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Design of Oligosaccharide Libraries to Characterize Heparan Sulfate – Protein InteractionsKurup, Sindhulakshmi January 2006 (has links)
<p>Heparan sulfates (HSs) are a class of anionic carbohydrate chains found at cell surfaces and in the extracellular matrix where they interact with a number of proteins. HS is characterized by extreme structural heterogeneity, and has been implicated in a number of biological phenomenon like embryogenesis, morphogen gradient formation and signalling of growth factors such as FGF, PDGF etc. Despite the characteristic structural heterogeneity, evidence from compositional studies show that the HS structure is expressed in a tightly regulated manner, implying a functional significance, which is most likely in the modulation of cell behaviour through HS-protein interactions. The lack of molecular tools has, however, hampered the understanding of HS structures with functional significance. This work therefore aims at characterizing the structural requirements on HS involved in the interaction with the anti-HS phage display antibodies HS4C3, AO4B08 and HS4E4 and a selected growth factor PDGF-BB. The characterization was done with the help of tailored oligosaccharide libraries generated from sources bearing structural resemblance to HS.</p><p>The work has thus made available tools that preferentially recognize certain structural features on the HS chain and will aid in the further study of HS structure and its regulation. Evidence is also provided to support the notion that HS protein interactions can occur in multiple manners, utilizing any of the structural features on the HS chain.</p>
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Formation,Storage and Secretion of Prostasomes in Benign and Malignant Cells and Their Immunogenicity in Prostate Cancer PatientsSahlén, Göran January 2007 (has links)
<p>Prostasomes are submicron-sized, membrane-bound organelles produced by the epithelial cells of the prostate and normally found in the secretion in the gland ducts. Their physiological role is in the promotion of sperm-function in human reproduction. This thesis contains four papers dealing with the production of prostasomes and some possible applications in clinical urology of the prostasome. </p><p>Paper I and II provided an ultrastructural description of the synthesis, storage and secretion of prostasomes in benign as well as in malignant tissue. Most notable were the extracellular appearances of prostasomes in metastatic lesions whereby the prostasomes become exposed to the immune system of the patient. This supported findings in earlier studies in which patients with advanced prostate cancer had elevated levels of anti-prostasome antibodies. The results of paper III reinforced the view of the prostate-unique origin of the prostasome. In particular, there were no indications in SDS-PAGE patterns or flow-cytometric studies of material from seminal vesicle secretion that it contained components that could be associated with a production of prostasomes. </p><p>Some possible clinical functions of the prostasomes were investigated in paper IV. Exposure of prostasomes to the immune system through mechanical and thermal trauma to the prostate did not induce an evident formation of anti-prostasome autoantibodies. Furthermore, the serum levels of anti-prostasome antibodies registered by assays with preparations of prostasomes from seminal plasma as antigen did not correlate with existing prostate cancer. Seminal prostasomes seemed not to function as substitute markers for prostate cancer in the test kit used. A possible explanation could be underestimated differences in antigen properties between seminal or prostate gland-derived prostasomes and prostasomes from tumor tissue.</p>
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The Significance of IgG Antibodies against Tissue Transglutaminase in Coeliac DiseaseDahlbom, Ingrid January 2008 (has links)
<p>Coeliac disease (CD) is a multifactorial disease of the small intestine. In genetically predisposed individuals the, ingestion of cereals leads to a remodulation of the mucosal architecture, and the production of autoantibodies against tissue transglutaminase (tTG). The treatment is a lifelong gluten-free diet.</p><p>The diagnostic procedure relies on the examination of a small-bowel biopsy that displays villous atrophy. A spectrum of clinical manifestations is associated with CD, ranging from overt enteropathy to atypical and silent symptoms. Approximately 1% of the general population has CD, and the majority is undiagnosed. Although most patients with active CD can be detected by the assessment of elevated IgA-tTG, some patients lack these antibodies. Moreover, individuals with IgA-deficiency cannot be identified by means of IgA serology. </p><p>The aim of this thesis was to investigate the clinical utility of IgG-tTG for the detection and follow-up of subjects with active CD. The included studies showed that IgG-tTG was highly prevalent in IgA-deficient and IgA-competent patients with CD, whereas non-CD patients rarely had these antibodies. During a gluten-free diet, IgG-tTG decreased, demonstrating that IgG-tTG can be used to follow the patient’s adherence such a diet. Furthermore, 10% of healthy IgA deficient blood donors had elevated IgG-tTG, indicating that they had silent CD. </p><p>In IgA-competent subjects, high IgG-tTG levels correlated with a severe mode of CD and profound mucosal deterioration, suggesting that IgG-tTG might be involved in the disease progression. Moreover, we found that although a considerable percentage of IgA-competent patients lack IgG-tTG, the presence of these antibodies in conjunction with high levels of IgA-tTG was highly predictive of a severe small-intestine villous atrophy. It was also demonstrated that IgG-tTG normalisation coincided with clinical remission in IgA-competent CD patients on a gluten-free diet. </p>
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Development of Nanomechanical Sensors for Environmental Contaminate Screening Using Protein Functionalized MicrocantileversHill, Kasey L 01 May 2010 (has links)
The development of real time, label-free biosensors based on ligand-induced nanomechanical responses of microcantilevers (MCs) allows for sensitive and selective detection. High sensitivity is afforded by the MCs small dimensions. Immobilizing biomolecular recognition phases imparts selectivity from bioaffinity interactions. Biological sensors on a MC platform utilize various proteins, such as antibodies and nuclear receptors, which can be used to detect and screen for potential environmental contaminants.
The interaction between contaminants and immobilized receptors induces an apparent surface stress that leads to static bending of the MC, which is monitored by an optical beam bending technique. Biofunctionalized MCs can provide high sensitivity and selectivity on a relatively inexpensive platform that requires small amounts of analyte. The goal of this research is to develop and optimize MCs as biosensors to detect low concentrations of contaminants.
Initially, the research utilized specific receptors and antibodies to detect and screen for contaminants that are deemed endocrine disrupting chemicals (EDCs). Immobilizing estrogen receptors and specific antibodies on the MC surface may provide information on the ever expanding list of EDCs, along with fundamental endocrine studies.
Then, the MC surface was morphologically and chemically optimized. This optimization included the thickness and metal ratio of the dealloyed surface. The concentration, reaction time, and pH of chemical immobilization reagents, which include aminoethanethiol and glutaraldehyde, were optimized by using an anti-body test system. Antibody and protein functionalization conditions, which are incubation time and concentration, were optimized using the anti-immunoglobulin G (anti-IgG) receptor: IgG and an anti-biotin:biotin test systems. The optimized immobilization conditions were applied to the detection of thyroid disrupting chemicals (TDCs) using MCs functionalized with the transport protein thyroxine-binding globulin.
The final project involved developing a nanomechanical transducer to study xenobiotic and EDC interactions with the bioreceptor PXR’s ligand binding domain (LBD). The combination of immobilized LBD PXR with a nanostructured microcantilever (MC) platform allows for the study of ligand interaction with the receptor’s binding domain. PXR shows real-time, reversible responses when exposed to specific pharmaceutical, EDC, and xenobiotic ligands. Three binding interactions that involve EDCs are tested, which include phthalic acid, nonylphenol, and bisphenol A, with PXR.
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Design of Oligosaccharide Libraries to Characterize Heparan Sulfate – Protein InteractionsKurup, Sindhulakshmi January 2006 (has links)
Heparan sulfates (HSs) are a class of anionic carbohydrate chains found at cell surfaces and in the extracellular matrix where they interact with a number of proteins. HS is characterized by extreme structural heterogeneity, and has been implicated in a number of biological phenomenon like embryogenesis, morphogen gradient formation and signalling of growth factors such as FGF, PDGF etc. Despite the characteristic structural heterogeneity, evidence from compositional studies show that the HS structure is expressed in a tightly regulated manner, implying a functional significance, which is most likely in the modulation of cell behaviour through HS-protein interactions. The lack of molecular tools has, however, hampered the understanding of HS structures with functional significance. This work therefore aims at characterizing the structural requirements on HS involved in the interaction with the anti-HS phage display antibodies HS4C3, AO4B08 and HS4E4 and a selected growth factor PDGF-BB. The characterization was done with the help of tailored oligosaccharide libraries generated from sources bearing structural resemblance to HS. The work has thus made available tools that preferentially recognize certain structural features on the HS chain and will aid in the further study of HS structure and its regulation. Evidence is also provided to support the notion that HS protein interactions can occur in multiple manners, utilizing any of the structural features on the HS chain.
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Formation,Storage and Secretion of Prostasomes in Benign and Malignant Cells and Their Immunogenicity in Prostate Cancer PatientsSahlén, Göran January 2007 (has links)
Prostasomes are submicron-sized, membrane-bound organelles produced by the epithelial cells of the prostate and normally found in the secretion in the gland ducts. Their physiological role is in the promotion of sperm-function in human reproduction. This thesis contains four papers dealing with the production of prostasomes and some possible applications in clinical urology of the prostasome. Paper I and II provided an ultrastructural description of the synthesis, storage and secretion of prostasomes in benign as well as in malignant tissue. Most notable were the extracellular appearances of prostasomes in metastatic lesions whereby the prostasomes become exposed to the immune system of the patient. This supported findings in earlier studies in which patients with advanced prostate cancer had elevated levels of anti-prostasome antibodies. The results of paper III reinforced the view of the prostate-unique origin of the prostasome. In particular, there were no indications in SDS-PAGE patterns or flow-cytometric studies of material from seminal vesicle secretion that it contained components that could be associated with a production of prostasomes. Some possible clinical functions of the prostasomes were investigated in paper IV. Exposure of prostasomes to the immune system through mechanical and thermal trauma to the prostate did not induce an evident formation of anti-prostasome autoantibodies. Furthermore, the serum levels of anti-prostasome antibodies registered by assays with preparations of prostasomes from seminal plasma as antigen did not correlate with existing prostate cancer. Seminal prostasomes seemed not to function as substitute markers for prostate cancer in the test kit used. A possible explanation could be underestimated differences in antigen properties between seminal or prostate gland-derived prostasomes and prostasomes from tumor tissue.
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The Significance of IgG Antibodies against Tissue Transglutaminase in Coeliac DiseaseDahlbom, Ingrid January 2008 (has links)
Coeliac disease (CD) is a multifactorial disease of the small intestine. In genetically predisposed individuals the, ingestion of cereals leads to a remodulation of the mucosal architecture, and the production of autoantibodies against tissue transglutaminase (tTG). The treatment is a lifelong gluten-free diet. The diagnostic procedure relies on the examination of a small-bowel biopsy that displays villous atrophy. A spectrum of clinical manifestations is associated with CD, ranging from overt enteropathy to atypical and silent symptoms. Approximately 1% of the general population has CD, and the majority is undiagnosed. Although most patients with active CD can be detected by the assessment of elevated IgA-tTG, some patients lack these antibodies. Moreover, individuals with IgA-deficiency cannot be identified by means of IgA serology. The aim of this thesis was to investigate the clinical utility of IgG-tTG for the detection and follow-up of subjects with active CD. The included studies showed that IgG-tTG was highly prevalent in IgA-deficient and IgA-competent patients with CD, whereas non-CD patients rarely had these antibodies. During a gluten-free diet, IgG-tTG decreased, demonstrating that IgG-tTG can be used to follow the patient’s adherence such a diet. Furthermore, 10% of healthy IgA deficient blood donors had elevated IgG-tTG, indicating that they had silent CD. In IgA-competent subjects, high IgG-tTG levels correlated with a severe mode of CD and profound mucosal deterioration, suggesting that IgG-tTG might be involved in the disease progression. Moreover, we found that although a considerable percentage of IgA-competent patients lack IgG-tTG, the presence of these antibodies in conjunction with high levels of IgA-tTG was highly predictive of a severe small-intestine villous atrophy. It was also demonstrated that IgG-tTG normalisation coincided with clinical remission in IgA-competent CD patients on a gluten-free diet.
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Studies of immunological and molecular biological techniques with infectious laryngotracheitis virus of chickensAbbas, Ferhat, 1962- 22 November 1994 (has links)
Monoclonal antibodies (MCA) produced against infectious
laryngotracheitis virus (ILTV) of chickens reacted in
western blotting experiments with several different ILTV
protein bands in the absence of tunicamycin which inhibits
carbohydrate synthesis. Most of the MCA lost their
reactivity in western blotting experiments when extracts of
tunicamycin-treated ILTV CELC were used, suggesting their
specificity for carbohydrate-based epitopes. In an indirect
immunofluorescence test most of the MCA bound primarily to
cytoplasmic antigens except some MCA which bound primarily
to nuclear antigens. Additivity ELISA was also performed to
study whether MCA are against the same epitope or different
epitopes.
The polymerase chain reaction (PCR) was developed as a
diagnostic technique for detection of ILTV using primers
made from a portion of the ILTV thymidine kinase gene. The
647-basepair amplified ILTV PCR product was labeled to
create a non-radioactive, biotinylated DNA probe.
Hybridization was performed using the probe to detect ILTV.
Both PCR and hybridization detected ILTV, and neither
hybridization nor PCR gave positive results with any other
pathogen. Hybridization was specific for ILTV, However,
slight hybridization occurred with CELC DNA when relatively
relaxed conditions were used.
In another experiment, diagnostic tests to detect ILTV
in tracheas of experimentally-infected chickens, including
the indirect fluorescent antibody test (IFAT),
immunoperoxidase (IP), virus isolation (VI), histopathology,
PCR, and hybridization, were performed and compared. Using
virus isolation as a reference, the sensitivity and
specificity of the tests were calculated. The IP test and
IFAT performed better than any other test used in this
study. / Graduation date: 1995
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Enzyme associations in deoxyribonucleotide biosynthesis : anti-idiotypic antibodies as probes for direct protein-protein interactionsYoung, James Patrick 11 May 1992 (has links)
The ability to faithfully replicate DNA is dependent upon the maintenance
and regulation of its precursors, the deoxyribonucleoside triphosphates.
Enzymes encoded by the bacteriophage T4 have been widely used as models
of biochemical processes. A body of evidence supports the concept that the
bacteriophage T4 enzymes involved in deoxyribonucleotide biosynthesis are
associated as a complex within the infected Escherichia coli. This dissertation
describes the continued examination of the protein-protein interactions
involved in deoxynucleotide biosynthesis of bacteriophage T4.
My studies on the protein-protein interactions involved in
deoxyribonucleotide biosynthesis focused on two unique phage proteins, the
dCMP hydroxymethylase enzyme and the translational regulator RegA. An
initial study was undertaken to determine if the generation of anti-idiotypic
antibodies would prove useful in the identification of direct interactions
between dCMP hydroxymethylase and other proteins of the
deoxyribonucleotide synthetase complex.
For the initial generation of anti-idiotypic antibodies, polyclonal rabbit
antibodies were generated to affinity purified anti-dCMP hydroxymethylase
polyclonal rabbit IgG. The anti-anti-dCMP hydroxymethylase antibody was
found to be specific in binding to the bacteriophage T4 dTMP synthase.
A second method to generate anti-idiotypic antibodies was attempted with
the translational regulator RegA. The generation of anti-idiotypic antibodies to
the RegA protein involved the purification of anti-RegA rabbit Fab fragments
and the generation of anti-anti-RegA polyclonal antibodies within mice. This
alternative method was determined to be inferior to the initial method for
generating anti-idiotypic antibodies. Additional studies involved the
examination of RegA protein-protein interactions using affinity chromatography.
A number of bacteriophage T4 early proteins were determined to associate
with an immobilized RegA column. / Graduation date: 1992
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Isolation, chemical modification and applications of flax cyclolinopeptides2013 June 1900 (has links)
Oil from flaxseed (Linum usitatisssimum L.) contains hydrophobic cyclic peptides or cyclolinopeptides (CLs) comprising eight or nine amino acids. These bioactive compounds have potential therapeutic applications and may be used as scaffolds for increased utility. Two steps were undertaken to increase the potential utility of these compounds. Initially multigram quantities of flax CLs were highly enriched from flax oil. Subsequently new synthetic procedures were developed for modification of the CLs through the methionine group (Met). Finally, the utility of the modified CLs was tested in a number of applications. CLs were recovered from a crude oil extract that contain five CLs (CLA, CLC, CLE, CLJ and CLK). Oxidation of this mixture reduced the complexity of the mix to just three CLA, CLJ and CLK. CLJ and CLK were enriched then characterized by NMR and MS-MS methods. CLs containing methionine sulfoxide groups (Mso), CLC and CLE were isolated from crude mixture then selectively reduced to afford Met containing analogs: CLB and CLE'. The Met of modified CLs was used as a point for attachment of tags and couplers for various applications. Cyclic peptide modification through Met groups has not been reported previously. Synthetic methods were devised to introduce activating functional groups such as -CN, -COOH, -OH and -NH2 to the sulfur atom of Met. The modified CL conjugates were characterized using spectrometric techniques including 1D and 2D NMR spectrometry, as well as mass spectrometry. After activation the CLs were covalently linked to molecules or materials of interest including fluorescence tags (coumarin), affinity chromatography media and bovine serum albumin (BSA) for production of polyclonal antibodies. Fluorescence studies were performed in methanol, ethanol, dimethylformamide and acetonitrile to study the solvent effect. CLs attached to solid affinity matrix showed specific binding to apolipoprotein A1 after incubation with chicken serum. These CLs also act as hapten and have been used to couple BSA to produce polyclonal antibodies. Met modification was a satisfactory approach to produce a range of useful peptide products where more conventional methods of molecule attachment are not available.
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