With the growing trend of globalization and rapid development of high technologies, emerging economies face more challenges in technology development because they are chasing a fast-moving frontier. They need to identify global technology trends and adapt to local needs and capabilities. Strategies for technology development differ among countries at different developmental stages. In this research, a technology policy choice framework is developed to link prospective high-tech areas, technology development strategies, and various innovative resources. The research approach is to develop a hierarchical decision model (HDM) and apply the analytic hierarchical process (AHP). Experts are invited from diverse sources to provide a balanced perspective representing different stakeholders. This research focuses on the fast developing Chinese biopharmaceutical industry as a case study. The results of this research have identified thirteen prospective biotech areas that China should invest more resources for development. These technology areas include: recombinant therapeutic proteins, recombinant vaccines, monoclonal antibody technology, cell and tissue engineering, gene therapy, antisense therapy, RNAi, nanobiotechnology, synthetic biology, bioinformatics, pharmacogenetics, gene sequencing, and biotechnology diagnostics. For most of these technology areas, the results have indicated an imitative innovation strategy should be taken as a better strategy under current technological conditions in China. The research has further found that high-tech small-to-medium companies and multinational corporations are major innovation contributors in the Chinese biopharmaceutical sector. The research outcomes can serve as guidelines in resource allocation and policy making for technology development. Based on the overall research findings, policy-makers can apply more specific policy instruments to support innovation activities. Appropriate policy measures may help the country to construct an innovative ecosystem that can serve as the driving force for future technology development.

This report reviews the field of bispecific antibodies, artificially engineered antibodies thathave the ability to bind two or more different antigen simultaneously. Historical as well asrecently developed techniques are demonstrated, together with formats in preclinical andclinical development. We studied the field with the future needs of the developers in mind,when it comes to the processes and tools that can be offered by GE Healthcare BiosciencesAB. The development of bispecific antibodies gave rise to new challenges and product-relatedimpurities, which are handled by various methods. We argue for, based on the formats inclinical and preclinical development, that the methods already used to purify monospecificantibodies remain the most successful methods for the purification of bispecific antibodies.This, together with the design strategies that resolve the initial bottle-necks, ensures that theneeds of the developers are met to the same extent as for monoclonal antibodies. The methodsand formats demonstrated here do not represent all that are available or under trial.

Carbohydrate recognition is one of the most sophisticated recognition processes in biological systems, mediating many important aspects of cell-cell recognition, such as inflammation, cell differentiation, and metastasis. Consequently, lectin-glycan interactions have been intensively studied in order to mimic their actions for potential bioanalytical and biomedical applications. Galectins, a class of ß-galactoside-specific animal lectins, have been strongly implicated in inflammation and cancer. Galectin-3 is involved in carbohydrate-mediated metastatic cell heterotypic and homotypic adhesion via interaction with Thomsen-Friedenreich (TF) antigen on cancer-associated MUC1. However, the precise mechanism by which galectin-3 recognizes TF antigen is poorly understood. Our thermodynamic studies have shown that the presentation of the carbohydrate ligand by MUC1-based peptide scaffolds can have a major impact on recognition, and may facilitate the design of more potent and specific galectin-3 inhibitors that can be used as novel chemical tools in dissecting the precise role of galectin-3 in cancer and inflammatory diseases. Another lectin, odorranalectin (OL), has been recently identified from Odorrana grahami skin secretions as the smallest cyclic peptide lectin, has a particular selectivity for L-fucose and very low toxicity and immunogenicity, rendering OL an excellent candidate for drug delivery to targeted sites, such as: (1) tumor-associated fucosylated antigens implicated in the pathogenesis of several cancers, for overcoming the nonspecificity of most anticancer agents; (2) the olfactory epithelium of nasal mucosa for enhanced delivery of peptide-based drugs to the brain. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2015.

伴隨著納米科技的發展，越來越多的納米顆粒系統已經應用於生物醫學领域。其中，二氧化硅納米顆粒因其簡單易操作的表面化學性質和在生理環境中的良好穩定性，已被廣泛認為是最有前途的治療和診斷載體之一。 / 在本論文中，我首先對二氧化硅納米顆粒與人類細胞之間的作用進行了系統研究。這些作用包括了以下主要特點: 胞吞和胞吐被分別確定為納米顆粒主要的進入和離開細胞的主要途徑; 大部份的納米顆粒被發現存在於有膜結構的細胞器里，這些細胞器相當穩定（不易破損），只有很少的一部份納米顆粒被釋放到細胞質里; 納米顆粒和細胞之間的作用是動態的，它們進入細胞內的數量由其在細胞培養液中的數量和形態（聚集的程度）所決定。正是這些特點決定了二氧化硅顆粒在低濃度時的低細胞毒性。 / 緊接著我比較了兩種最常見的二氧化硅納米顆粒（晶體態和無定型態）引入細胞后對細胞所帶來的影響。儘管兩種形態的納米顆粒所造成的細胞毒性都比較低，但是更細緻的分析揭示了它們對細胞及其衍化途徑的不同影響。細胞吞入晶體態的二氧化硅納米顆粒后，其內部的活性氧物質含量顯著提高，這種變化會導致細胞線粒體功能受損（表現為線粒體增生）並且最終將細胞導向死亡。不過只有在p53基因缺失的細胞中才有這種由活性氧物質水平升高導致的細胞損傷，p53正常的細胞卻能抵禦這種來自晶體態二氧化硅納米顆粒的刺激。而無定型態二氧化硅納米顆粒對生物系統無損害，因而有發展為藥物載體的巨大潛力。 / 基於對二氧化硅顆粒細胞毒性研究的理解，我們設計了一種新型納米載體--金核/二氧化硅殼層（Au@SiO₂）納米顆粒用於藥物輸運。在這一體系中，無定形態二氧化硅和金納米顆粒的優勢被整合在一起，同時光敏劑（PS）藥物分子被裝載在二氧化硅殼層內。對比於自由形式的PS，裝載在Au@SiO₂納米顆粒中的PS展示出增強的藥效。需要強調的是，用這種納米顆粒處理的細胞以阻梗壞死為主要的死亡途徑，代替了凋亡這種不太有效的方式。在光照下，金的等離子體效應被發現能促進PS的光響應過程，這使得細胞殺死率得到了大幅度增強。這一效應得益于我們把PS束縛在金核的表面，同時保證金表面等離子體振盪能量和PS吸收能量的配對。此外，把PS裝載在二氧化硅中會引起PS有益的光化學改變。這些作用結合在一起導致了藥效的提高。這些機理能被普遍應用於納米顆粒裝載藥物分子的設計中，為最優化設計提供指導。 / With recent development of nanotechnology, various nanoparticulate systems have been proposed to serve as functional units for biomedical applications in many innovative ways. Among various possible choices, silica nanoparticles (NPs) enjoys easily modifiable surface chemical characteristics and excellent stability in physiological environment. Therefore, it is considered as one of the most promising carrier candidate for therapeutic and diagnostic applications. / A systematic study on the interaction between silica nanoparticles and human cells is first carried out in the present thesis work. Endocytosis and exocytosis are identified as major pathways for NPs entering, and exiting the cells, respectively. Most of the NPs are found to be enclosed in membrane bounded organelles, which are fairly stable (against rupture) as very few NPs are released into the cytoplasma. The nanoparticle-cell interaction is a dynamic process, and the amount of NPs inside the cells is affected by both the amount and morphology (degree of aggregation) of NPs in the medium. These interaction characteristics determine the low cytotoxicity of SiO₂ NPs at low feeding concentration. / Experiments were then designed to compare the the biological consequence of two most common form of SiO₂ nanoparticles, i.e., crystalline and amorphous NPs, when they were introduced to human cells. Although the apparent cytotoxicity of both types of NPs seems to be low, more detailed characterizations disclose the profound difference induced by the crystalline and amorphous ones, resulting in significantly different cell evolution pathways. Crystalline NPs but not amorphous ones are found to drastically increase the recative oxygen species (ROS) level in the cells, which can cause mitochondria dysfunction (being expressed as mitochondria proliferation), and eventually direct the cell into apoptosis. Nonetheless, only p53 deficient cells are subjective to such ROS induced cell damage, while p53 proficient cells can accommodate the stimulation from crystalline SiO₂ NPs. The amorphous SiO₂ NPs are found to be benign in the biological systems, and have great potential to be developed as nanomedicine. / Base on the understanding obtained from the toxicology study of the SiO₂ NPs, we have designed a special nanocarrier system for drug delivery. We have combined advantages of both SiO₂ and Au NPs by constructing Au-core/SiO₂-shell (Au@SiO₂) nanocarriers with the photosensitizer (PS) drug embedded in the SiO₂ shell layer. Compared with free PS, PS loading in the Au@SiO₂ NPs showes a enhanced drug efficacy. In particular, the cells treated with the NP drug take necrosis as a major death path instead of apoptosis, which is a much less effective route. The Au plasmonic effect is found to promote the photo-response of the PS drug under light irradiation, contributing to the largely decreased cell viability. Nevertheless, one shall note that spatial confinement of the drug moledules to the close proximity of the Au core and an energy match between the drug absorption and the Au surface plasmon resonance are critical in manifesting the plasmonic effect. At the same time, embedding the drug in the SiO₂ matrix leads to favorable change in the photochemical process. The combined effects brought by the Au@ SiO₂ NP carrier is responsible for the high drug efficacy. These mechanisms can be generally valid in engineering drug molecule incorporation into NP carriers and also give guidance for the optimum design of the NP drug carrier. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chu, Zhiqin = 二氧化硅納米顆粒與人類細胞的作用及其在生物醫學方面的應用 / 褚智勤. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 120-137). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Chu, Zhiqin = Er yang hua gui na mi ke li yu ren lei xi bao de zuo yong ji qi zai sheng wu yi xue fang mian de ying yong / Chu Zhiqin. / Table of contents --- p.VIII / List of figures --- p.XIII / List of tables --- p.XIX / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Chapter 2 --- Background --- p.4 / Chapter 2.1 --- Overview of the silica-based nanoparticles for bio-medical applications --- p.4 / Chapter 2.2 --- Health issue on the silica-base nanoparticles --- p.5 / Chapter 2.3 --- Understanding the nano-bio interface --- p.6 / Chapter 2.3.1 --- Nano-bio interface in vitro --- p.7 / Chapter 2.3.2 --- Nano-bio interface in vivo --- p.10 / Chapter 2.4 --- Bio-application of silica-based nanoparticles --- p.11 / Chapter 2.4.1 --- Use of silica nanoparticle as imaging agent --- p.11 / Chapter 2.4.2 --- Use of silica nanoparticle as drug carrier --- p.12 / Chapter 2.4.3 --- Use of silica nanoparticle as coating media --- p.12 / Chapter 2.5 --- Surface plasmon of gold nanostructures and its bio-application --- p.13 / Chapter 2.5.1 --- Introduction to the SPR of gold nanostructures --- p.13 / Chapter 2.5.2 --- Synthesis of gold NRs and their SPR effect --- p.13 / Chapter 2.5.3 --- SPR of gold NRs in bio-application --- p.16 / Chapter Chapter 3 --- Experimental --- p.18 / Chapter 3.1 --- Standard methodologies for nanoparticle preparation and their feeding to the cells --- p.18 / Chapter 3.2 --- Cell sampling for room temperature TEM study --- p.18 / Chapter 3.3 --- Developing methods to distinguish NPs in cell sample under TEM --- p.20 / Chapter 3.4 --- Confocal microscopy study --- p.21 / Chapter 3.4.1 --- Study the photoluminescence of various dye molecules --- p.21 / Chapter 3.4.2 --- Study the two photon luminescence (TPL) of Au NRs --- p.23 / Chapter 3.5 --- UV-Vis-NIR spectrophotometer and fluorescence spectrophotometer --- p.25 / Chapter 3.6 --- Flow-cytometry --- p.26 / Chapter 3.7 --- Western plot --- p.28 / Chapter 3.8 --- Colormetric assays and other biological labels --- p.28 / Chapter 3.8.1 --- 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test --- p.28 / Chapter 3.8.2 --- Mitochondria, lysosome and nucleus staining --- p.30 / Chapter 3.8.3 --- Detection of apoptosis --- p.31 / Chapter 3.8.4 --- Detection of various reactive oxygen species (ROS) --- p.31 / Chapter Chapter 4 --- Silica NPs interact with human cells --- p.34 / Chapter 4.1 --- Introduction --- p.34 / Chapter 4.2 --- Characterization of silica nanoparticles --- p.36 / Chapter 4.3 --- General description of the NPs’ uptaking and excreting process --- p.42 / Chapter 4.4 --- Tracking of NPs inside the cells --- p.53 / Chapter 4.5 --- Factors influencing the NP-cell interaction and exocytosis process --- p.55 / Chapter 4.5.1 --- The effect of serum (in the incubation medium) on cellular uptake --- p.55 / Chapter 4.5.2 --- Crystallinity effectdistribution of amorphous and crystalline SiO₂ NPs in the cells --- p.57 / Chapter 4.5.3 --- Factors affecting the exocytosis process --- p.59 / Chapter 4.6 --- Cytotoxic effect of silica NPs --- p.60 / Chapter 4.7 --- Conclusion --- p.63 / Chapter Chapter 5 --- Genotoxic effect specifically induced by crystalline SiO₂ nanoparticles in p-53 deficient human cells --- p.65 / Chapter 5.1 --- Introduction --- p.65 / Chapter 5.2 --- The difference between crystalline and amorphous silica NPs --- p.66 / Chapter 5.2.1 --- Mitochondria multiplication specially induced by crystalline silica NPs --- p.68 / Chapter 5.2.2 --- DNA fragmentation specially observed in crystalline silica NPs treated cells --- p.71 / Chapter 5.3 --- The cell line sensitive cytotoxicity of crystalline silica NPs --- p.79 / Chapter 5.3.1 --- A general phenomenon of mitochondria increase in p-53 negative cell lines --- p.80 / Chapter 5.3.2 --- General biological consequence of such mitochondria increase --- p.82 / Chapter 5.4 --- Conclusion --- p.83 / Chapter Chapter 6 --- Surface plasmon enhanced drug efficacy for PDT using core shell Au@SiO₂ nanoparticle carrier --- p.84 / Chapter 6.1 --- Introduction --- p.84 / Chapter 6.1.1 --- Brief introduction to the photodynamic therapy (PDT) and photosensitizer (PS) --- p.84 / Chapter 6.1.2 --- Brief introduction to the SPR enhanced generation of ROS --- p.86 / Chapter 6.2 --- Using Au@SiO₂ NPs as drug carrier --- p.88 / Chapter 6.2.1 --- Growth of gold NRs and their controllable oxidation --- p.88 / Chapter 6.2.2 --- Preparation and characterization of Au@(SiO₂-MB) NPs --- p.90 / Chapter 6.2.3 --- Confirmation of MB loading into silica shell --- p.92 / Chapter 6.3 --- Enhanced PDT drug (MB) efficacy when loaded in Au@SiO₂ NPs --- p.95 / Chapter 6.3.1 --- Cellular uptake pathway of free MB and Au@SiO₂ NPs --- p.95 / Chapter 6.3.2 --- Comparing the efficacy of free MB, SiO₂-MB NPs and Au@(SiO₂MB) NPs --- p.98 / Chapter 6.4 --- Studying the behavior of free MB and Au@(SiO₂-MB) NPs as PDT agent --- p.100 / Chapter 6.4.1 --- Comparing the ability of generating ROS by free MB and Au@(SiO₂MB) NPs --- p.100 / Chapter 6.4.2. --- Comparing the types of ROS generated by free MB and Au@(SiO₂MB) NPs --- p.103 / Chapter 6.4.3 --- Comparing the cellular death pathway triggered by free MB and Au@(SiO₂-MB) NPs --- p.105 / Chapter 6.5. --- Discussion on the mechanism for the enhanced efficacy --- p.109 / Chapter 6.5.1 --- Excluding the photothermal effect of Au NRs core --- p.109 / Chapter 6.5.2 --- The role of SiO₂ in the Au@SiO₂ NPs carrier --- p.111 / Chapter 6.5.3 --- Attributing the enhanced efficacy to plasmonic effect of Au NRs core --- p.112 / Chapter 6.6 --- Exploring the potential of using Au@SiO₂ NP carrier in vivo --- p.114 / Chapter 6.7 --- Conclusion --- p.116 / Chapter Chapter 7 --- Conclusion --- p.118 / References --- p.120

The genus Cymbopogon (Poaceae) is composed of more than 100 species found in tropical countries, and about 56 species have essential oils with aromatic characteristics and some ones have mediinal, pharmaceutical and industrial importance. The essential oils of the genus are rich in mono-and sesquiterpenes. Among the species of the genus Cymbopogon winterianus Jowitt, popularly known as |citronella|, |Java citronella| or |jacapé| is used in folk medicine as a repellent, antimicrobial, analgesic, anxiolytic and for the treatment of epilepsy. The aim of this study was to evaluate pharmacological and toxicological properties of the essential oil extracted from the leaves of Cymbopogon winterianus (EOC) in experimental protocols. Analysis by GC-MS showed the major components are citronellal, citronellol and geraniol. In pharmacological screening the main changes observed were: ptosis, sedation and decreased ability to raise. In the test of acute toxicity LD 50 was calculated in 1953.8 mg / kg (1580.9 to 2326.7) (p.o.) and in 567.3 mg / kg (395.8 to 758.2) (ip). The OEC decreased the number of crossings in the test of spontaneous movement and increased the sleep time induced by thiopental. In the the rota-rod task EOC did not interfere the motor coordination of the animals. Antinociceptive, anti-inflammatory and antioxidant effects were performed using the tests of writhing induced by acetic acid, nociception induced by formalin into the paw hot plate, the carrageenan-induced inflammation and sequestering activity of free radicals (DPPH). The results showed that the OEC reduced the response to pain, the migration of neutrophils to the abdominal cavity and is very efficient in sequestering the free radicals. In the evaluation of acute and subchronic toxicity, the animals were treated respectively with EOC in a single dose (900 mg / kg - po) and daily doses of 50mg/kg (po). In the acute test, the main behavioral changes were ptosis, sedation, decreased response to touch, decreased ability to raise and analgesia. No changes suggestive of toxicity in acute and subchronic tests were found in the assessment of body weight, water intake and feed and organ weights. In the evaluation of biochemical parameters, there were significant changes in the dosage of aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase (p <0.05) in both acute and subchronic tests. The results together suggest that EOC possess depressant, antinociceptive, antiinflammatory and antioxidant properties without loss of motor coordination. In addition, EOC showed toxicity, since histological changes were found in kidney and liver in the acute assay and in liver in the subchronic assay. / O gênero Cymbopogon (Poaceae) é composto por mais de 100 espécies encontradas em países tropicais, e por volta de 56 espécies são aromáticas e algumas têm importância medicinal, farmacológica e industrial, sendo ricas em óleos essenciais. Os óleos essenciais extraídos do gênero são ricos em mono e sesquiterpenos. Dentre as espécies do gênero o Cymbopogon winterianus Jowitt, conhecido popularmente como citronela , citronela de Java ou jacapé , é utilizado na medicina popular como repelente, antimicrobiano, analgésico, ansiolítico e para o tratamento da epilepsia. O objetivo desse trabalho foi estudar aspectos farmacológicos e toxicológicos do óleo essencial extraído das folhas de Cymbopogon winterianus (OEC) em protocolos experimentais. Análise realizada através de GC-MS demonstrou como componentes majoritários o citronelal, citronelol e geraniol. Na triagem farmacológica as principais alterações registradas foram: ptose, sedação e levantar diminuído. No teste da toxicidade aguda a DL50 calculada foi de 1.953,8 mg/kg (1.580,9 2.326,7) (v.o.) e 567,3 mg/kg (395,8-758,2) (i.p.). O OEC promoveu redução no número de cruzamentos no teste da movimentação espontânea e aumento no tempo de sono induzido pelo tiopental. No teste do rota rod observou-se que o OEC não interfere a coordenação motora dos animais. Os testes para se determinar propriedades antinociceptiva, antiinflamatória e antioxidante foram realizados utilizando-se os testes de contorção abdominal induzido por ácido acético, de nocicepção na pata induzido por formalina, da placa quente, de inflamação induzido por carragenina e atividade de sequestro dos radicais livres (DPPH). Os resultados demonstraram que o OEC reduziu a resposta a dor, a migração de neutrófilos para a cavidade abdominal e foi muito eficiente no seqüestro dos radicais livres. Na avaliação da toxicidade aguda e subcrônica, os animais foram tratados respectivamente com o OEC em dose única (900 mg/kg - v.o.) e doses diárias de 50mg/kg (v.o.). No teste agudo, as principais alterações comportamentais apresentadas foram ptose palpebral, sedação, resposta ao toque diminuída, levantar diminuído e analgesia. Em relação à avaliação do peso corpóreo, ingesta de água e ração e peso dos órgãos não ocorreram alterações sugestivas de toxicidade nos ensaios agudo e subcrônico. Na avaliação dos parâmetros bioquímicos, observaram-se alterações significativas na dosagem de aspartato aminotransferase, alanina aminotransferase e fosfatase alcalina (p<0.05) em ambos os ensaios agudo e subcrônico. Os resultados em conjunto sugerem propriedades depressora, antinociceptiva, antiinflamatória e antioxidantes sem que esses efeitos promovam perda de coordenação motora. Além disso, o OEC possui toxicidade na dose e via testada, visto que alterações histológicas foram encontradas nos rins e no fígado no ensaio agudo e no fígado no ensaio subcrônico.

In Brazil leishmaniasis reach 19 states, and more than 90% of human cases of the disease are concentrated in Northeast region, there is still important regions foci in the Midwest, North and Southeast regions. Studies point for the occurrence of about 20.000 new cases annually of the disease. The need for new drugs more effective, safe and accessible for the treatment of leishmaniasis, visceral in particular, later on, affects liver, spleen, reticuloendothelial system, bone marrow and lymph nodes becomes relevant. Furthermore, according to the World Health Organization, the plant species are the best and major source of drugs for humanity and Brazil has 60.7% of its territory of natural and planted forests, representing the second largest forest area in the world, only behind from Russia. Thus, this study aimed investigated the activities of the essential oil of Cymbopogon citratus and Citral against Leishmania (L.) chagasi. In order for this to happen, oil from fresh leaves were harvested on the Mother Earth farm, located in Santana do Sao Francisco/SE. The Citral was acquired from Sigma-Aldrich (Darmstadt, Germany). Gaseous Chromatography and Detections by Flame Ionization were carried to identify its chemical constituents, just like an evaluation of inhibitory concentrations 50% in promastigotes and amastigotes in Leishmania mentioned beforehand, cytotoxicity assays, nitric oxide production and fluorescence to detect possible increase of membrane permeability in the presence of Citral were developed. Pharmacological activities were found in promastigotes (CI50/48 hours 25 Êg/mL for oil and 30 Êg/mL for Citral), however, activities were not found in amastigotes. The cytotoxicity (CI50 was 26.25 Êg/mL for oil and 33.96 Êg/mL for Citral). As for the increased production of nitric oxide, this did not occur, however, pore formation was observed in cell membranes of promastigotes in the presence of Citral. Therefore, the essential oil of Cymbopogon citratus and Citral may come to be used as preventive measure against visceral leishmaniasis. Since the promastigotes are the ways that infect vertebrates, including man. / No Brasil as leishmanioses atingem 19 estados, sendo que mais de 90% dos casos humanos da doenca concentram-se na regiao Nordeste, havendo ainda focos importantes nas regioes Centro-Oeste, Norte e Sudeste. Estudos sinalizam para a ocorrencia de cerca de 20.000 casos anuais da doenca. A necessidade de novos farmacos mais eficazes, seguros e acessivel para o tratamento das leishmanioses, em especial a visceral, posto, acomete figado, baco, sistema reticuloendotelial, medula ossea e linfonodos torna-se relevante. De acordo com a Organizacao Mundial de Saude, as especies vegetais sao fontes de farmacos importantes para humanidade e o Brasil possui 60,7% do seu territorio de florestas naturais e plantadas, representando a segunda maior area florestal do mundo, atras apenas da Russia. Assim, esse estudo objetivou investigar as atividades do oleo essencial de Cymbopogon citratus e Citral contra Leishmania (L.) chagasi. Para tal, obteve-se o oleo das folhas fresca colhidas na fazenda Mae Terra, localizada em Santana do Sao Francisco/SE, ja o Citral foi adquirido da Sigma-Aldrich (Darmstadt, Germany). Foram realizadas suas Cromatografias Gasosas e Deteccoes por Ionizacao de Chama para identificar seus constituintes quimicos, bem como avaliacao das concentracoes inibitorias 50% nas promastigotas e amastigotas na Leishmania supracitada, ensaios de citotoxicidade, producao de oxido nitrico e fluorescencia para detectar possivel aumento da permeabilidade de membrana em presenca do Citral foram desenvolvidos. Atividades farmacologicas foram constatadas em promastigotas (CI50/48 horas 25 Êg/mL para oleo e 30 Êg/mL para Citral), entretanto, nao foi encontrada acao nas amastigotas. A citotoxicidade (CI50 foi de 26,25 Êg/mL para oleo e 33,96 Êg/mL para Citral). Quanto ao aumento na producao de oxido nitrico, essa nao ocorreu, todavia, foi observada formacao de poros nas membranas celulares nos promastigotas em presenca de Citral. Portanto, o oleo essencial de Cymbopogon citratus e Citral pode vir a ser usados como medida preventiva contra leishmaniose visceral. Uma vez que as promastigotas sao as formas que infectam os animais vertebrados, inclusive o homem.

In recent years, advances in biotechnology has allowed the development of synthetic membranes associated with nanocomposites, which has shown promising results as dermal burns dressings. In this sense, silver nanoparticles (NPAg) has been the focus of interest because of their biological properties such as antimicrobial and antiinflammatory effect. The incorporation of NPAg in biological membranes of different natures, such as chitosan, polyester, polymethacrylate methyl and cellulose, has been successfully tested in several biological models. The association between NPAg and polymers produced by the micro-organism presents important advantages, such as water solubility and lack of toxicity. Recently we developed a technique for producing NPAg associated with xanthan (GX), a biopolymer with potential application in various sectors of the petrochemical industry, food and pharmaceutical, through fermentation by Xanthomonas sp performed in the presence of silver nitrate. Therefore, this study aimed to develop, characterize and evaluate the potential antimicrobial and healing membranes nanocomposite xanthan: silver on second-degree burns in the porcine model. Therefore, xanthan biocomposites: silver were used for fabrication of membranes (for casting process, which were subsequently characterized for thickness, mechanical properties (stress, strain, Young´s modulus) and the thermal profile (DSC, TG and DTG). Activity antimicrobial was tested against strains of Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923). analysis for tissue repair were made two dermal burns on the back nine male pigs breed Yorkshire (25 ± 5 kg), treated with Xanthan biosensor membrane: silver (XNPAg) with topical application of silver sulfadiazine 1% (SDZ). After eight, 18 and 30 days the wounds were examined macroscopically determined for each lesion area, and the animals euthanized for Microscopic study of the scar area observed that XNPAg membranes showed a significant increase in the values of thickness (P <0.05), density (p <0.01) and Young´s modulus (p <0.001) and reduced strength strain (p <0.05) when compared to membranes of xanthan. Were revealed changes in the thermal profile of the two membranes suggesting the incorporation of silver nanoparticles in the polymer xanthan. XNPAg The membrane induced the formation of inhibition zones 9, 7 mm and 9.6 mm and death rate of 89% and 100% for Staphylococcus aureus and Escherichia coli respectively. Histological analysis showed quantitative and qualitative increase in the reaction granulation and best architectural arrangement of collagen fibers along the the healing process of wounds covered with membranes XNPAg. Could be concluded that the membranes nanocomposite xanthan:silver showed satisfactory mechanical properties for its handling, transportation and storage, as well as important antimicrobial activity and pro-healing in dermal burns using porcine model. / Nos últimos anos, os avanços na área da biotecnologia tem propiciado o desenvolvimento de membranas sintéticas associados à nanocompostos, que vem apresentando resultados promissores como curativos de queimaduras dérmicas. Nesse sentido, as nanopartículas de prata (NPAg) tem sido foco de interesse devido as suas propriedades biológicas como atividade antimicrobiana e efeito antiinflamatório. A incorporação de NPAg em membranas biológicas de diferentes naturezas, como quitosana, poliéster, polimetacrilato de metila e celulose, vem sendo testada com sucesso em diversos modelos biológicos. A associação entre NPAg e polímeros produzidos por microrganismo apresenta importantes vantagens, como solubilidade em água e ausência de toxicidade. Recentemente foi desenvolvida uma técnica de produção de NPAg associadas a goma xantana (GX), um biopolímero com aplicação potencial em vários setores da indústria petroquímica, alimentícia e farmacêutica, através de processo fermentativo realizado pela Xanthomonas sp na presença de nitrato de prata. Diante disso, o presente estudo teve como objetivo desenvolver, caracterizar e avaliar o potencial antimicrobiano e cicatrizante de membranas de bionanocompósito xantana:prata sobre queimaduras de segundo grau em modelo suíno. Para tanto, biocompósitos xantana:prata foram utilizadas para confecção de membranas (por casting process, que foram posteriormente caracterizadas quanto a espessura, propriedades mecânicas (tensão, deformação, módulo de Young) e perfil termoanalítico (DSC, TG e DTG). A atividade antimicrobiana foi avaliada frente a cepas de Escherichia coli (ATCC 25922) e Staphylococcus aureus (ATCC 25923). Para análise do reparo tecidual foram confeccionadas duas queimaduras dérmicas no dorso de nove suínos machos, da raça Yorkshire (25 ± 5 kg), tratadas com membrana de biocompósito xantana:prata (XNPAg) e com aplicação tópica da sulfadiazina de prata a 1% (SDZ). Após oito, 18 e 30 dias, as feridas foram analisadas macroscopicamente, determinada a área de cada lesão, e os animais eutanasiados para estudo microscópico da área cicatricial. Observou-se que as membranas XNPAg apresentaram aumento significativo nos valores de espessura (p<0,05), deformação (p<0,01) e módulo de Young (p<0,001), e redução da força de tensão (p<0,05) quando comparados a membranas de xantana. Foram evidenciadas alterações no perfil termoanalítico das duas membranas sugestivas da incorporação das nanopartículas de prata no polímero de xantana. As membrana XNPAg induziram a formação de halos de inibição de 9,7 mm e 9,6 mm e Taxa de letalidade de 89% e 100% para Staphylococcus aureus e Escherichia coli respectivamente. A análise histológica mostrou incremento quantitativo e qualitativo na reação de granulação, bem como melhor disposição arquitetural das fibras de colágeno ao longo do processo cicatricial das feridas cobertas com membranas XNPAg. Pôde-se concluir que as membranas de bionanocompóstico xantana:prata apresentaram propriedades mecânicas satisfatórias para sua manipulação, transporte e armazenamento, bem como importante atividade antimicrobiana e pró-cicatrizante em queimaduras dérmicas utilizando modelo suíno.

Non-steroidal anti-inflammatory drugs (NSAIDs) are one of the most commonly used classes of drugs. They target the cyclooxygenases (COX) 1 and 2 to reduce the physiological responses of pain, fever, and inflammation. Due to their role in inducing angiogenesis, COX proteins have also been identified as targets in cancer therapies. In this thesis, I describe computational protocols of molecular docking, molecular dynamics simulations and free energy calculations. These methods were used in this thesis to determine structure-activity relationships of a diverse set of NSAIDs in binding to their target proteins COX-1 and 2. Binding affinities were calculated and used to predict the binding modes. Based on combinations of molecular dynamics simulations and free energy calculations, binding mechanisms of sub-classes of NSAIDs were also proposed. Two stable conformations of COX were probed to understand how they affect inhibitor affinities. Finally, a brief discussion on selectivity towards either COX isoform is discussed. These results will be useful in future de novo design and testing of third-generation NSAIDs.

Quality by Design (QbD) is a systematic approach that has been recommended as suitable for the development of quality pharmaceutical products. The QbD approach commences with the definition of a quality target drug profile and predetermined objectives that are then used to direct the formulation development process with an emphasis on understanding the pharmaceutical science and manufacturing principles that apply to a product. The design space is directly linked to the use of QbD for formulation development and is a multidimensional combination and interaction of input variables and process parameters that have been demonstrated to provide an assurance of quality. The objective of these studies was to apply the principles of QbD as a framework for the optimisation of a sustained release (SR) formulation of salbutamol sulphate (SBS), and for the establishment of a design space using Response Surface Methodology (RSM) and Artificial Neural Networks (ANN). SBS is a short-acting ♭₂ agonist that is used for the management of asthma and chronic obstructive pulmonary disease (COPD). The use of a SR formulation of SBS may provide clinical benefits in the management of these respiratory disorders. Ashtalin®8 ER (Cipla Ltd., Mumbai, Maharashtra, India) was selected as a reference formulation for use in these studies. An Ishikawa or Cause and Effect diagram was used to determine the impact of formulation and process factors that have the potential to affect product quality. Key areas of concern that must be monitored include the raw materials, the manufacturing equipment and processes, and the analytical and assessment methods employed. The conditions in the laboratory and manufacturing processes were carefully monitored and recorded for any deviation from protocol, and equipment for assessment of dosage form performance, including dissolution equipment, balances and hardness testers, underwent regular maintenance. Preliminary studies to assess the potential utility of Methocel® Kl OOM, alone and in combination with other matrix forming polymers, revealed that the combination of this polymer with xanthan gum and Carbopol® has the potential to modulate the release of SBS at a specific rate, for a period of 12 hr. A central composite design using Methocel® KlOOM, xanthan gum, Carbopol® 974P and Surelease® as the granulating fluid was constructed to fully evaluate the impact of these formulation variables on the rate and extent of SBS release from manufactured formulations. The results revealed that although Methocel® KlOOM and xanthan gum had the greatest retardant effect on drug release, interactions between the polymers used in the study were also important determinants of the measureable responses. An ANN model was trained for optimisation using the data generated from a central composite study. The efficiency of the network was optimised by assessing the impact of the number of nodes in the hidden layer using a three layer Multi Layer Perceptron (MLP). The results revealed that a network with nine nodes in the hidden layer had the best predictive ability, suitable for application to formulation optimisation studies. Pharmaceutical optimisation was conducted using both the RSM and the trained ANN models. The results from the two optimisation procedures yielded two different formulation compositions that were subjected to in vitro dissolution testing using USP Apparatus 3. The results revealed that, although the formulation compositions that were derived from the optimisation procedures were different, both solutions gave reproducible results for which the dissolution profiles were indeed similar to that of the reference formulation. RSM and ANN were further investigated as possible means of establishing a design space for formulation compositions that would result in dosage forms that have similar in vitro release test profiles comparable to the reference product. Constraint plots were used to determine the bounds of the formulation variables that would result in the manufacture of dosage forms with the desired release profile. ANN simulations with hypothetical formulations that were generated within a small region of the experimental domain were investigated as a means of understanding the impact of varying the composition of the formulation on resultant dissolution profiles. Although both methods were suitable for the establishment of a design space, the use of ANN may be better suited for this purpose because of the manner in which ANN handles data. As more information about the behaviour of a formulation and its processes is generated during the product Iifecycle, ANN may be used to evaluate the impact of formulation and process variables on measureable responses. It is recommended that ANN may be suitable for the optimisation of pharmaceutical formulations and establishment of a design space in line with ICH Pharmaceutical Development [1], Quality Risk Management [2] and Pharmaceutical Quality Systems [3]

The hypothesis that similar chemicals exert similar biological activities has been widely adopted in the field of drug discovery and development. Quantitative Structure-Activity Relationship (QSAR) models have been used ubiquitously in drug discovery to understand the function of chemicals in biological systems. A common QSAR modeling method calculates similarity scores between chemicals to assess their biological function. However, due to the fact that some chemicals can be similar and yet have different biological activities, or conversely can be structurally different yet have similar biological functions, various methods have instead been developed to quantify chemical similarity at the functional level. Quantmap is one such method, which utilizes biological databases to quantify the biological similarity between chemicals. Quantmap uses quantitative molecular network topology analysis to cluster chemical substances based on their bioactivities. This method by itself, unfortunately, cannot assign new chemicals (those which may not yet have biological data) to the derived clusters. Owing to the fact that there is a lack of biological data for many chemicals, deep learning models were explored in this project with respect to their ability to correctly assign unknown chemicals to Quantmap clusters. The deep learning methods explored included both convolutional and recurrent neural networks. Transfer learning/pretraining based approaches and data augmentation methods were also investigated. The best performing model, among those considered, was the Seq2seq model (a recurrent neural network containing two joint networks, a perceiver and an interpreter network) without pretraining, but including data augmentation.