Spelling suggestions: "subject:"molekylärbiologi"" "subject:"molekylärbiologin""
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Antibodies for better or worse or Antibody variability in an egg-laying mammal and a novel strategy in the treatment of allergiesJohansson, Jeannette January 2002 (has links)
<p>Antibodies are a central part of the immune defense system, and a large variability in their specificity is needed in order to be able to react against all possible foreign substances we may encounter during our lives. In this thesis, results are presented from investigations into how an egg-laying mammal, the Australian duck-billed platypus (<i>Ornithorhynchus anatinus</i>) creates antibody variability. Our results show that despite the lack of many V gene families the antibody repertoire in the platypus seems to be well developed. A long and highly variable complementarity-determining region (CDR) 3 compensates for the limited germline diversity. Interestingly, the presence of additional cysteine residues in the CDRs may form stabilizing disulfide bridges in the antigen binding loops and thereby increasing the affinity of the antibody-antigen interaction. </p><p>Although the immune system is necessary for survival, it must be strictly controlled since it may otherwise over-react and cause more harm than benefits. Allergies and autoimmune diseases are examples of such over-reactions by the immune system. Allergies are increasing in the western world and have become one of the main medical issues of the 21<sup>st</sup> century. IgE is the central mediator in atopic allergies such as hay fever, eczema and asthma; it is therefore a prime target in the development of allergen-independent preventative treatments. Here we present results from several studies of a novel vaccine strategy aimed at reducing the levels of IgE antibodies. The vaccine results in the induction of anti-IgE antibodies, and the skin reactivity upon allergen challenge was significantly reduced in vaccinated animals. Our results suggest that active immunization against IgE has the potential to become a therapeutic method for humans. In addition, an evaluation of possible adjuvants that could be used as immune stimulators and thus help break self-tolerance at the time of vaccination is presented.</p>
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The rise and fall of IgEVernersson, Molly January 2002 (has links)
<p>Immunoglobulin E (IgE) occurs exclusively in mammals and is one of five immunoglobulin (Ig) classes found in man. Unlike other isotypes, IgE is best known for its pathological effects, whereas its physiological role remains somewhat elusive. </p><p>To trace the emergence of IgE and other post-switch isotypes we have studied Ig expression in two monotreme species, the duck-billed platypus (<i>Ornithorhynchus anatinus</i>) and the short-beaked echidna (<i>Tachyglossus aculeatus</i>), leading to the cloning of IgE, two IgG isotypes in platypus and echidna IgE. The presence of IgE and the conservation of the overall structure in all extant mammalian lineages indicates an early appearance in mammalian evolution and a selective advantage of structural maintenance. Furthermore, both of the two highly divergent platypus γ-chains have three constant domains. Hence, the major evolutionary changes that gave rise to the IgE and IgG isotypes of present day mammals occurred before the separation of monotremes from the marsupial and placental lineages, estimated to have occurred 150-170 million years ago.</p><p>As the central mediator in atopic allergy, IgE is a prime target in the development of preventive treatments. This thesis describes an active immunization strategy that has the potential to reduce IgE to a clinically significant extent. The active vaccine component is a chimeric IgE molecule, Cε2-Cε3-Cε4. The receptor-binding target domain, Cε3, is derived from the recipient species, whereas the flanking domains, acting both as structural support and to break T-cell tolerance, are derived from an evolutionarily distant mammal. Vaccination of ovalbumin-sensitized rats resulted in a substantial reduction in total IgE in three out of four strains, accompanied by a significant reduction in skin-reactivity upon allergen challenge. No cross-linking activity was observed and the response to vaccination was reversible with time. The apparent safety and efficacy of the vaccine suggest that active immunization against IgE has the potential to become a therapeutic method for humans. </p><p>Furthermore, the cloning and expression of the pig (<i>Sus scrufa</i>) ε-chain will facilitate the development of sensitive and specific assays for pig IgE, thus increasing the possibilities of using the pig model in future studies of IgE-mediated reactions.</p>
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Protein Tyrosine Phosphatases as Regulators of Receptor Ryrosine KinasesPersson, Camilla January 2003 (has links)
<p>Tyrosine phosphorylation is a crucial mechanism in cellular signaling and regulates proliferation, differentiation, migration and adhesion. The phosphorylation reaction is reversible and is governed by two families of enzymes: protein tyrosine kinases and protein tyrosine phosphatases (PTPs). This thesis investigates the role of PTPs in regulating receptor protein tyrosine kinases (RTKs), and explores a mechanism for regulation of phosphatase activity.</p><p>Most receptor tyrosine kinases are activated by ligand induced dimerization, which results in an increase in receptor phosphorylation. Preparations of ligand-stimulated dimeric PDGF β-receptors were shown to be less susceptible to dephosphorylation compared with unstimulated receptors. This revealed that reduced receptor dephosphorylation contributes to ligand-induced increase in RTK phosphorylation.</p><p>The receptor-like phosphatase DEP-1 site-selectively dephosphorylates the PDGF β-receptor. One of the most preferred sites is the PLC-γ binding phosphotyrosine pY1021, and the autoregulatory pY857 is one of the least preferred sites. By using chimeric phospho-peptides derived from these two sites as substrate for DEP-1, it was shown that a lysine residue at position +3 acts as a negative determinant for DEP-1 and that an aspartic acid residue at position –1 is a positive determinant.</p><p>The modulatory effect of TC-PTP on PDGF β-receptor signaling was explored by using mouse embryonic fibroblasts derived from TC-PTP knockout mice. PDGF β-receptors derived from knockout cells exhibited a higher level of ligand-induced phosphorylation compared to receptors from wildtype cells. The increase was unevenly distributed between different autophosphorylation sites. The PLC-γ binding site, previously implicated in chemotactic response, displayed the largest increase. Consistently, a cell migration assay revealed hyper-responsiveness to PDGF of TC-PTP knockout cells as compared to wildtype cells.</p><p>Reversible oxidation of the active site cysteine in PTPs is a mechanism, which have been postulated to regulate phosphatase specific activity. An antibody-based generic method for detection of oxidized PTPs was developed. Using this method it was revealed for the first time that UV-induced inactivation of PTPs involves oxidation of the active site cysteine.</p>
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Indirect Radiohalogenation of Targeting Proteins : Labelling Chemistry and Biological CharacterisationOrlova, Anna January 2003 (has links)
<p>In about half of all newly diagnosed cancer cases, conventional treatment is not adequately curative, mainly due to the failure of conventional techniques to find and kill residual cells and metastases, which might consist of only a few malignant cells, without causing unacceptable complications to healthy tissue. To solve the problem a more selective delivery of cytotoxic substances to tumour cells is needed. The approach applied here is called ‘tumour targeting’ and implies the use of biomolecules that recognise specific molecular structures on the malignant cell surface. Such molecules are then used for a selective transport of toxic agents to the cancer cells. </p><p>The use of radionuclides as cytotoxic substances has a number of advantages: 1) radiation does not cause severe resistance; 2) there is a cross-fire effect and 3) smaller amounts of nuclides are required than other cytotoxic substances to cause the same damage. Such an approach is called radionuclide tumour therapy. Several factors are important for the success of radionuclide therapy, such as the pharmacokinetics of the radiolabelled substance and its radiocatabolites, as well as the physical and chemical properties of the radiolabel used.</p><p>Nuclear properties of the label should be consistent with the problem to be solved: primary diagnostics; quantification of pharmacokinetics and dose planning; or therapy. From this point of view, radiohalogens are an attractive group of radiolabels. Halogens have nuclides with a variety of physical properties while the chemical and biological properties of halogens are very similar. The same labelling procedures can be used for all heavy halogens, i.e. bromine, iodine and astatine. It has been demonstrated that the biodistribution of proteins labelled with different heavy halogens is quite similar. </p><p>The main goal of the study was to develop protein radiohalogenation methods that provide a stable halogen-protein bond, convenient labelling chemistry that preserves the binding properties of proteins, long intracellular retention of radioactivity in targeted cells and quick release of radiohalogenated catabolites from the blood circulation. Radiohalogenation of proteins using indirect methods was studied, including optimisation of labelling chemistry and biological characterisation of some labelled conjugates. Two groups for indirect radiohalogenation were used, representing two different labelling principles: activated ester of benzoic acid (1) and the derivative of <i>closo</i>-dodecaborate anion (2). The non-phenolic linker (1) as well as the borate-halogen moiety (2) probably prevent dehalogenation. The negative charge of the potential catabolic products of (2) might trap radiohalogens intracellularly. </p>
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Cbl in Regulation of Growth Factor Receptor Endocytosis and Actin DynamicsSzymkiewicz, Iwona January 2003 (has links)
<p>Proteins belonging to the Cbl family are multidomain scaffolds that participate in numerous processes, assembling signaling complexes and mediating attachment of ubiquitin to receptor and non-receptor tyrosine kinases.</p><p>We characterized a novel role for Cbl and Cbl-b in ligand-dependent internalization of growth factor receptors. Upon stimulation with epidermal growth factor (EGF), Cbl proteins associate with EGF receptor, become phosphorylated, and bind to the three SH3 domains of CIN85, which brings endophilins to the complex with active receptors. Endophilins can induce internalization of the plasma membrane, contributing to formation of clathrin-coated pits. We identified a minimal binding domain for CIN85 in the carboxyl termini of Cbl/Cbl-b and observed constitutive association between CIN85, Cbl/Cbl-b and oncogenically stimulated receptor tyrosine kinases. In addition to functioning as a ubiquitin ligase, Cbl forms a complex with CIN85 and endophilin, which is required for efficient endocytosis and downregulation of membrane receptors.</p><p>In EGF stimulated cells, we observed inducible modification of CIN85 and related CMS proteins by attachment of a single ubiquitin molecule. Monoubiquitination of CIN85 was mediated by the RING finger and dependent on the carboxyl terminal part of Cbl/Cbl-b, and demanded an intact carboxyl terminus of CIN85. Prolonged stimulation with EGF induced concomitant degradation of EGF receptors, Cbl, and monoubiquitinated forms of CIN85 in lysosomes.</p><p>Cbl regulates cytoskeletal processes in a variety of cell systems. We identified SH3P2, a protein with SH3 domain and ankyrin repeats, as a Cbl partner and described its phosphorylation by Src and its distribution in fibroblasts and osteoclasts. SH3P2 formed inducible complexes with Cbl and actin in spread cells and colocalized with dynamic actin structures.</p><p>Our data contribute to better understanding of the role of Cbl in downregulation of receptor tyrosine kinases as well as in controlling actin rearrangement.</p>
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Viruses as a Model System for Studies of Eukaryotic mRNA ProcessingLindberg, Anette January 2003 (has links)
<p>Viruses depend on their hosts for the production and spread of new virus particles. For efficient virus replication, the viral genes have adapted the strategy of being recognized and processed by the cellular biosynthetic machineries. Viruses therefore provide an important tool to study the cellular machinery regulating gene expression. In this thesis, we have used two model DNA viruses; herpes simplex virus (HSV) and adenovirus, to study RNA processing at the level of pre-mRNA splicing in mammalian cells. </p><p>During a lytic infection, HSV cause an almost complete shut-off of host cell gene expression. Importantly, HSV infection cause inhibition of pre-mRNA splicing which is possibly advantageous to the virus, as only four HSV genes contain introns. </p><p>The HSV immediate early protein, ICP27, has been shown to modulate several post-transcriptional processes such as polyadenylation and pre-mRNA splicing. We have studied the role of ICP27 as an inhibitor of pre-mRNA splicing.</p><p>We show that ICP27 inhibits pre-mRNA splicing <i>in vitro</i> in the absence of other HSV proteins. We further show that ICP27 inhibits splicing at the level of spliceosome assembly. Importantly, ICP27 induced inhibition of splicing can be reversed, either by the addition of purified SR proteins or by the addition of an SR protein specific kinase, SRPK1. We propose that SR proteins are prime candidates as mediators of the inhibitory effect of ICP27 on pre-mRNA splicing. </p><p>In order to learn more about how splicing is organized in the cell nucleus <i>in vivo</i>, we investigated how cellular splicing factors are recruited to sites of transcription and splicing in adenovirus infected cells using confocal microscopy. Our results showed that the SR proteins, ASF/SF2 and SC35, are efficiently recruited to sites in the nucleus where adenovirus genes are transcribed and the resulting pre-mRNAs are processed. Our results demonstrate that only one of the two RNA recognition motifs (RRMs) present in the ASF/SF2 protein is required for its recruitment to active sites of splicing. The arginine/serine rich (RS) domain in ASF/SF2 is redundant and insufficient for the translocation of the protein to active viral polymerase II genes in adenovirus infected cells.</p>
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Human Papillomavirus Load and Cervical CarcinomaMoberg, Martin January 2004 (has links)
<p>Human Papillomavirus (HPV) is a key factor in the development of cervical cancer. Out of the more than 100 known HPV types 13 are considered oncogenic. In addition to presence of the virus several other factors have been proposed to influence risk of cervical cancer. This thesis focuses on viral load and HLA class II alleles as risk factors for cervical cancer.</p><p>To enable quantification of the most common oncogenic HPV types, a real-time PCR-based assay was developed and evaluated in terms of technical sensitivity and specificity.</p><p>This assay was then employed on archival smears from 457 cases and 552 controls to assess associations between viral load and cervical carcinoma <i>in situ</i> (CIS). Whereas the data indicate a pronounced dose dependent effect of HPV 16 load on the risk of CIS, other HPV types only seem to increase CIS risk at higher viral loads. These effects were observed even when cytology indicated that cells were normal.</p><p>We then investigated viral load as a risk factor for invasive cervical carcinoma (ICC) in a retrospective study comprising 139 cases and 550 controls. Viral load contributed similarly to the risk of ICC as to the risk of CIS.</p><p>Finally, associations between HLA class II alleles, viral load and CIS were investigated. Carriers of the DRB1*1301 allele were less prone to infections and high viral loads of HPV 31 and -18/45. Moreover, DRB1*1301 had a protective effect against CIS among women infected by HPV 31 or -18/45. In contrast, carriers of DRB1*1501 and DQB1*0602 were more susceptible to infections and high viral loads of HPV 16.</p><p>These results indicate that HPV load may have HPV-type specific effects on cervical cancer risk. Furthermore, HLA class II alleles may confer either susceptibility or protection against cervical cancer by acting on the HPV infections preceding tumor development.</p>
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Structure-Assisted Design of Drugs Towards HIV-1 and Malaria Targets : Applied on Reverse Transcriptase and Protease from HIV-1 and Plasmepsin II from <i>Plasmodium falciparum</i>Lindberg, Jimmy January 2004 (has links)
<p>Globally of today, acquired immunodeficiency syndrome (AIDS) and malaria are two of the most threatening diseases known to mankind. The World Health Organization estimated that AIDS and malaria together claimed nearly 4 million lives in 2003 and many more were infected by the causative agent human immunodeficiency virus (HIV) and the <i>Plasmodium falciparum</i> (<i>P. falicparum</i>) parasite. Current treatment regims for HIV and <i>P. falicparum</i> infections are undermined by rapid emergence of drug-resistant strains and severe drug side-effects.</p><p>A resistance mechanism of the commonly selected K103N RT mutant towards three second generation non-nucleoside RT inhibitors (NNRTIs) is presented based on X-ray structures. Subtle changes in contacts between inhibitor and residue in position 103 aided the design of improved inhibitors. For the PR target, attempts have been made to structurally assist the development of diol-based protease inhibitors (PIs) with the aim of improving the anti-viral potency without reducing the inhibitory efficacy. It was shown that ortho- and meta-fluoro-substituted P1/P1’-benzyloxy side chains improved the anti-viral potency without affecting the accommodation to the S1/S1’ subsites. </p><p>The apparent increase in malaria resistance makes drug interventions of current targets increasingly complicated. A prominent new drug target is found in the parasite’s hemoglobin degradation pathway – the aspartic protease plasmepsin II (Plm II). The usefulness of Plm II as an anti-malarial target is presented supported by Plm II complexed with a novel inhibitor. Structurally it is shown that bulky P1- and P3-side-chains adopt a novel binding mode to the Plm II binding cleft with implications for further inhibitor development.</p>
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Translational regulation of genes in salmonella typhimurium by vitamin B12Ravnum, Solveig January 2000 (has links)
<p>In this thesis I have studied the mechanism by which vitamin B12 regulates the expression of the <i>cob</i> operon and the <i>btuB</i> gene in <i>Salmonella typhimurium</i>. The <i>cob</i> operon encodes most of the 25 genes required for the <i>de novo</i> synthesis of vitamin B12, and the <i>butB</i> gene encodes the outer membrane protein needed for transport of exogenous vitamin B12 into the cell. Vitamin B12 is used as a cofactor in four enzymatic reactions in <i>Salmonella typhimurium</i>. The regulation by vitamin B12 of the <i>cob</i> operon and the <i>btuB</i> gene requires sequences in the long leader regions of the respective mRNAs. Proper folding of the reader mRNA is essential for normal repression, in particular a hairpin structure that sequesters the ribosomal binding site (RBS). The upstream leader region contains two conserved sequence elements that are required for the vitamin B12 regulation; the translational enhancer (TE) element element and the B12 box. The TE element confers its enhancer function by resolving the downstream inhibitory RBS hairpin through basepairing with nucleotides in the stem. In the presence of vitamin B12, either B12 itself, or a B12 regulatory factor binds to the upstream reader region and prevents the enhancer function. This will inhibit unfolding of the RBS hairpin and repress translation.</p>
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Finding the unknowns in <i>trans-</i>translation / Hitta de okända faktorerna för <i>trans-</i>translationIvanova, Natalia January 2005 (has links)
<p>Ribosomes stalled on problematic mRNAs can be rescued by a mechanism called <i>trans</i>-translation. This mechanism employs a dual transfer-messenger RNA molecule (tmRNA) together with a helper protein (SmpB). </p><p>In this work we have used an <i>in vitro</i> translation system with pure components to further clarify the roles of tmRNA and SmpB in <i>trans-</i>translation. </p><p>We found that SmpB binds ribosomes <i>in vivo</i> and <i>in vitro</i> independently of tmRNA presence and is essential for tmRNA binding and <i>trans-</i>peptidation. We show that two SmpB molecules can bind per ribosome, that SmpB does not leave the ribosome after <i>trans-</i>peptidation and that SmpB pre-bound to the ribosome can trigger <i>trans-</i>translation. </p><p>We demonstrated that the rate of <i>trans-</i>transfer of a peptide from the P-site tRNA to Ala-tmRNA and the efficiency by which Ala-tmRNA competes with peptide release factors decrease with increasing the mRNA length downstream from the P site of the ribosome. We showed that <i>trans-</i>translation is strongly stimulated by RelE cleavage of A-site mRNA. We concluded that tmRNA action<i> in vivo</i> must always be preceded by mRNA truncation.</p><p>We showed that rapid release of truncated mRNAs from the ribosome requires translocation of the peptidyl-tmRNA into the ribosomal P site, which is strictly EF-G dependent. mRNA release is slowed down by strong Shine and Dalgarno like sequences upstream the A site and by long 3’-extensions downstream from the P-site codon. </p><p>Footprinting was used to monitor SmpB binding to tmRNA, ribosomes and subunits and to study tmRNA interactions with the ribosome at distinct <i>trans-</i>translation stages. We confirmed that two SmpB molecules bind per ribosome and interact with nucleotides below the L7/L12-stalk on the 50S subunit and near the subunit interface on the 30S. We showed that tmRNA is mostly in complex with SmpB <i>in vivo</i> and during <i>trans-</i>translation. Specific cleavage patterns of tmRNA were observed at different stages of <i>trans-</i>translation, but the overall tmRNA conformation seems to be maintained during the whole process.</p>
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