Cell surface carbohydrates play an important role in a wide variety of biological processes such as inflammation, tumor metastasis, and viral and bacterial infection. The goal of our research has been two-fold. The first objective was the synthesis of antimycobacterial compounds. A mannose containing tetrasaccharide from the mannan core of lipoarabinomannan (LAM) of Mycobacterium tuberculosis has been synthesized using α-D-methylmannopyranoside as starting material and Koeings-Knorr reactions to couple saccharides. The synthesis was completed in nine steps and in 14% total yield. This compound should be useful in competitive inhibition studies with macrophages or as an immunological marker. We have successfully synthesized nonsulfated mimics of the aminosterol antibiotic from 5α-cholestan-3-one in two steps in 40-70% total yield. The critical step in this synthesis is the addition of the boronic acid functional group using 2-o-formylphenylboronic acid. It is hypothesized that the addition of boronic acids will improve the antibacterial and anti-angiogenic activity of these compounds. The second objective was the synthesis of a simple fluorescent receptor for simple carbohydrates. A receptor using anthracene as the fluorophore has been completed demonstrating an improved yield over previous methods. This receptor is the first to show selectivity for myo-inositol over other saccharides.

by Luk Choi Wan. / Thesis submitted in: November 2005. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 125-132). / Abstracts in English and Chinese. / Abstract --- p.iv / 摘要 --- p.vi / Acknowledgements --- p.viii / Tabel of contents --- p.x / List of Figures --- p.xii / List of Tables --- p.xiv / List of Abbreviations --- p.xv / List of Abbreviations --- p.xv / Chapter Chapter One --- Literature review --- p.1 / Chapter 1.1 --- General introduction --- p.1 / Chapter 1.2 --- General aspects of plant lectins --- p.4 / Chapter 1.3 --- Monocot mannose-binding lectins --- p.5 / Chapter 1.3.1 --- General introduction --- p.5 / Chapter 1.3.2 --- Sugar specificity --- p.6 / Chapter 1.3.3 --- Isolation and purification --- p.7 / Chapter 1.3.4 --- Molecular cloning --- p.9 / Chapter 1.3.5 --- Molecular structure and modifications --- p.10 / Chapter 1.3.6 --- Molecular evolution --- p.12 / Chapter 1.3.7 --- Transformation --- p.12 / Chapter 1.3.8 --- Physiological roles --- p.14 / Chapter 1.3.9 --- Application --- p.19 / Chapter 1.4 --- Dendrobium nobile --- p.31 / Chapter 1.4.1 --- Background --- p.31 / Chapter 1.4.2 --- Chemical analysis --- p.32 / Chapter Chapter Two --- Biological activities of a mannose-binding lectin isolated from Dendrobium mobile --- p.33 / Chapter 2.1 --- Introduction --- p.33 / Chapter 2.2 --- Materials and methods --- p.35 / Chapter 2.2.1 --- Mannose-binding lectin from D. nobile --- p.35 / Chapter 2.3 --- Biological activities of mannose-binding lectin from D. nobile --- p.36 / Chapter 2.3.1 --- Hemagglutinating activity --- p.36 / Chapter 2.3.2 --- In vitro anti-proliferative assay --- p.37 / Chapter 2.3.3 --- In vitro antiviral assay --- p.40 / Chapter 2.3.4 --- Statistical Analysis --- p.42 / Chapter 2.4 --- Results --- p.43 / Chapter 2.4.1 --- Physiochemical properties of D. nobile lectins --- p.43 / Chapter 2.4.2 --- Cytotoxicity to cancer cell lines --- p.53 / Chapter 2.4.3 --- Antiviral activity --- p.59 / Chapter 2.5 --- Discussion --- p.60 / Chapter Chapter Three --- Molecular cloning of lectin gene of Dendrobium nobile --- p.65 / Chapter 3.1 --- Introduction --- p.65 / Chapter 3.2 --- Methods --- p.67 / Chapter 3.2.1 --- RNA extraction --- p.67 / Chapter 3.2.2 --- RT-PCR synthesis of D. nobile lectin cDNA --- p.68 / Chapter 3.2.3 --- RACE of D. nobile agglutinin gene --- p.69 / Chapter 3.2.4 --- Generation of DNL full-length lectin gene sequence --- p.70 / Chapter 3.2.5 --- Cloning and sequencing of PCR product --- p.71 / Chapter 3.2.6 --- Data analyses --- p.72 / Chapter 3.2.7 --- Synthesis of single-stranded DIG-labeled DNA probe --- p.74 / Chapter 3.2.8 --- Northern blot analysis --- p.74 / Chapter 3.2.9 --- Genomic DNA extraction --- p.75 / Chapter 3.2.10 --- Southern blot analysis --- p.76 / Chapter 3.2.11 --- Expression of DNL in E. coli --- p.76 / Chapter 3.2.12 --- Western blot analysis --- p.78 / Chapter 3.3 --- Results --- p.80 / Chapter 3.3.1 --- Isolation and characterization of DNL gene --- p.80 / Chapter 3.3.2 --- Sequence analysis of DNL --- p.89 / Chapter 3.2.3 --- Secondary and tertiary structure --- p.96 / Chapter 3.2.4 --- Southern blot analysis --- p.99 / Chapter 3.2.5 --- Northern blot analysis --- p.99 / Chapter 3.2.6 --- Expression of fusion protein in E.coli --- p.102 / Chapter 3.4 --- Discussion --- p.104 / Chapter Chapter Four --- General Discussion --- p.110 / Chapter 4.1 --- General discussion --- p.110 / Chapter 4.2 --- Isolation and Characterization of monocot mannose-binding lectin of D. nobile --- p.111 / Chapter 4.3 --- Molecular cloning of monocot mannose-binding lectin of D. nobile --- p.115 / Chapter 4.4 --- Further investigations --- p.122 / Chapter Chapter Five --- Conclusion --- p.124 / References: --- p.125 / Appendix --- p.133

A leishmaniose visceral (LV) ou calazar é uma doença endêmica, crônica, grave e de alta letalidade se não tratada. Os estudos apontam a proteína Lectina Ligante de Manose (MBL), codificada pelo gene MBL2, como uma peça-chave na imunidade inata, dada a sua função no reconhecimento microbiano, na eliminação, inflamação e morte celular. Neste trabalho realizamos um estudo do tipo caso-controle que teve como objetivo investigar a associação entre variantes no gene MBL2 e a suscetibilidade à LV em indivíduos residentes em áreas endêmicas da Ilha de São Luís-MA. A amostra foi constituída por 322 indivíduos, sendo 161 casos com LV, não aparentados, de ambos os sexos, residentes em áreas endêmicas da doença na Ilha de São Luís e 161 controles saudáveis, não infectados e não aparentados da mesma região. A identificação dos casos de LV se deu por meio do contato constante com os principais hospitais e ambulatórios de referência para a doença na cidade. Também foram feitas buscas de pacientes com LV em ambiente domiciliar, a partir de registros da FUNASA-MA. A análise molecular consistiu na genotipagem de 6 variantes localizadas na região promotora [posições -550 (C>G), -221(G>C), +4(C>T)] e codificadora [códons 52 (C>T), 54 (G>A) e 57 (G>A)] do gene MBL2, através da reação em cadeia da polimerase e sequenciamento automático. A dosagem da proteína MBL no soro foi realizada pelo teste de ELISA. Verificamos que os fenótipos MBL dependem do conjunto de alelos presentes no gene MBL2, sendo nítido o efeito que as variantes defectivas causam nos níveis da proteína. Não encontramos diferença significativa entre casos e controles em relação à distribuição dos genótipos MBL2 e dos níveis séricos de MBL. As frequências alélicas das variantes exônicas na amostra total mostram que o alelo A é o mais comum (74,8%) e que os alelos defectivos (B, C e D) se encontram principalmente em heterozigose (36,6%), o que reforça a ideia de que alelos MBL2 defectivos são mantidos na população por conferirem vantagem seletiva aos heterozigotos. Em relação aos 3 principais polimorfismos existentes na região promotora, verificamos ser a variante -221G (Y) a mais frequente (88%) seguida de +4C (P) (73%) e de -550C (L) (67%). Identificamos oito haplótipos em MBL2 num total de 644 cromossomos avaliados, em 30 combinações diferentes, sendo HYPA e LYQA os mais frequentes e HYPD e HYPB os mais raros. Todos os portadores de combinações de haplótipos homozigotos para alelos defectivos apresentaram níveis séricos de MBL indetectáveis. Os genótipos LYQA/LYQA e HYPA/HYPA apresentaram as maiores concentrações médias de MBL no soro. A combinação entre SNPs no éxon 1 e na região promotora do gene MBL2 resulta em grande variação nas concentrações de MBL em indivíduos saudáveis. Consideramos que o conjunto de dados gerados é uma contribuição valiosa que poderá ser expandida para outros cenários. / Visceral leishmaniasis (VL), also known as kalazar, is an endemic, chronic, severe and highly lethal disease when not treated. Studies have shown that the protein Mannose-biding lectin (MBL), encoded by the gene MBL2, is the major player in innate immune system due its role in microbial recognition, elimination and inflammation as well as in the cell death. In the current work, we conducted a case-control study which aimed to investigate the association between variants in the gene MBL2 and the susceptibility to VL in individuals living in endemic areas of the São Luís - MA. 322 individuals participated in this study. Of these, 161 were VL cases being unrelated individuals of both sexes, and inhabitants from endemic areas of the disease in São Luís. The other 161 individuals were uninfected healthy controls, being unrelated and from the same region. The identification of VL cases occurred by visiting reference hospitals and clinics in the city. VL patients were identified in the household environment through the records of FUNASA-MA. Molecular analysis consisted in genotyping six variants located in the promoter region [positions -550 (C> G), -221 (G> C), +4 (C> T)] and coding region [codons 52 (C> T), 54 (G> A) and 57 (G> A)] of the MBL2 gene by polymerase chain reaction and automated DNA sequencing. The concentrations of MBL protein in the serum was performed by ELISA. We found that MBL phenotypes depend on the number of alleles present in the gene MBL2, being clear the consequence of the defective variants in the protein levels. There was no significant difference between cases and controls regarding the distribution of MBL2 genotypes and MBL serum levels. The allele frequencies of exon variants in the overall sample showed that the A allele is the most common (74.8%) and that the defective alleles (B, C and D) are mainly heterozygous (36.6%). This highlights the idea that defective MBL2 alleles are maintained in the population to confer selective advantage to heterozygotes. Concerning the three main existing polymorphisms in the promoter region, we noticed that the variant-221G (Y) is more frequent (88%) followed by +4 C (P) (73%) and 550C-(L) (67%) variants. We identified eight haplotypes in MBL2 in a total of 644 chromosomes evaluated in 30 different combinations, being the HYPA and LYQA the most frequent haplotypes and HYPD and HYPB the rarest ones. All carriers with combinations of homozygous haplotypes for defective alleles had undetectable serum levels of MBL. Genotypes LYQA / LYQA and HYPA / HYPA had the highest mean concentrations of MBL in the serum. Combination between SNPs in exon 1 and in the promoter region of the gene MBL2 results in a great variation of MBL concentrations in healthy individuals. We consider that the data set that was generated is a valuable contribution that can be expanded to others cenarios.

Cancer immunotherapy is an approach to cancer therapy that involves the enhancement of the cancer patient’s own innate and/or adaptive immune systems to attack their own cancer. Clinically available cancer immunotherapies rely on different strategies: infusion of ex vivo manipulated autologous dendritic cells (DCs), infusion of genetically engineered autologous cytotoxic CD8+ T lymphocytes, stimulation of T lymphocyte proliferation, or inhibition of immunosuppressive pathways to improve T lymphocyte effector function. Nonetheless, only a small percentage of cancer patients receive benefit from immunotherapies and thus further improvements in clinical outcomes are required. Among numerous other therapeutic immunotherapies strategies being developed and tested, adenovirus serotype 5-based vectors (Ad5) have been well studied in preclinical and clinical settings. Preclinical research has shown that vaccination of mice with Ad5-OVA (an Ad5 encoding a model tumor antigen, chicken ovalbumin (OVA)) results in activation and proliferation of OVA-specific CD8+ T lymphocytes capable of specific killing of tumor cells that express OVA. This dissertation evaluates the potential of polymeric nanoparticles (NP) loaded with all-trans retinoic acid (ATRA), a vitamin A derivative with potent immunostimulatory effects, to improve the immunostimulatory and therapeutic effects of Ad5-OVA in a murine E.G7-OVA tumor model, a well described model that can be used for studying the immune response to Ad5-based immunotherapies. In the first part of this work, poly(lactide-co-glycolide) NP loaded with ATRA (ATRA-PLGA-NP) were prepared and characterized. Next, the antitumor effect and the magnitude of the OVA-specific immune response due to Ad5-OVA vaccination versus ATRA-PLGA-NP (or ATRA soluble) plus Ad5-OVA combination treatment were compared in vivo. The results showed that the combination treatment using ATRA-NP, but not ATRA soluble, resulted in enhanced survival and enhanced levels of OVA-specific CD8+ T lymphocytes in peripheral blood, spleen, and tumor. Next, cRGD- and mannose-functionalized PLGA-PEG NP were developed in an attempt to actively target the tumor neovasculature and DC-rich organs, respectively. The functionalization efficacy was confirmed by ex vivo fluorescence imaging studies. In vivo studies using E.G7-OVA-challenged mice showed that treatment with ATRA-loaded cRGD-functionalized PLGA-PEG-NP + Ad5-OVA, despite not enhancing the levels of OVA-CD8+ T lymphocytes in peripheral blood, substantially enhanced survival compared to either the combination of Ad5-OVA + non-functionalized ATRA-PLGA-PEG-NP or Ad5-OVA + conventional ATRA-PLGA-NP. On the contrary, treatment with mannose-functionalized PLGA-PEG-NP + Ad5-OVA, despite optimally enhancing the levels of OVA-CD8+ T lymphocytes in peripheral blood (compared to all other treatment groups), did not lead to enhanced survival compared to either the combination of Ad5-OVA + non-functionalized ATRA-PLGA-PEG-NP, Ad5-OVA + conventional ATRA-PLGA-NP, and over Ad5-OVA treatment alone. Although not investigated further in this dissertation, it was speculated that the observed trend in survival benefit provided by ATRA-PLGA-PEG-cRGD-NP + Ad5-OVA over the other NP formulations may have been due to higher levels of ATRA within the TME due to actively targeting the tumor vasculature, corroborating previous studies which demonstrated that ATRA functions as a potent stimulator of anti-tumor cellular immune responses within the tumor. The paradoxical results obtained with mannose-functionalized PLGA-PEG-NP are less readily explained. In conclusion, it was demonstrated in this work that the co-administration of Ad5-OVA and ATRA-loaded NP formulations enhanced the tumor specific cellular immune response and the survival of tumor challenged mice compared to vaccination with Ad5-OVA alone.

Candida albicans, endo-saprophyte du tube digestif, est responsable d’infections superficielles ou invasives. Les candidoses invasives (CI) constituent un problème persistant de santé publique avec une mortalité attribuable de 40% en réanimation et une morbidité exponentielle liée à la consommation d’antifongiques ou aux séjours prolongés des patients. Cet impact médico-économique est principalement lié aux difficultés diagnostiques. En effet, ni la symptomatologie, ni l’imagerie ne sont spécifiques des CI. Le diagnostic biologique demeure complexe et souvent tardif. Les tests conventionnels (hémoculture) sont peu sensibles, et les tests alternatifs à la culture manquent souvent de sensibilité ou de spécificité. Dans ce contexte, il est nécessaire de développer de nouvelles stratégies diagnostiques en mesure d’améliorer la précocité du diagnostic, d’identifier les sujets à haut risque d’infection et de permettre la mise en place d’un traitement antifongique approprié. Notre travail a porté sur deux médiateurs circulants de l’immunité anti-Candida, la mannose-binding lectin (MBL) et les anticorps anti- proteines (Ab) en relation le diagnostic des CI.En ce qui concerne la détection d’anticorps, nous avons produit 6 protéines recombinantes (Hwp1, Eno1, Hsp90, Fba1, Mp65 et Sod5), surexprimées pendant la phase pathogène de C. albicans et développés 5 prototypes de tests EIA. L’association de ces biomarqueurs avec la détection du mannane circulant (Mn) par EIA a permis d’identifier 3 protéines à fort potentiel diagnostique. Pour une spécificité fixée arbitrairement à 80%, la sensibilité des associations Fba1-Ab/Mn, Hwp1-Ab/Mn et Hsp90-Ab/Mn étaient de 84%, 84% et 81% respectivement. Le délai moyen de positivité était de 5 jours avant l’isolement de Candida d’hémoculture. Les Odds ratios associés aux trois combinaisons de marqueurs étaient de 108, 65 et 77 pour Fba1-Ab/Mn, Hwp1-Ab/Mn et Hsp90-Ab/Mn.En ce qui concerne la MBL, nous avons montré i) une évolution dynamique de la MBL au cours des CI, ii) une interaction directe avec le mannane pariétal de levure ainsi qu’une iii) prédisposition à la colonisation chez les patients déficients en MBL. Outre les aspects quantitatifs, nous avons mis en place un modèle d’évaluation fonctionnels de la MBL par resonance plasmonique de surface (SPR) en impliquant des cellules entières ou des glycannes fongiques issus de la paroi de C. albicans ou des oligommanosides de synthèse. Ce modèle nous a permis de mieux caractériser les épitopes reconnus par la MBL et de montrer l’interaction de cette lectine avec un panel d’espèces Candida communément isolées en pathologie humaine.Ces médiateurs de l’immunité humorale ou innée permettent d’envisager des perspectives cliniques en termes diagnostique ou pronostique. Ils permettent également d’améliorer notre compréhension de la physiopathologie des CI et d’optimiser la prise en charge des patients. / Candida albicans, digestive tract endo-saprophyte, is responsible of superficial and invasive infections. Invasive candidiasis (IC) is a persistent public health with an attributable mortality of 40% in intensive care unit and an exponential morbidity related to the antifungals consumption and extended hospital stays of patients. This medico-economic impact is mainly related to diagnostic difficulties. Indeed, neither clinical symptoms nor the imaging are specific of IC. Biological diagnosis remains complex and often late. Conventional tests (blood culture) have a low sensitivity, and non-culture based methods often lack sensitivity or specificity. In this context, it’s necessary to develop new diagnostic strategies to improve the early diagnosis, to identify patients with high risk of infection and to lead the prescription of an appropriate antifungal therapy. Our work has focused on two circulating anti-Candida immunity mediators, mannose-binding lectin (MBL) and anti-protein antibodies (Ab) in relation to IC diagnosis. Regarding the detection of antibodies, we produced six recombinant proteins (Hwp1, Eno1, Hsp90, FBA1, Mp65 and Sod5) overexpressed during the C. albicans pathogen phase and developed five prototypes of EIA. The association of these biomarkers with the detection of circulating mannan (Mn) revealed three proteins with high diagnostic performances. For a specificity fixed arbitrarily at 80%, the sensitivity of combinations of Fba1-Ab/Mn, Hwp1-Ab/Mn and Hsp90-Ab/Mn were 84%, 84% and 81%, respectively. The average delay of positivity was 5 days before isolation of Candida from blood culture. Odds ratios associated with these three combinations of markers were 108, 65 and 77 for Fba1-Ab/Mn, Hwp1-Ab/Mn and Hsp90-Ab/Mn, respectively. Our findings on serum MBL have shown i) dynamic evolution of MBL levels during IC, ii) direct interaction with circulating cell wall mannan and iii) susceptibility to colonization in MBL deficient patients. Besides quantitative aspects, we have developed a model for assessing the MBL functionality by surface plasmon resonance (SPR) involving whole cells, fungal cell wall glycans from C. albicans cell wall or synthetic oligommanosides. This model was used to better characterize the epitopes recognized by MBL and showed the interaction of this lectin with a panel of Candida species commonly isolated in human pathology.These mediators of innate or humoral immunity open new clinical perspectives in terms of diagnostic or prognosis of IC. These findings may participate to improve our understanding of IC pathophysiology and to optimize management of patients.

Several lactic acid bacteria contain β-galactosidases. Beta galactosidases catalyze lactose hydrolysis and transfer acceptor sugars onto galactose, producing galactooligosaccharides. The aim of this work was to exploit β-galactosidases of lactic acid bacteria as crude cell extracts to produce novel oligosaccharides with mannose, N-acetylglucosamine, and fucose. Of 17 strains of lactic acid bacteria, transferase activity was the strongest in crude cell extracts of Lactobacillus delbrueckii subsp. bulgaricus, followed by Streptococcus thermophilus, Lactobacillus animalis, and Lactobacillus reuteri in a buffered 19% (w/w) lactose solution. Incorporation of 6 % (w/w) glycerol increased transferase activity and enzyme stability at higher incubation temperatures. Incorporation of 10% (w/w) mannose, N-acetylglucosamine and fucose as acceptor sugars yielded three distinct oligosaccharides with mannose and two with N-acetylglucosamine and fucose, with the composition confirmed using gas chromatography-mass spectrometry. This is the first public report indicating production of oligosaccharides containing N-acetylglucosamine and fucose from β-galactosidases of lactic acid bacteria.

Cancer vaccines have shown little success in clinic. Dendritic cells (DCs) are of particular interest in cancer vaccination due to their role in cell-mediated immunity. Active targeting of DCs, through PLGA nanoparticles (PLGA-NPs) decorated with ligands for DC-expressed mannose receptor (MR) can enhance internalization, processing and presentation of antigens and subsequent immnuostimulation. In this study we have shown PLGA-NPs decorated with mannan and the synthetic hydrophobized mannan, especially those with covalent attachment, can target DCs leading to increased uptake of nanoparticles and DC maturation. This approach may be used for improved delivery of antigens and adjuvants to DCs and development of more efficient cancer vaccines. Moreover, significant progress in cancer vaccination requires immunomonitoring. Live imaging using a Positron Emission Tomography (PET) probe encapsulated in PLGA-NPs can elucidate dynamics of recruitment and fate of DCs to develop successful vaccines. The PET-nanoprobe prepared by radio-iodinated 5-IDFPdR demonstrated uncontrolled high burst release implying low quality images. / Pharmaceutical Sciences

No description available.

Thesis (Ph. D.)--Institute of Paper Chemistry, 1956. / Includes bibliographical references (p. 58-60).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 96-101).