Orientador: Flavio Antonio Maes dos Santos / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-08T20:13:46Z (GMT). No. of bitstreams: 1 Fonseca_MarisaGesteira_D.pdf: 4573597 bytes, checksum: e5724ae76f277692db551ed06e0f66d4 (MD5) Previous issue date: 2007 / Resumo: Apesar da importância atual e crescente da atividade madeireira em florestas naturais na Amazônia, a ecologia populacional das espécies exploradas é praticamente desconhecida. O presente trabalho tem como objetivo geral contribuir para o entendimento da ecologia populacional e dos efeitos da exploração madeireira convencional (EC) e de impacto reduzido (EIR) sobre a demografia de três espécies arbóreas, Lecythis lurida (Lecythidaceae), Pseudopiptadenia psilostachya (Leguminosae) e Chrysophyllum lucentifolium subsp. pachycarpum (Sapotaceae), em Paragominas, na Amazônia oriental. As espécies foram estudadas em três áreas de 24,5 ha, sendo duas áreas submetidas à exploração madeireira em 1993 (uma EIR e outra EC) e uma área não explorada (NE). Os indivíduos com diâmetro à altura do peito (DAP) 10 cm foram acompanhados nas três áreas de 1993 a 2003 e os jovens (comprimento 30 cm e DAP < 10 cm) foram amostrados em 5,25 ha de 2003 a 2005. Em 2004 foram tiradas fotografias hemisféricas para avaliação da cobertura do dossel nas três áreas. A frutificação foi avaliada durante um período reprodutivo de L. lurida e P. psilostachya e durante três períodos reprodutivos no caso de C. lucentifolium. Foram realizados experimentos de germinação de sementes e crescimento inicial de plântulas de P. psilostachya em laboratório e em viveiro, de forma a subsidiar iniciativas de produção de mudas da mesma. Os resultados evidenciaram a ampla variação das características ecológicas das três espécies e de suas respostas à exploração madeireira. Estimou-se que, adotando-se um único diâmetro mínimo de corte (DAP 50 cm) e retendo-se a mesma proporção de árvores sementeiras (10% daquelas com DAP 50 cm), o efeito em termos de porcentagem de indivíduos reprodutivos retirados da população pode variar entre espécies na ordem de aproximadamente cinco vezes. P. psilostachya apresentou características associadas com baixa tolerância à sombra e bom potencial para manejo sob EIR em ciclos de corte de 30 anos. A facilidade de produção de mudas dessa espécie sugere que ela pode ser usada com sucesso no enriquecimento de capoeiras e na recomposição de áreas degradadas. C. lucentifolium, por outro lado, apresentou maior tolerância à sombra e menor potencial para manejo sustentável, necessitando de ciclos de corte aproximadamente três vezes mais longos. Apesar de jovens de L. lurida apresentarem sobrevivência e potencial de crescimento intermediários aos das outras espécies, a alta freqüência de quebra dos mesmos contribui de forma significativa para o baixo potencial da espécie para o manejo. Tanto a análise do padrão de distribuição espacial da abertura do dossel quanto da dinâmica populacional das espécies indicam a importância da aplicação das técnicas de EIR para a manutenção da composição e estrutura florestal. No entanto, os resultados sugerem que baixas intensidades de exploração e longos ciclos de corte são necessários para compatibilizar a preservação e utilização da floresta / Abstract: Despite the current and increasing importance of logging in natural forests of the Brazilian Amazon, the population ecology of commercial species is largely unknown. The objective of this study was to contribute to the understanding of the population ecology and the effects of conventional (CL) and reduced impact logging (RIL) on the population dynamics of Lecythis lurida (Lecythidaceae), Pseudopiptadenia psilostachya (Leguminosae) and Chrysophyllum lucentifolium subsp. pachycarpum (Sapotaceae), three commercial tree species, in eastern Amazonia. These species were studied in three plots (24.5 ha each), two of them subjected to logging in 1993 (CL and RIL) and a control plot (CP). All trees 10 cm in diameter at breast height (dbh) were studied between 1993 and 2003, and juveniles (height 30 cm and dbh < 10 cm) were sampled in 5.25 ha between 2003 and 2005. Canopy openness was estimated in 2004 using hemispherical photographs. Fruiting was assessed over one reproductive season of L. lurida and P. psilostachya, and three reproductive seasons of C. lucentifolium. Seed germination and the initial growth of P. psilostachya seedlings were assessed in laboratory and shade-house experiments in order to provide information about seedling production. The results highlight the variation in the species ecology and their response to logging. The effects of adopting a single minimum cutting diameter (dbh 50 cm) and of setting aside the same proportion of seed trees (10% of the trees 50 cm dbh) on the proportion of remaining reproductive individuals after harvesting may vary by five-fold among species. P. psilostachya showed characteristics associated with low shade-tolerance and promising potential for management under RIL in 30-year cutting cycles. Given that it is relatively easy to produce seedlings of this species, these seedlings may probably be successfully used in secondary forest enrichment and in the recovery of degraded areas. C. lucentifolium, however, presented a higher shade tolerance and lower potential for management, requiring three-fold longer cutting cycles. Although L. lurida juveniles have an intermediate survival and potential growth rate when compared to the other species, the high frequency of damage to individuals significantly decreases its management potential. The spatial distribution of canopy openness and the population dynamics of the study species indicate the importance of RIL techniques to maintain forest composition and structure. However, results suggest that long cutting cycles and low harvest intensities are necessary in order to achieve both utilization and conservation of forests / Doutorado / Doutor em Biologia Vegetal

Orientador: Mary Anne Heidi Dolder, Clovis Andrade Neves / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T19:24:09Z (GMT). No. of bitstreams: 1 Cruz_LilianCota_M.pdf: 6744301 bytes, checksum: 728821086751ef70a46cd6df9152a7a4 (MD5) Previous issue date: 2008 / Resumo: As abelhas são insetos holometábolos, pois apresentam metamorfose completa. Na metamorfose destes insetos, o trato gastrointestinal é completamente remodelado. Poucos estudos enfatizam os aspectos que envolvem a reconstituição do epitélio do intestino médio de abelhas durante a metamorfose. Os trabalhos que destacam a proliferação das células digestivas durante a metamorfose carecem de documentação das figuras de mitose. O presente trabalho contribui para o conhecimento da biologia dos Hymenoptera, gerando dados que possam esclarecer aspectos da fisiologia digestiva durante a metamorfose dos insetos sociais. Comprovamos a necessidade de aumento do número de células digestivas do intestino médio de Melipona quadrifasciata anthidioides ao final da metamorfose sem, contudo, observar figuras de mitose que comprovassem a proliferação destas células, como notado em Nasutitermes rotundatus (Holmgren) (Isoptera). É possível que as células regenerativas tenham origem externa ao intestino médio e migrem através da membrana basal para se estabelecerem no epitélio. Além disso, nesse trabalho descrevemos a morfologia do epitélio do intestino médio com auxílio da microscopia eletrônica de varredura, demonstrando que somente a membrana peritrófica diferiu entre as castas / Abstract: Bees are holometabolus insects, since they present complete metamorphosis. During the metamorphosis of the insects the gut is completely remodeled. Few studies emphasize aspects of the renewal of the epithelium of bee¿s midgut during metamorphosis. Studies that describe digestive cell proliferation are lacking in images of mitosis. This research is a contribution to the understanding of Hymenoptera biology in relation to some aspects of digestive physiology during metamorphosis of social insects. We have established the necessity of increasing the number of cells in the median intestine of Melipona quadrifasciata anthidioides at the end of metamorfosis without having, however, observed mitosis, as was found for Nasutitermes rotundatus (Holmgren) (Isoptera). Possibly the regenerative cells arisen outside the midgut migrated through the basal membrane, to establish themselves in the epithelium. Also, in this study we describe the morphology of the median intestine epithelium, using scanning electron microscopy, showing that only the peritrophic membrane presented differences in the casts studied. / Mestrado / Biologia Celular / Mestre em Biologia Celular e Estrutural

Orientador: Ivanira Jose Bechara / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-11T09:34:23Z (GMT). No. of bitstreams: 1 Bockelmann_PetraKarla_D.pdf: 29179947 bytes, checksum: 8c02cdd38976a29e54d61610a6bfd9fe (MD5) Previous issue date: 2008 / Resumo: As nadadeiras caudais de teleósteos, quando parcialmente amputadas, passam por um rápido processo de regeneração chamado de regeneração epimórfica, caracterizado pela formação de uma massa de células indiferenciadas, diferenciação dessas células, síntese e deposição de matriz extracelular e restauração morfológica. A regeneração da nadadeira é extremamente sensível a fatores físicos e químicos externos, tais como variações na temperatura, intensidade da luz, ação de alguns agentes contaminantes ambientais e ação de algumas drogas que podem interferir na capacidade regenerativa das nadadeiras dos peixes teleósteos. Existem relatos na literatura, que drogas anti-inflamatórias não esteróides, podem interferir de alguma forma na restauração tecidual de diversos organismos, uma vez que inibem a ação da enzima ciclooxigenase e, conseqüentemente, a conversão do ácido araquidônico em prostaglandina, elementos que desempenham funções importantes na proteção celular, crescimento, angiogênese e produção de matriz extracelular. Em vista disso, este trabalho teve como objetivo avaliar os efeitos de drogas antiinflamatórias não esteróides, naproxeno e indometacina, durante o processo regenerativo da nadadeira caudal de peixe teleósteo Cyprinus carpio (carpa). Para isso, foram montados experimentos com cinco grupos: grupo formado com peixes que serviram como controle, grupo formado com peixes que entraram em contato com o naproxeno, na dose de 15,6 mg/L, e três grupos formados por peixes que tiveram contato com a indometacina nas doses 10, 20 e 30 mg/L cada. Os peixes foram anestesiados e suas nadadeiras caudais foram amputadas transversalmente e, após a amputação os peixes foram divididos entre os cinco grupos e permaneceram nos aquários até que a regeneração ocorresse. Os animais foram anestesiados, sacrificados e as nadadeiras em regeneração foram excisadas e fixadas em intervalos de 1, 2, 4, 5, 6, 8, 10 e 12 dias após a amputação. As amostras foram processadas para permitir uma análise histoquímica, ultra-estrutural e morfométrica das possíveis alterações no processo regenerativo da nadadeira caudal de teleósteo em contato com as drogas em questão. Os grupos tratados com o naproxeno e a indometacina utilizada na dose de 10 mg/L apresentaram o processo de regeneração de forma semelhante ao grupo controle, ou seja, não afetaram a formação da capa epidermal, a formação do blastema, a diferenciação das células blastemais, bem como a síntese, deposição, organização e mineralização dos componentes da matriz lepidotriquial e a síntese das actinotriquias durante o processo regenerativo da nadadeira caudal. No entanto, os peixes tratados com a indometacina nas doses de 20 e 30 mg/L apresentaram um atraso no processo de regeneração da lepidotriquia e da actinotriquia quando comparados com os peixes do grupo controle. Estudos mais detalhados sobre os mecanismos de ação das drogas anti-inflamatórias não esteróides e a ação dessas drogas sob a expressão ou a inibição da expressão de alguns genes envolvidos no processo de regeneração da nadadeira caudal de teleósteo talvez possam responder a razão das diferenças de efeitos entre essas duas drogas / Abstract: The fins of teleosts, when partially amputated, they pass for a quick regenerative process called epimorphic regeneration, characterized by the formation of a mass of undifferentiated cells, by the differentiation of these cells, by the synthesis and the deposition of the extracellular matrix and morphological restoration. The regeneration of the fin is extremely sensitive to external physical and chemical factors such as temperature variations, light intensity, the action of some environmental contaminants and the action of some drugs that can interfere in the regenerative capacity of teleost fins. There are some studies that show that nonsteroids anti-inflammatory drugs can interfere somehow in the tissue restoration of many organisms, as they inhibit the action of ciclooxygenase enzyme and, consequently, the conversion of arachidonic acid in prostaglandins, elements that execute important roles in cell protection, growth, angiogenesis and in the production of extracellular matrix. In this sense, this study aimed to evaluate the effects of nonsteroid anti-inflammatory drugs, naproxen and indomethacin, during the regenerative process of the teleost tail fin Cyprinus carpio (carp). Therefore, experiments were undertaken in five groups: the control group fish, group of fish in touch with naproxen in doses of 15.6 mg/L, and three groups of fish in contact with indomethacin in doses of 10, 20 and 30 mg/L each. The fish were anesthetized and their fins transversally amputated and, after amputations the fish were divided among the five groups described above and were left in the aquaria until the occurrence of regeneration. The animals were anesthetized, sacrificed and the regenerating fins excised and fixed in intervals of 1, 2, 4, 5, 6, 8, 10 and 12 days after amputation. The samples were processed in order to permit a histochemical, ultra-structural and morphometric analysis of the possible alterations in the regenerative process of the tail fin of the teleosts in contact with the drugs mentioned above. The group treated with naproxen and indomethacin in a 10 mg/L dose showed a regenerative process similar to the control group, thus, it did not affect the formation of the epidermal layer, the formation of blastema, and the differentiation of blastemal cells, as well as its synthesis, deposition, organization and mineralization of the lepidotrichial matrix and the synthesis of actinotrichia during the process of regeneration of the tail fin. However, the fish treated with indomethacin in doses of 20 and 30 mg/L presented a delay in the regenerative process of the lepidotrichia and actinotrichia when compared to the control group fish. Detailed studies about the mechanisms of nonsteroids anti-inflammatory drugs action and the action of these drugs under the expression or inhibition of expression of some genes involved in the teleost tail fin regenerative process could explain more precisely the reason of the differences of effect between these two drugs / Doutorado / Histologia / Doutor em Biologia Celular e Estrutural

Orientador: Gisela de Aragão Umbuzeiro / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Tecnologia / Made available in DSpace on 2018-08-21T02:00:16Z (GMT). No. of bitstreams: 1 Ribeiro_AlysonRogerio_M.pdf: 3701819 bytes, checksum: b2f0a69f435398e5de38f031d75649e3 (MD5) Previous issue date: 2012 / Resumo: As planarias de água doce possuem vasta distribuição geográfica, plasticidade biologica, facilidade de cultivo em laboratório, sensibilidade a contaminantes diversos e capacidade regenerativa, por isso vem sendo utilizadas como organismos-teste em ensaios ecotoxicologicos. Porem, ainda não ha normatização internacional ou nacional que padronize esses ensaios. O objetivo deste trabalho foi verificar o potencial do uso de planarias de agua doce (Girardia tigrina) na avaliação de alguns contaminantes e amostras ambientais utilizando protocolos padronizados avaliando-se mortalidade e atraso na regeneração encefálica. Foram empregados 15 organismos por concentração-teste nos ensaios de mortalidade de filhotes (96h) e 20 planarias adultas regenerantes por concentração-teste nos ensaios de regeneração encefálica (120h). Avaliou-se cromo e zinco como substancias de referencia e empregou-se cobre, azo corante têxtil Disperse Red 1, herbicida atrazina, nanotubos de carbono de parede múltipla, alem de amostras da ETE Carioba e do Ribeirão dos Cristais. Carta controle com substancia de referencia foi realizada, sendo estabelecido um protocolo para o monitoramento da sensibilidade das culturas de G. trigrina. O zinco foi a substancia mais apropriada que o cromo, com a vantagem do ensaio de sensibilidade poder ser finalizado em 24h. Os métodos utilizados para realização do ensaio de regeneração encefálica e de mortalidade de filhotes foram eficazes para a observação de efeitos adversos das substancias analisadas. Todas as amostras testadas apresentaram toxicidade as planarias, exceto os nanotubos de carbono. Atraso na regeneração encefálica foi o endpoint mais sensível para os contaminantes avaliados. Os protocolos utilizados neste estudo podem ser utilizados como subsídios na elaboração futura de normas técnicas de testes ecotoxicologicos com planarias de agua doce / Abstract: Freshwater planarians are worldwide distributed, have biological plasticity and a unique body regenerative capacity. They are easily maintained under laboratorial conditions and are sensitive for several compounds. For these reasons they have been used in ecotoxicological testing although the lack of national or international guidelines. This research aimed to verify the potential use of freshwater planarians (Girardia tigrina) in mortality and regeneration delay endpoints to assess the toxicity of environmental contaminants using standardized protocols. The culture sensitivity of G. tigrina was monitored with zinc, which was the most suitable substance for this purpose, providing responses in only 24h. The adopted protocols for regeneration and mortality tests were efficient to assess the adverse effects of the chemical agents (copper, an azo textile dye, atrazine), carbon nanotubes and wastewater and water samples from Carioba WWTP and Cristais River. The regeneration test was the most sensitive endpoint and all analyzed substances showed toxicity for planarians, except carbon nanotubes. The methods adopted in this research can be used as basis for the development of a testing guideline using freshwater planarians / Mestrado / Tecnologia e Inovação / Mestre em Tecnologia

Orientadores : Enilson Antonio Sallum, Francisco Humberto Nociti Jr / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-04T03:40:27Z (GMT). No. of bitstreams: 1 Pimentel_SuzanaPeres_D.pdf: 763565 bytes, checksum: c250595a718942b97ba05ce3bd89f569 (MD5) Previous issue date: 2004 / Resumo: Objetivo ¿ O propósito deste trabalho foi avaliar, histometricamente, o processo de cura de defeitos periodontais do tipo deiscência, criados cirurgicamente em cães e tratados pela técnica de regeneração tecidual guiada (RTG) com o uso de membranas reabsorvíveis (copolímero de ácido polilático e poliglicólico) e proteínas derivadas da matriz do esmalte (PME) em animais que receberam nicotina de forma subcutânea. Material e Métodos - Defeitos do tipo deiscência (4 X 6 mm) foram criados sobre as raízes mesiais dos 3os e 4os pré-molares mandibulares, bilateralmente. Durante três meses os defeitos foram cronificados com tiras metálicas permaneceram adaptadas aos defeitos promovendo acúmulo de placa. Após este período, os defeitos foram aleatoriamente designados a receber um dos tratamentos cirúrgicos: 1) raspagem e alisamento radicular (CO); 2) regeneração tecidual guiada com membranas reabsorvíveis (RTG) e 3) proteínas derivadas da matriz do esmalte (PME). Durante quatro meses após os tratamentos os animais receberam nicotina subcutânea (2mg/kg duas vezes ao dia com intervalo de 12 horas entre as aplicações). Após este período, os cães foram sacrificados e os espécimes processados histológicamente para a análise histométrica, que incluiu os seguintes parâmetros: extensão do defeito, extensão do epitélio, extensão da adaptação conjuntiva, extensão do novo cemento, extensão de novo osso e recessão gengival. Resultados ¿ Observou-se uma maior extensão linear de novo cemento, estatisticamente significante (p<0,05), nos sítios tratados com PME (5,32 mm) quando comparados ao grupo CO (3,60mm), entre os grupos PME e RTG (4,05mm) não houve diferença estatística. Todos os outros parâmetros não tiveram diferenças estatísticas. A extensão de epitélio foi de 1,71+0,61mm para no grupo CO; 1,23+0,99mm para o grupo RTG e 0,63+ 0,35mm para o grupo PME. Quanto à adaptação conjuntiva o grupo CO apresentou uma extensão de 0,15+0,22mm; o grupo RTG 0,15 +0,21mm e o grupo PME 0,36+ 0,27mm. A formação de novo osso foi de 1,55 +0,93mm no grupo controle; 1,56+ 0,96mm no grupo RTG e de 2,52 +1,34mm no grupo PME. A recessão gengival foi de 0,36+ 0,48mm no grupo CO; 0,25+0,35mm no grupo RTG e de 0,02+ 0,07mm no grupo PME. Conclusão - Dentro dos limites deste estudo, conclui-se que mesmo na presença da nicotina houve reparo em todos os grupos e a utilização da proteína derivada da matriz do esmalte foi o procedimento regenerativo mais efetivo para se obter maior formação cementária quando comparado com o controle / Abstract: Objectives: The goal of this investigation was to evaluate, histologically and histometrically, the healing process of dehiscence-type defects treated by enamel matrix derivative (EMD) or guided tissue regeneration (GTR) in dogs that received nicotine. Methods: Eight mongrel dogs were used. Buccal osseous dehiscences were surgically created on the mesial roots of the mandibular third and fourth premolars. The defects were exposed to plaque accumulation for 3 months. After this period, the defects were randomly assigned to one of the treatments: open flap debridement (OFD), enamel matrix derivative (EMD) and guided tissue regeneration with resorbable membrane (GTR). During 4 months afther the treatments the dogs received subcutaneous administration of nicotine (2mg/kg twice a day with a twelve-hour interval between the applications). After this period, the animals were sacrificed and the blocks were processed. The histometric parameters evaluated included: gingival recession, epithelial length, connective tissue adaptation, new cementum and new bone. Results: A superior length of new cementum statistically significant was observed in the sites treated by EMD (5.32mm) in comparison with OFD (3.60mm) (P<0.05) There was no statistically difference in the EMD group compared with GTR (4.05mm). All others parameters was not statistically significant. The extension of the epithelium was 1.71+0.61mm for the CO group, 1.23+0.99mm for the GTR group and 0.63+0.35mm for the EMD. Regarding the conective tissue adaptation was 0.15+0.22mm in the CO group, 0.15+0.21mm for GTR and 0.36+0.27mm for EMD. The new bone formation was 1.55+0.93mm for CO, 1.56+0.96mm for GTR and 2.52+1.34mm for EMD group. Gingival recession was 0.36+0.48mm for CO group, 0.25+0.35mm in GTR and 0.02+0.07mm in the PME. Conclusions: Within the limits of this study, it can be concluded that healing was uneventful in all the treatments and EMD alone seems to be the most effective way to promote new cementum formation in the presence of nicotine compared to control / Doutorado / Periodontia / Doutor em Clínica Odontológica

Regeneration, the replacement of lost or damaged body parts, and biomineralisation, the biologically controlled formation of minerals, are important and widespread abilities in the animal kingdom. Both phenomena have a complex evolutionary history; thus their study benefits from investigations in diverse animals. Spirobranchus (formerly Pomatoceros) lamarcki is a small tube-dwelling polychaete worm of the serpulid family. Serpulids have evolved a novel head appendage, the operculum, which functions as a defensive tube plug and regenerates readily when lost. In S. lamarcki, the end of the operculum is reinforced by a calcareous plate; thus, the operculum is a good system in which to study both regeneration and biomineralisation. This thesis explores several aspects of these important processes in the adult operculum. First, a time course of normal regeneration is established. Next, cell proliferation patterns are described, suggesting a combination of proliferation-dependent and proliferation-independent elements in opercular regeneration. The formation of the calcareous opercular plate is examined using both microscopic observations of whole opercular plates and X-ray diffraction analysis of isolated plate mineral, revealing a large shift in mineralogy over the course of regeneration. Histochemical study of alkaline phosphatase enzyme activity indicates the importance of these enzymes in the operculum, although their precise functions are as yet unclear. Finally, a preliminary survey of three opercular transcriptomic datasets is presented, with a broad sampling of gene families with regeneration- or biomineralisation-related roles in other animals. The opercular transcriptome constitutes the first biomineralisation transcriptome from any annelid, and one of the first transcriptomic datasets related to annelid regeneration. Many of the candidate genes examined here display interesting behaviour and suggest targets for further investigation. The work presented here establishes the S. lamarcki operculum as a promising model system in the field of evolutionary developmental biology.

HA (Hyaluronan, hyaluronic acid) is a predominant component of the extracellular matrix and plays diverse roles in the process of wound healing. The major significance of the biological functions of HA in wound healing arise from early studies showing that rich and prolonged existence of HA is associated with the fetal scarless wound healing. It has been hypothesized that adult wound healing can be modulated by the application of exogenous HA to make the process of wound repair more like regeneration. / In this thesis, the effects of molecular weight and concentration of HA on the biological behavior of human fibroblasts and keratinocytes have been investigated in both monolayer and 3D culture models. In addition, the mechanism for the HA-induced biological effects has also been investigated. Previous laboratory studies have primarily focused on individual cell type, but in wound healing in vivo there is considerable interaction between the keratinocytes and fibroblasts. The role of HA in keratinocyte-fibroblast interactions is not clear, so an organotypical culture model was used to investigate the keratinocyte differentiation and stratification and basement membrane formation with and without fibroblasts to explore the effect of HA on keratinocyte-fibroblast interactions. / The experimental data suggest that exogenous application of HA modulates the process of wound healing so that it becomes more like regeneration by regulating the biological functions of human keratinocytes and fibroblasts, including their interaction. By further understanding the HA-induced cell biological phenomena and involved mechanisms new approaches for wound care may be revealed. / The skin is the largest organ in the body. It consists of an outer layer, the epidermis supported by a connective tissue matrix, the dermis. The keratinocyte and fibroblast are the major cell types in the epidermis and dermis respectively. When the skin is damaged, the body will active a cascade of mechanisms to restore the tissue integrity. If the damaged tissue is limited to the epidermis, it will be restored by the process of regeneration. Regeneration involves the replacement of injured tissue by new tissue which has both form and function identical to the original tissue. If the defect involves the dermis, healing is achieved by repair which results in permanent scar formation. Wound healing is an extremely complex biological process which involves several overlapping steps; inflammation, granulation tissue formation and remodeling which are coordinated by various cell types, growth factors, cytokines and extracellular matrix components including hyaluronan. / Whilst there have been many studies looking at the effect of HA on different cell types using varying molecular parameters, concentrations and sources of HA, the result are often contradictory. Even the endogenous or exogenous application of HA have been reported to have opposite effects on cell behavior. / Gu, Hua. / Adviser: Andrew Bard. / Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0216. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 116-135). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

El procés de regeneració permet als organismes refer parts o teixits del seu cos que han sofert algun dany. Els discs imaginals de Drosophila melanogaster, primordis larvaris de les estructures adultes, tenen capacitat de promoure processos de cicatrització i proliferació, regenerant el teixit i donant lloc a estructures adultes completament normals. Hem estudiat el perfil d’expressió de discs d’ala a 0, 24 i 72h de regeneració. Els resultats obtinguts mostren un enriquiment significatiu de l’expressió de Factors de Transcripció lligats a importants vies de senyalització com Wingless (Wg), Notch (N) i Jun N-terminal Kinasa (JNK). Mitjançant la cerca computacional de motius d’unió d’AP-1 (factor de transcripció de la via de la JNK) en els promotors dels gens amb canvis d’expressió, hem descrit els gens diana d’aquesta la via procés durant la regeneració. Entre aquests gens es troba cabut (cbt), l’expressió del qual augmenta durant les primeres 24h i disminueix a les 72h, recuperant els seus nivells basals. Hem confirmat aquests resultats per qRT-PCR i amb tècniques d’hibridació in situ, demostrant a més, que l’increment de l’expressió de cbt està associat a cèl•lules del blastema. També hem comprovat que l’expressió de cbt augmenta per sobreactivació de la via de la JNK en el disc d’ala, tal i com s’havia vist prèviament en embrió. Mitjançant la tècnica EMSA hem validat la unió in vitro de les proteïnes Jun i DFos (que formen el dímer AP-1) a la seqüència predita en el promotor del gen cbt confirmant la regulació directe de la via de la JNK. L’anàlisi de discs mutants per cbt indica que l’expressió d’aquest gen és necessària per al tancament de la ferida i proliferació durant la regeneració. Cabut (Cbt) és un factor de transcripció Zn finger de la família dels Krüpple like Factors i els seus ortòleg en vertebrats són els gens TGFβ Inducible Early Genes (TIEG). Aquest gen s’ha descrit com a modulador de les vies Dpp i JAK/STAT en el disc d’ala. Una aproximació per descriure les funcions d’un factor de transcripció com Cbt és determinar els seus gens diana. Amb aquest objectiu hem realitzat un ChIP-Seq amb un anticòs contra la proteïna Cbt. Els resultats obtinguts mostren que Cbt es troba localitzat principalment als promotors dels gens. Aquest estudi ha reportat 2060 gens diana de Cbt en el disc d’ala. Les categories funcionals GO associades a aquest grup de gens són: Regulació de la transcripció, cicle cel•lular, desenvolupament dels discs imaginals i desenvolupament neuronal. També hem trobat un enriquiment en gens lligats a vies de senyalització com la JNK, Wg i N. L’anàlisi de les seqüències de les dianes de Cbt i altres estudis publicats apunten els factors Sin3A i GAF com a possibles cofactors de Cbt. Finalment hem analitzat el paper de Cbt en la regulació de la via de N, els resultats obtinguts mostren que Cbt regula positivament la via de N promovent el creixement i la formació del marge Dorso/Ventral del disc d’ala. Per tal d’establir quins factors de transcripció s’uneixen al promotor de cbt i regulen la seva expressió, hem realitzat un cribratge a gran escala utilitzant la tècnica de Yeast-One-Hybrid, que permet descriure la unió de factors de transcripció a una seqüència concreta. Hem obtingut factors de transcripció lligats al desenvolupament neural, a la segmentació i a la resposta immune amb capacitat d’unir-se al promotor de cbt. Aquestes dades no només ens donen informació sobre la regulació de Cbt sinó que també suggereixen la seva implicació en aquests processos. En conjunt el nostre treball mostra que Cbt regula el creixement i l’homeòstasi del disc d’ala durant el desenvolupament. I que quan es produeix un dany en aquest teixit l’expressió de Cbt incrementa, de manera depenent de la via de la JNK, per garantir la restauració del teixit perdut. / Regeneration is the ability to rebuild a body part that has been damaged or amputated. Drosophila imaginal discs are able to undergo wound healing and regenerative growth after ablation. Genome-wide expression profiling of regenerating wing discs at 0, 24 and 72 hours after fragmentation have revealed a significant enrichment of transcription factors and chromatin regulators. To identify JNK target genes among the differentially expressed genes we performed a computational search of AP-1 binding sites in the promoter regions of these gens. Among them we identified cabut (cbt), the Drosophila ortholog of TGFβ Inducible Early Genes (TIEG). During regeneration cbt expression is induced between 0 and 24 and down-regulated between 24 and 72 hours, suggesting a tight and controlled mechanism of regulation. qRT-PCR and in situ hybridization experiments confirmed cbt expression changes and highlighted that cbt upregulation is associated to cells located in the blastema region. Moreover the analysis of regenerated cbt mutant discs demonstrated that cbt upregulation is required for wound healing and growth during regeneration. Cbt is a Zn finger transcription factor related to JNK and Dpp pathways, but few is known about its target genes. In order to identify Cbt target genes in development, we performed a Cbt ChIP-Seq analysis of wing discs from larvae III. This study reveal that Cbt is located mostly in the promoter regions of its target genes. We identified 2060 Cbt target genes which were related to functional categories such imaginal disc development, transcription regulation, neuron development or cell cycle. Furthermore several Cbt target genes were associated to signaling pathways like JNK, Wg or Notch (N). The analysis of cbt targets and previous reports pointed the factors GAF and Sin3A as a possible cofactors of Cbt. Finally, genetic interactions between cbt and N mutants demonstrated that Cbt regulates positively N signaling contributing to growth and Dorso/Ventral wing margin regulation. Although we have demonstrated that JNK is upstream of cbt, there is no much data available about cbt regulation in normal development. To approach this question we performed a high throughput Yeast-One-Hybrid screening that allowed us to defined factors that bind to cbt proximal promoter. The identified factors were related to immune response, neuron development and segmentation. Our results contributed to decipher cbt regulation network and suggested possible new roles for Cbt. In summary Cbt regulates growth and homeoastasis of wing disc during development. However in front of injury cbt is upregulated in a JNK dependent manner to ensure complete regeneration.

Coronary artery stent placement has been a significant advance in the percutaneous treatment of atherosclerotic disease, and tissue engineered vascular grafts may provide a viable alternative to autologous segments for small diameter vessels. However, in-stent restenosis remains an important limitation, and tissue engineered grafts have poor patency and high risk of thrombus formation due to their inability to maintain a confluent, adherent, and quiescent endothelium. While animal models provide insight into the pathophysiology of these situations, elucidation of the relative importance of stent or graft components, hemodynamic factors, and molecular factors is difficult. Very little research has focused on bridging gaps in knowledge concerning blood/biomaterial interactions, blood/endothelial cell interactions, and endothelial cell/smooth muscle cell cross-talk. The work presented within this thesis will do just that. The objective of this thesis research was to elucidate the influence of biomaterial-induced activation of leukocytes on endothelial cell or smooth muscle cell phenotype, as well as endothelial cell/smooth muscle cell cross-talk in co-culture systems. Towards this goal, two complimentary in vitro endothelial cell/smooth muscle cell co-culture models with divergent smooth muscle cell phenotype were developed and characterized. Using these systems, it was found that the presence of more secretory smooth muscle cells (as would be seen in wound healing or disease) in general enhanced endothelial cell activation in response to biomaterial-pretreated monocytes, while the presence of less secretory smooth muscle cells (to model more quiescent smooth muscle cells found in uninjured healthy vessels) suppressed endothelial cell activation in response to biomaterial-pretreated monocytes (and neutrophils to a small degree). Additionally, biomaterial-pretreated monocytes and neutrophils amplified a smooth muscle cell phenotypic shift away from a more quiescent state. It is likely that the compounding effect of secretory smooth muscle cells and biomaterial-activated leukocytes are responsible for altered vascular wound healing upon implantation of stents or vascular grafts. Understanding the specific signals causing these effects, or signals delivered by contractile smooth muscle cells that limit these effects help to provide design criteria for development of devices or grafts capable of long term patency.

Embryonic stem cells (ESCs) can differentiate into all somatic cells, and as such, are a promising cell source for therapeutic applications. In vitro, ESCs spontaneously differentiate via the aggregation of cells into embryoid bodies (EBs), which recapitulate aspects of early embryogenesis and harbor a unique reservoir of cues critical for tissue formation and morphogenesis. Embryonic healing responses employ similar intrinsic machinery used for tissue development, and these morphogenic cues may be captured within the EB microenvironment. Recent studies have shown that when injected into injury or defect models in vivo, ESCs synthesize and secrete extracellular factors that ultimately contribute to repair, suggesting that these molecules may be as important for regenerative therapies as functional differentiation of the cells. The overall objective of this project was to develop novel acellular matrices derived from differentiating ESCs undergoing morphogenesis. The central hypothesis was that embryonic matrices contain complex mixtures of extracellular factors that, when isolated, retain bioactivity and enhance wound healing in an adult environment. The overall objective was accomplished by: (1) investigating the production of extracellular matrix (ECM) by differentiating ESCs as a function of differentiation time; (2) assessing the ability of solvents to efficiently decellularize EBs; and (3) evaluating the healing response elicited by acellular matrices derived from EBs in a murine dermal wound healing model. Endogenous ECM synthesis by EBs varied with time and was associated with specific differentiation events. Novel techniques were developed to effectively remove cell components from EBs in order to extract complex, bioactive acellular matrices. EB-derived acellular matrices significantly enhanced the healing of excisional dermal wounds in mice, indicating the potency of extracellular factors synthesized by ESCs. All together, these studies demonstrate that acellular matrices derived from ESCs retain morphogenic factors capable of influencing tissue repair. In addition, this work lays the foundation for future studies to further examine the functional role of endogenous matrix molecules on ESC differentiation and to evaluate the utility of a stem cell-derived matrix for a variety of regenerative medicine applications.