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Made available in DSpace on 2014-06-11T19:33:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-11-30Bitstream added on 2014-06-13T20:45:01Z : No. of bitstreams: 1 kim_yj_dr_arafo.pdf: 1176074 bytes, checksum: 46110c11d5e206c41d176d1fef40ea28 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Artin M é uma lectina purificada de sementes de Artocarpus heterophyllus que, recentemente, mostrou-se capaz depromover aceleração da cicatrização de lesões por queimadura de pele ou por abrasão da córnea em ratos e coelhos. O objetivo desta tese foi avaliar os efeitos da Artin M no processo de reparação da mucosa mastigatória bucal, por meio de modelos de estudo in vivo. Primeiro estudo: Três feridas cirúrgicas circulares de 6 mm de diâmetro foram criadas na mucosa palatina de 20 cães, e divididas aleatoriamente em 3 grupos de acordo com os tratamentos: C – controle (não tratados), A - Artin M, V - veículo. Quatro animais de cada grupo foram sacrificados após 2, 4, 7, 14 e 21 dias póstratamento e suas maxilas analisadas clinicamente quanto ao padrão de cicatrização, seguido de análise histológica, imuno-histoquímica para antígeno nuclear de proliferação celular (PCNA) e atividade de mieloperoxidase. Clinicamente, o grupo A demonstrou melhor cicatrização em todos os períodos quando comparada aos outros grupos (p<0,05). A análise histológica mostrou ter havido no grupo A maior estimulação na produção de fibras colágenas, maturação do tecido de granulação e organização do epitélio. A imunolocalização de PCNA mostrou uma maior tendência na proliferação celular em lesões do grupo A principalmente nos dias iniciais (p<0,05). O influxo de neutrófilos mostrou-se estatisticamente aumentado no grupo A quando comparado aos outros grupos nos dias 2 e 4 (p<0,05). Conclui-se que a Artin M promoveu aceleração na reparação das feridas na mucosa mastigatória em cães, via recrutamento de neutrófilos e indução da proliferação celular. É bem conhecido o envolvimento de mediadores biológicos como citocinas... / Artin M, lectin from Artocarpus heterophyllus seeds, has been demonstrated to stimulate recruitment and activation of neutrophil and mast cells. Furthermore, it has been shown to accelerate the process of wound healing on burn injuries and corneal epithelial lesions in rats and rabbits, respectively. The aim of this study was to evaluate the effects of Artin M on wound healing in palatal mucosa in two animal models. Study 1: Three wounds of 6 mm full thickness were surgically created in the hard palate mucosa of twenty dogs. The wounds of each animal were randomly divided into three groups according to one of the treatment assigned: C– Control (nontreated), A- Artin M gel, and V– Vehicle (carboxymethylcellulose 3% gel). Four animals per group were sacrificed at 2, 4, 7, 14 and 21 days post-surgery. Before sacrifice, wounded areas were photographed and scored for macroscopic healing evaluation. Afterwards, maxillary tissue were harvested and divided to perform histological analysis, immunohistochemical staining against Proliferating Cell Nuclear Antigen (PCNA) and measurement of myeloperoxidase activity. Clinical analysis showed that wound closure was accelerated in group A in comparison to other groups in all periods. Histological features showed enhanced reepithelization and collagen fiber formation resulting in faster maturation of granular tissue in group A compared to the other groups, by day 14. Artin M treatment significantly induced cells proliferation at day 2 and 4 (p<0.05). Neutrophils infiltration in the group A was significantly higher than in the other groups at days 2 and 4 (p<0.05). The results suggest that Artin M gel may potentially facilitate wound healing on palatal mucosa via the recruitment of neutrophils and promotion of cells proliferation. It is well known that cytokines and growth... (Complete abstract click electronic access below)

Thesis (Masters Diploma (Medical Technology) -- Cape Technikon, Cape Town, 1992 / Gastric cancer, a fatal malignancy, is endemic in the Coloured population of the Western Cape region of South Africa. Diagnosis is based mainly on histologic investigation with patients of either sex being mainly between 40-60 years of age. The extent to which genetic and environmental influences play a role in the aetiology of the disease is unknown. This study is an attempt to biochemically characterise gastric mucins or mucus glycoproteins, (the main gel forming components of crude mucus scrapings off the mucosal surface), in carcinoma of the stomach (HCA), as compared to those in ulcer disease (HGU), post mortem specimens (PM) and samples obtained from organ transplant donor stomachs (HD). The aim of this study is the development of a diagnostic marker within mucus secretions for the detection of pre-malignant disease amongst the high risk population of the Western Cape region of South Africa. Mucins were extracted from crude mucus gel scrapings according to a carefully designed technique in which proteolytic inhibitors were used to minimise the possibility of endogenous proteolysis in the laboratory through possible contamination. Two density gradient ultra-centrifugation steps for 48 hours each at 105,000g in caesium chloride, a well established standard isolation procedure for mucins, gave a yield of pure mucins which fractionated at a density of approximately 1.41gjml in all groups. These mucins, from the HO, PH, HGU and HCA groups eluted mainly in the included volume of a Sepharose 2B column as broad, polydisperse peaks, suggesting that they were degraded and comprised mainly lower molecular weight PAS positive material in relation to large polymeric gel forming mucin.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / O cÃncer de mama representa o tipo de cÃncer mais comum entre as mulheres no mundo e no Brasil, depois do cÃncer de pele nÃo melanoma. Caracterizando-se como uma doenÃa clÃnica e biologicamente heterogÃnea, a detecÃÃo precoce do cÃncer de mama, uma caracterizaÃÃo fidedigna da doenÃa e um diagnÃstico preciso sÃo de fundamental importÃncia para reduÃÃo da mortalidade. Assim, o objetivo do presente trabalho foi realizar anÃlises qualitativas e quantitativas de proteÃnas plasmÃticas de mulheres saudÃveis e mulheres com cÃncer de mama tipo ductal, em diferentes estÃgios. Para isso, inicialmente, as amostras de plasma foram depletadas de albumina e IgG e depois reunidas em pools representativos para cada grupo em estudo. Os pools foram entÃo fracionados por meio de cromatografias de afinidade em matrizes de Sepharose 4B imobilizadas com as lectinas vegetais &#945;-galactose-ligante de Artocarpus incisa - Frutalina e glucose/manose-ligante de Dioclea altÃssima â DAL. Posteriormente, tantos os pools como as fraÃÃes cromatogrÃficas obtidas foram digeridas e submetidas à anÃlise independente de dados (MSE) e quantificadas (label-free) por espectrometria de massas. A utilizaÃÃo das cromatografias de afinidade com lectinas vegetais a priori da anÃlise por espectrometria de massas foi fundamental para reduzir a complexidade das amostras e estender o intervalo dinÃmico de proteÃnas, e frutalina apresentou os melhores resultados de fracionamento. Um somatÃrio de todas as anÃlises realizadas revelou a identificaÃÃo de cerca de 445.000 peptÃdeos e mais de 30.000 proteÃnas, com redundÃncia entre isoformas do mesmo grupo e entre grupos. Ademais, alÃm de fracionar, as cromatografias de afinidade com lectinas vegetais foram eficientes em isolar glicoformas especÃficas que refletem um padrÃo de glicosilaÃÃo alterado associado à doenÃa. Diversas proteÃnas diferencialmente expressas, algumas delas com mudanÃas na glicosilaÃÃo, foram identificadas, tais como apolipoproteÃna A2, apolipoproteÃna C3, fatores do sistema complemento (C3, C4b e C4A), clusterina, &#945;-1-2-Ãcido glicoproteÃna, haptoglobina, hemopexina, paraoxonase arilesterase sÃrica, proteÃna plasmÃtica de ligaÃÃo ao retinol, plasminogÃnio e vitronectina. Dentre as proteÃnas identificadas, algumas estÃo relacionadas com a decomposiÃÃo da matriz extracelular, metabolismo lipÃdico, estresse oxidativo e outras caracterizam-se como proteÃnas de fase aguda. Finalmente, os dados de expressÃo diferencial e possÃveis alteraÃÃes de glicosilaÃÃo das proteÃnas contribuem para o desenvolvimento de um perfil protÃico, alvo para posterior validaÃÃo, que apresenta uma associaÃÃo com o desenvolvimento e a caracterizaÃÃo do cÃncer de mama em seus diferentes estÃgios. / The breast cancer presents one of the most commonly diagnosed types of cancer among women worldwide and in Brazil, after nonmelanoma skin cancer. Itâs a clinically and biologically heterogeneous disease. Therefore, early detection of breast cancer, a reliable characterization of the disease and an accurate diagnosis are crucial to reducing mortality. The aim of this work was perform a qualitative and quantitative analyzes of plasma proteins from healthy women and from women with ductal breast cancer in different stages. For this, initially, plasma samples were depleted of IgG and albumin and then pooled into samples representative for each study group. The pools were then fractionated by immobilized plant lectins (frutalin and DAL)-affinity chromatography. Subsequently, the pools and chromatographic fractions were digested and submited to and data-independent label-free mass spectrometric analysis. The use of affinity chromatography with plant lectins prior to analysis by mass spectrometry was essential to reduce sample complexity and extend the dynamic range of proteins, and frutalin showed the best results from fractionation. A sum of all analyzes performed revealed the identification of about 445,000 peptides and more than 30,000 proteins, with redundancy between isoforms of the same group and between groups. Furthermore, in addition to fractionation, the chromatography of affinity with plant lectins were effective in isolating specific glycoforms that reflect an altered glycosylation pattern associated with the disease. Several differentially expressed proteins, some of which changes in glycosylation were identified, such as apolipoprotein A2, apolipoprotein C3, complement factors (C3, C4b and C4A), clusterin, 1-2-&#945;-acid glycoprotein, haptoglobin, hemopexin, serum paraoxonase arylesterase, plasma retinol binding protein, plasminogen and vitronectin. Among the proteins identified, some are related to the breakdown of the extracellular matrix, lipid metabolism, oxidative stress and other characterized as acute phase proteins. Finally, the data of differential expression and possible changes in the glycosylation patterns of proteins contributes to the development of a protein profile, subject to later validation, presenting an association with the development and characterization of breast cancer in its different stages.

Envelope proteins of the human immunodefiency virus (HIV) use the cell surface CD-4 molecule of target cells to initiate infection which eventually lead to the acquired immunodeficiency syndrome (AIDS). HIV-1 strains form three groups, namely the M, N and O, with the former group further divided into at least ten equidistant subtypes or clades (i.e. A through J) classified on the basis of sequence homologies in the envelope gene. Recombinant envelope proteins expressed in transfected Chinese hamster ovary (CHO) cells were isolated and purified here (~ 0.01 mg yield). An economical but efficient purification procedure using affinity chromatography and freeze-drying was developed. The results obtained through SDS-PAGE, western blotting, specific ELISA (using Galanthus nivalis a lectin with affinity for ENV glycoproteins) and partial sequencing confirmed the purity (~ 85 - 90 %) and identity of the proteins. Since these proteins were derived in a clade A (Uganda) and B (USA) environment we anticipated limited crossreactivity with immune responses induced in a subtype C (RSA) environment. This was assessed using ELISA (titers of 1000) and western blot analysis. The ability to induce apoptosis was used to demonstrate functionality of the purified protein (Results showed that in-vitro induction of apoptosis (65 %) using the continuous cell line PM1 was achieved). / Dr. Debra Meyer

The aim of the present study was to assess whether the changes in the epithelial glycoproteins seen in the mucosa adjacent to tumors are specific premalignant markers or secondary reactive phenomena. A secondary objective was to assess whether ulcerative colitis and Crohn's Disease could be distinguished from one another histochemically. The carbohydrate prosthetic groups from colonic epithelial glycoproteins were characterized histologically and histochemically from 17 cases of ulcerative colitis, 21 cases of Crohn's Disease and 19 cases of diverticular disease. Two histochemical parameters - the relative proportion of sulpho- and sialomucin and the side-chain substitution pattern of O-acetylated sialic acid - were assessed using a battery of seven histochemical techniques. Serial sections from each specimen were also evaluated morphologically, using hematoxylin and eosin. In addition, the patterns of O-acetylated side-chain sialic acid from the three inflammatory bowel diseases were compared to data previously acquired from the mucosa adjacent to colonic tumors. Results indicate that neither focal changes nor the predominance of sialomucins are specific to the mucosa adjacent to tumors. As well, changes in one histochemical parameter were independent of changes in the other parameter. No histochemical class of epithelial glycoproteins was specific to any of the inflammatory bowel diseases and, therefore, it was not possible to distinguish between ulcerative colitis and Crohn's Disease on the basis of the histochemical techniques used in the present study. It was also noted that the histochemical changes in ulcerative colitis, Crohn's Disease and diverticular specimens were not related to the degree of inflammation. Finally, as a group, Crohn's Disease specimens showed a loss of sulphomucin-sialomucin gradient along the length of the crypts. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Tissues are heterogeneous ecosystems comprised of various cell types. For example, in tumor tissues, malignant cancer cells are surround by various non-malignant stromal cells. Proteins, especially N-linked glycoproteins, are key players in tumor microenvironment and respond to many extracellular stimuli for involving and regulating intercellular signaling. Understanding the human proteome and glycoproteome in heterogeneous tissues with spatial resolution are meaningful for exploring intercellular signaling networks and discovering protein biomarkers for various diseases, such as cancer. In this study, we aimed to develop new liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based analytical methods for spatially-resolved proteome and glycoproteome profiling in tissue samples, and apply them for profiling potential biomarkers for pancreatic cancer. We first systematically and synchronously optimized the LC-MS parameters to increase peptide sequencing efficiency in data dependent proteomics. Taking advantage of its hybrid instrument design with various mass analyzer and fragmentation strageties, the Orbitrap Fusion mass spectrometer was used for systematically comparing the popular high-high approach by using orbitrap for both MS1 and MS2 scans and high-low approach by using orbitrap for MS1 scan and ion trap for MS2 scans. High-high approach outperformed high-low approach in terms of better saturation of the scan cycle and higher MS2 identification rate. We then systematically optimized various MS parameters for high-high approach. We investigated the influence of isolation window and injection time on scan speed and MS2 identification rate. We then explored how to properly set dynamic exclusion time according to the chromatography peak width. Furthermore, we found that the orbitrap analyzer, rather than the analytical column, was easily saturated with higher peptide loading amount, thus limited the dynamic range of MS1-based quantification. Finally, by using the optimized LC-MS parameters, more than 9000 proteins and 110,000 unique peptides were identified by using 10 hours of effective LC gradient time. The study therefore illustrated the importance of synchronizing LC-MS precursor targeting and high-resolution fragment detection for high-efficient data dependent proteomics. Understanding the tumor heterogeneity through spatially resolved proteome profiling is meaningful for biomedical research. Laser capture microdissection (LCM) is a powerful technology for exploring local cell populations without losing spatial information. Here, we designed an immunohistochemistry (IHC)-based workflow for cell type-resolved proteome analysis of tissue samples. Firstly, targeted cell type was stained by IHC using antibody targeting cell-type specific marker to improve accuracy and efficiency of LCM. Secondly, to increase protein recovery from chemically crosslinked IHC tissues, we optimized a decrosslinking procedure to seamlessly combine with the integrated spintip-based sample preparation technology SISPROT. This newly developed approach, termed IHC-SISPROT, has comparable performance with traditional H&E staining-based proteomic analysis. High sensitivity and reproducibility of IHC-SISPROT was achieved by combining with data independent proteomic analysis. This IHC-SISPROT workflow was successfully applied for identifying 6660 and 6052 protein groups from cancer cells and cancer- associated fibroblasts (CAFs) by using only 5 mm 2 and 12 μm thickness of hepatocellular carcinoma tissue section. Bioinformatic analysis revealed the enrichment of cell type-specific ligands and receptors and potentially new communications between cancer cells and CAFs by these signaling proteins. Therefore, IHC-SISPROT is sensitive and accurate proteomic approach for spatial profiling of cell type-specific proteome from tissues. N-linked glycoproteins are promising candidates for diagnostic and prognostic biomarkers and therapeutic targets. They often locate at plasma membrane and extracellular space with distinct cell type distribution in tissue microenvironment. Due to access to only low microgram of proteins and low abundance of glycoproteins in tissue sections harvested by LCM, region- and cell type-resolved glycoproteome analysis of tissue sections remains challenging. Here we designed a fully integrated spintip-based glycoproteomic approach (FISGlyco) which achieved all the steps for glycoprotein enrichment, digestion, deglycosylation and desalting in a single spintip device. Sample loss is significantly reduced and the total processing time is reduced to 4 hours, while detection sensitivity and label-free quantification precision is greatly improved. 607 N-glycosylation sites were successfully identified and quantified from only 5 μg of mouse brain proteins. By seamlessly combining with LCM, the first region-resolved N-glycoproteome profiling of four mouse brain regions, including isocortex, hippocampus, thalamus, and hypothalamus, was achieved, with 1,875, 1,794, 1,801, and 1,417 N-glycosites identified, respectively. Our approach could be a generic approach for region and even cell type specific glycoproteome analysis of tissue sections. Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with five year survival rate of around 8%. No effective biomarkers and targeted therapy are one of the major reasons for this urgent clinical situation. To explore potential protein biomarkers and drug targets located at intercellular space of pancreatic tumor microenvironment, we established chemical proteomic approach for deep glycoproteome profiling of PDAC clinical tissue samples based on the above- mentioned new proteomic methods. Taking advantage of a long chain biotin- hydrazide probe with less space hindrance, the new method outperformed traditional hydrazide chemistry method in terms of sensitivity, time efficiency and glycoproteome coverage. The method was successfully applied to enrich and validate LIF and its receptors as potential biomarkers for PDAC. In addition, to explore the full map of pancreatic tumor microenvironment glycoproteome with diagnostic and therapeutic values, we collected 114 pancreatic tissues, including 30 PDAC tumor tissues, 30 adjacent non-tumor (NT) tissues, 32 chronic pancreatitis tissues and 22 normal pancreatic tissues, and systematically profiled their glycoprotein expression pattern by using the developed glycoproteomic strategy. The deepest glycoproteome of PDAC was achieved, which covered the majority of previously reported glycoprotein biomarkers and drug targets for PDAC. Importantly, we discovered many new glycoproteins with differential expression in PDAC and normal tissue types. Moreover, LCM-based cell-type proteome profiling was achieved for 13 PDAC tissue samples, which covered more than 8000 proteins for both pancreatic stromal cells and pancreatic cancer cells in each sample. We therefore provided a valuable resource for screening novel and cancer specific glycoprotein biomarkers for pancreatic cancer with spatial resolution

The present program of research, therefore, was designed to attempt to gain new insights into the possible etiology of inflammatory states - for when new information concerning the etiology of these diseases states becomes available, more meaningful screening procedures for anti-inflammatory drugs will certainly be devised. Two preliminary studies have been published.

Thesis advisor: Welkin E. Johnson / Tetherin/BST2 is an interferon-inducible antiviral factor that restricts the egress of numerous enveloped viruses including HIV-1. Consequently, many viruses have evolved mechanisms to actively or passively evade restriction by tetherin. Most studies conducted to date focused on the tetherin-evasion mechanism of complex retroviruses like HIV and SIV, which encode accessory proteins like Vpu and Nef respectively to counteract tetherin-mediated restriction. However, there is a wide gap in knowledge in understanding how simple retroviruses (that includes alpharetroviruses, some betaretroviruses and gammaretroviruses) that lack obvious accessory proteins like HIV-1 Vpu and SIV-Nef, evade restriction by tetherin. In this dissertation, I have established that Simian retrovirus type-3, a prototypical type-D betaretrovirus, isolated from Asian macaques, is restricted by human tetherin but not by rhesus macaque tetherin. This differential sensitivity indicated that SRV-3 has a mechanism to evade tetherin-mediated restriction. I have identified the SRV-3 envelope (Env) glycoprotein as the viral determinant of tetherin antagonism, and have also found that SRV-3 envelope expression in-trans was sufficient to rescue a heterologous virus from tetherin. SRV-3 Env resulted in cell-surface down-modulation of rhesus tetherin, and this mechanism of tetherin-antagonism is independent of the SRV-3 Env trafficking pathway. The target specificity of SRV-3 Env overlapped a stretch of five residues (G14DIWK18) in the rhesus tetherin cytoplasmic tail that are absent from human tetherin. Additionally, I was able to show that SRV-3 Env physically interacts with rhesus tetherin by targeting the G14DIWK18 motif. SRV-3 belongs to a large supergroup of retroviruses, called the RDR Interference Supergroup. Due to this reason, I screened additional RDR envelope glycoproteins for their ability to antagonize a panel of tetherin homologs. All the RDR envelopes tested were sensitive to human tetherin but exhibited anti-tetherin activity when tested against a panel of tetherin homologs from squirrel monkey, baboon, dog and cat. I also found that several non-RDR gammaretroviral envelope glycoproteins also have anti-tetherin function. Thus, tetherin-antagonism is not just restricted to the envelope glycoproteins of retroviruses in the RDR interference supergroups but extends to other non-RDR gammaretroviruses as well. To my knowledge, this is the first characterization of gamma-type envelopes as tetherin antagonists. Thus, in the absence of a dedicated tetherin antagonist, many simple retroviruses in the beta- and gammaretrovirus genera may evade tetherin-mediated restriction through neo-functionalization of their envelope glycoproteins. We speculate that the evolutionary success of the gamma-type envelope may be due, at least in part, to this anti-tetherin function. / Thesis (PhD) — Boston College, 2018. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

A nanoHILIC-MS platform was developed to provide a means for top-down and middle-up analysis of glycoproteins. A mechanistic approach was taken in evaluating characteristics of the stationary phase of the chromatography column. Results from this evaluation informed the selection of a polyacrylamide brush layer-based bonded phase, which was applied to nonporous silica nanoparticles. Fluorescence microscopy was used for direct analysis of labeled proteins during nanoHILIC separations, with the platform subsequently adapted for mass spectrometry-based detection of intact and semi-intact glycoproteins. It was found that the standard technique of linking a hollow needle emitter to the capillary chromatography column for mass spectrometry-based detection yielded an unacceptable level of band broadening, necessitating the development of a column with an integrated needle emitter. The resulting nanoHILIC-MS method yielded peak widths as sharp as 3.5 seconds, enabling ultra-sensitive detection and identification of 28 glycoforms of an IgG1κ monoclonal antibody standard.