Serum levels of apolipoprotein ApoC1 have been described in a number of systemic tumor entities as potential biomarkers, but little is known about ApoC1 in neurosurgical patients. A total of 230 serum samples from 96 patients were analyzed using an ELISA technique. Patient diagnoses comprised 70 glioblastomas WHO IV◦ , 10 anaplastic astrocytomas III◦ , one anaplastic oligodendroglioma III◦ , one oligodendroglioma II◦ , one diffuse astrocytoma II◦ , one pilocytic astrocytoma I◦ , and a single case of a spindle cell tumor without WHO grading, as well as 11 spinal interventions. The mean ApoC1 level of the 230 samples was 132.03 µg/mL (median 86.83, SD 292.91). In the 176 glioblastoma samples, the mean ApoC1 level was 130.0 µg/mL (median 86.23, SD 314.9), which was neither different from the whole group nor from patients with spinal interventions (215.1 µg/mL, median 63.6, SD 404.9). In the postoperative samples, the mean ApoC1 level was significantly lower (85.81 µg/mL) than in the preoperative samples (129.64 µg/mL) and in samples obtained during adjuvant chemotherapy (168.44 µg/mL). While absolute ApoC1 serum levels in a patient do not allow for the distinction between neurosurgical histological entities, future analyses will examine whether the time course of ApoC1 in an individual patient can be related to certain treatment stages.

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Individer med homozygot familjär hyperkolesterolemi(FH), har höga halter av Low Density Lipoprotein (LDL) vilket leder till ökad risk för kardiovaskulära sjukdomar. Behandling av dessa individer kan göras med extrakorporal elimination av LDL med hjälp av specifika reningskolonner. Syftet med studien var att utvärdera några agarosmodifierade adsorbenter för denna applikation. Adsorbenterna, modifierad polyakrylat (DALI), agaros (Zetaros), direkt sulfateradZetarose och taurin immobiliserad Zetarose, inkuberades med humant plasma spädd med PBS, och en volyms förhållande mellan matris och plasman på 1:5. Inkubering utfördes i rumstemperatur under 60 min med kontinuerlig blandning i rotator. Efter inkubation centrifugerades proverna och LDL bestämdes i såväl supernatant som pellet. Totalmängd adsorberade proteiner analyserades också i eluat från erhållen pellet. LDL bestämdes indirekt med hjälp av Friedewaldsformel (LDL = totalkolesterol (TC) –highdensitylipoprotein (HDL) - (0,45 x Triglycerider(TG)). TC och TG bestämdes enzymatiskt medan HDL kvantifierades som TC efter utfällning av LDL med dextransulfat. Resultaten visar tydligt att DALI har god adsorptionsförmåga.Dock uppvisar de modifierade Zetaroserna begränsad adsorptionskapacitet för LDL. Vid desorption av adsorbenterna visar SDS en bättre elueringsförmåga än NaCl relaterad till protein, vilket tyder hydrofoba proteiner. Metodiken som används i studien är lämplig för vidare studier av andra adsorbenter som förväntas användas i kliniska applikationer för elimination av LDL hos FH patienter. / Individuals that suffer from homozygote Familiar Hyperkolesterolemia (FH), has increased amounts of Low Density Lipoproteins (LDL) which leads to a higher risk of cardiovascular diseases. Treatment of these individuals can be achieved by extracorporeal elimination of LDL using specific columns. The aim of this study was to evaluate different agarose-modified adsorbents ability to adsorb LDL from human plasma. The adsorbents (DALI, Zetarose, sulphonated Zetarose and taurine immobilized onto Zetarose) were incubated for 60 minutes with human plasma diluted with PBS, in a ratio of 1:5 between the matrix and the plasma during rotation with a rotator. After incubation the samples were centrifuged and the LDL content was determined in both the supernatant and the pellet. The amount proteins adsorbed were assayed by eluting the pellets. LDL was determined indirectly using Friedwalds equation; LDL= Total cholesterol (TC) - High density lipoprotein (HDL)-(0,45x Triglycerides (TG). The values of TC and TG in the sample were determined enzymatically, whilst HDL was quantified as TC after LDL-precipitation by dextran sulfate. The results clearly show that DALI has good adsorption capacity, but none of the modified Zetaroses shows any capacity to absorb LDL from human plasma. Desorption of the adsorbents using SDS gave higher amounts of eluated protein compared to NaCl elution, indicating hydrofobic proteins. However, the methods used in this study could be used to evaluate new adsorbents for LDL-elimination applications in patients with chronic hyperlipemia.

Le virus de l’hépatite C (HCV) infecte les hépatocytes. Il est remarquable par le fait q’ilperturbe fortement le metabolisme lipidique de l’hôte, conduisant à des dysfonctions majeures telles qu’une stéatose ou une résistance à l’insuline. In vivo, les virions sériques présentent une densité faible et variable reflétant leur association aux lipoprotéines de faible et de tres faible densité (LDL et VLDL). Ainsi, l'existence de lipo-viro-particules (LVP), contenant à la fois les constituants viraux et les apolipoprotéines B (apoB) et E (apoE) a été suggerée. Ces LVP joueraient un rôle important dans la persistance virale. Cependant, cette association entre HCV et apoB n'a pas été retrouvée in vitro avec les modeles cellulaires disponibles.Mes travaux de thèse se sont donc concentrés sur la mise en place d'un nouveau modèle deculture cellulaire capable de produire a la fois des VLDL et des particules virales HCV, permettantainsi d’étudier l’interaction entre les deux voies de synthèse. Dans un premier temps, lacaracterisation de la production de lipoproteines par differentes lignees d'hepatocytes a permis demontrer l'existence d'un défaut de secretion de VLDL en cellules Huh7.5, classiquement utiliséespour étudier HCV in vitro, alors que les cellules HepG2 et HepaRG sont capables de produire des VLDL physiologiques. Dans un second temps, des cellules HepG2 repliquant HCV de manière persistante ont été utilisées pour caractériser les particules virales produites. Etonnamment, a l'instar des cellules Huh7.5 et malgré leur capacité a produire des VLDL, les cellules HepG2 ne secreteraient pas de LVP mais des particules virales positives pour apoE et négatives pour apoB. / Hepatitis C virus (HCV) mainly infects hepatocytes. It is unique in its ability to impair host lipidmetabolism, leading to major liver dysfunctions as, for instance, hepatic steatosis or insulinresistance. In vivo, serum virions have a low and variable density, reflecting their association withlow- and very-low-density lipoproteins (LDL and VLDL). Hence, the existence of lipo-viro-particles(LVP), containing both viral components as well as apolipoprotein B (apoB) and E (apoE), has beensuggested. These LVPs could play an important role in viral persistence. However, this associationbetween HCV and apoB has not been observed in vitro, using the currently available cell culturemodels.Therefore, during my PhD, I have worked at setting up a new cell culture model, which wascapable of producing both VLDL and HCV particles, and therefore would enable the study of theinterplay between both synthesis pathways. First of all, we characterized lipoprotein production indifferent hepatocyte cell lines and confirmed that Huh7.5 cells, usually used to study HCV in vitro,were deficient for VLDL secretion, whereas two other cell lines, HepG2 and HepaRG, were able toproduce quasi-physiological VLDLs. Therefore, in a second time, we used HepG2 cells to replicate aHCV strain containing a selection gene and to characterize viral particle production. Surprisingly,VLDL-producing HepG2 cells were also unable to secrete LVPs, but rather secreted apoE-positive andapoB-negative viral particles, which were similar to ones Huh7.5 cells produced. This suggests thatthe ability to produce LVPs does not correlate with the ability to produce VLDL.

Introduction: Cognitive impairment is converted for 15% to Alzheimer’s disease (AD), which is incurable. The aim of this study is to investigate whether Herpes simplex virus type 1 (HSV1) and its interaction with allele ε4 of Apolipoprotein E (ApoE-ε4) may be possible risk factors for cognitive impairment. Here, suggestions for implementation of Precision Public Health (PPH) is also presented; population studies of relevant biomarkers of infectious burden and data from health outcomes with the aim of remedying public health crises, monitoring diseases, anticipating risks and using public health initiatives adapted to risk groups to increase understanding of diseases, such as AD. Method: An analysis of 1013 people aged 75 from the cohort, Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS), was performed. Linear mixed models (LMM) were used to investigate whether anti-HSV1 IgG and ApoE-ε4 are associated with risk of developing cognitive impairment over 5 years. Anti-HSV1 IgG in serum was detected by Enzyme-linked immunosorbent assays and cognitive impairment was examined by Mini Mental State Examination. Results: Neither anti-HSV1 IgG positivity nor its interaction with ApoE-ε4 were associated with cognitive impairment. Discussion: Studies of HSV1 without HSV2 or HSV show mixed results. Therefore, we do not yet have sufficient evidence to implement PPH-interventions against these risks. Other cognitive tests that are more sensitive to early changes and adjustment for underrepresented groups may potentially contribute to a more accurate analysis. / Bakgrund: Kognitiv nedsättning övergår för 15 % till Alzheimers sjukdom (AD) som är obotlig. Denna studie har målsättningen att undersöka om Herpes simplex virus typ 1 (HSV1) och dess interaktion med riskvarianten allel ε4 av genen Apolipoprotein E (ApoE-ε4) kan vara möjliga riskfaktorer för kognitiv nedsättning. I arbetet diskuteras även hur man genom Precision Public Health (PPH) kan studera befolkningen med avseende på relevanta biomarkörer och data från hälsoutfall med syfte att avhjälpa folkhälsokriser, övervaka sjukdomar och förutse risker såsom sjunkande kognition och med folkhälsoinsatser anpassade till riskgrupper öka förståelsen av dessa tillstånd. Metod: En analys av 1013 personer 75 år ur kohorten ”Prospecive Investigation of the Vasculature in Uppsala Seniors” (PIVUS). Linear mixed models (LMM) användes för att undersöka om anti-HSV1 IgG och ApoE-ε4 är associerade med kognitiv nedsättning under 5 år. Anti-HSV1 IgG i serum detekterades med ”Enzyme-linked immunosorbent assays” och kognitiv nedsättning undersöktes med Mini Mental State Examination. Resultat: Anti-HSV1 IgG positivitet var inte associerat med kognitiv nedsättning. Anti-HSV1 IgG positivitet och samtidig ApoE-ε4 var inte heller associerat med kognitiv nedsättning. Diskussion: Andra studier av endast HSV1 utan HSV2 eller HSV visar blandade resultat, varför kunskapsläget idag inte stödjer tillämpning av PPH-interventioner mot dessa risker. Andra kognitiva mått som är känsligare för små förändringar nära intakt kognition samt justering för underrepresenterade grupper kan möjligen bidra till säkrare analys.

The field of proteomics studies the structure and function of proteins in a large scale and high throughput manner. My work in the field of proteomics focuses on identifying interactions between proteins and discovering novel interactions. The identification of these interactions provides new information on metabolic and disease pathways and the working proteome of a cell. Cells are lysed and purified using antibody based affinity purification followed by digestion and identification using an HPLC coupled to a mass spectrometer. In my studies, I looked at the interaction networks of several AD related genes (Apolipoprotein E, Clusterin variant 1 and 2, Low-density lipoprotein receptor, Phosphatidylinositol binding clathrin assembly protein, Alpha-synuclein and Platelet-activating factor receptor) and an endosomal recycling pathway involved in cholesterol metabolism (Eps15 homology domain 1,2 and 4, Proprotein convertase subtilisin/kexin type 9 and Low-density lipoprotein receptor). Several novel and existing interactors were identified and these interactions were validated using co-immunopurification, which could be the basis for future research.

Tsoi Lo Ming. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 99-109). / Abstracts in English and Chinese. / Acknowledgements --- p.I / Abstract --- p.II / Abstract in Chinese --- p.III / List of Abbreviations --- p.IV / List of Figures --- p.V / List of Tables --- p.VI / Table of Contents --- p.VII / Chapter Chapter 1 : --- Introduction / Chapter 1.1. --- Apolipoprotein and Lipoprotein Metabolism --- p.1 / Chapter 1.2. --- Molecular Information of ApoE --- p.2 / Chapter 1.3. --- Tissue Distribution of ApoE --- p.2 / Chapter 1.4. --- Functions of ApoE --- p.4 / Chapter 1.5. --- Genetic Polymorphism of ApoE --- p.7 / Chapter 1.6. --- Protein Structure and Characteristics of ApoE Isoforms --- p.9 / Chapter 1.7. --- Plasma and Cellular Expression Level of ApoE Isoforms --- p.12 / Chapter 1.8. --- Association between ApoE Isoforms and Plasma Lipid Profiles --- p.13 / Chapter 1.9. --- ApoE Polymorphisms and Pathophysiological Conditions / Chapter 1.9.1. --- Type III Hyperlipoproteinemia (Type III HLP) --- p.14 / Chapter 1.9.2. --- Alzheimer's Disease --- p.15 / Chapter 1.9.3. --- Atherosclerosis / Chapter 1.9.3.1. --- Atherosclerosis - An Inflammatory Process --- p.15 / Chapter 1.9.3.2. --- Role of ApoE in Atherosclerosis --- p.18 / Chapter (a) --- Functions Associated to Lipid Metabolism --- p.19 / Chapter (b) --- Functions Independent to Lipid Metabolism --- p.20 / Chapter 1.9.3.3. --- TNF-α and IL-6 in Atherosclerosis --- p.25 / Chapter 1.10. --- Macrophage Cytokine Expression and MAPKs / Chapter 1.10.1. --- Organization of MAPKs Signaling Pathway --- p.26 / Chapter 1.10.2. --- Lipopolysaccharide and MAPKs in Macrophage Cytokine Expression --- p.28 / Chapter 1.10.3. --- Regulation of Macrophage Cytokine Expression / Chapter 1.10.3.1. --- ERK1/2 and p38 MAPK Pathway --- p.30 / Chapter 1.10.3.2. --- Arachidonic Acid Metabolism --- p.30 / Chapter 1.11. --- Aim and Hypothesis --- p.31 / Chapter Chapter 2 : --- Materials and Methods / Materials / Chapter 2.1 --- Culture of ApoE-isoform-expressing J774A.1 Macrophage Cell Line --- p.32 / Chapter 2.2 --- RNA Extraction and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.33 / Chapter 2.3 --- Protein Extraction and Quantification --- p.37 / Chapter 2.4 --- Enzyme-linked Immunosorbent Assay (ELISA) --- p.38 / Chapter 2.5 --- Western Blotting --- p.39 / Chapter 2.6 --- LPS Treatment --- p.42 / Chapter 2.7 --- MAPK Inhibitor Experiment --- p.43 / Methods / Chapter 2.8 --- Study on the Effect of Endogenously Expressed ApoE Isoforms on Macrophage Cytokine Secretion / Chapter 2.8.1. --- Establishment of ApoE-isoform-expressing Macrophages --- p.44 / Chapter 2.8.2. --- Semi-quantification of ApoE mRNA Level by RT-PCR / Chapter 1) --- Isolation of Total RNA --- p.45 / Chapter 2) --- RT-PCR --- p.46 / Chapter 2.8.3. --- Determination of ApoE Protein Expression Level by ELISA and Western Blot --- p.47 / Chapter 1) --- Quantification of Total Proteins --- p.48 / Chapter 2) --- ELISA --- p.48 / Chapter 3) --- Western Blot --- p.49 / Chapter 2.8.4. --- LPS Treatment --- p.51 / Chapter 2.8.5. --- MEK1/2 Inhibitor Experiment --- p.53 / Chapter 2.8.6. --- p38 Inhibitor Experiment --- p.54 / Chapter 2.9 --- Study on the Effect of Exogenous ApoE Isoform on Macrophage Cytokine Secretion --- p.55 / Chapter 2.10 --- Statistical Analysis --- p.55 / Chapter Chapter 3: --- Results / Changes of Inflammatory Properties Associated with Endogenous ApoE Isoform Expression in Macrophages / Chapter 3.1 --- Characterization of ApoE-isoform-expressing Macrophages --- p.56 / Chapter 3.1.1. --- Cell Lines with Stable Expression of ApoE Isoforms --- p.56 / Chapter 3.2 --- Cell Morphology Study --- p.58 / Chapter 3.3 --- Changes of IL-6 and TNF-α Secretion Associated with Endogenous ApoE Isoforms Expression / Chapter 3.3.1. --- In the Presence of Lipoproteins --- p.60 / Chapter 3.3.2. --- Serum/Lipoprotein-independent Effects of ApoE Isoforms --- p.63 / Chapter 3.4 --- The Effects of Endogenous ApoE Isoform Expression on the Activities of MAPK Signaling Pathways / Chapter 3.4.1. --- Study on the Activation Status and Expression of MAPKs --- p.66 / Chapter 1) --- ERK1/2 MAPK Pathway --- p.66 / Chapter 2) --- p38 MAPK Pathway --- p.69 / Chapter 3.4.2. --- IL-6 and TNF-a Secretion Among ApoE Isoforms in the Presence of MEK1/2 mhibitor --- p.72 / Chapter 3.4.3. --- IL-6 and TNF-α Secretion Among ApoE Isoforms in the Presence of p38 Inhibitor --- p.75 / Chapter Chapter 4 : --- Discussions / Chapter 4.1. --- Mouse Peritoneal Macrophage Cell Line J774A.1 as Cell Model --- p.79 / Chapter 4.2. --- Inflammatory Properties Associated with Endogenous ApoE Isoform Expression in Macrophages / Chapter 4.2.1. --- Expression Level of ApoE Isoform Transgenes in Mouse Peritoneal Macrophages --- p.80 / Chapter 4.2.2. --- Macrophage Activation by LPS --- p.81 / Chapter 4.2.3. --- Effect of Endogenous ApoE Isoform Expression on Cytokine Secretion and Signal Transduction in Macrophages --- p.82 / Chapter 4.3. --- Conclusions and Future Prospects / Chapter 4.3.1. --- Conclusions --- p.90 / Chapter 4.3.2. --- Future Prospects --- p.91 / Chapter Chapter 5 : --- Appendices / Chapter 5.1 --- Changes of Inflammatory Properties of Macrophages Supplemented with Exogenous ApoE Isoforms / Chapter 5.1.1. --- Changes of IL-6 and TNF-a Secretion in Macrophages Supplemented with Exogenous ApoE Isoforms --- p.92 / Chapter 5.1.2. --- Changes of Signal Transduction in Macrophages Supplemented with Exogenous ApoE Isoforms / Chapter 5.1.2.1. --- Study on the Activation Status and Expression of MAPKs / Chapter 1) --- ERK1/2 MAPK Pathway --- p.95 / Chapter 2) --- p38 MAPK Pathway --- p.97 / Chapter Chapter 6: --- Bibliography --- p.99

A corticoterapia tem sido utilizada frequentemente nas éguas suscetíveis. O uso de isuflupredona melhora a taxa de prenhez e altera o perfil proteico do líquido endometrial em relação a éguas não tratadas. A utilização de dexametasona diminui o acúmulo de líquido pós-cobertura, reduz o edema do útero, porém, desconhecem-se seus efeitos no perfil proteico do líquido endometrial. O objetivo do presente estudo foi analisar o efeito da dexametasona em éguas suscetíveis à endometrite persistente póscobertura sobre perfil proteico do líquido endometrial na presença ou ausência de infecção. Nove éguas suscetíveis foram utilizados, com idade entre 7 a 30 anos. Após a verificação dos sinais de estro as éguas foram submetidas a quatro tratamentos: (C) éguas não receberam nenhum tipo de tratamento e serviram como controle; (D) éguas receberam 40mg de dexametasona (IV), no momento da cobertura, com coleta da amostras após 6 horas, (I-6 e I-24) infusão intra-uterina de 1 x 109 de S. zooepidemicus/mL, com coleta da amostra após 6 e 24 horas; (I/D-6 e I/D-24) infusão intra-uterina de 1 x 109 S. zooepidemicus/mL e administração de 40mg de dexametasona (IV), com coleta da amostra após 6 e 24 horas. Todas as éguas foram submetidas a todos os tratamentos. As amostras foram coletadas e submetidas à eletroforese bidimensional para separação proteica e espectrometria de massa para a identificação das bandas proteicas relevantes. A corticoterapia provocou alteração na proteômica do líquido endometrial de éguas suscetíveis, caracterizada pelo aumento (TTR) e/ou diminuição (ApoA1) na densidade óptica de proteínas da fase aguda da inflamação. Conclui-se que a utilização da dexametasona em éguas com e sem presença de infecção altera a proteômica do fluido endometrial de éguas suscetíveis. Sugere-se que a dosagem ou a frequência de aplicação da dexametasona deva ser aumentada. / Corticotherapy has often been used in susceptible mares. The use of isuflupredona improves pregnancy rate and alters the protein profile of the endometrial fluid in relation to untreated mares. The use of dexamethasone decreases the post breeding fluid accumulation, reduces the uterine edema, however is unaware of its effects on the protein profile of endometrial fluid. The aim of the present study was analyze the effect of dexamethasone in mares susceptible to post-breeding persistent endometritis on the protein profile of endometrial fluid in the presence or absence of infection. Nine susceptible mares were used, aged 7-30 years old. After checking the signs of estrus, mares were subjected to four treatments: (C) mares received no treatment and served as controls; (D) mares received 40 mg of dexamethasone at breeding time, with collection of samples after 6 hours; (I-6 and I-24) intrauterine infusion of 1 x 109 S. zooepidemicus/ml and the sample was collected after 6 and 24 hours; (I/D-6 and I/D-24) intrauterine infusion of 1 x 109 S. zooepidemicus/ml and 40 mg of dexamethasone administration, collecting the sample after 6 and 24 hours. All of the mares were subjected to all treatments. Samples were collected and subjected to two-dimensional electrophoresis for protein separation and mass spectrometry for the identification of relevant protein bands. Corticotherapy resulted in alteration of the protein profile of the endometrial fluid of susceptible mares, characterized by an increase (TTR) and/or decrease (ApoA1) in optical density of the acute phase of proteins of inflammation. We conclude that the use of dexamethasone in mares with and without the presence of infection alters the protein profile of endometrial fluid of susceptible mares. It is suggested that the dosage or frequency of application of dexamethasone should be increased.