose levels. Despite of development of implantable glucose sensors in long periods, none of the biosensors are capable of continuously monitoring glucose levels during long-term implantation reliably. Progressive loss of sensor function occurs due in part to biofouling and to the consequences of a foreign body response such as inflammation, fibrosis, and loss of vasculature. In order to improve the function and lifetime of implantable glucose sensors, a new 3D porous and bio-stable collagen scaffold has been developed to improve the biocompatibility of implantable glucose sensors. The novel collagen scaffold was crosslinked using nordihydroguaiaretic acid (NDGA) to enhance biostability. NDGA-treated collagen scaffolds were stable without any physical deformation in the subcutaneous tissue of rats for 4 weeks. The scaffold application does not impair the function of our sensor. The effect of the scaffolds on sensor function and biocompatibility was examined during long-term in vitro and in vivo experiments and compared with control bare sensors. The sensitivity of the short sensors was greater than the sensitivity of long sensors presumably due to less micro-motions in the sub-cutis of the rats. The NDGA-crosslinked scaffolds induced much less inflammation and retained their physical structure in contrast to the glutaraldehyde (GA)-crosslinked scaffolds. We also have developed a new dexamethasone (Dex, anti-inflammatory drug)-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres/porous collagen scaffold composite for implantable glucose sensors. The composite system showed a much slower and sustained drug release than the standard microspheres. The composite system was also shown to not significantly affect the function of the sensors. The sensitivity of the sensors with the composite system in vivo remained higher than for sensors without the composites (no scaffold, scaffold without microspheres). Histology showed that the inflammatory response to the Dex-loaded composite was much lower than for the control scaffold. The Dex-loaded composite system might be useful to reduce inflammation to glucose sensors and therefore extend their function and lifetime.

Erbin belongs to the LAP (leucine-rich repeat and PDZ domain) family of scaffolding proteins that play important roles in orchestrating cell signaling. Here, we show that Erbin functions as a tumor suppressor in colon cancer. Analysis of Erbin expression in patient specimens reveals that Erbin is downregulated at both mRNA and protein levels in tumor tissues. Functionally, knockdown of Erbin disrupts epithelial cell polarity and increases cell proliferation in 3D culture. In addition, silencing Erbin results in an increase in the amplitude and duration of signaling through Akt and RAS/RAF pathways. Moreover, Erbin-loss induces epithelial-mesenchymal transition (EMT), which coincides with a significant increase in cell migration and invasion. Erbin interacts with KSR1 and displaces it from the RAF/MEK/ERK complex to prevent signaling propagation. Furthermore, genetic deletion of Erbin in Apc knockout mice promotes tumorigenesis and significantly reduces survival. Tumor organoids derived from Erbin/Apc double knockout mice have increased tumor initiation potential along with increased Wnt target gene expression as seen by qPCR. Collectively, the studies within this dissertation identify Erbin as a negative regulator of EMT and tumor progression by directly suppressing Akt and RAS/RAF signaling in vivo.

In 2005, 1.6 million Americans lived with a debilitating amputation and this figure is predicted to double by 2050. But the ability of a mammal to recapitulate a complex limb structure is not impossible. Evidence of children and mice re-growing digit tips following amputation midway through the terminal phalanx (P3) exists. The hallmark of this phenomenon is development of a blastema housing undifferentiated cells capable of being re-programmed to replicate the missing part. Our central goal is to understand specific components of this process for application into pro-scarring injuries. The mouse digit anatomy is prominently outlined by microfilaments containing ER-TR7, and antigen derived from fibroblast reticular cells (FRCs) of the thymus shown to facilitate intercellular communication to promote lymphoid organogenesis. A unique blastema characteristic is the upregulation of an ER-TR7+ scaffold stemming from half of the blastema population which reverts to its pre-existing pattern after regenerate differentiation concludes. We measured a correlation between ER-TR7 and type III collagen (COL3) at the transcriptional and protein levels both in vitro during induction of ER-TR7 in primary P3 cells and throughout digit regeneration. Co-expression with COL3 sheds light on ER-TR7 identity and allows testing various approaches to manipulation of the scaffold through the better understood mechanism of COL3 regulation. Furthermore, we aimed at determining the origin of ER-TR7+ blastema FRCs. Using bone marrow (BM) transplantation, we generated eGFP+ BM chimeras to study the fate of BM-derived cells (BMDCs) after amputation based on the hypothesis that in the regenerate, multipotent BMDCs contribute to various cellular phenotypes including FRCs. So we tested co-immunolocalization of eGFP with antigens particular to fibroblastic, hematopoietic, endothelial, osteoblastic, and mural cells. Many BMDCs homed to the injury throughout regeneration. But hematopoietic BMDCs were limited to inflammation whereas mesenchymal BMDCs expanded and were primed as ER-TR7+ FRCs in the P3 BM niche prior to homing to the blastema site, where they amounted to nearly 50% of cells. Moreover, BMDCs differentiated into endothelial, osteoblastic, and smooth muscle subpopulations and although diluted by pre-existing progenitors by the endpoint of regeneration, BMDCs persisted as part of various structures thus contributing to long-term function. / acase@tulane.edu

<p>This thesis describes the design, synthesis and structure-activity relationships analysis of potential inhibitors targeting the hepatitis C virus (HCV) NS3 protease. Also discussed is the disease caused by HCV infection and the class of enzymes known as proteases. Furthermore are explained why such enzymes can be considered to be suitable targets for developing drugs to combat diseases in general and in particular HCV, focusing on the NS3 protease. Moreover, some strategies used to design protease inhibitors and the desired properties of potential drug candidates are briefly examined. Synthesis of linear and macrocyclic NS3 protease inhibitors comprising a designed trisubstituted cyclopentane moiety as an <em>N</em>-acyl-(4<em>R</em>)-hydroxyproline bioisostere is also addressed, and several very potent and promising compounds are evaluated.</p> / Report code: LIU-TEK-LIC-2006:46.

This thesis describes the design, synthesis and structure-activity relationships analysis of potential inhibitors targeting the hepatitis C virus (HCV) NS3 protease. Also discussed is the disease caused by HCV infection and the class of enzymes known as proteases. Furthermore are explained why such enzymes can be considered to be suitable targets for developing drugs to combat diseases in general and in particular HCV, focusing on the NS3 protease. Moreover, some strategies used to design protease inhibitors and the desired properties of potential drug candidates are briefly examined. Synthesis of linear and macrocyclic NS3 protease inhibitors comprising a designed trisubstituted cyclopentane moiety as an N-acyl-(4R)-hydroxyproline bioisostere is also addressed, and several very potent and promising compounds are evaluated. / Report code: LIU-TEK-LIC-2006:46.

Long-term survival of implanted cells requires oxygen and nutrients, the need for which is met by vasculari- zation of the implant. The use of scaffolds with surface-attached heparin as anchoring points for angiogenic growth factors has been reported to improve this process. We examined the potential role of surface modification of gelatin scaffolds in promoting endothelial cell infiltration by using a unique macromolecular conjugate of heparin as a coating. Compared to other heparin coatings, this surface modification provides flexible heparin chains, representing a new concept in heparin conjugation. In vitro cell infiltration of scaffolds was assessed using a three-dimensional model in which the novel heparin surface, without growth factors, showed a 2.5-fold increase in the number of infiltrating endothelial cells when compared to control scaffolds. No additional improvement was achieved by adding growth factors (vascular endothelial growth factor and/or fibroblast growth factor-2) to the scaffold. In vivo experiments confirmed these results and also showed that the addition of angiogenic growth factors did not significantly increase the endothelial cell infiltration but increased the number of inflammatory cells in the implanted scaffolds. The endothelial cell-stimulating ability of the heparin surface alone, combined with its growth factor-binding capacity, renders it an interesting candidate surface treatment to create a prevascularized site prepared for implantation of cells and tissues, in particular those sensitive to inflammation but in need of supportive revascularization, such as pancreatic islets of Langerhans. / <p>De två sista författarna delar sistaförfattarskapet.</p>

A degradable/polar/hydrophobic/ionic (D-PHI) polyurethane with properties intended to promote tissue regeneration in a small diameter peripheral artery vascular graft was evaluated for cell biocompatibility and growth. Films were cast in polypropylene 96 well plates for monocyte/endothelial cell (EC) co-culture in vitro studies and porous scaffold discs were implanted in an in vivo subcutaneous mouse model. After 7 days in culture the co-culture demonstrated cell adhesion and growth, low esterase activity (a measure of degradative potential and cell activation), no detectable release of pro-inflammatory cytokine (tumour necrosis factor -α) but measurable anti-inflammatory interleukin (IL)-10. The EC and the co-culture expressed the EC biomarker CD31, whereas the monocyte monoculture did not. Cytokine array analysis of the in vivo characterization of D-PH supported an anti-inflammatory phenotype of cells at the site of the implant. Levels of IL-6 significantly decreased over time while IL-10 was significantly higher at 6 weeks post implant. TNF-α levels did not change significantly from 24 hours onwards, however the trend was towards lesser amounts following the initial time point. Histological analysis of the explanted scaffolds showed excellent tissue ingrowth and vascularization. A live/dead stain showed that the cells infiltrating the scaffolds were viable. Both the in vitro and in vivo results of this thesis indicate that D-PHI is a good candidate material for tissue engineering a peripheral artery vascular graft.