O presente estudo utilizou o modelo fatia-dental/matriz-polimérica para avaliar a influência do tratamento dentinário e das BMPs dentinárias na diferenciação das células-tronco da polpa de dentes decíduos (SHED). Secções transversais (1mm) foram preparadas a partir de terceiros molares humanos extraídos. Matrizes poliméricas a base de ácido poli-L-lático (PLLA) foram criadas no interior da cavidade pulpar das secções dentinárias, tratadas com solução de EDTA a 10%; NaOCl a 5.25%; ou permanecendo sem tratamento. Matrizes poliméricas confeccionadas sem as fatias dentais foram utilizadas como controle. As células (5x104) foram semeadas nas matrizes e, após 7, 14, 21 e 28 dias de cultura in vitro, a expressão de marcadores de diferenciação odontoblástica (DSPP, DMP1 e MEPE) e a proliferação celular (WST-1) foram avaliadas. Células (5x105) semeadas nas matrizes foram transplantadas em camundongos imunodeficientes e cultivadas in vivo por um período de 14 e 28 dias. Para avaliar a atividade das BMPs dentinárias, 5x104 células foram semeadas em matrizes poliméricas com fatia dental e cultivadas na presença de anticorpos anti-BMP- 2, -4, ou -7 (2 μg/ml) durante 14 dias. Adicionalmente, 5x105 células foram tratadas com rhBMP-2, -4, ou -7 (100ng/mL) por 24hs. As células cultivadas in vitro e in vivo alteraram sua expressão genética durante o curso do tempo. DSPP, DMP-1 e MEPE foram expressos por células cultivadas in vitro após 14 dias (tratamento com EDTA e dentina sem tratamento) e in vivo após 28 dias (EDTA), não sendo detectados nos grupos NaOCl e nas células cultivadas nas matrizes sem fatia dental. A proliferação foi reduzida com a diferenciação celular (p<0.05). A utilização de BMP-2/4Ab no meio de cultura exerceu um efeito inibitório na expressão dos marcadores de diferenciação celular, não ocorrendo quando do cultivo das SHED na presença de BMP-7Ab. DSPP, DMP-1 e MEPE foram expressos por células tratadas com rhBMP-2, e DSPP e DMP-1 por células tratadas com rhBMP-4 e -7. Células sem tratamento não expressaram os marcadores. O modelo fatia-dental/matriz-polimérica demonstrou ser adequado para o estudo da diferenciação de células-tronco pulpares, sugerindo que a dentina possa fornecer um microambiente favorável para a diferenciação de celular. As proteínas ósseas morfogenéticas dentinárias BMP-2 e BMP-4 parecem exercer um papel relevante nesse processo. / The effect of dentin pre-treatments and dentin-derived BMPs on SHED differentiation was tested using the Tooth-Slice Scaffold model (TSS). Dentin slices (1mm thickness) were prepared from extracted human third molars. Biodegradable PLLA scaffolds were prepared inside the pulp chamber of the tooth-slices, treated alternatively with a 5.25% NaOCl or 10% EDTA solution, or remaining untreated (WO-T). PLLA sponge scaffolds with no tooth-slice (PSS) were used as control. SHED (5x104) were seeded in TSS and PSS and after 7, 14, 21 and 28 days in culture, RT-PCR (DSPP, DMP1 and MEPE) and WST-1 proliferation assay were performed. Additionally, cells (5x105) were seeded in TSS and PSS and transplanted into SCID mice (14 and 28 days). To verify the dentinderived BMPs bioactivity, SHED (5x104) were cultured in TSS in the presence of antihuman BMP-2, -4, and -7 antibodies for 14 days. Besides, cells in culture were treated with rhBMP-2; -4; or -7 for 24 hours. After in vitro and in vivo time course, SHED altered their genetic expression. The cells cultured in vitro in the TSS (EDTA or WO-T) expressed the differentiation markers after 14 days and maintained expression thereafter. Cell proliferation rate was reduced following the differentiation (p<0.05). Cells transplanted in vivo expressed DSPP, DMP-1 and MEPE after 28 days (EDTA). No transcripts were found in tooth-slices treated with NaOCl or in PSS groups. BMP-2/4Ab prevented the differentiation process and no inhibitory effect was detected for BMP-7Ab. After 24 hours, expression of DSPP, DMP-1 and MEPE was found for rhBMP-2, and DSPP and DMP-1 for rhBMP-4 and rhBMP-7 treated SHED, but not for untreated cells. The tooth slice scaffold model suggests that dentin can provide the environment for SHED differentiation and dentin-derived morphogenic signals BMP-2 and BMP-4 play an important role in this process.

Danos ao sistema nervoso central (SCN) resultam em perda de conexões axonais, das funções motoras e sensoriais. Uma das estratégias para seu reparo é o transplante de células-tronco mesenquimais (CTMs). Porém essa alternativa requer uma adequada via de aplicação. Nesse sentido, o uso de matrizes alinhadas pode ser usado para apoiar o crescimento e diferenciação das CTMs e, quando incorporadas com fatores de crescimento, otimizam o processo de regeneração tecidual. O objetivo desse trabalho foi avaliar a diferenciação neural das CTMs cultivadas sobre matrizes de nanofibras orientadas com o fator de crescimento epidermal (EGF) incorporado. Os scaffolds com fibras alinhadas foram produzidos por electrospinning de emulsão e avaliados conforme a sua morfologia, o diâmetro das nanofibras, a degradabilidade e a liberação do EGF. As CTMs utilizadas foram provenientes da polpa de dentes decíduos esfoliados humanos. Essas células foram cultivadas nos scaffolds e avaliadas conforme os testes biológicos: adesão, viabilidade, proliferação, citotoxicidade e diferenciação neural. Os scaffolds com fibras alinhadas controle (AC) e contendo o EGF (AE) apresentaram morfologia, diâmetro das nanofibras e tempo de degradação semelhantes. Com base no total de EGF presente na matriz AE, 90,14% foi liberado após 28 dias. O citoesqueleto e o núcleo das CTMs cultivadas nos scaffolds AC e AE estavam mais alongados e alinhados quando comparado com as CTMs cultivadas no poço de cultura (controle). As CTMs aderiram mais nas matrizes AE em relação às matrizes AC, porém a proliferação e viabilidade celular foram similares, exceto no tempo de 72 horas, o qual a viabilidade no grupo controle foi maior, em comparação aos demais grupos. Os scaffolds AC e AE não foram tóxicos para as CTMs. Em relação aos resultados da neuro-diferenciação, a expressão de nestina e neurofilamentos consideravelmente maior em todos os grupos analisados quando comparado ao grupo controle. A expressão de βIII-tubulina e GFAP foi maior em todos os grupos diferenciados quando comparada ao grupo controle. A maioria das CTMs cultivadas nas matrizes AC e AE, induzidas ou não à diferenciação neural, apresentaram correntes dependente de voltagem para sódio. O valor de condutância máxima foi maior para todos os grupos analisados quando comparado ao grupo controle onde as células não foram diferenciadas. Portanto, as matrizes com nanofibras orientadas induzem à diferenciação neural das CTMs em neurônios funcionais tanto na ausência como na presença de EGF incorporado. As matrizes AE ainda mostraram ser capazes de melhorar a adesão celular. Dessa forma, conclui-se que as matrizes de nanofibras estudadas são uma possível estratégia para otimização da regeneração de lesões neurológicas. / Damage to the central nervous system (CNS) results in loss of axonal connections and motor and sensory functions. One of the strategies for its repair is the transplantation of mesenchymal stem cells (MSCs). However, this requires a suitable application route. Accordingly, the use of scaffolds support the growth of MSCs and, when incorporated with growth factors, optimize the regeneration process. The purpose of this study was to evaluate the neural differentiation of MSCs cultured on nanofiber matrices oriented with epidermal growth factor (EGF) incorporated. Aligned scaffolds were produced by electrospinning emulsion and evaluated according to their degradation, the morphology and diameter of the nanofibers, and release of EGF from the nanofibers. MSCs used were from human exfoliated deciduous teeth (SHED). These cells were cultured on the scaffolds and evaluated according to biological tests: adhesion, viability, proliferation, cytotoxicity and neural differentiation. The aligned control scaffolds (AC) containing EGF (AE) presented similar morphology, diameter of nanofibers and degradation time. Based on the total EGF present in the scaffold AE, 90.14% was released after 28 days. The cytoskeleton and the core of the MSCs cultured on scaffolds AC and AE were more aligned and elongated when compared to the MSCs grown on plate wells (control). MSCs adhered more to matrices AE when compared to matrices AC, although proliferation and cell viability were similar, except after 72 hours. In this period, the viability of the control group was higher when compared to the rest of the groups. Scaffolds AC and AE were not toxic to MSCs. In regard to the results of neuro-differentiation, the expression of nestin and neurofilament was much higher in all groups than the control group. The expression of βIII tublin and GFAP was higher in all differentiated groups than the control group. Most of the MSCs grown in matrices AC and AE, induced or not to neural differentiation, showed voltage-dependent sodium currents. The maximum value of conductance of these groups was higher for the cells in all groupscompared to the control group, where the cells were not differentiated. Therefore, oriented nanofiber matrices induce neural differentiation of MSCs into functional neurons both in the absence and in the presence of incorporated EGF. The matrices AE also showed improved cell adhesion. Thus, these matrices are a possible strategy for optimizing the regeneration of neurologic lesions.

Made available in DSpace on 2016-06-02T20:18:11Z (GMT). No. of bitstreams: 1 2456.pdf: 6548607 bytes, checksum: 486173aee7443e35a2910af043a12256 (MD5) Previous issue date: 2009-04-03 / Universidade Federal de Sao Carlos / Bioactive materials have the ability to bond and to integrate with bone tissue by forming a biologically active bonelike apatite layer, which has chemical and structural properties equivalent to the mineral phase of living bone. This process is determined by chemical reactions, whose products also influence the attachment, the proliferation, the differentiation and the mineralizing capacity of bone cells. Cellular responses contribute to the bioactive behavior, which is known for being higher in glass materials. However, as low mechanical properties are also inherent characteristics of glasses, researchers from Federal University of Sao Carlos were stimulated to develop nucleation and growth thermal treatments for the obtainment of the Biosilicate®, a fully-crystallized bioactive glassceramic of the quaternary system P2O5-Na2O-CaO-SiO2. Although a high in vitro osteogenic potential of this novel glass-ceramic has been previously demonstrated, its in vivo effects have not been investigated yet. To contribute to this knowledge, two studies were developed. The first one aimed to investigate the in vivo biological performance of Biosilicate® in bone defects of rat tibias, by means of hystomorphometric and biomechanical analyses 20 days after the surgical procedure. This study revealed that the fully-crystallized Biosilicate® has good bone-forming and bone-bonding properties. Hence, the second study aimed to compare the kinetics of the bone reactions to two different granulometric distributions of this novel glass-ceramic. Although they were both efficient for bone formation, smaller-sized particles of Biosilicate® showed partial reabsortion, which was accompanied by a more pronounced osteogenic activity within the period of time studied. Since positive results were obtained, the search for scaffolds that could serve as supports for the guided bone regeneration had started. A third study preliminarily evaluated cell culture and cocultures in porous structures made of Biosilicate® and of other chemical compositions that were specifically developed for this purpose. The findings suggest that, when in adjusted conditions, the scaffolds can create favorable cellular responses for bone tissue engineering purposes. Taken togheter, these studies point to a promising potential and provide directives for the use of Biosilicate® in bone regenerative processes. / Materiais bioativos possuem a capacidade de se ligar ao tecido ósseo por meio da formação de uma interface apatítica que apresenta similaridade química e estrutural com a fase mineral dos ossos. Esse processo ocorre devido a uma série de reações químicas, cujos produtos também influenciam a adesão, a proliferação, a diferenciação e a capacidade de mineralização da matriz pelas células ósseas. As respostas celulares contribuem para o comportamento bioativo, que é conhecido por ter maiores índices em materiais vítreos. No entanto, como baixas propriedades mecânicas também são características inerentes aos vidros, pesquisadores da Universidade Federal de São Carlos foram estimulados a empregarem nucleação e tratamentos térmicos especiais para o desenvolvimento do Biosilicato®, uma vitrocerâmica biotiva, totalmente cristalina, pertencente ao sistema quaternário P2O5-Na2O-CaO-SiO2. Embora um elevado potencial osteogênico in vitro tenha sido demonstrado para esta vitrocerâmica, seus efeitos in vivo ainda não são conhecidos. Para auxiliar este entendimento, foram desenvolvidos dois estudos. O primeiro teve como objetivo investigar o desempenho biológico in vivo do Biosilicato® particulado em defeitos ósseos em tíbias de ratos, por meio de análises histomorfométricas e biomecânicas 20 dias após o procedimento cirúrgico. Este estudo evidenciou que o Biosilicato® parece favorecer a formação óssea in vivo e o estabelecimento de fortes ligações com o tecido neoformado. Com isso, o objetivo do segundo estudo foi comparar a cinética das reações ósseas frente a duas diferentes distribuições granulométricas desta nova vitrocerâmica. Embora ambas tenham sido eficientes para a formação óssea, as partículas de Biosilicato® com menores diâmetros demonstraram reabsorção parcial no período estudado, que foi acompanhada de uma maior atividade osteogênica. Com os resultados positivos obtidos nestas investigações, iniciou-se uma busca para o desenvolvimento de matrizes porosas que pudessem servir de suporte para a regeneração guiada do tecido ósseo. Um terceiro estudo preliminarmente avaliou monoculturas e coculturas celulares em matrizes porosas de Biosilicato® e de outras novas composições químicas desenvolvidas especificamente para este propósito. Os achados sugerem que, quando em condições adequadas, as matrizes avaliadas podem produzir respostas celulares favoráveis ao seu emprego na engenharia do tecido ósseo. Estes estudos, de maneira conjunta, apontam para um potencial promissor e fornecem diretrizes para o emprego do Biosilicato® no favorecimento de processos regenerativos ósseos.

O presente estudo utilizou o modelo fatia-dental/matriz-polimérica para avaliar a influência do tratamento dentinário e das BMPs dentinárias na diferenciação das células-tronco da polpa de dentes decíduos (SHED). Secções transversais (1mm) foram preparadas a partir de terceiros molares humanos extraídos. Matrizes poliméricas a base de ácido poli-L-lático (PLLA) foram criadas no interior da cavidade pulpar das secções dentinárias, tratadas com solução de EDTA a 10%; NaOCl a 5.25%; ou permanecendo sem tratamento. Matrizes poliméricas confeccionadas sem as fatias dentais foram utilizadas como controle. As células (5x104) foram semeadas nas matrizes e, após 7, 14, 21 e 28 dias de cultura in vitro, a expressão de marcadores de diferenciação odontoblástica (DSPP, DMP1 e MEPE) e a proliferação celular (WST-1) foram avaliadas. Células (5x105) semeadas nas matrizes foram transplantadas em camundongos imunodeficientes e cultivadas in vivo por um período de 14 e 28 dias. Para avaliar a atividade das BMPs dentinárias, 5x104 células foram semeadas em matrizes poliméricas com fatia dental e cultivadas na presença de anticorpos anti-BMP- 2, -4, ou -7 (2 μg/ml) durante 14 dias. Adicionalmente, 5x105 células foram tratadas com rhBMP-2, -4, ou -7 (100ng/mL) por 24hs. As células cultivadas in vitro e in vivo alteraram sua expressão genética durante o curso do tempo. DSPP, DMP-1 e MEPE foram expressos por células cultivadas in vitro após 14 dias (tratamento com EDTA e dentina sem tratamento) e in vivo após 28 dias (EDTA), não sendo detectados nos grupos NaOCl e nas células cultivadas nas matrizes sem fatia dental. A proliferação foi reduzida com a diferenciação celular (p<0.05). A utilização de BMP-2/4Ab no meio de cultura exerceu um efeito inibitório na expressão dos marcadores de diferenciação celular, não ocorrendo quando do cultivo das SHED na presença de BMP-7Ab. DSPP, DMP-1 e MEPE foram expressos por células tratadas com rhBMP-2, e DSPP e DMP-1 por células tratadas com rhBMP-4 e -7. Células sem tratamento não expressaram os marcadores. O modelo fatia-dental/matriz-polimérica demonstrou ser adequado para o estudo da diferenciação de células-tronco pulpares, sugerindo que a dentina possa fornecer um microambiente favorável para a diferenciação de celular. As proteínas ósseas morfogenéticas dentinárias BMP-2 e BMP-4 parecem exercer um papel relevante nesse processo. / The effect of dentin pre-treatments and dentin-derived BMPs on SHED differentiation was tested using the Tooth-Slice Scaffold model (TSS). Dentin slices (1mm thickness) were prepared from extracted human third molars. Biodegradable PLLA scaffolds were prepared inside the pulp chamber of the tooth-slices, treated alternatively with a 5.25% NaOCl or 10% EDTA solution, or remaining untreated (WO-T). PLLA sponge scaffolds with no tooth-slice (PSS) were used as control. SHED (5x104) were seeded in TSS and PSS and after 7, 14, 21 and 28 days in culture, RT-PCR (DSPP, DMP1 and MEPE) and WST-1 proliferation assay were performed. Additionally, cells (5x105) were seeded in TSS and PSS and transplanted into SCID mice (14 and 28 days). To verify the dentinderived BMPs bioactivity, SHED (5x104) were cultured in TSS in the presence of antihuman BMP-2, -4, and -7 antibodies for 14 days. Besides, cells in culture were treated with rhBMP-2; -4; or -7 for 24 hours. After in vitro and in vivo time course, SHED altered their genetic expression. The cells cultured in vitro in the TSS (EDTA or WO-T) expressed the differentiation markers after 14 days and maintained expression thereafter. Cell proliferation rate was reduced following the differentiation (p<0.05). Cells transplanted in vivo expressed DSPP, DMP-1 and MEPE after 28 days (EDTA). No transcripts were found in tooth-slices treated with NaOCl or in PSS groups. BMP-2/4Ab prevented the differentiation process and no inhibitory effect was detected for BMP-7Ab. After 24 hours, expression of DSPP, DMP-1 and MEPE was found for rhBMP-2, and DSPP and DMP-1 for rhBMP-4 and rhBMP-7 treated SHED, but not for untreated cells. The tooth slice scaffold model suggests that dentin can provide the environment for SHED differentiation and dentin-derived morphogenic signals BMP-2 and BMP-4 play an important role in this process.

The Human Immunodeficiency Virus (HIV) is a member of the retroviridae family from lentivirus genus which primarily infects CD4+ T cells and also to lesser degree monocytes, macrophages, and dendritic cells causing progressive failure of the immune system, ultimately leading to development of acquired immunodeficiency syndrome (AIDS). Currently ~ 37 million people are infected with HIV-1 with approximately 2 million new infections occurring every year (UNAIDS, 2016). Developing safe, effective, and affordable vaccines to prevent HIV infection is the best hope for controlling the HIV/AIDS pandemic. Envelope glycoprotein (Env) on the HIV-1 virion surface is synthesized as a single precursor protein gp160 which is cleaved by furin to form the gp120 and gp41 subunits. gp41 is inserted into the membrane, while gp120 remains non-covalently associated with the ectodomain of gp41 to form a trimer of heterodimers. gp120 binds to the CD4 receptor on CD4+ T cells, which triggers a series of conformational changes leading to the exposure of co-receptor binding sites on gp120. Subsequent binding to the co-receptor (CXCR4 or CCR5) on T-cells initiates fusion of cellular and viral membranes via gp41 subunit. The envelope glycoprotein gp120, on the virion surface is the most accessible component of HIV-1 to the host immune system, and the target of most of the neutralization response. However, the virus has evolved many efficient ways to escape this immune surveillance. Extensive glycosylation of gp120 is one way by which it masks critical neutralization epitopes and the presence of immunodominant long variable loops focuses the immune response away from conserved regions. Certain conserved epitopes are cryptic and get exposed only after gp120 binds to its receptor. Also gp120 and gp41 are highly flexible molecules, attached in a non-covalent fashion to form a trimer of heterodimers, leading to inherent metastability of the Env. This results in exposure of a large number of non-native conformations to the immune system and thus minimizes elicitation of neutralizing antibodies. Despite these defense mechanisms, about 20-30% of HIV-1 patients do generate a broad neutralization response. Although these bNAbs and their epitopes have been identified, eliciting similar bNAbs through immunization is challenging. Monomeric gp120 when used as an immunogen elicits non neutralizing antibodies. This indicates that the epitopes of bNAbs are not present in the right conformation on this molecule. A rational design approach which focuses the immune response towards specific epitopes targeted by bNAbs is required, with the aim to maximize the exposure of conserved neutralization epitopes and to simultaneously ensure minimal exposure of variable non neutralizing epitopes. This can likely be achieved either by (a) stabilization of native Env trimers, or/and by (b) protein fragment design. Chapter 1 gives a brief description of HIV-1 virus. Structural features of the Env protein are described along with epitopes targeted by various bNAbs. Various strategies employed towards structure based vaccine design are discussed. One of the strategies towards rational vaccine design is using protein fragment based approaches. Grafting epitopes onto heterologous scaffolds is a promising approach which can provide more structural stability to the epitope, helps focus immune response on the epitope of interest and can be employed in a prime boost strategy for immunization studies. In a scaffold based approach we used crystal structure information of gp120 in complex with bNAb b12 to define the epitope of this antibody. In Chapter 2 we use this epitope information to graft the epitope on an unrelated scaffold protein to design unique epitope scaffolds. We report a computational strategy to graft the discontinuous epitope of b12 antibody onto different scaffold proteins. Our strategy focuses on identifying the best match of the target scaffold to the query protein so as to cause the least structural disturbance in the scaffold protein. The best hits were screened for binding to b12 using Yeast Surface Display (YSD). Random mutant libraries were also generated to screen for better b12 binders using YSD. We further characterized a few of these epitope scaffolds after purifying them from bacterial systems. One of the epitope scaffolds 1mkh_E2 bound to b12 with a KD value of 7.5µM. 2bodx_03, an unoptimized epitope scaffold reported previously (Azoitei et al, 2011) binds b12 with a KD value of 300μM. Thus our epitope scaffold 1mkh_E2 shows reasonable binding to b12 without any optimization. We are currently purifying other b12 epitope scaffolds and will be characterizing them for binding to b12. We have previously used a protein minimization strategy to design fragments of gp120, called b122a and b121a comprising a compact beta barrel on the lower part of the outer domain in order to focus the immune response towards the b12 epitope. (Bhattacharyya et al, 2013). These were bacterially expressed, found to be partially folded, however, could bind the broadly neutralizing antibody b12 with micromolar affinity. In rabbit immunization studies sera obtained following four primes with the b122a fragment protein and two boosts with full-length gp120 showed broad neutralization of a panel of multiple viruses across different clades (Bhattacharyya et al, 2013). In the present work, These designs were further stabilised by introducing various disulphides. One of the disulphide mutants b122a1-b showed better binding to b12 compared to b122a and increased protection to protease digestion. However these are partially structured as assessed by CD. In Chapter 3 we attempted to evolve stabilized versions of b122a1-b by using a genetic selection based on antibiotic resistance described previously (Foit et al, 2009). We were successfully able to show an in-vivo stability difference between b122a and b122a1-b. From the library generated in the background of b122a1-b using random mutagenesis, a few apparently stabilized mutants were isolated. Most of these mutations were hydrophobic to polar substitutions at exposed positions while a few of the mutations were substitutions with similar side chain chemistry as in wildtype. In future studies we will measure mutant stabilities and binding affinity to b12. A set of similar fragment immunogens were also designed based on subtype C CAP210 gp120 sequences. In Chapter 4 we describe various immunization studies comprising of different sets of b12 epitope based fragment immunogens. In one study we displayed some of these immunogens on Qβ VLPs. In another study, we tested subtype C based fragment immunogens. The humoral immune response was probed in terms of generation of antibodies against the immunogens using ELISA. Neutralization activity of the sera was measured in a standard TZM-bl assay. Sera raised against these particles in rabbit immunization studies could neutralize Tier1 viruses across different subtypes. The group primed with particles displaying b122a1-b and the group primed with b122a conjugated to particle in the presence of adjuvant contained significantly higher amounts of antibodies directed towards the CD4bs than sera from the group primed with empty particles and boosted with gp120. This study demonstrates the overall utility of the particle based display approach. In immunization studies with subtype C derived fragment immunogens as primes, no significant neutralization was seen even for Tier 1 viruses. In this study, the group primed and boosted with full length gp120 performed better than other groups suggesting that antibodies elicited against regions present in these subtype C priming immunogens are non-neutralizing. One of the rational vaccine design strategies is by stabilization of native Env trimers. In previous studies, a disulfide bond was engineered between gp120 and gp41 of Env to stabilize the interactions (SOS gp140). An I559P mutation was also introduced to stabilize the native gp41 conformation in the context of disulfide engineered Env (SOSIP gp140). The purified, soluble SOSIP gp140 immunogens were trimeric and cleaved properly and are believed to be one of the closest mimics of native Env trimers. However, these immunogens have so far failed to elicit broad neutralizing responses. In Chapter 5, we use structural information derived from high resolution atomic structure of native like cleaved gp140 BG505-SOSIP, to provide an alternate strategy to form uncleaved trimeric gp140s by cyclic permutation to design molecules that mimic cleaved trimers. The structure reveals that the gp41 C-terminus is in very close proximity (~8Å) to the N-terminus of gp120 from an adjacent subunit. We have designed a cyclic permutant of gp140 from JRFL strain where the gp41 C terminus is now connected to the gp120 N-terminus with a short linker. This novel connectivity results in preservation of the native gp41 N-terminus along with a much shorter linker length than in conventional gp140. This might promote trimer folding and stabilization because of the resulting decreased magnitude of conformational entropy change during folding. The structure also reveals that the gp120 C-terminus is close to the trimer axis, and due to cyclic permutation, this becomes the new C-terminus of gp140. To further stabilize the trimeric form, we have attached a foldon trimerization domain at the C terminus. The protein has been expressed and purified from mammalian cells. The protein exists primarily as a trimer in solution as assessed by SEC-MALS. It shows better binding to broadly neutralizing antibody b12 when compared to b6, a non-neutralizing antibody. Further biophysical characterization of the protein is in progress. We have previously described design of a bacterially expressed outer domain derivative of gp120 (ODEC) that had V1/V2 and V3 loops deleted and bound CD4 (Bhattacharyya et al, 2010). To improve the initial ODEC design, three different rational design strategies were used. In the first approach, residue frequency based methods were used to design a construct named ODECConsensus. In another approach, a cyclic permutant of ODEC (CycV4OD) was designed with new N and C termini in the flexible V4 loop. In the third approach the bridging sheet (BS) region was deleted from ODEC to form ODECΔBS. In Chapter 6 we have used hydrogen deuterium exchange-mass spectrometric analysis (HDX-MS) to study conformational flexibility of these fragment immunogens. These studies revealed that all the three immunogens show reduced conformational flexibility compared to ODEC. 5-7 protons remain protected up to 2 hours whereas for ODEC, exchange completes at 20 minutes. This reduced flexibility correlates with 6-20 fold tighter VRC01 binding relative to ODEC. In rabbit immunizations, all three constructs elicit significant gp120 titers as early as week 6 in the absence of any gp120 boost whereas ODEC shows significant gp120 titers only after two gp120 boosts. Week 24 sera elicited after immunization with ODECΔBS, ODECConsensus and CycV4OD boosted with gp120 show neutralization of multiple Tier 1 viruses from subtype B and C, whereas corresponding ODEC immunized animals failed to show a neutralizing response. This study demonstrates that reduced conformational flexibility correlates with better antigenicity and an improved immunogenicity profile for these fragment immunogens. Also we have used HDX-MS studies to one of the stem based HA fragment immunogen pH1HA10-foldon described previously (Mallajosyula et al, 2014) to do peptide finger printing and find regions of protein showing increased protection to hydrogen deuterium exchange and thus derive some structural insights about this trimeric fragment immunogen. Peptide mapping experiments show that the HA stem fragment peptides are exchanging rapidly with more than 90% exchange completing by 30 s for most of the peptides. The well folded foldon trimerization domain peptide shows a very slow exchange profile. A few of the HA peptides exchange slowly with 1-2 protons exchanging after 30 s. Fast exchange seen for this fragment immunogen may be due to truncation of the stem region leading to greater solvent accessibility of the trimer interface.

Les objectifs de cette thèse concernent la caractérisation des polymères à l’état fondu via la rhéologie non linéaire dans les modes de cisaillement ou en élongationnel et les procédés faisant intervenir de fortes élongations tel que l’électrospinning en voie solvant et en voie fondue. Pour atteindre le premier objectif, nous nous sommes concentrés sur la caractérisation des polymères fondus enchevêtrés dans les régimes viscoélastiques linéaires et non linéaires. L'influence de la masse moléculaire, Mw et de sa distribution MWD, la présence de longues chaînes branchées (LCB) ou encore l'addition de nanoparticules dans la matrice de polymère à l'état fondu ont été étudiées en utilisant des techniques rhéologiques en cisaillement et en élongationnel. Dans le cas des écoulements de cisaillement oscillatoires à grandes amplitudes (LAOS), nous avons proposé de nouveaux paramètres mécaniques qui ont permis de définir les relations structure-propriétés des différents systèmes étudiés. / The goals of this thesis are the characterization of polymer melts using mainly non-linear shear and extensional rheological techniques. The fabrication of scaffolds with excellent physical and mechanical properties using solution electrospinning technology for tissue engineering applications and the development of melt electrospinning equipment to facilitate the fabrication of solvent free scaffolds. To achieve the first goal, we focused on the characterization of entangled polymer melts in the linear and nonlinear viscoelastic regimes. The influence of molecular weight, Mw, molecular weight distribution (MWD), long-chain branching (LCB) and addition of particles to the polymer matrix on polymer melt properties were investigated using shear and extensional rheological techniques. The resulting structure-property relationships were established using newly introduced mechanical parameters under large amplitude oscillatory shear (LAOS) flow.

Danos ao sistema nervoso central (SCN) resultam em perda de conexões axonais, das funções motoras e sensoriais. Uma das estratégias para seu reparo é o transplante de células-tronco mesenquimais (CTMs). Porém essa alternativa requer uma adequada via de aplicação. Nesse sentido, o uso de matrizes alinhadas pode ser usado para apoiar o crescimento e diferenciação das CTMs e, quando incorporadas com fatores de crescimento, otimizam o processo de regeneração tecidual. O objetivo desse trabalho foi avaliar a diferenciação neural das CTMs cultivadas sobre matrizes de nanofibras orientadas com o fator de crescimento epidermal (EGF) incorporado. Os scaffolds com fibras alinhadas foram produzidos por electrospinning de emulsão e avaliados conforme a sua morfologia, o diâmetro das nanofibras, a degradabilidade e a liberação do EGF. As CTMs utilizadas foram provenientes da polpa de dentes decíduos esfoliados humanos. Essas células foram cultivadas nos scaffolds e avaliadas conforme os testes biológicos: adesão, viabilidade, proliferação, citotoxicidade e diferenciação neural. Os scaffolds com fibras alinhadas controle (AC) e contendo o EGF (AE) apresentaram morfologia, diâmetro das nanofibras e tempo de degradação semelhantes. Com base no total de EGF presente na matriz AE, 90,14% foi liberado após 28 dias. O citoesqueleto e o núcleo das CTMs cultivadas nos scaffolds AC e AE estavam mais alongados e alinhados quando comparado com as CTMs cultivadas no poço de cultura (controle). As CTMs aderiram mais nas matrizes AE em relação às matrizes AC, porém a proliferação e viabilidade celular foram similares, exceto no tempo de 72 horas, o qual a viabilidade no grupo controle foi maior, em comparação aos demais grupos. Os scaffolds AC e AE não foram tóxicos para as CTMs. Em relação aos resultados da neuro-diferenciação, a expressão de nestina e neurofilamentos consideravelmente maior em todos os grupos analisados quando comparado ao grupo controle. A expressão de βIII-tubulina e GFAP foi maior em todos os grupos diferenciados quando comparada ao grupo controle. A maioria das CTMs cultivadas nas matrizes AC e AE, induzidas ou não à diferenciação neural, apresentaram correntes dependente de voltagem para sódio. O valor de condutância máxima foi maior para todos os grupos analisados quando comparado ao grupo controle onde as células não foram diferenciadas. Portanto, as matrizes com nanofibras orientadas induzem à diferenciação neural das CTMs em neurônios funcionais tanto na ausência como na presença de EGF incorporado. As matrizes AE ainda mostraram ser capazes de melhorar a adesão celular. Dessa forma, conclui-se que as matrizes de nanofibras estudadas são uma possível estratégia para otimização da regeneração de lesões neurológicas. / Damage to the central nervous system (CNS) results in loss of axonal connections and motor and sensory functions. One of the strategies for its repair is the transplantation of mesenchymal stem cells (MSCs). However, this requires a suitable application route. Accordingly, the use of scaffolds support the growth of MSCs and, when incorporated with growth factors, optimize the regeneration process. The purpose of this study was to evaluate the neural differentiation of MSCs cultured on nanofiber matrices oriented with epidermal growth factor (EGF) incorporated. Aligned scaffolds were produced by electrospinning emulsion and evaluated according to their degradation, the morphology and diameter of the nanofibers, and release of EGF from the nanofibers. MSCs used were from human exfoliated deciduous teeth (SHED). These cells were cultured on the scaffolds and evaluated according to biological tests: adhesion, viability, proliferation, cytotoxicity and neural differentiation. The aligned control scaffolds (AC) containing EGF (AE) presented similar morphology, diameter of nanofibers and degradation time. Based on the total EGF present in the scaffold AE, 90.14% was released after 28 days. The cytoskeleton and the core of the MSCs cultured on scaffolds AC and AE were more aligned and elongated when compared to the MSCs grown on plate wells (control). MSCs adhered more to matrices AE when compared to matrices AC, although proliferation and cell viability were similar, except after 72 hours. In this period, the viability of the control group was higher when compared to the rest of the groups. Scaffolds AC and AE were not toxic to MSCs. In regard to the results of neuro-differentiation, the expression of nestin and neurofilament was much higher in all groups than the control group. The expression of βIII tublin and GFAP was higher in all differentiated groups than the control group. Most of the MSCs grown in matrices AC and AE, induced or not to neural differentiation, showed voltage-dependent sodium currents. The maximum value of conductance of these groups was higher for the cells in all groupscompared to the control group, where the cells were not differentiated. Therefore, oriented nanofiber matrices induce neural differentiation of MSCs into functional neurons both in the absence and in the presence of incorporated EGF. The matrices AE also showed improved cell adhesion. Thus, these matrices are a possible strategy for optimizing the regeneration of neurologic lesions.

O presente estudo utilizou o modelo fatia-dental/matriz-polimérica para avaliar a influência do tratamento dentinário e das BMPs dentinárias na diferenciação das células-tronco da polpa de dentes decíduos (SHED). Secções transversais (1mm) foram preparadas a partir de terceiros molares humanos extraídos. Matrizes poliméricas a base de ácido poli-L-lático (PLLA) foram criadas no interior da cavidade pulpar das secções dentinárias, tratadas com solução de EDTA a 10%; NaOCl a 5.25%; ou permanecendo sem tratamento. Matrizes poliméricas confeccionadas sem as fatias dentais foram utilizadas como controle. As células (5x104) foram semeadas nas matrizes e, após 7, 14, 21 e 28 dias de cultura in vitro, a expressão de marcadores de diferenciação odontoblástica (DSPP, DMP1 e MEPE) e a proliferação celular (WST-1) foram avaliadas. Células (5x105) semeadas nas matrizes foram transplantadas em camundongos imunodeficientes e cultivadas in vivo por um período de 14 e 28 dias. Para avaliar a atividade das BMPs dentinárias, 5x104 células foram semeadas em matrizes poliméricas com fatia dental e cultivadas na presença de anticorpos anti-BMP- 2, -4, ou -7 (2 μg/ml) durante 14 dias. Adicionalmente, 5x105 células foram tratadas com rhBMP-2, -4, ou -7 (100ng/mL) por 24hs. As células cultivadas in vitro e in vivo alteraram sua expressão genética durante o curso do tempo. DSPP, DMP-1 e MEPE foram expressos por células cultivadas in vitro após 14 dias (tratamento com EDTA e dentina sem tratamento) e in vivo após 28 dias (EDTA), não sendo detectados nos grupos NaOCl e nas células cultivadas nas matrizes sem fatia dental. A proliferação foi reduzida com a diferenciação celular (p<0.05). A utilização de BMP-2/4Ab no meio de cultura exerceu um efeito inibitório na expressão dos marcadores de diferenciação celular, não ocorrendo quando do cultivo das SHED na presença de BMP-7Ab. DSPP, DMP-1 e MEPE foram expressos por células tratadas com rhBMP-2, e DSPP e DMP-1 por células tratadas com rhBMP-4 e -7. Células sem tratamento não expressaram os marcadores. O modelo fatia-dental/matriz-polimérica demonstrou ser adequado para o estudo da diferenciação de células-tronco pulpares, sugerindo que a dentina possa fornecer um microambiente favorável para a diferenciação de celular. As proteínas ósseas morfogenéticas dentinárias BMP-2 e BMP-4 parecem exercer um papel relevante nesse processo. / The effect of dentin pre-treatments and dentin-derived BMPs on SHED differentiation was tested using the Tooth-Slice Scaffold model (TSS). Dentin slices (1mm thickness) were prepared from extracted human third molars. Biodegradable PLLA scaffolds were prepared inside the pulp chamber of the tooth-slices, treated alternatively with a 5.25% NaOCl or 10% EDTA solution, or remaining untreated (WO-T). PLLA sponge scaffolds with no tooth-slice (PSS) were used as control. SHED (5x104) were seeded in TSS and PSS and after 7, 14, 21 and 28 days in culture, RT-PCR (DSPP, DMP1 and MEPE) and WST-1 proliferation assay were performed. Additionally, cells (5x105) were seeded in TSS and PSS and transplanted into SCID mice (14 and 28 days). To verify the dentinderived BMPs bioactivity, SHED (5x104) were cultured in TSS in the presence of antihuman BMP-2, -4, and -7 antibodies for 14 days. Besides, cells in culture were treated with rhBMP-2; -4; or -7 for 24 hours. After in vitro and in vivo time course, SHED altered their genetic expression. The cells cultured in vitro in the TSS (EDTA or WO-T) expressed the differentiation markers after 14 days and maintained expression thereafter. Cell proliferation rate was reduced following the differentiation (p<0.05). Cells transplanted in vivo expressed DSPP, DMP-1 and MEPE after 28 days (EDTA). No transcripts were found in tooth-slices treated with NaOCl or in PSS groups. BMP-2/4Ab prevented the differentiation process and no inhibitory effect was detected for BMP-7Ab. After 24 hours, expression of DSPP, DMP-1 and MEPE was found for rhBMP-2, and DSPP and DMP-1 for rhBMP-4 and rhBMP-7 treated SHED, but not for untreated cells. The tooth slice scaffold model suggests that dentin can provide the environment for SHED differentiation and dentin-derived morphogenic signals BMP-2 and BMP-4 play an important role in this process.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Treating lesions in living tissues has become a health issue due to the late natural healing process, limited acceptance and compatibility with grafts. The development of biomaterials emerges as a new strategy. Thus, this study aimed to obtain bioactive porous composite, of different compositions and the evaluation of the influence of the composition on the physical, chemical, structural, morphological and biological properties for possible application in tissue engineering. The project started with the preparation and characterization of hydroxyapatite nanoparticles (HAP), via a wet precipitation route, and the modification of their surface by stearic acid (SA) (reflux). The presence of SA in the surface of HAP promoted significant changes in their general characteristics, especially in their morphology, size and thermal stability (TG/DTG). For the formation of composites, chitosan with different degrees of purity were chosen. To the homogeneous dispersion of ceramic in the polymer dispersion has required the incorporation of a viscosity agent, hydroxyethyl cellulose (HEC), which prevented the settling of HAP particles. Thus, six groups were evaluated and characterized by techniques that allowed to infer basic information about the degree of swelling, porosity, surface morphology, thermal stability, crystallinity and chemical environment (chemical groups). The cellular viability evaluated under the composites allows suggesting the biocompability of these. The study of the structural organization during lyophilization, using the small angle X-ray scattering, and evaluation of internal morphological structure of the composites under the influence of the composition was performed using X-ray Tomography. These analyzes showed that the presence of SA in the surface composition of nanoparticles inserted in composites influenced the agglomeration and deposition of HAP nanometric in the polymer structure, determining the structural characteristics of these composites. / Tratar lesões em tecidos vivos tornou-se uma problemática em saúde devido ao tardio processo de cicatrização natural, a uma limitada aceitação e compatibilidade com enxertos. O desenvolvimento de biomateriais surge como nova estratégia. Deste modo, este trabalho teve como objetivo principal a obtenção de compósitos bioativos porosos e avaliar a influência da composição nas características físicas, químicas, estruturais, morfológicas e biológicas para uma possível aplicação em tecido ósseo. A proposta iniciou-se com a obtenção e caracterização de nanopartículas de hidroxiapatita (HAP) (precipitação por via úmida) e com a modificação de sua superfície pelo ácido esteárico (AE) (refluxo). A presença do AE sob a superfície da HAP promoveu alterações significativas em suas características gerais, principalmente em suas morfologias, tamanhos e estabilidade térmica (TG/DTG). Para os compósitos, quitosanas com diferentes graus de pureza foram escolhidas. Com o intuito de homogeneizar a cerâmica sob a dispersão polimérica, fez necessária a incorporação de um agente de viscosidade, o hidroxietilcelulose (HEC). Sendo assim, seis grupos de compósitos foram avaliados e caracterizados por técnicas que permitiram inferir informações básicas a respeito do grau de intumescimento, da porosidade, da morfologia de superfície, da estabilidade térmica, da cristalinidade e do ambiente químico. A viabilidade celular avaliada sob os compósitos permitiu sugerir a biocompatibilidade destes. O estudo da organização estrutural durante a liofilização, utilizando-se o espalhamento de raios X a baixos ângulos, e a avaliação da estrutura morfológica interna dos compósitos sob influência da composição foi realizada através da tomografia de raios X. Tais análises permitiram inferir que a presença do AE na composição de superfície das nanopartículas inseridas nos compósitos influenciou na aglomeração e deposição das nanocargas de HAP sob a estrutura polimérica, determinando as características estruturais destes compósitos.

Des résultats récents ont montré les premières preuves d’un effet multivalent puissant des iminosucres sur l’inhibition des glycosidases, avec des gains d’affinité allant jusqu’à 10000. Afin de comprendre les différentes caractéristiques de cet « effet de cluster » et d’en poursuivre l’optimisation, de nouvelles charpentes doivent être conçues. Le premier axe de recherche a donc consisté en la mise au point d’un ensemble de techniques d’ingénierie « click » pour la synthèse de systèmes multivalents, avec le développement d’une stratégie Sweet LEGO®. Cette approche permettrait un accès simple à une grande variété de néocyclodextrines préfonctionnalisées. Le second axe a consisté en une étude de relation structure-activité autour de charpentes moléculaires polyalcynes utilisées pour préparer de nouveaux clusters par chimie « click ». Une partie des clusters a été préparée en utilisant de nouvelles charpentes cyclopeptoïdes. Ils ont permis de poursuivre l’optimisation de l’effet multivalent des iminosucres sur l’inhibition de glycosidases. En particulier, un composé portant 30 ligands a montré le meilleur effet multivalent connu sur une enzyme modèle. / Recent reports have demonstrated the first pieces of evidence of a strong multivalent effect in glycosidase inhibition by iminosugars, with affinity enhancements close to 10000. In order to understand the different parameters of this “cluster effect” and to continue its optimization, new scaffolds must be designed. The first research topic was thus to develop a set of « Click » Chemistry engineering techniques for the synthesis of multivalent systems, with the development of a Sweet LEGO® strategy. In the end, it would allow an easy access to a broad range of prefunctionalized neocyclodextrins. The second research topic consisted in a structure-activity relationship study by varying the molecular polyalkyne scaffold used for the preparation of new clusters by way of « Click » Chemistry. They allowed to investigate the specific features of the iminosugar cluster effect in the inhibition of glycosidases. In particular, a compound of unprecedented valency bearing 30 iminosugar units demonstrated an unprecedented dramatic affinity enhancement for the inhibition of a model enzyme (Jack bean alpha-mannosidase).