Neointimal hyperplasia (NIH) is a cardiovascular disease characterised by increased smooth muscle cell (SMC) inflammation and proliferation. Suppressor of cytokine signalling 3 (SOCS3) limits Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathways involved in vascular remodelling but is limited by its short biological half-life. Therefore, mutation of all 9 Lys residues that are potential sites of ubiquitylation to Arg should produce a mutated SOCS3 resistant to ubiquitin-mediated proteasomal degradation (“Lys-less” SOCS3). This study hypothesise that enhancing SOCS3 stability and limiting JAK/STAT signalling may provide sustained inhibition of the vascular remodelling in NIH. Lentiviral transduction of WT and Lys-less SOCS3 in human saphenous vein (HSVSMCs) was highly efficient after 48 hours (>97%) and was sustained over 2 weeks. Lys-less SOCS3 was resistant to ubiquitylation contrary to WT-transduced HSVECs, and Lys-less SOCS3 was more stable (t1/2=4h) than WT (t1/2<4h) (n=6, P<0.001) in HSVSMCs. In HSVSMCs, both Lys-less SOCS3 and WT inhibited sIL-6Rα/IL-6 mediated STAT3 activation but not extracellular signal regulated protein kinase 1/2 (ERK1/2) by 80±7% (Lys-lessSOCS3/pSTAT3) and 74±6% (WT/pSTAT3) (n=3, P<0.05) and similarly inhibited PDGF-mediated STAT3 activation but not ERK1/2 by 67±17% (Lys-less SOCS3/pSTAT3) and 72±18% (WT/pSTAT3) (n=3, P<0.05). Functionally, Lys-less SOCS3 and WT were equivalent in inhibiting sIL-6Rα/IL-6 and PDGF-induced proliferation, whilst having no effects on PDGF-induced migration in HSVSMCs. Lys-less SOCS3 can be successfully transduced into primary HSVSMCs. It is more stable than WT yet retains its functional ability to ameliorate pro-inflammatory signalling and SMC proliferation, making it an attractive option for developing treatment of NIH. / University of Botswana

<p>Crystal structure, morphology and crystallization kinetics of melt-crystallized polypropylene and poly(propylene-stat-ethylene) fractions with 2.7 to 11.0 mol% of ethylene were studied by differential scanning calorimeter, wide- and small-angle X-ray scattering, polarized light microscopy, transmission electron microscopy and infrared spectroscopy. With increasing ethylene content the poly(propylene-stat-ethylene) fractions showed unchanged crystallinity, increased unit cell volume and constant crystal thickness in combination with a shortened helix length. This indicated that a fraction of ethylene defects were incorporated into the crystal structure. During the isothermal crystallization both α- and γ-crystals could be formed. The γ-crystal fraction increased with increasing ethylene content and increasing crystallization temperature. For samples with α- and γ-crystal contents, multimodal melting was observed and a noticeable γ- to α-crystal conversion was observed on slow heating. The spherulitic structure of the copolymers was coarser than that for the homopolymer.</p><p>The crystalline lamellae in copolymers exhibited profound curvature in contrast to the straighter cross-hatched α-crystals typical to the homopolymer. Area dependence of electrical breakdown strength was studied for thin polypropylene homopolymer films. The measurements were performed with an automatic measurement system equipped with a scanning electrode arm. Five different electrodes having areas between 0.045 cm2 and 9.3 cm2 were used and typically 40-80 breakdowns per sample and electrode area were collected. All measurements were performed on dry samples in air at room temperature. The data was analyzed statistically and the Weibull function parameters α and β, the first one related to 63% probability for the sample to break down and the second one to the width of the distribution were fitted to the obtained data. Different features concerning the measurement system and conditions, e.g. criteria for the automatic detection of the breakdowns, effect of electrode edge design, partial discharges, DC ramp speed and humidity were critically analyzed. It was concluded that the obtained α-parameter values were stable and repeatable over several years of time. The β-parameter values, however, varied ± 10-30%, more for the large than the small electrodes, and were also sensitive to the changes both in the sample itself and in the measurement conditions.</p><p>Breakdown strengths of over 50 capacitor grade polypropylene films were analyzed. The obtained α-parameter values were between 450 and 850 V/μm, depending on the film grade and electrode area. In addition to the high breakdown strengths, reflected by the obtained α-values, another, sparse distribution consisting of low breakdown strengths was revealed when the amount of measurement points was high enough. This means that more than one Weibull distribution could be needed to describe the breakdown strength behavior of a polypropylene film. Breakdown values showed decreasing area dependence with decreasing electrode area. Breakdown strengths for larger sample areas were predicted from the small area data by area- and Weibull extrapolation. The area extrapolation led to predicted α-values 50% higher than measured at 4 m² whereas the Weibull extrapolation showed an accuracy of ±15 % when predicted and measured values were compared.</p><p>Breakdown strengths were also extrapolated for film areas similar to those in impregnated power capacitors. It turned out that the power capacitors, tested at the factory, performed much better than predicted by the extrapolation. However, a few weak spots with very low breakdown values were also found. For the poly(ethyelene terephtalate) dielectric, which is not swelled by the impregnation liquid, the large area breakdown strength was predictable. This indicates that for polypropylene film processing and impregnation led, in addition to the improved large area breakdown performance, also to sparse weak spots with low breakdown probabilities. Different Weibull distributions were responsible for the breakdown strengths for the processed and impregnated polypropylene than for the dry film samples.</p>

Introdução: A modulação da resposta imune em muitas situações clínicas persiste como um dos problemas mais desafiadores em imunologia. Citocinas são fundamentais para esta regulação e são amplamente estudadas. Após a ligação com receptores específicos na superfície das células alvo, uma das principais vias de sinalização é o sistema JAKs/STATs. No entanto, em contraste com as citocinas, não existem estudos detalhados sobre a cinética expressão intracelulares de STAT in vitro. Objetivo: Determinar por citometria de fluxo, a cinética expressão de proteínas STAT 1, 3, 4, 5 e 6 fosforiladas, a produção de citocinas associadas e expressão de seus receptores em PBMC humanas estimuladas in vitro com fito-hemaglutinina (PHA) e antígeno de citomegalovírus (CMV). Metodologia: Foram avaliados CMNs de 23 doadores saudáveis em relação à cinética de expressão STATs (12 doadores estimulados com PHA e 11 estimulados com CMV), secreção de citocinas e expressão de seus receptores. Resultados: Em células estimulada com PHA e CMV, pSTAT1 e 6 tiveram sua expressão aumentada precocemente (4h e três dias, respectivamente). pSTAT3 teve sua expressão aumentada em momentos posteriores (respectivamente 36h e 6 dias). A indução de expressão de pSTAT4 e 5 foi observada nos tempos mais tardios da cinética em células estimuladas com PHA (24-36h), enquanto observamos constante baixo nível de expressão em todos os tempos analisados em células estimuladas com CMV. No que diz respeito á secreção de citocinas em células estimuladas com PHA, níveis maiores de IL-6, IL-10 e IL-4 foram detectados a partir de 12h, enquanto aumento da secreção de IFN-y e IL-2 ocorreu a partir de 24h. Com CMV, apenas IL-6 mostrou um aumento da secreção nos dias 4 e 6. Os receptores de citocinas CD119 (IFN-g), CD126 (IL-6), CD210 (IL-10), CD212 (IL-12), CD25 (IL -2) e CD124 (IL-4) tiveram um aumento da expressão após 24-36h de estimulação com PHA. Com estímulo de CMV, CD126, CD212, CD25, também aumentaram a expressão em tempos tardios (5-6 dias), enquanto os outros receptores mantiveram níveis baixos de expressão em todos os momentos estudados. Discussão: Houve uma correlação entre a cinética de expressão de pSTAT3 e 5, e a cinética das citocinas associadas e de seus receptores (IL-6 / CD126, IL-10 / CD210 e IL-2 / CD25). A cinética de expressão de pSTAT4 se correlacionou com a expressão de CD212. (IL-12p70 não foi detectada no presente estudo). Entretanto a expressão de pSTAT1 e 6 precedeu à de IFN-y / CD119 e IL-4 / CD124. Conclusão: A determinação da cinética de expressão pSTAT in vitro pode contribuir para a compreensão da regulação da resposta imune a patógenos distintos e, potencialmente, ajudar no desenvolvimento de novos alvos terapêuticos bem como de novas estratégias destinadas a modular as vias de sinalização em diversas condições clínicas associadas à desregulação imunológica / Introduction: Modulation of immune responses in many clinical situations persists as one of the most challenging issues in immunology. Cytokines are fundamental to this regulation and have already been extensively studied. After binding with its specific receptors on the surface of the target cells, the main signaling pathway is the JAKs/STATs system. However, in contrast to cytokines, there are no detailed studies on the in vitro intracellular expression kinetics of STATs. Objective: To determine by flow cytometry the kinetics of phosphorylated STAT1, 3, 4, 5 and 6 proteins expression in human PBMCs in vitro stimulated with phytohemagglutinin (PHA) and cytomegalovirus antigen (CMV), and the associated cytokine production and cytokine receptors expression. Methodology: We evaluated PBMCs from 23 healthy donors regarding the kinetics of STATs expression (12 donors stimulated with PHA and 11 stimulated with CMV), cytokine secretion and respective receptors expression. Results: In PHA and CMV stimulated cells, pSTAT 1 and 6 expression increased early, 4h and 3days respectively). pSTAT3 expression augmented at later times (respectively 36h and 6 days). pSTAT4 and 5 expression were observed late in PHA stimulated cells (24-36h), while there was a constantly low level of expression in all times analyzed. Regarding cytokine release In PHA stimulated cells, IL-6, IL10 and IL-4 secretion started to increase at 12h while IFN-y and IL-2 increased at 24h. With CMV, only IL-6 showed increased expression at days 4 and 6. The cytokines receptors CD119 (IFN-g), CD126 (IL-6), CD210 (IL-10), CD212 (IL-12), CD25 (IL-2) and CD124 (IL-4) had increased expression at 24-36h with PHA stimulation. With CMV stimulation, CD126, CD212, CD25 also had increased expression at late times (5-6 days) while the other receptors maintained low expression levels at all times. Discussion: There was a correlation in between the pSTAT3 and 5 expression kinetics and the associated cytokines and cytokine receptors kinetics (IL-6/CD126, IL-10/CD210 and IL-2/CD25. pSTAT4 expression kinetics correlated with that of CD212 expression (IL-12p70 was not detected in the present study). The higher pSTAT1 and 6 expressions preceded that of IFN-y/CD119 and IL-4/CD124, respectively. Conclusion: Determination of pSTAT expression kinetics in vitro can contribute to the understanding of the regulation of immune responses to distinct pathogens and potentially help in the design of new therapeutic targets as well as new strategies aimed at modulating signaling pathways

Ma Kit Wan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 189-206). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Table of Contents --- p.iii / Abstract --- p.xi / 摘要 --- p.xiv / Abbreviation List --- p.xv / List of Figures --- p.xvii / List of Tables --- p.xxiii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Ubiquitination --- p.1 / Chapter 1.1.1 --- Ubiquitin --- p.1 / Chapter 1.1.2 --- Ubiquitin Pathway --- p.3 / Chapter 1.1.3 --- Functions of Ubiquitination --- p.5 / Chapter 1.1.4 --- Ubiquitin Like Proteins --- p.8 / Chapter 1.2 --- SUMO Proteins --- p.10 / Chapter 1.2.1 --- SUMO Isoforms --- p.10 / Chapter 1.2.2 --- SUMO Structure --- p.11 / Chapter 1.3 --- Sumoylation --- p.14 / Chapter 1.3.1 --- Functions of Sumoylation --- p.14 / Chapter 1.3.1.1 --- General Functions of Sumoylation --- p.15 / Chapter 1.3.1.2 --- Function of Sumoylation on Transcription Factors / Chapter 1.3.1.3 --- Specific Function of SUMO-2/3 Conjugation / Chapter 1.3.2 --- Sumoylation Pathway --- p.19 / Chapter 1.4 --- E3 Ligases in Sumoylation --- p.24 / Chapter 1.4.1 --- Types and Functions of E3 Ligases --- p.23 / Chapter 1.4.2 --- Structure of PI AS --- p.23 / Chapter 1.4.3 --- Function of PI AS --- p.27 / Chapter 1.5 --- Aims of Study --- p.29 / Chapter Chapter 2 --- Materials & Methods --- p.30 / Chapter 2.1 --- Polymerase Chain Reaction (PCR) Screening of Multiple Human Tissue cDNA (MTC´ёØ) Panel --- p.30 / Chapter 2.1.1 --- Primer Design --- p.30 / Chapter 2.1.2 --- Semi-quantitative PCR --- p.31 / Chapter 2.1.2.1 --- Human MTC´ёØ Panel --- p.31 / Chapter 2.1.2.2 --- PCR --- p.32 / Chapter 2.2 --- DNA Cloning --- p.34 / Chapter 2.2.1 --- "Amplification of El, E3 (PIAS), PIAS1 Fragments" --- p.34 / Chapter 2.2.1.1 --- Primer Design --- p.34 / Chapter 2.2.1.2 --- PCR --- p.36 / Chapter 2.2.1.3 --- Purification of PCR Product --- p.37 / Chapter 2.2.2 --- Restriction Digestion --- p.37 / Chapter 2.2.3 --- Ligation --- p.40 / Chapter 2.2.4 --- Transformation --- p.40 / Chapter 2.2.4.1 --- Preparation of Chemically Competent Cells'(DH5α) --- p.40 / Chapter 2.2.4.2 --- Transformation of Ligation Product --- p.41 / Chapter 2.2.5 --- Plasmid Preparation --- p.42 / Chapter 2.2.6 --- Screening for Recombinant Clones --- p.43 / Chapter 2.2.7 --- Sequencing of Recombinant Plasmid --- p.43 / Chapter 2.3 --- Subcellular Localization Study --- p.45 / Chapter 2.3.1 --- Midi Scale Plasmid Preparation --- p.45 / Chapter 2.3.2 --- Transfection of GFP Recombinant Plasmids --- p.46 / Chapter 2.3.2.1 --- Cell Culture of WRL-68 & HepG2 Cell Lines --- p.46 / Chapter 2.3.2.2 --- LipofectAMINE Based Transfection --- p.47 / Chapter 2.3.3 --- Immunostaining of Endogenous SUMO-1 & -2/-3 --- p.48 / Chapter 2.3.4 --- Nucleus Staining by DAPI --- p.48 / Chapter 2.3.5 --- Fluorescent Microscopic Visualization --- p.49 / Chapter 2.3.6 --- Western Blotting --- p.49 / Chapter 2.3.6.1 --- LipofectAMINE Based Transfection --- p.49 / Chapter 2.3.6.2 --- Protein Extraction --- p.50 / Chapter 2.3.6.3 --- Protein Quantification --- p.51 / Chapter 2.3.6.4 --- SDS-PAGE Analysis --- p.51 / Chapter 2.3.6.5 --- GFP Fusion Proteins Detection --- p.52 / Chapter 2.4 --- Two-Dimensional Gel Electrophoretic Analyses --- p.54 / Chapter 2.4.1 --- Sample Preparation --- p.54 / Chapter 2.4.1.1 --- Protein Extraction from the Nucleus --- p.54 / Chapter 2.4.1.2 --- Clean Up of Extracted Nuclear Fraction --- p.55 / Chapter 2.4.2 --- First Dimensional Isoelectric Focusing (IEF) --- p.55 / Chapter 2.4.3 --- Second Dimension SDS-PAGE --- p.57 / Chapter 2.4.3.1 --- SDS-PAGE Analysis --- p.57 / Chapter 2.4.3.2 --- Silver Staining --- p.58 / Chapter 2.4.4 --- Image Analysis --- p.59 / Chapter 2.4.5 --- Protein Identification by Mass Spectrometry --- p.60 / Chapter 2.4.5.1 --- Sample Preparation --- p.60 / Chapter 2.4.5.2 --- Data Acquisition --- p.62 / Chapter 2.4.5.3 --- Data Analysis of Protein Fingerprinting --- p.62 / Chapter 2.5 --- Confirmation of the Differentially Expressed Proteins by RT-PCR & Western Blotting --- p.63 / Chapter 2.5.1 --- RT-PCR Analysis --- p.63 / Chapter 2.5.1.1 --- RNA Extraction --- p.63 / Chapter 2.5.1.2 --- First Strand cDNA Synthesis --- p.64 / Chapter 2.5.1.3 --- Normalization of cDNA Template --- p.64 / Chapter 2.5.1.4 --- PCR Amplification of the Target Genes --- p.65 / Chapter 2.5.2 --- Western Blotting --- p.66 / Chapter 2.6 --- Expression of Human PIAS and PIAS1 Fragments in Prokaryotic System --- p.67 / Chapter 2.6.1 --- Preparation of Competent Cells --- p.67 / Chapter 2.6.2 --- Small Scale Expression --- p.67 / Chapter 2.6.2.1 --- Transformation --- p.67 / Chapter 2.6.2.2 --- IPTG Induced Protein Expression --- p.68 / Chapter 2.6.3 --- Large Scale Expression of PIAS1 Fragments --- p.70 / Chapter 2.6.3.1 --- Transformation --- p.70 / Chapter 2.6.3.2 --- IPTG Induced Protein Expression --- p.70 / Chapter 2.6.4 --- Purification Trial of MBP-PIAS1-321-410 --- p.71 / Chapter 2.6.4.1 --- Binding of Amylose Resin & On Column Cleavage (with Low Concentration of DTT) --- p.71 / Chapter 2.6.4.2 --- Elution from the Amylose Resin & Cleavage (with Low Concentration of DTT) --- p.73 / Chapter 2.6.4.3 --- Elution from the Amylose Resin & Cleavage (with High Concentration of DTT) --- p.73 / Chapter 2.6.4.4 --- Purification of PIAS1-321-410 by Size ExclusionChromatography --- p.73 / Chapter 2.6.5 --- Purification of MBP-PIAS1 Fragments --- p.74 / Chapter 2.6.5.1 --- Purification by Affinity Column (Amylose) --- p.74 / Chapter 2.6.5.2 --- Amylose Resin Regeneration --- p.74 / Chapter 2.6.5.3 --- Purification by Both Affinity and Ion Exchange (Heparin) --- p.75 / Chapter 2.6.5.4 --- Regeneration of Heparin Column --- p.76 / Chapter 2.6.5.5 --- Purification by Size Exclusion Chromatography --- p.76 / Chapter 2.6.5.6 --- Regeneration of Size Exclusion Chromatography --- p.77 / Chapter 2.6.6 --- Co-expression & Purification of PIAS1 Fragment with E2 (Ubc9) --- p.77 / Chapter 2.6.6.1 --- Co-transformation of pMAL-PIASl (Fragments) & pET-Ubc9 --- p.77 / Chapter 2.6.6.2 --- Co-expression of PIAS1 Fragments & Ubc9 --- p.78 / Chapter 2.6.6.3 --- Purification by Affinity Column (Amylose Resin) --- p.78 / Chapter 2.6.6.4 --- Purification by Both Affinity & Ion Exchange (Heparin) --- p.79 / Chapter 2.6.6.5 --- Purification by Size Exclusion Chromatography --- p.79 / Chapter 2.6.7 --- Urea Treatment for the Purification of PIAS 1 Fragments --- p.80 / Chapter 2.6.7.1 --- Transformation --- p.80 / Chapter 2.6.7.2 --- IPTG Induced Protein Expression --- p.80 / Chapter 2.6.7.3 --- Purification by Affinity Column (Amylose Resin) --- p.80 / Chapter 2.6.7.4 --- Purification by Both Affinity & Ion Exchange (Heparin) --- p.80 / Chapter 2.6.7.5 --- Purification by Size Exclusion Chromatography --- p.81 / Chapter Chapter 3 --- Results --- p.82 / Chapter 3.1 --- Tissue Distribution of Human PIAS Genes --- p.82 / Chapter 3.1.1 --- Determination of the Number of Cycles for PCR --- p.82 / Chapter 3.1.2 --- General Expression Pattern of All PIAS Genes --- p.82 / Chapter 3.1.3 --- Tissue Distribution of PIAS1 --- p.83 / Chapter 3.1.4 --- Tissue Distribution of PIAS3 --- p.83 / Chapter 3.1.5 --- Tissue Distribution of PIASxa --- p.83 / Chapter 3.1.6 --- Tissue Distribution of PIASxp --- p.84 / Chapter 3.1.7 --- Tissue Distribution of PIASy --- p.84 / Chapter 3.2 --- Subcellular Localization of SUMO Pathway Components --- p.90 / Chapter 3.2.1 --- Overexpression Confirmation --- p.90 / Chapter 3.2.2 --- Multiple Bands Detected After Overexpression of EGFP- SUMO-1 --- p.91 / Chapter 3.2.3 --- Subcellular Localization of EGFP --- p.94 / Chapter 3.2.4 --- Subcellular Localization of El Subunits --- p.94 / Chapter 3.2.5 --- Subcellular Localization of E2 (Ubc9) --- p.95 / Chapter 3.2.6 --- Subcellular Localization of PIAS Proteins --- p.95 / Chapter 3.2.7 --- Subcellular Localization of PIAS1 Fragments --- p.96 / Chapter 3.2.8 --- Subcellular Localization of SUMO-1 --- p.97 / Chapter 3.3 --- Differential Protein Expression Pattern after Transient Transfection of SUMO-1 --- p.112 / Chapter 3.3.1 --- Protein Expression Profiles after Transient Transfection / Chapter 3.3.2 --- Identification of the Differential Expressed Proteins --- p.113 / Chapter 3.4 --- Confirmation of Differentially Expressed Proteins in Cells Overexpressing SUMO-1 --- p.124 / Chapter 3.4.1 --- RT-PCR Analyses --- p.124 / Chapter 3.4.1.1 --- Downregulation of RNA Transcript of hnRNP A2/B1 isoform B1 --- p.124 / Chapter 3.4.1.2 --- No Significant Change in the Transcription Level of UDG --- p.125 / Chapter 3.4.2 --- Western Blotting --- p.128 / Chapter 3.4.2.1 --- Upregulation of hnRNP A2/B1 at the Protein Level --- p.128 / Chapter 3.4.2.2 --- Different Molecular Weight of hnRNP A2/B1 Was Detected --- p.129 / Chapter 3.4.2.3 --- Upregulation of UDG at the Protein Level --- p.129 / Chapter 3.5 --- Expression & Purification of Human PIAS Proteins & PIAS1 Fragments --- p.133 / Chapter 3.5.1 --- Expression of Human PIAS Proteins --- p.133 / Chapter 3.5.2 --- Expression of PIAS1 Fragments --- p.135 / Chapter 3.5.3 --- A Trial of Purification of MBP-PIAS1-321-410 --- p.137 / Chapter 3.5.3.1 --- On Column Cleavage of MBP Tag --- p.137 / Chapter 3.5.3.2 --- Cleavage after Elution --- p.137 / Chapter 3.5.3.3 --- High Concentration of DTT Used --- p.138 / Chapter 3.5.3.4 --- Separation of the Cleaved and Non Cleaved Proteins --- p.138 / Chapter 3.5.4 --- Purification of the PIAS 1 Fragments --- p.141 / Chapter 3.5.4.1 --- Purified by Affinity Column (Amylose Resin) --- p.141 / Chapter 3.5.4.2 --- Purified by Heparin Column --- p.141 / Chapter 3.5.4.3 --- Purified by Gel Filtration --- p.143 / Chapter 3.5.5 --- Co-expression & Purification of PIAS1 Fragments & E2 --- p.147 / Chapter 3.5.5.1 --- Co-expression of PIAS1 Fragments & E2 --- p.147 / Chapter 3.5.5.2 --- Co-purification of PIAS1 Fragments & E2 Amylose --- p.147 / Chapter 3.5.5.3 --- Co-purification of PIAS1 Fragments & E2 by Heparin --- p.148 / Chapter 3.5.5.4 --- Co-purification of PIAS 1 Fragments with Ubc9 by Gel Filtration --- p.148 / Chapter 3.5.6 --- Urea Treatment for Purification of PIAS1 Fragments --- p.153 / Chapter 3.5.6.1 --- Purification by Amylose Resin --- p.153 / Chapter 3.5.6.2 --- Purification by Heparin --- p.153 / Chapter 3.5.6.3 --- Purification by Gel Filtration --- p.154 / Chapter Chapter 4 --- Discussion --- p.157 / Chapter 4.1 --- Tissue Specificity of PIAS Proteins --- p.157 / Chapter 4.1.1 --- Principle of Tissue Specificity Study --- p.157 / Chapter 4.1.2 --- Importance of Sumoylation --- p.158 / Chapter 4.1.3 --- Role of Sumoylation in Reproduction --- p.159 / Chapter 4.1.4 --- Functional Role of Sumoylation in Other Tissue --- p.160 / Chapter 4.2 --- Subcellular Localization of SUMO Pathway --- p.162 / Chapter 4.2.1 --- SUMO Conjugation Occurs in the Nucleus --- p.162 / Chapter 4.2.2 --- Does Sumoylation Occur Outside the Nucleus --- p.163 / Chapter 4.2.3 --- Dots-like Structure Formed by the PIAS --- p.164 / Chapter 4.2.4 --- SAP Domain and PINIT Motif Are Not Essential for Nuclear Targeting --- p.165 / Chapter 4.2.5 --- Signal Involves in the Formation of Nuclear Speckles --- p.167 / Chapter 4.3 --- Differentially Expressed Proteins under SUMO-1 Overexpression --- p.169 / Chapter 4.3.1 --- Increase in High Molecular Weight Proteins --- p.169 / Chapter 4.3.2 --- Upregulation of hnRNP A2/B1 & UDG in Protein Level --- p.170 / Chapter 4.3.3 --- Variants of hnRNP A2/B1 Formed --- p.172 / Chapter 4.3.4 --- Possibility of Sumoylation on hnRNP A2/B1 isoform B1 & UDG --- p.172 / Chapter 4.3.5 --- Possible Roles of SUMO-1 on hnRNP A2/B1 isoform B1 --- p.174 / Chapter 4.3.6 --- Mechanism of Sumoylation on mRNA Processing --- p.175 / Chapter 4.3.7 --- Possible Roles of SUMO-1 on UDG --- p.176 / Chapter 4.3.8 --- Important of SUMO on Genome Integrity --- p.178 / Chapter 4.3.9 --- Sumoylation and Carcinogenesis --- p.178 / Chapter 4.4 --- Protein Purification of the Human PIAS Proteins & PIAS1 Fragments --- p.180 / Chapter 4.4.1 --- Low Expression Level & Solubility of the PIAS Proteins --- p.180 / Chapter 4.4.2 --- High Expression Level & Solubility of PIAS 1 Fragments --- p.181 / Chapter 4.4.3 --- Incorrect Disulfide Bond Formation of the PIAS1 Fragments --- p.182 / Chapter 4.4.4 --- MBP-PIAS1 Fragments Formed Soluble Aggregates --- p.182 / Chapter 4.4.5 --- A Low Concentration of Urea Cannot Dissociate the Soluble Aggregates --- p.183 / Chapter 4.4.6 --- Aggregation May Weaken the Interaction between the PIAS1 Fragments & Ubc9 --- p.184 / Chapter 4.5 --- Conclusion --- p.185 / Chapter 4.6 --- Future Perspectives --- p.187 / Chapter 4.6.1 --- Identification of the Role of SUMO Interacting Motif in the Nuclear Speckle Formation --- p.187 / Chapter 4.6.2 --- Investigation of Sumoylation on Liver Cancer --- p.187 / Chapter 4.6.3 --- Optimization of the Expression & Purification of the PIAS Proteins --- p.188 / References --- p.189 / Appendix --- p.207

<p>Pancreatic cancer is the fourth most lethal cancer and ranks as the eighth most commonly diagnosed cancer worldwide. This is due to its rapid proliferation, strong metastatic potential and its delayed detection. One major risk factor for developing pancreatic cancer is the aggressive inflammatory disease chronic pancreatitis. Chronic inflammation frequently precedes the development of certain pancreatic cancers.</p><p>Inflammation is a protective and necessary process by which the body can alert the immune system of the existence of a wound or infection and mount an immune response to remove the harmful stimuli and start wound healing. The cross-talking of cells of the immune system and infected cells happens through cytokines, soluble proteins that activate and recruit other immune cells to increase the system’s response to the pathogen. Failure to resolve the injury can result in persistent cytokine production that in turn allows a cell that is damaged or altered to survive when in normal conditions it would be killed. Inflammation is thought to create a microenvironment that facilitates the initiation and/or growth of pancreatic cancer cells.</p><p>Cytokines use two important kinases for their signaling: Janus Kinases (JAKs) and Signal Transducers and Activators of Transcription (STATs). The JAKs are activated upon the binding of cytokines to their corresponding receptors. When activated, the JAKs activate STATs through tyrosine phosphorylation. The STATs transduce signals to the nucleus of the cells to induce expression of critical genes essential in normal physiological cellular events such as differentiation, proliferation, cell survival, apoptosis and angiogenesis. STAT3 (a member of the STAT family) is constitutively activated in some pancreatic cancers, promoting cell cycle progression, cellular transformations and preventing apoptosis. Therefore, STAT3 is a promising target for cancer treatment. Novel therapies that inhibit STAT3 activity in cancers are urgently needed. Natural products are a very good resource for the discovery of new drugs against pancreatic cancer.</p><p>Covering more than 70% of the Earths surface, The Ocean is an excellent source of bioactive natural products. Harbor Branch Oceanographic Institute’s Center for Marine Biomedical and Biotechnology Research (HBOI-CMBBR) situated in Florida, aims to find new marine natural products useful in disease prevention and drug therapy. Their current focus is to look for novel treatments for preventing both the formation of new pancreatic tumors and the metastasis of existing tumors.</p><p>The hypothesis of this degree project was that novel inhibitors of STAT3 useful in the treatment of pancreatitis and/or pancreatic cancer could be found from marine-natural products. The first specific aim of this degree project was to set up an assay to identify bioactive marine natural products as inhibitors of inflammation. Furthermore the assay was validated using a commercially available inhibitor of inflammation (Cucurbitacin I). The last aim was to further validate the assay by screening pure compounds and peak library material from the HBOI marine specimen collection.</p><p>At the end of the experimentation time, the assay still was not set-up as there were difficulties in proper cell culture techniques and the cell line did not respond as advertised. While the results were not as expected, the work performed resulted in familiarization with research laboratory practices and increased laboratory skills. Moreover, the results from the assays point to future directions to accomplish this project.</p> / Development of a screening assay for inhibitors of inflammation useful against pancreatic cancer

<p>The means that the Swedish State has to legitimize and standardize different perspectives of women and their sexuality is significant. Persecution of gender and sexuality is today something that can grant refugee status according to the Swedish Aliens Act. By studying the documents that forestall the amendment of the laws is it possible to unveil the concept and ideas about women sexuality that the state manifests and reproduces through these laws and official documents.</p><p>The aim of this essay is to look upon the sexual policy as it occurs in official the documents that deal with lesbian refugees. The study cover and maps present research on this group of women and gives an answer to how norms about women sexuality are expressed in these documents.</p><p>Throughout the survey of the research field not much previous research was found, therefore even research that only partly covers the field is included and reviewed.</p><p>As method a detailed reading of the official documents and a discourse analysis with it’s emphasize on theoretical themes like gender and sexuality, lesbianism and state feminism, was made. Lesbians are missing in the official state documents and the one time the Migration Board’s documents refer to lesbians they are ascribed to as gender and not as sexuality.</p><p>The essay concludes that the confusion about the category gender and the category sexuality has the result that the lesbians become invisible. Lesbians tend throughout the material to be ascribed to the category gender and gay men to the category sexuality.</p><p>The standardized perspective and normative discourse that has been found in the official documents has the effect that women is looked a pound as gender and therefore, by the same logic loose their sexuality. In this way the Swedish State discriminates lesbian.</p><p>Another conclusion is that the Swedish State has a somewhat ambivalence perspective on their own work on promoting gender equality.</p>

Crystal structure, morphology and crystallization kinetics of melt-crystallized polypropylene and poly(propylene-stat-ethylene) fractions with 2.7 to 11.0 mol% of ethylene were studied by differential scanning calorimeter, wide- and small-angle X-ray scattering, polarized light microscopy, transmission electron microscopy and infrared spectroscopy. With increasing ethylene content the poly(propylene-stat-ethylene) fractions showed unchanged crystallinity, increased unit cell volume and constant crystal thickness in combination with a shortened helix length. This indicated that a fraction of ethylene defects were incorporated into the crystal structure. During the isothermal crystallization both α- and γ-crystals could be formed. The γ-crystal fraction increased with increasing ethylene content and increasing crystallization temperature. For samples with α- and γ-crystal contents, multimodal melting was observed and a noticeable γ- to α-crystal conversion was observed on slow heating. The spherulitic structure of the copolymers was coarser than that for the homopolymer. The crystalline lamellae in copolymers exhibited profound curvature in contrast to the straighter cross-hatched α-crystals typical to the homopolymer. Area dependence of electrical breakdown strength was studied for thin polypropylene homopolymer films. The measurements were performed with an automatic measurement system equipped with a scanning electrode arm. Five different electrodes having areas between 0.045 cm2 and 9.3 cm2 were used and typically 40-80 breakdowns per sample and electrode area were collected. All measurements were performed on dry samples in air at room temperature. The data was analyzed statistically and the Weibull function parameters α and β, the first one related to 63% probability for the sample to break down and the second one to the width of the distribution were fitted to the obtained data. Different features concerning the measurement system and conditions, e.g. criteria for the automatic detection of the breakdowns, effect of electrode edge design, partial discharges, DC ramp speed and humidity were critically analyzed. It was concluded that the obtained α-parameter values were stable and repeatable over several years of time. The β-parameter values, however, varied ± 10-30%, more for the large than the small electrodes, and were also sensitive to the changes both in the sample itself and in the measurement conditions. Breakdown strengths of over 50 capacitor grade polypropylene films were analyzed. The obtained α-parameter values were between 450 and 850 V/μm, depending on the film grade and electrode area. In addition to the high breakdown strengths, reflected by the obtained α-values, another, sparse distribution consisting of low breakdown strengths was revealed when the amount of measurement points was high enough. This means that more than one Weibull distribution could be needed to describe the breakdown strength behavior of a polypropylene film. Breakdown values showed decreasing area dependence with decreasing electrode area. Breakdown strengths for larger sample areas were predicted from the small area data by area- and Weibull extrapolation. The area extrapolation led to predicted α-values 50% higher than measured at 4 m2 whereas the Weibull extrapolation showed an accuracy of ±15 % when predicted and measured values were compared. Breakdown strengths were also extrapolated for film areas similar to those in impregnated power capacitors. It turned out that the power capacitors, tested at the factory, performed much better than predicted by the extrapolation. However, a few weak spots with very low breakdown values were also found. For the poly(ethyelene terephtalate) dielectric, which is not swelled by the impregnation liquid, the large area breakdown strength was predictable. This indicates that for polypropylene film processing and impregnation led, in addition to the improved large area breakdown performance, also to sparse weak spots with low breakdown probabilities. Different Weibull distributions were responsible for the breakdown strengths for the processed and impregnated polypropylene than for the dry film samples. / QC 20101027

Protesterna i samband med WTO mötet i Seattle, USA, 1999 är allmänt betraktade som startskottet för våld i samband med stora demonstrationer rörande globalisering. I juni 2001 stod Göteborg, Sverige, som värd för EU:s ministermöte samt George W Bush besök. I dessa samt ett flertal andra stora demonstrationer rörande globalisering kom våld att överskugga mötets såväl som demonstranternas sakfrågor. Våldet utövades både av aktivister samt av polisen som statens ordningsmakt. Denna uppsats fokuserar på våldsproblematiken från statens sida; hur polisen kan upprätthålla statens säkerhet och samtidigt fungera som medborgarnas garanti för att skydda deras rättigheter. Sverige har haft socialdemokratiskt styre under nästan hela 1900-talet och att säga att det har påverkat samhällsutvecklingen torde knappast vara en överdrift. USA å andra sidan benämns ofta som Frihetens land och liberalismens principer kan sägas vara inbäddade i det amerikanska samhället. Med anledning av detta syftar denna uppsats att undersöka om det är möjligt att i Seattle och i Göteborg förstå staternas agerande i våldsproblematiken från ett liberalt respektive socialdemokratiskt perspektiv. Uppsatsen har formen av en kvalitativ idéanalys med fallstudie som metod. Fallstudien har passat mycket bra i denna uppsats då den fokuserar på relationer och processer samt präglas av ett holistiskt synsätt. Syftet kan sägas vara besvarat då flera exempel tas upp för att visa hur man kan förstå statens agerande med hjälp av ideologierna. Exempelvis försvarades användningen av våld i Göteborg med att säkerheten ansågs bli bättre. Detta hänvisas i uppsatsen till den socialdemokratiska idén om statens funktion att se till det gemensamma goda. Även i Seattle försvarades användningen av våld, genom de undantagstillstånd som inrättades, av att säkerheten höjdes. Denna gång hänvisades istället till den liberala idén om staten som främjare av den individuella friheten. Slutsatsen är därmed att denna uppsats ej kanförklara USA:s eller Sveriges ageranden med hjälp av liberalismen eller socialdemokratin, man kan däremot förstå dem. Ideologier är idéer som man med rätt medel kan urskilja där man letar efter dem.

The development of multi-cellular organisms requires extensive cell-cell communication to coordinate cell functions. However, only a handful of signaling pathways have emerged to mediate all the intercellular communications; therefore, each of them is under an array of regulations to achieve signaling specificity and diversity. One such signaling pathway is the Janus Kinase/ Signal Transducer and Activator of Transcription (JAK/STAT) pathway, which is the primary signaling cascade responding to a variety of cytokines and growth factors in mammals and involved in many developmental processes. This signaling pathway is highly conserved between mammals and Drosophila, but the Drosophila JAK/STAT pathway possesses only three ligands: Unpaired (Upd), Upd2 and Upd3. Co-localized expression patterns of the ligands at several developmental stages raise the possibility that they physically interact. This work was aimed at testing the protein-protein interactions between Upd-family ligands and exploring possible outcomes of ligand oligomerization. Physical interactions between Upd-family ligands were tested using a Bimolecular Fluorescence Complementation (BiFC) assay. The data suggested that homotypic interactions of Upd2 and Upd3 were stronger than their respective heterotypic interactions with Upd, and the homotypic interaction between Upd molecules was the weakest. In addition, the homotypic interaction of Upd3 was confirmed using yeast two-hybrid interaction assays. To identify protein domains critical for Upd3/Upd3 interaction, a series of poly-alanine substitutions were made to target the 6 conserved domains of Upd3. All 6 substitutions altered the strength of Upd3/Upd3 interaction and drastically reduced Upd3-induced JAK signaling activity. In addition, poly-alanine substitutions of some domains also affected Upd3 extracellular localization or protein accumulation. Potential outcomes of interactions between Upd-family ligands were tested both in vitro and in vivo. The interaction between Upd and Upd3 did not significantly change the level of JAK signaling activity. However, loss of Upd3 restricted the distribution of Upd in egg chambers and consequently altered the follicle cell composition. Therefore, Upd/Upd3 interaction is likely to affect the range rather than the intensity of JAK signaling in egg chambers. In summary, this study suggested the possibility of ligand oligomerization as a mechanism for regulating signaling pathways in order to achieve signaling specificity and diversity during development.

Drug resistant strains of Clostridium difficile are a major health concern with over 3 million cases costing over 1 billion $ per year in the United-States. The diseases associated with these bacteria (CDAD) are toxin-mediated which offers a mean of treating and lessening the severity of CDAD symptoms. Toxin inactivation via antibodies therapy can drastically reduce CDAD morbidity and this project was aiming at investigating the large-scale production and recovery of a novel llama single domain antibody (pSJF2H-ToxA5.1) in recombinant Escherichia coli TG1 targeting C. difficile enterotoxin A (TcdA). In order to achieve these objectives, the project was divided into four segments: 1) ToxA5.1 being an intracellular recombinant protein, obtaining a high biomass production was the first step towards large-scale production. To achieve HCDC, effects of initial glucose concentration and pH-stat feeding strategy were studied; 2) Upon achieving HCDC, effects of parameters such as temperature, induction timing and media supplementation with complex nitrogen sources were investigated; 3) Once large-scale production of ToxA5.1 was obtained, the recombinant protein needed to be recovered and a selective cell lysis scheme where synergistic lysis effects of Triton X-100 and temperature were studied. And finally 4) Single-step purification using nickel nanoparticles (NNP) synthesized via a modified polyol method was studied. Combining the HCDC strategy with a temperature shift and yeast extract addition at the time of induction, ToxA5.1 concentration of 127 mg/L was obtained. Synergistic and selective cell lysis using Triton X-100 and temperature was achieved where 95% of the available ToxA5.1 was recovered and still functional while ToxA5.1 fraction in the resulting lysate increased to 27% in the cell lysate. Single-step purification was achieved using the synthesized NNP which proved to be highly selective and could be used up to five times. Diameter of the NNP synthesized was controlled by using various concentration of ranging from 131 ± 80 nm to 47 ± 20 nm. Using experimental data from binding isotherm, the ToxA5.1-NNP system was modeled.