Thesis (Ph. D.)--Columbia University, 1939. / Vita. Includes bibliographical references.

No description available.

Os carboidratos são moléculas diversas e complexas que são empregadas por organismos vivos em funções biológicas, como eventos energéticos, estruturais e de sinalização. L-Fucose é um monossacarídeo presente em diversos grupos biológicos, como mamíferos, insetos e plantas. Umas das modificações póstraducionais mais conhecidas contendo L-Fucose são os antígenos do sistema sanguíneo ABO e o leite humano. Alterações no padrão de fucosilação estão relacionadas a patologias como câncer e fucosidose. Essas alterações estão relacionadas ao balanço entre as atividades de α-L-fucosidases e fucosiltransferases. As -L-fucosidases são glicosídeo hidrolases que catalisam a hidrólise de ligações entre resíduos de L-Fucose ligados a outras moléculas. Fucosiltransferases são glicosiltransferases que transferem L-Fucose de GDPfucose para um substrato receptor específico. Embora a importância do metabolismo da fucose, existem poucas informações sobre este assunto em Arthropoda. A literatura indica que fucose, α-L-fucosidases e fucosiltransferases estão envolvidas na interação parasita-hospedeiro em carrapatos, sugerindo que o metabolismo da fucose é essencial para Arachnida. Nosso objetivo foi estudar o metabolismo de fucose em duas espécies de Arachnida: a aranha Nephilingis cruentata e o carrapato Amblyomma sculptum. Ele foi realizado através da caracterização das fucosidases nativas e recombinantes, estrutura e análise in silico, mutagênese sitio-dirigida, padrão de expressão, especificidade e efeito nas células tumorais. Além disso, analisamos as sequências de fucosiltransferases e a expressão por qPCR. As enzimas envolvidas em caminhos metabólicos de fucose também foram investigadas. NcFuc e AsFuc têm um pH ótimo de 5, são inibidas por fucose e fuconojirimicina e apresentam processo de oligomerização dependente de pH. Nós produzimos com sucesso as formas recombinantes dessas enzimas, e elas apresentam as mesmas propriedades cinéticas das formas nativas. A hidrólise de substratos naturais por NcFucr e AsFucr sugerem especificidades diferentes, e ambas conseguiram remover resíduos de fucose de celulares tumorais, reduzindo a invasão celular. Além disso, elas catalisaram reações de transfucosilação. A produção recombinante permitiu a identificação dos resíduos D214 e E59 como díade catalítica em NcFuc. As análises filogenéticas, os dados cinéticos, a modelagem molecular e a especificidade sugerem que os sítios ativos das α-Lfucosidases são diferentes em cada espécie de Arachnida e indicaram que o significado fisiológico da remoção de fucose é diferente entre os organismos. Os ensaios de qPCR evidenciaram que, embora as α-L-fucosidases possam ser enzimas lisossômicas, elas são principalmente expressas no sistema digestório em Arachnida e estão envolvidas na digestão. Representantes de todas as famílias conhecidas de fucosiltransferases foram identificados nos dados do transcriptoma de aranha. No entanto, POFUT1 também foi identificado no nível proteômico e sua análise de expressão indicou uma maior expressão no MG. Isso pode estar relacionado à regeneração de células após a secreção de enzimas digestivas. A análise transcriptômica e proteômica indica que o Arachnida usa vias salvage e de novo para a síntese de fucose. Considerando todos os dados obtidos, concluímos 11 que o metabolismo da fucose está relacionado à digestão em Arachnida, uma vez que eles podem obter a fucose da dieta devido à presença de α-L-fucosidases muito ativas. / Carbohydrates are diverse and complex molecules being employed by living organisms in biological functions such as energetic, structural and signalling events. L-Fucose is a monosaccharide component of many glycans present in a variety of biological groups, such as mammals, insects, and plants. Some of the best-known examples of post-translational modified molecules containing L-Fucose are the ABO blood antigen system and human milk. Changes in the fucosylation pattern are related to pathologies like cancer and fucosidosis. These changes are related to the balance between the activities of α-L-fucosidases and fucosyltransferases. α-Lfucosidases are glycoside hydrolases that catalyse the hydrolysis of glycosidic bonds between residues of L-Fucose to other molecules. Fucosyltransferases are glycosyltransferases which transfer L-Fucose from a GDP-fucose to a specific acceptor. Although the importance of fucose metabolism, there is few information about this subject in Arthropoda. Literature indicates that fucose, α-L-fucosidases and fucosyltransferases are involved in host-pathogen interaction in ticks, suggesting that fucose metabolism is essential to Arachnida. Our aim was to study the metabolism of fucose in two Arachnida species: the spider Nephilingis cruentata and the tick Amblyomma sculptum. This was accomplished through the characterization of native and recombinant fucosidases, structure and in silico analyses, site-directed mutagenesis, expression pattern, specificity and, effect on tumour cells. Besides that, we analysed fucosyltransferases sequences and expression by qPCR. The enzymes involved in fucose metabolic pathways were also investigated. NcFuc and AsFuc have a pH optimum of 5.0, are competitively inhibited by fucose and fuconojirimycin and present an oligomerisation process pH dependent. We successfully produced the recombinant form of these enzymes, and they have the same kinetic properties of native forms. Natural substrate hydrolysis by NcFucr and AsFucr suggest different specificities and they were able to remove fucose residues from tumour cell lines, reducing cell invasion. Moreover, they catalysed transfucosylation reactions. The recombinant production allowed the identification of the residues D214 and E59 as the catalytic dyad in NcFuc. Phylogenetic analysis, kinetic data, molecular modelling, and specificity assays suggest that α-L-fucosidase active sites are different to each Arachnida species and indicated that the physiological significance of fucose removal is different among organisms qPCR assays evidenced that although fucosidases might be lysosomal enzymes, they are mainly expressed at the digestive system in Arachnida and are involved in digestion. Representatives of all known families of fucosyltransferases were identified in the spider MG transcriptome data. However, POFUT1 was also identified at the proteomic level and its expression analysis indicated a higher expression at MG. This might be related to the regeneration of cells after secretion of digestive enzymes. Transcriptomic and proteomic analysis indicate that Arachnida uses both salvage and de novo pathways to fucose synthesis. Considering all the obtained data we concluded that fucose metabolism is related to digestion in Arachnida since they are able to salvage fucose from diet due to the presence of very active α-L-fucosidases.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from PDF title page (viewed on July 16, 2010). Includes bibliographical references (p. 98-111).

The biosynthesis of GDP-Fucose, the activated donor for fucose, has been recently shown to be essential in the parasite Trypanosoma brucei. Fucose is a common sugar modification on eukaryotic glycan structures, but it has not been well described in trypanosomatids. To elucidate the role of fucose in T. brucei we searched for putative fucosyltransferases in this parasite. A single putative T. brucei fucosyltransferase (TbFT) was identified and recombinantly expressed in Escherichia coli. The protein was active and structural characterization of its reaction product identified it as a GDP-Fuc: ß-D-galactose a-1,2-fucosyltransferase with preference for Galß1,3GlcNAc containing structures as glycan acceptors. A procyclic form conditional null mutant for TbFT was generated and this glycosyltransferase shown to be essential for parasite growth in vitro, with the mutant cells displaying a slightly abnormal morphology and an apparent reduction in the surface high molecular weight glycoconjugate complex. Here we also describe the various experimental approaches that were used to try to identify the fucosylated glycocojugates in T. brucei. Lastly, to better understand the biosynthesis of GDP-Mannose, the starting metabolite for the biosynthesis of GDP-Fuc, we biochemically characterized T. brucei phosphomannomutase (TbPMM). Here we show this enzyme could interconvert not only mannose-phosphates, but also glucose-phosphates.

Melanoma is the deadliest form of skin cancer. Prognosis for early stage melanoma patients is excellent, and surgery is often curative for these patients. However, once patients have presented with invasive disease, the average 5-year survival rate drops significantly from over 90% to between 10 and 15%. Several therapies have been developed to target a commonly mutated oncogene BRAF, or its downstream effectors. Unfortunately, while these treatments show robust initial response, most patients relapse within a year. Moreover, therapy-resistant tumors are often more invasive and metastatic. Therefore, it is important to investigate the molecular mechanisms underlying melanoma invasion and metastasis, and to prevent melanoma cell dissemination and metastatic progression. Invadopodia are proteolytic membrane protrusions used by metastatic cancer cells to degrade the extracellular matrix and to facilitate cancer cell invasion and metastasis. In my thesis research I have focused on protein fucosylation and store-operated calcium entry, two separate mechanisms involved in invadopodial regulation. Post translational modifications of proteins are essential for their structure and function. Many cell surface proteins require modifications such as glycosylation for protein-protein interactions, cell adhesion, and signal transduction. Fucosylation is a form of glycosylation that adds L-fucose on glycan structures of proteins. There is evidence indicating that fucosylation plays an important but cancer-type and branching dependent role in cancer progression. Emerging evidence indicates that the fucose salvage pathway and protein fucosylation are altered during melanoma progression and metastasis. Here, we report that the fucose salvage pathway inhibits invadopodia formation and extracellular matrix degradation by promoting α(1,2) fucosylation of cell surface proteins. The activation of the fucose salvage pathway decreases invadopodia numbers and inhibits the proteolytic activity of invadopodia in WM793 melanoma cells. Inhibiting fucokinase, one of the critical enzymes in the fucose salvage pathway, in melanoma cells abrogates L-fucose-mediated inhibition of invadopodia, suggesting dependence on the fucose salvage pathway. The inhibition of invadopodia formation by L-Fucose treatment or fucokinase overexpression could be rescued by treatment with α(1,2), but not α(1,3/4) fucosidase, implicating an α(1,2) fucose linkage-dependent inhibitory effect. The ectopic expression of FUT1, an α(1,2) fucosyltransferase, is sufficient to inhibit invadopodia formation and ECM degradation. Our findings indicate that the fucose salvage pathway can inhibit invadopodia formation, and consequently, invasiveness in melanoma via α(1,2) fucosylation. Re-activation of this pathway in melanoma could be useful for preventing melanoma invasion and metastasis. Calcium is a critical second messenger involved in a multitude of biological processes from cell proliferation to muscle contraction. In melanoma, previous studies have found that activation of the store operated calcium entry (SOCE) channel promotes tumor invasion and metastasis, in vitro and in xenograft models. The expression levels of STIM1, an essential component of the store operated calcium channels, has been found to increase with later stages of melanoma. In melanoma cell lines, the over expression of STIM1 enhances invadopodia number whereas STIM1 knockdown inhibits invadopodia formation. Similarly, gelatin degradation activity is enhanced with STIM1 overexpression and abrogated with STIM1 knockdown, implicating STIM1 as an important factor in the regulation of invadopodia formation and melanoma invasion. Though the studies published have shown a significant role of STIM1 in tumor progression, a robust transgenic animal model has not yet been established. Here, we developed a novel transgenic mouse model which, upon 4-hydroxytamoxifen (4OHT) treatment, induces the BRAFV600E mutation and PTEN, STIM1, and STIM2 deletions in melanocytes via an inducible Cre-lox system. Our investigation found that the loss of STIM1 exacerbates tumor growth and results in tumor formation significantly more quickly than STIM1 wild type mice. Whereas PCR analysis of 4OHT-treated skin showed deletion of STIM1 and PTEN, immunohistochemical staining of these genes in tumors did not convincingly demonstrate complete deletion. Therefore, it remains to be determined whether the effects we observed are due to STIM1 and STIM2 loss. These findings need to be corroborated in the future. Our studies focus on two important mechanisms required for melanoma progression and metastasis. We found that α(1,2) fucosylation is able to inhibit invadopodia formation, and melanoma cell invasion. The reestablishment of α(1,2) fucosylation in melanoma could potentially be exploited to inhibit melanoma metastasis. Additionally, early evidence points to STIM1 having a tumor suppressive role in melanoma oncogenesis and tumor growth based on the transgenic mouse model. Although the phenotype is unexpected, further investigation of this model will likely provide important insight for the complicate roles of SOCE in melanoma initiation and progression.

One important goal of this thesis is the hydrogenation of the glycosyl cyanide, which has never been mentioned by any of the groups who have done work in this area, except for B. Coxon and G. Fletcher, who in 1964, reduced a tetra-O-acetyl-β-D-galactopyranosyl cyanide with lithium aluminum hydride. We were hoping to obtain, by reduction with hydrogen on Pd/C, an aminomethyl C-glycoside. It is believed that these aminomethyl C-glycosides are of potential biological importance especially in the area of AIDS and HIV therapy.

No description available.

Bacterial microcompartments (BMCs) are proteinaceous metabolic compartments found in a wide range of bacteria, whose function it is to encapsulate pathways for the breakdown of various carbon sources, whilst retaining toxic and volatile intermediates formed from substrate breakdown. Examples of these metabolic processes are the 1,2- propanediol-breakdown pathway in Salmonella enterica (Pdu microcompartment), as well as the ethanolamine breakdown pathway in Clostridium difficile (Eut microcompartment). Some of the major challenges to exploiting BMCs as a tool in biotechnology are understanding how enzymes are targeted to microcompartments, as well as being able to engineer the protein shell of BMCs to make synthetic microcompartments that allow specific enzyme pathways to be targeted to their interior. Finally, the metabolic burden imposed by the production of large protein complexes requires a detailed knowledge of how the expression of these systems are controlled. This project explores the structure and biochemistry of an essential BMC pathway enzyme, the acylating propionaldehyde dehydrogenase. With crystal structures of the enzyme with the cofactors in the cofactor binding site and biochemical data presented to confirm the enzyme's substrate. The project also focuses on the creation of synthetic biology tools to enable BMC engineering with a modular library of BMC shell protein parts; forward engineered ribosome binding sites (RBS) fused to BMC aldehyde dehydrogenase localisation sequences. The parts for this library were taken from the BMC loci found in Clostridium phytofermentans and Salmonella enterica. Using a synthetic biology toolkit will allow the rapid prototyping of BMC constructs for use in metabolic engineering. The shell protein parts were used to generate a number of transcriptional units, to assess the effect of overexpression of individual BMC shell components on the morphology of BMCs and the effect these had on their host chassis. Different strength forward engineered RBS and localisation constructs have been designed to assess the possibility of controlling the levels of heterologous proteins targeted to the interior of microcompartment shell to allow metabolic engineering of encapsulated pathways. Along with looking at overexpression of a single shell protein, to assess viability of BMCs as scaffold-like structures, recombinant BMCs can be explored for their utility in bioengineering and their potential role in generating biofuels.

Blood group glycolipids are important tools in the study of microbial receptor interactions and other biological phenomena. Presently blood group glycolipids of interest are isolated from biological samples. However, all glycolipids are not readily available due to the low frequency of some phenotypes in the general population. The ability to acquire the rare glycolipids from the degradation of common glycolipids would be a useful alternative to trying to obtain the molecules from biological sources.This research set out to establish the ability of blood group glycolipids to be degraded into useful glycolipids in a controlled manner by acid hydrolysis and possibly metal catalysis. The initial experiments investigated the degradation/hydrolysis of the more readily available glycolipid globoside with a range of salts and acids to establish degradation concepts such as; temperature, type of acid, acid concentration, and the role of metal ions in glycolipid degradation. These concepts then led to a series of degradation experiments with the blood group glycolipids Leb and ALeb. These glycolipids were incubated with a range of acid concentrations and varying temperatures. Thin layer chromatography separation and chemical and immunochemical staining were the main methods used to identify the products of degradation.It was established that metal ions were not directly involved in the catalysis of glycolipids in the short-term, however some metal ions were indirectly implicated in their degradation due to their ability to form acid solutions. Acid hydrolysis was established as the principle mechanism for glycan chain degradation. In general it was found that the glycan chain primarily lost its fucose groups (in no particular order) and was then followed by sequential degradation of the remaining glycan chain. The glycan chain also appeared to have a protective function on the ceramide moiety. Degradation of globoside established a simple sequential pathway of glycan chain reduction from the non-reducing end. Blood group glycolipids ALeb and Leb first lost their fucose side groups followed by sequential reduction of the glycan chain. Although not fully controllable, degradation of Leb was able to produce Lea, Led and Lec. In contrast degradation of ALeb did not produce any Lea or Led. Instead A-type 1 and two novel A-like structures, 'linear A' and 'GalNAc-Lea' were generated. Lec was only produced from ALeb in extremely acidic conditions. This research established the ability to generate, by acid hydrolysis, a range of rare and "unnatural" novel glycolipids from more commonly available structures. It is of interest that the so-called unnatural glycolipids obtained from the acid hydrolysis of ALeb may, in theory, occur naturally in the acid environment of the stomach, and as such could have the potential to be implicated in disease. It is probable that by applying the principles learned here, a range of novel and natural structures suitable for use in the study of biological interactions can be obtained.