MicroRNAs (miRNAs) are short, non-coding RNAs involved in the regulation of several processes associated with inflammatory diseases and infection. Bacterial infection modulates miRNA expression to subvert innate immune response. In this study, we analyzed bacterial modulation of miRNAs in bone-marrow-derived macrophages (BMMs), in which activity was induced by infection with Porphyromonas gingivalis (Pg) through a microarray analysis. Several miRNA expressions levels were modulated 3 hours post infection (at a multiplicity of infection (MOI) of 25). A bioinformatics analysis was performed to further identify pathways related to the innate immune host-response pathways that are under the influence of the selected miRNAs. To assess the effects of the identified miRNAs on cytokines secretion (pro inflammatory TNF-α and anti-inflammatory IL-10), BMMs were transfected with selected miRNAs mimics or inhibitors. Transfection with mmu-miR-155 and mmu-miR- 2137 did not modify TNF-α secretion while their inhibitors increased it. Inhibitors of mmumiR-2137 and mmu-miR-7674 increased the secretion of the anti-inflammatory IL-10. In Pginfected BMMs, mmu-miR-155-5p significantly decreased TNF-α secretion while inhibitor of mmu-miR-2137 increased IL-10 secretion. In vivo, in a Pg-induced calvarial bone resorption mouse model, injection of mmu-miR-155-5p or anti-mmu-miR-2137 reduced the size of the lesion significantly. Furthermore, anti-mmu-miR-2137 significantly reduced inflammatorycell infiltration, osteoclast activity and bone loss. Bioinformatics analysis demonstrated that pathways related to cytokines and chemokines related pathways but also osteoclast differentiation may be involved in the observed effects. The study highlights the potential therapeutic merits of targeting mmu-miR-155-5p and mmu-miR-2137 to control inflammation induced by Pg infection. To assess the regenerative process in the same animal model, we aimed to compare the effect of Bone Morphogenic Protien 2 (BMP2), Platelets Rich Plasma (PRP), Leukocyte-Platelets Rich Fibrin (L-PRF), and Polygucosamine (PGIcNAc) on bone formation in critical size bone defects in mice. One-hundred-thirty-eight mice were divided into 23 groups (n=6), negative control, different combinations of the PGIcNAc with or without of BMP2, Collagen Sponge (SurgiFoam), PRP, and L-PRF. The 5mm defect, then, was allowed to heal. After six weeks, samples were analyzed for bone formation utilizing radiographs, H&E staining, alkaline phosphatase staining. Our results show that BMP2 were able to produce 90-95% healing of critical size defects after six weeks histologically and radiographically. However, SurgiFoam, PRP and L-PRF with or without PGIcNAc were able to close 60% of the original defect. This study supports that BMP2 is more effective for bone regeneration than SurgiFoam, PRP, L-PRF and PGIcNAc.

A análise química do extrato bruto de esponja marinha é um método clássico de descoberta de novas drogas. Metabólitos secundários e peptídeos são geralmente os resultados neste tipo de pesquisa, utilizando diferentes e consecutivos protocolos de purificação. Neste estudo de uma amostra de extrato aquoso esponja separada numa coluna Sephadex®-G25 foi purificado por HPLC, numa tentativa de isolar uma única molécula. Com uma base de dados de RNA e a análise in silico espera-se que o RNAm pode ser relacionado com a molécula precursora do composto bioativo ou mesmo o seu gene. A dissociação de células a partir da matriz da esponja é um método importante para a extração de RNA optimizado em quantidade e qualidade. Frações extrato bruto do organismo e dados de RNAm a partir de células isoladas da esponja podem ser comparados por uma abordagem de comparação dos dados. Ensaios de MTS foram utilizados para analisar a bioatividade e microscopia confocal foi a principal ferramenta para examinar os danos em células tumorais (T47D). / The chemical analysis of sea sponge crude extract is a classic method in drug discovery. Secondary metabolites and peptides are usually the results in this type of research using different and consecutives purification protocols. In this study a sample of sponge aqueous extract separated in a sephadex-G25 column was purified by HPLC in an attempt to isolate a single molecule. With a RNA databank and in silico analysis is expected that the mRNA may be related with the precursor molecule of the bioactive compound or even its gene. The dissociation of cells from the sponge matrix was an important method to the optimized RNA extraction in quantity and quality. Crude extract fractions of the organism and mRNA data from sponge-isolated cells were obtained to a next step cross-data approach. MTS assays were used to analyze the bioactivity and confocal microscopy was the main tool to scan the damage in tumor cells (T47D).

A descoberta de metabólitos secundários de esponjas marinhas decorre da enorme diversidade de entidades químicas e atividades biológicas que estes animais apresentam. O presente projeto origina-se da coleta de dois gêneros de esponja (Dictyonella e Agelas) em expedição a bordo do Navio Cruzeiro do Sul (Marinha do Brasil) ao longo da margem equatorial da Foz Rio do Amazonas (Pará). Esta região é submetida a um forte ciclo sazonal no padrão de dispersão da pluma do Rio Amazonas. Purificação extensiva do extrato aquoso da Dictyonella sp. através de vários passos cromatográficos de separação, levou ao isolamento e identificação de 7 alcalóides bromopirrólicos: himenidina (16), clathrodina (17) e monobromoisofakelina (20) são compostos já reportados na literatura. A 4-desbromooroidina (28), 5-desbromo-seco-isofakelina (30), 4-desbromo-seco-isofakelina (31) e 5-bromopalau\'amina (32) não foram reportados na literatura até então, sendo compostos inéditos. Da esponja Agelas sventres, foi isolado a oroidina (15), o primeiro composto da classe dos alcalóides pirrólicos a ser isolado reportado na literatura. / The discovery of secondary metabolites from marine sponges is consequential from the enormous diversity of chemical entities and biological activities presented by these animals. This project originates from the collection of two genera of sponges (Dictyonella and Agelas), in an expedition aboard the Cruzeiro do Sul ship (Brazilian Navy) along the equatorial margin of Amazon river mouth (Pará). This region is subject to a strong seasonal cycle related to the pattern of dispersion of the plume of the Amazon River. Extensive purification of the MeOH extract of Dictyonella sp. through several chromatographic steps of separation, led to the isolation and identification of 7 bromopyrrole alkaloids: hymenidin (16), clathrodin (17) and monobromoisophakellin (20), compounds already reported in the literature. 4-Desbromooroidin (28), 5-debromo-seco-isophakellin (30), 4-debromo-seco-isophakellin (31) and 5-bromopalau\'amine (32) were not yet reported in the literature. The purification of the sponge Agelas sventres extract led to the isolation of oroidin is the first compound of pyrrole alkaloids ever isolated.

Made available in DSpace on 2015-04-22T22:14:07Z (GMT). No. of bitstreams: 1 DISSERTACAO-ALEXANDRE.PDF: 10763079 bytes, checksum: 1fa828c53b0ab96a7d096e8f63ec9af0 (MD5) Previous issue date: 2008-10-15 / Fundação de Amparo à Pesquisa do Estado do Amazonas / Sponges are filters animals fixed in stones submerged and/or on branches of trees in places of periodical floods. The freshwater poriferans, known in the Amazon region as cauixi, cauxi or cauí, are reported from the beginning of the twentieth century as causing of dermatosis. Differently from marine sponges, which are studied in a such relation to their biological properties, the chemistry of sponges freshwater in the Amazon region, are not known, this way this gap still open to research. With the objective to define which part of the poriferan is causing the dermatological reaction, it was done an experimental study using three forms of tillage freshwater sponges (sponges undamaged, macerated and the isolated spikes). To perform the experiment was used the freshwater Sponge Drulia uruguayensis. The experiment was divided in two stages: The first one, was to determined the kinetics of dermatosis caused by sponge (using a macerating sponge), obtaining as control, a hypoallergenic microporous plaster; as second action, were tested the other two forms of process: full sponge and isolated spikes using the schedules 2, 4 and 12 hours came from kinetics. The cells of common action in an inflammatory reaction (mast cells, eosinophils, neutrophils) more intra-epithelial lymphocytes and degranulated mastocytes, were counted, to serve as a parameter, in order to determine which of the forms of trim induced a higher reaction of sponges. Histopathological studies evaluated the effects of the sponge in the tegument. The results obtained by ANOVA, showed that the full sponge caused more inflammatory reaction (p = 0.000005) stimulating mainly, the mastocytes (p = 0.0018). The Histopathological analysis showed small loss of continuity of the epidermis in applications with isolated spikes and full sponge, and streamlining in the application with macerating sponge besides the mild thickening on the epidermis in the plaster control. These studies allow to deduce that, in a first contact, a full structurally sponge, may cause major inflammatory reaction by its capacity to cause drilling in the skin and allowing the entrance of inflammatory agents. The macerating sponge, does not achieve bore the tegument, caused streamlining of the epidermis with lower inflammatory reaction, possibly by containing rigid fragments and possible by unknow chemical process. The isolated spikes, cleaned and autoclaved, do not cause dermal reaction resulting the activity in a restricted mechanical action. The studies suggests the using of undamaged sponges, cleaned and autoclaved to verify the maintenance of the capacity to induce dermatosis, beyond the use of the other parts of sponges to verify the participation of these components in the inflammatory reaction. / Esponjas são animais filtradores, fixados em pedras submersas e/ou galhos de árvores em locais de enchentes periódicas. As poríferas de água doce, conhecidas na região Amazônica como cauixi, cauxi ou cauí, são relatadas desde o início do século XX como causadoras de dermatose. Diferentemente das esponjas marinhas, que são estudadas de forma corrente em relação às suas propriedades biológicas, a química das esponjas dulciaqüícolas, na Amazônia, não é conhecida, permanecendo aberta esta lacuna. Com o objetivo de determinar a parte da porífera que provoca a reação dermatológica, foi realizado um estudo experimental utilizando três formas de preparo de esponjas de água doce (esponjas íntegras, maceradas e suas espículas isoladas). Para o experimento foi utilizada a esponja dulciaqüícola Drulia uruguayensis. O experimento foi dividido em duas fases: primeiro, foi determinada a cinética da dermatose causada pela esponja (utilizando a esponja macerada), tendo, como controle, esparadrapo microporoso hipoalérgico; e segundo, foram testados as outras duas formas de preparo: esponja íntegra e espículas isoladas, utilizando os horários de 2, 4 e 12 horas, provenientes da cinética. As células de ação comum em uma reação inflamatória (mastócitos, eosinófilos, neutrófilos) mais linfócitos intra-epiteliais e mastócitos degranulados, foram contados, para servir de parâmetro, a fim de determinar qual das formas de preparo das esponjas induziu maior reação. Estudo histopatológico avaliou os efeitos da esponja no tegumento. Os resultados obtidos, utilizando ANOVA fatorial, mostraram que a esponja íntegra provocou maior reação inflamatória (p = 0,000005) ativando, principalmente, mastócitos (p = 0,0018). A análise histopatológica mostrou pequena perda de continuidade da epiderme nas aplicações com espículas isoladas e esponja íntegra, e adelgaçamento na aplicação com esponja macerada, além de leve espessamento da epiderme no controle-esparadrapo. Esses achados permitem deduzir que, em um primeiro contato, a esponja estruturalmente íntegra, pode causar maior reação inflamatória pela sua capacidade de causar perfurações na pele e permitir a entrada de agentes inflamatórios. A esponja macerada, por não conseguir perfurar o tegumento, provocou adelgaçamento da epiderme com menor reação inflamatória, possivelmente por conter fragmentos rígidos e possíveis químicos não conhecidos. As espículas isoladas, limpas e autoclavadas, não causam reação dérmica, ficando seu papel restrito à ação mecânica. O estudo sugere o uso de esponjas íntegras, limpas e autoclavadas, para verificar a manutenção da capacidade de induzir dermatose. Além do uso dos outros componentes da esponja para verificar a participação destes na reação inflamatória.

A descoberta de metabólitos secundários de esponjas marinhas decorre da enorme diversidade de entidades químicas e atividades biológicas que estes animais apresentam. O presente projeto origina-se da coleta de dois gêneros de esponja (Dictyonella e Agelas) em expedição a bordo do Navio Cruzeiro do Sul (Marinha do Brasil) ao longo da margem equatorial da Foz Rio do Amazonas (Pará). Esta região é submetida a um forte ciclo sazonal no padrão de dispersão da pluma do Rio Amazonas. Purificação extensiva do extrato aquoso da Dictyonella sp. através de vários passos cromatográficos de separação, levou ao isolamento e identificação de 7 alcalóides bromopirrólicos: himenidina (16), clathrodina (17) e monobromoisofakelina (20) são compostos já reportados na literatura. A 4-desbromooroidina (28), 5-desbromo-seco-isofakelina (30), 4-desbromo-seco-isofakelina (31) e 5-bromopalau\'amina (32) não foram reportados na literatura até então, sendo compostos inéditos. Da esponja Agelas sventres, foi isolado a oroidina (15), o primeiro composto da classe dos alcalóides pirrólicos a ser isolado reportado na literatura. / The discovery of secondary metabolites from marine sponges is consequential from the enormous diversity of chemical entities and biological activities presented by these animals. This project originates from the collection of two genera of sponges (Dictyonella and Agelas), in an expedition aboard the Cruzeiro do Sul ship (Brazilian Navy) along the equatorial margin of Amazon river mouth (Pará). This region is subject to a strong seasonal cycle related to the pattern of dispersion of the plume of the Amazon River. Extensive purification of the MeOH extract of Dictyonella sp. through several chromatographic steps of separation, led to the isolation and identification of 7 bromopyrrole alkaloids: hymenidin (16), clathrodin (17) and monobromoisophakellin (20), compounds already reported in the literature. 4-Desbromooroidin (28), 5-debromo-seco-isophakellin (30), 4-debromo-seco-isophakellin (31) and 5-bromopalau\'amine (32) were not yet reported in the literature. The purification of the sponge Agelas sventres extract led to the isolation of oroidin is the first compound of pyrrole alkaloids ever isolated.

<p>Marine invertebrates, in particular sponges, represent a source of a wide range of secondary metabolites, many of which have been attributed various defensive capabilities against environmental stress factors. In this thesis sponge-derived low-molecular peptide-like compounds and associated analogs are investigated for bioactivity and pharmacological targets. </p><p>The compound bromobenzisoxazolone barettin (cyclo[(6-bromo-8-(6-bromo-benzioxazol -3(1H)-one)-8-hydroxy)tryptophan)]arginine) was isolated from the sponge <i>Geodia barretti</i> and its ability to inhibit larval settlement of the barnacle <i>Balanus improvisus</i> was determined. With an EC₅₀ value of 15 nM, this compound’s antifouling effect was higher than those of the previously reported brominated dipeptides from <i>Geodia barretti</i>, i.e., barettin and 8,9-dihydrobarettin; moreover, this antifouling effect was demonstrated to be reversible. However, the compound lacked affinity for 5-HT_1-7 receptors, whereas barettin possessed specific affinity to 5-HT_2A, 5-HT_2C and 5-HT₄, while 8,9-dihydrobarettin interacted with 5-HT₄. In an attempt to evaluate structure-activity relationships synthesized analogs with barettin and dipodazine scaffolds were investigated for antifouling activity. The analog benso[g]dipodazine, with an EC₅₀ value of 34 nM, displayed the highest settlement inhibition.</p><p>The studies of the structure-activity relationships of sponge-derived compounds were extended to cover analogs of agelasines and agelasimines originally isolated from sponges of the genus <i>Agelas</i>. Synthesized (+)-agelasine D and two structurally close analogs were investigated for cytotoxic and antibacterial activity. The profound cytotoxicity and broad spectrum antibacterial activity found prompted a further investigation of structure-activity relationships in 42 agelasine and agelasimine analogs and several characteristics that increased bioactivity were identified.</p><p>In conclusion this work has produced new results regarding the potent bioactivity of compounds derived from the sponges <i>Geodia barretti</i> and <i>Agelas</i> spp. and increased SAR knowledge of the fouling inhibition, cytotoxicity and antimicrobial activity of these compounds.</p>

Lymph node metastasis is a critical prognostic factor for lung cancer. Effective therapy to control lymphatic metastasis may improve survival. The work described in this thesis focuses on the development of a microparticulate lymphatic targeting system, which can be applied as an adjuvant therapy in the control of lymphatic metastasis in lung cancer. The study shows that intrapleural administered colloidal particulates are predominantly taken up by regional lymphatic tissue in rat models including healthy rats, rats bearing orthotopic lung tumours and rats following pneumonectomy. The effect of particle size on lymphatic particle distribution was examined by intrapleural administration of 111In-aminopolystyrene beads. Approximately 2 µm is a suitable size for intrapleural lymphatic targeting. Biodegradable polylactide-co-glycolide (PLGA) microparticles containing the anticancer agent paclitaxel (PTX) were subsequently formulated in the desired size by spray drying. PLGA-PTX microspheres were incorporated into a biodegradable and biocompatible gelatin sponge matrix to form an implantable lymphatic targeted drug delivery system. The system was characterized in vitro and its lymphatic targeting ability was examined in vivo. Fluorescence labeled microspheres embedded within the sponge were selectively taken up by regional lymphatics as the sponge matrix disintegrated following intrapleural implantation. A pharmacokinetic study showed that the total PTX exposure in lymphatic tissue was dramatically higher than that achieved through intravenous administration. The peak plasma drug concentration, which governs systemic toxicity, was significantly reduced. The low but persistent detection of plasma PTX indicates that PTX was control released from the system after intrapleural implantation. In a therapeutic efficacy study performed in the H460 orthotopic lung cancer model, gelatin sponges containing PLGA-PTX microspheres were placed in the pleural cavity as an adjuvant treatment after surgical resection of the primary lung tumour. Trans-lymphatic chemotherapy resulted in a significantly lower incidence of lymphatic tumour recurrence (20%) compared to no treatment and placebo control animals (100%). PLGA-PTX microspheres were seen in regional lymphatic tissue over 4 weeks after the sponge placement. It is concluded that the trans-lymphatic targeting drug delivery system described in this thesis may improve the control of lymphatic metastasis in lung cancer.

Lymph node metastasis is a critical prognostic factor for lung cancer. Effective therapy to control lymphatic metastasis may improve survival. The work described in this thesis focuses on the development of a microparticulate lymphatic targeting system, which can be applied as an adjuvant therapy in the control of lymphatic metastasis in lung cancer. The study shows that intrapleural administered colloidal particulates are predominantly taken up by regional lymphatic tissue in rat models including healthy rats, rats bearing orthotopic lung tumours and rats following pneumonectomy. The effect of particle size on lymphatic particle distribution was examined by intrapleural administration of 111In-aminopolystyrene beads. Approximately 2 µm is a suitable size for intrapleural lymphatic targeting. Biodegradable polylactide-co-glycolide (PLGA) microparticles containing the anticancer agent paclitaxel (PTX) were subsequently formulated in the desired size by spray drying. PLGA-PTX microspheres were incorporated into a biodegradable and biocompatible gelatin sponge matrix to form an implantable lymphatic targeted drug delivery system. The system was characterized in vitro and its lymphatic targeting ability was examined in vivo. Fluorescence labeled microspheres embedded within the sponge were selectively taken up by regional lymphatics as the sponge matrix disintegrated following intrapleural implantation. A pharmacokinetic study showed that the total PTX exposure in lymphatic tissue was dramatically higher than that achieved through intravenous administration. The peak plasma drug concentration, which governs systemic toxicity, was significantly reduced. The low but persistent detection of plasma PTX indicates that PTX was control released from the system after intrapleural implantation. In a therapeutic efficacy study performed in the H460 orthotopic lung cancer model, gelatin sponges containing PLGA-PTX microspheres were placed in the pleural cavity as an adjuvant treatment after surgical resection of the primary lung tumour. Trans-lymphatic chemotherapy resulted in a significantly lower incidence of lymphatic tumour recurrence (20%) compared to no treatment and placebo control animals (100%). PLGA-PTX microspheres were seen in regional lymphatic tissue over 4 weeks after the sponge placement. It is concluded that the trans-lymphatic targeting drug delivery system described in this thesis may improve the control of lymphatic metastasis in lung cancer.

Resolving diversity patterns and their underlying drivers has application for both ecological theory and ocean management. Because seafloor characteristics are often used to assess bottom habitat, I examined the relationship between deep-sea benthic (bottom-living) diversity and multi-scale topographic heterogeneity. Most work occurred on the Canadian Pacific continental shelf at Learmonth Bank with additional sites in Strait of Georgia (BC) and Gulf of Maine (Atlantic shelf). High-resolution species distribution and seafloor data were annotated from remotely operated vehicle benthic imagery surveys while large-scale seafloor data were derived from multibeam sonar. New method development to address problems of current methods and to facilitate comparison among ecosystems is a major outcome. My new MiLS method (microtopographic laser scanning) can profile the deep seafloor at a resolution of ~1-2 cm with high accuracy and precision. I also developed a new ACR (arc-chord ratio) rugosity index as a measure of 3-D topographic heterogeneity that is simple, accurate and highly versatile. Model systems and scales vary among my studies but results consistently yield a positive relationship between diversity and topographic heterogeneity and identify bottom hydrodynamics as an important underlying driver. Rockfish Sebastes spp. associate with higher seafloor rugosity non-randomly and select for deep-sea corals and sponges over inert substrata alone. Data indicate that degradation of biogenic structures is a long-term detriment to rockfish species. Gorgonian coral- and sponge-dominant biotopes strongly associate with a single substratum type. These relationships were used to map coral and sponge distributions. This work, which collectively adds new information on the ecological relevance and distribution of corals and sponges, is pertinent to the conservation and management of fish stocks and vulnerable marine ecosystems. Epibenthic community variables abundance, richness, and Shannon diversity positively correlated with both the local microtopographic heterogeneity on a scale of 10 m2 and with the surrounding regional large-scale topographic heterogeneity on scales of 25 to 250,000 m2. Relationships were strongest between epibenthic community variables and the largest scale rugosity and were used to generate and test predictive diversity models. Where management strategies rely on surrogate measures in data-poor areas, mapping benthic diversity using ACR rugosity will provide good indicators. Although bottom hydrodynamics is consistently identified as an underlying driver of epibenthic patterns related to topographic heterogeneity, data suggest the nature of the relationship varies across spatial scales. At small scales, high topographic heterogeneity likely increases diversity by increasing the number of available niches (including hydrodynamic gradients; e.g., the abrupt vertical rugosity created by tall corals and sponges provides rockfish refuge from currents) while at large scales, high topographic heterogeneity increases local diversity less directly through distant hydraulic events that alter bottom flow hydrodynamics. / Graduate / 0329 / 0416 / 0799 / cdupreez@uvic.ca

Marine invertebrates, in particular sponges, represent a source of a wide range of secondary metabolites, many of which have been attributed various defensive capabilities against environmental stress factors. In this thesis sponge-derived low-molecular peptide-like compounds and associated analogs are investigated for bioactivity and pharmacological targets. The compound bromobenzisoxazolone barettin (cyclo[(6-bromo-8-(6-bromo-benzioxazol -3(1H)-one)-8-hydroxy)tryptophan)]arginine) was isolated from the sponge Geodia barretti and its ability to inhibit larval settlement of the barnacle Balanus improvisus was determined. With an EC50 value of 15 nM, this compound’s antifouling effect was higher than those of the previously reported brominated dipeptides from Geodia barretti, i.e., barettin and 8,9-dihydrobarettin; moreover, this antifouling effect was demonstrated to be reversible. However, the compound lacked affinity for 5-HT1-7 receptors, whereas barettin possessed specific affinity to 5-HT2A, 5-HT2C and 5-HT4, while 8,9-dihydrobarettin interacted with 5-HT4. In an attempt to evaluate structure-activity relationships synthesized analogs with barettin and dipodazine scaffolds were investigated for antifouling activity. The analog benso[g]dipodazine, with an EC50 value of 34 nM, displayed the highest settlement inhibition. The studies of the structure-activity relationships of sponge-derived compounds were extended to cover analogs of agelasines and agelasimines originally isolated from sponges of the genus Agelas. Synthesized (+)-agelasine D and two structurally close analogs were investigated for cytotoxic and antibacterial activity. The profound cytotoxicity and broad spectrum antibacterial activity found prompted a further investigation of structure-activity relationships in 42 agelasine and agelasimine analogs and several characteristics that increased bioactivity were identified. In conclusion this work has produced new results regarding the potent bioactivity of compounds derived from the sponges Geodia barretti and Agelas spp. and increased SAR knowledge of the fouling inhibition, cytotoxicity and antimicrobial activity of these compounds.