Immune thrombocytopenia (ITP) is a heterogeneous immune-mediated blood disorder with multiple pathologies that cause thrombocytopenia. The primary source of this thrombocytopenia is platelet destruction by antiplatelet autoantibodies. Although several treatment options are available for ITP, they are often transient, and responses can be difficult to predict. Different studies show ITP plasma and autoantibodies can also inhibit platelet production, but the mechanism and its impact in causing thrombocytopenia remains unknown. By identifying the different mechanisms causing ITP thrombocytopenia, it may be possible to identify more effective and patient specific treatment options, as well as identify patients who could be at an increased risk of bleeding. To study the antibody mediated inhibition of platelet production, I developed a peripheral blood based megakaryopoiesis assay that used the patient’s own hematopoietic stem and progenitor cells (HSPC) as a starting cell source to grow megakaryocytes. I demonstrate this assay can use a small amount of peripheral blood to grow mature megakaryocytes that are capable of thrombopoiesis. Using this assay, I investigated the effect patient plasma had on platelet production. As such, this study is the first autologous investigation of the effect ITP plasma has on platelet production. I found no inhibition of megakaryopoiesis, but did find an effect on thrombopoiesis, indicating that the plasma is affecting the end stages of platelet production. Secondary observations also show that some ITP HSPC have an enhanced megakaryopoiesis potential, generating more mature megakaryocytes than what was observed with healthy donors. While screening monoclonal antiplatelet antibodies, I discovered an anti-GPIb antibody that inhibited megakaryocyte maturation and found this affect was also present with the Fab antibody fragment. From my research I have developed several tools that can be used to investigate impaired platelet production in ITP and further our understanding of this pathology. / Thesis / Doctor of Philosophy (PhD) / Immune thrombocytopenia (ITP) is an immune mediated blood disorder where autoantibodies target and destroy platelets, cells that are crucial for preventing blood loss. Evidence from different studies show that ITP autoantibodies are also affecting the cells producing platelets (megakaryocytes), but the mechanism of this effect remains unknown. To study antibody mediated inhibition of megakaryopoiesis, I developed a peripheral blood based megakaryopoiesis assay that used the patient’s own cells as a starting source to grow megakaryocytes. With this assay, I investigated the effect patient plasma had on platelet production. I found no inhibition of megakaryocyte growth but did find an effect on their ability to produce platelets. I also found a model antibody that affected the maturity of the megakaryocytes during their development. These tools can now be used to further investigate impaired platelet production in ITP patients and determine the impact this inhibition has on ITP pathology.

Background: Primary immune thrombocytopenia (ITP) is an autoimmune disease involving autoantibody-mediated platelet destruction, suboptimal platelet production, and T-cell-mediated platelet lysis. These processes cause a decreased peripheral blood platelet count (< 150 x 109/L), resulting in an increased susceptibility to bleeding events. While thecourse of primary ITP is often acute (< 6 months) among children, it is generally chronic in adults. Despite a marked increase in epidemiological research over the past decade, unresolved questions remain with regard to disease pathogenesis, treatment effectiveness, and co-morbid burden among adults with primary ITP. Objectives: 1. To launch a registry for adults with primary ITP in the United Kingdom (UK); 2. To evaluate associations of candidate single nucleotide polymorphisms (SNPs) with primary ITP; 3. To assess the effectiveness of 111In-labelled platelet studies in predicting outcomes from splenectomy; 4. To document health-related lifestyle concerns among patients; 5. To gauge the effectiveness of Helicobacter pylori (H. pylori) eradication in elevating platelet counts; 6. To characterise associations of primary ITP with both arterial and venous thromboembolic events (TEs). Data sources: 1. The UK Adult ITP Registry (17 centres, 327 patients); 2. The UK Adult ITP Registry-affiliated, 111In-Labelled Platelet Study Database (117 centres, 256 patients); 3. The Wellcome Trust Case-Control Consortium (WTCCC) 1958 British Birth Cohort (3,000 individuals); 4. The General Practice Research Database (GPRD) (500+ centres, 4 million+ patients); 5. The UK ITP Support Association Health-Related Lifestyle Survey (790 patients); 6. Systematic reviews of published epidemiological studies. Methods and Results: SNP Study: Caucasian patients from the UK Adult ITP Registry were gender-matched (1:3) with healthy controls from the WTCCC 1958 British Birth Cohort. Six functional candidate SNPs in cytokine or cytokine receptor genes were measured in cases and retrieved for controls from the European Genome-phenome Archive. Associations were evaluated using logistic regression models. A statistically significant per allele odds ratio (OR) of 1.34 (95% confidence interval [CI], 1.03-1.75) was observed for TNFA -308 g>a, implicating increased disease susceptibility among carriers of the rare allele. 111In Study: The effectiveness of autologous 111In-labelled platelet sequestration studies in predicting short (1-3 months), medium (6-12 months), and long-term (last follow-up) complete response (CR; count < 100 x 109/L) to splenectomy in patients with primary ITP was evaluated using multivariable logistic regression models. Significant adjusted (gender, age at splenectomy, and mean platelet lifespan) ORs were uncovered for short (7.47 [95% CI, 1.89-29.43]), medium (4.85 [95%CI, 1.04-22.54]) and long-term (5.39 [95% CI, 1.34-21.65]) CR in patients with purely or predominantly splenic versus mixed or hepatic splenic platelet sequestration, highlighting the utility of platelet sequestration studies as an adjunct predictive tool prior to splenectomy. H. pylori Eradication Study: A systematic literature review was conducted of studies evaluating the effects of H. pylori eradication on platelet count in patients with primary ITP. Twenty-five studies were identified, encompassing 696 evaluable patients. The weighted mean complete response (count > 100 x 109/L) and overall response (platelet count > 30 x 109/L) were 42.7% (95% CI, 31.8%-53.9%) and 50.3% (95% CI, 41.6%-59.0%), respectively. Observed responses were higher in countries with a higher prevalence of H. pylori infection and in patients with milder thrombocytopenia. These findings support the benefits of H. pylori detection and eradication in patients with primary ITP. Health-Related Lifestyle Study: A 43-question, closed-field questionnaire was used to identify health-related lifestyle concerns among patient members of the UK Adult ITP Support Association. Completed surveys were returned by 790 (44.7%) members, with nearly one-third of adults reporting having an elective surgery delayed due to a low platelet count and experiencing difficulty in obtaining travel insurance. Notably, 12.5% of all patients reported 'always' or 'often' missing work or school due to fatigue. These results highlight several promising avenues for future health-related quality of life (HRQoL) research. Thromboembolic Event Studies: Using the GPRD, 1,070 adults with primary ITP were matched (1:4) with 4,280 ITP-free controls by age, gender, practice, and observation time. Comparative risks of TEs were assessed using Cox proportional hazards models. Over a median time of 47.6 months (range: 3.0-192.5 months), adjusted hazard ratios of 1.58 (95% CI, 1.01-2.48) and 1.37 (95% CI, 0.94-2.00) were found for venous and arterial TEs, respectively. A similar investigation was conducted comparing age and sex-stratified incidence rates of TEs among patients in the UK Adult ITP Registry with population-based estimates for the general adult population, yielding significant standardised rate ratios of 2.43 (95% CI, 1.01-5.83) and 2.45 (95% CI,1.48-4.06) for venous and arterial TEs, respectively. These results collectively highlight an increased risk of venous and arterial TEs in adults with primary ITP. Conclusions: Primary ITP is a pro-inflammatory (i.e., TH-1-mediated), pro-thrombotic disease in adults. Available evidence supports testing for and eradication of H. pylori infection in patients with primary ITP and the utility of autologous 111Inlabelled platelet sequestration studies in identifying patients likely to respond to splenectomy. The development of a prospective, international registry will help assemble the sample sizes needed for promising further research, namely genome-wide disease association studies and investigations into the effects of the eradication of strain-specific H. pylori infection on platelet count, the precise relative risk of non-response to splenectomy in patients with mixed or hepatic platelet sequestration, and the associations of TEs with primary ITP in antiphospholipid antibody-negative.

Immune thrombocytopenia (ITP) is an autoimmune disorder, mediated mainly by autoantibodies against platelet glycoprotein GPIIbIIIa and GPIbα resulting in enhanced platelet destruction. Decreased platelet production and cellular immunity also contribute to ITP. GPIIbIIIa and GPIbα are distinct platelet receptors. Previous studies suggested that anti-GPIbα (versus anti-GPIIbIIIa)-mediated ITP is less responsive to IVIG therapy. However, little information is available whether antibody specificities also dictate efficacy of Glucocorticosteroids (GC), which are the first-line ITP treatment. Here, I first induced ITP in mice by passive administration of anti-GPIbα or anti-GPIIbIIIa antibodies. Results suggest GCs were more effective at amelioration of anti-GPIIbIIIa-mediated thrombocytopenia. I repeated this observation in an active ITP model, in which splenocytes from wild-type platelet immunized GPIbα-/- or GPIIIa-/- mice were engrafted into wild-type mice, which developed ITP. Thus, I established new murine models of ITP for GC therapy and demonstrated that anti-GPIbα-mediated thrombocytopenia may be less responsive to GC therapy.

Immune thrombocytopenia (ITP) is an autoimmune disorder, mediated mainly by autoantibodies against platelet glycoprotein GPIIbIIIa and GPIbα resulting in enhanced platelet destruction. Decreased platelet production and cellular immunity also contribute to ITP. GPIIbIIIa and GPIbα are distinct platelet receptors. Previous studies suggested that anti-GPIbα (versus anti-GPIIbIIIa)-mediated ITP is less responsive to IVIG therapy. However, little information is available whether antibody specificities also dictate efficacy of Glucocorticosteroids (GC), which are the first-line ITP treatment. Here, I first induced ITP in mice by passive administration of anti-GPIbα or anti-GPIIbIIIa antibodies. Results suggest GCs were more effective at amelioration of anti-GPIIbIIIa-mediated thrombocytopenia. I repeated this observation in an active ITP model, in which splenocytes from wild-type platelet immunized GPIbα-/- or GPIIIa-/- mice were engrafted into wild-type mice, which developed ITP. Thus, I established new murine models of ITP for GC therapy and demonstrated that anti-GPIbα-mediated thrombocytopenia may be less responsive to GC therapy.

Abstract: Poly (vinylidene fluoride) (PVDF), thanks to its versatile properties, finds many applications ranging from water purification membranes (thermal and chemical stability) to electronic devices (piezoelectric, pyroelectric and ferroelectric properties). Block copolymers of PVDF with other polymers further expand its properties and, consequently, its applications. Toward this line, my thesis investigates the synthesis, molecular characterization and properties of novel PVDF-based copolymers mainly with poly(tert-butyl acrylate) (PtBuA), poly(methyl methacrylate) (PMMA) and polystyrene (PSt). To prepare the block copolymers a living polymerization is needed, which is compatible with the VDF and the comonomer (tBuA, MMA, St). For this purpose, we used iodine transfer polymerization (ITP) with the difunctional chain transfer agent (CTA) C4F8I2. Difunctional macroinitiator (I-PVDF-I) was first obtained by ITP of VDF monomer with C4F8I2, followed by addition of the comonomer tBuA, MMA or St to afford the triblock copolymers poly(tert-butyl acrylate)-block-poly(vinylidene fluoride)-block-poly(tert-butyl acrylate) (PtBuA-b-PVDF-b-PtBuA), poly(methyl methacrylate)-block-poly(vinylidene fluoride)-block-poly(methyl methacrylate) (PMMA-b-PVDF-b-PMMA) and polystyrene-block-poly(vinylidene fluoride)-block-polystyrene (PSt-b-PVDF-b-PSt). The structure of all intermediates and final products were characterized by Nuclear Magnetic Resonance (NMR) and Gel Permeation Chromatography (GPC). The microstructure and polymorphism of all triblock copolymers, characterized by XRD, shown that the PVDF in the first two copolymers exhibits the electroactive β-phase, while in the third copolymer there is the coexistence of α- and γ-phases. Linear PVDF homopolymers, using the free radical and IT polymerizations, were prepared for comparison purposes. All linear polymers possess the α-phase. The thesis is divided into the following five chapters: 1. Introduction, where the scope of this thesis is given with a brief background on PVDF; 2. Literature Review, where a summary of previously published works on PVDF synthesis and polymorphism is presented; 3. Experimental Section, where detailed procedures and characterization methods are given; 4. Results and Discussion, where outcomes of successful experiments are discussed; and 5. Conclusion and Perspective, where the outcomes of this work are summarized and perspective are discussed.

The autoimmune disease known as autoimmune thrombocytopenic purpura (ITP) is clinically defined by a low numbers of platelets in the circulation blood. Anti-platelet antibodies bind to glycoprotein molecules on the membranes of platelets and result in their dysfunction and destruction. Despite a growing body of information about ITP, it is difficult to isolate and characterise anti-platelet antibodies, because only limited monoclonal antibodies are available from ITP patients. This study used a phage display system to recognise Fab anti-platelet antibodies. Anti-platelet Fab-expressing phage was isolated by sequential panning of an ITP Fab library against normal non-ITP platelets. After isolation, the anti-platelet Fab-expressing phage was characterised by ELISA and Western blotting. The Fab-bearing phage pool obtained from five rounds of panning was analysed in order to determine its anti-platelet reactivity. Of the phage colonies obtained, 100 colonies of different sizes were randomly selected for reaction with whole platelets, using Ml3 phage as a negative control. 12 colonies of them had strong reactions against the whole platelet preparation, but only four colonies showed substantial reactivity against the lysed platelet preparation (lysate). Colony S7 showed highest the greatest degree of binding to both the lysate and the whole platelet preparation. The specificity of the four colonies (S2, S7, S8 and S9) that had strong positive reactions against platelet antigens was determined for the glycoprotein component GP Ilb/IIIa. Further characterisation of the proteins in the lysate preparation was carried out using blotting techniques. The protein content of the four Fab-bearing phage colonies was quantified under the non-reducing conditions of Western blotting to evaluate their ability to recognise platelet antigens. Three of the four colonies showed three bands representing proteins with different molecular weights. Each of these three colonies had one band that corresponded to a protein of molecular weight 92 kD. The fourth colony showed only a single band, but this band also corresponded to a 92-kD protein.

Detta examensarbete har syftat till att utforma ett informativt budskap om ITP 2 ålderspension. Målet har varit att lyfta fram specifik information om ITP 2 ålderspension för vår uppdragsgivares anställda. Budskapet har visualiserats genom en animerad informationsfilm och ska fungera som ett komplement till redan befintlig information. Baserat på tidigare forskning och resultat från intervjuer som vi genomfört har vi funnit riktlinjer som legat till grund för de designval och beslut vi tagit under processens gång. Resultatet blev en animerad informationsfilm med en avskalad och enkelt utformad design. / This project has aimed to design an informative message regarding ITP 2 retirement pension. The goal has been to highlight specific information regarding ITP 2 retirement pension for the employees of our client. The message has been visualized as an animated information video and will serve as a complement to existing information. Based on previous research and results from our conducted interviews, guidelines have been obtained which have formed the basis for the design choices and decisions we have taken during the process. The outcome of this project is an animated information video with a clean and simple design.

Pensionsområdet upplevs av många som svårt och jobbigt att sätta sig in i. Framtida pension består av de olika delarna allmän pension, tjänstepension och privat pension och det ser olika ut för alla beroende bland annat på arbetsplats och intjänande. Studiens fokus är tjänstepension och den grupp som har möjlighet att välja en alternativ pensionslösning. Studien är undersökande till sin natur och syftar till att utforska och beskriva medarbetares och experters syn på mötet mellan en medarbetare och en försäkringsförmedlare. Mötet ämnar ses som en potentiell arena för kunskapsutveckling kring tjänstepension och tjänstepensionsbeslut. Studien syftar också till att svara på frågan om vilka möjligheter och hinder, vad gäller lärande och kunskapsutveckling, pensionsmötet upplevs innebära. Utgångspunkt för den teoretiska referensramen är det sociokulturella perspektivet på lärande och några centrala begrepp inom teorin som upplevs särskilt aktuella för studien. Tidigare forskning och litteratur som ansetts relevanta är bland annat litteratur om pensionsmötet och det pensionsval mötet mynnar ut i. Då uppsatsen handlar om själva pensionsmötet belyses dessutom forskning som fokuserar på institutionella samtal samt liknande sociala sammanhang som mötet mellan pensionsrådgivare och medarbetare, så som möten mellan banktjänsteman och konsument. Forskningsstrategin som använts i studien är kvalitativ, och empirisk data har samlats genom intervjuer med medarbetare och experter inom området. Resultatet visar att arbetstagaren i många fall hindras få en neutral arena för kunskapsutveckling vad gäller beslut om pension. Hindren kan vara egna fördomar, krångligt upplägg eller rådgivarens roll. Arbetsgivarens roll lyfts också som viktig för framtida lärande i den här typen av möten.

The purpose of this study was to analyze students' specific ITP-related social skills goals, student self-reported social skills, and the relationship between teacher and employer ratings of vocational students social skills and problem behaviors. This study examined (48) vocational students, (24) general education vocational students and (24) special education vocational students in grades nine through twelve. The students' vocational teachers and employers also participated in the study. This represented (144) individual assessment of social skills and problem behaviors utilizing the Social Skills Rating System -Student version (SSRS-S) and the Social Skills Rating System Teachers -version (SSRS-T). The findings indicated no specific social skill goals were deliminated in the students' ITP's. However, the findings did indicate the general education vocational students rated themselves higher, on average, on the empathy subscale than did the special education students. The analysis of data comparing standardized social skill scores, social skill subscale scores, standardized problem behavior scores, and standardized problem behavior subscale scores between teachers and employers for general and special education vocational students indicated employers rated special education students higher on the cooperation subscale only. No other differences were found.

Isotachophoresis (ITP) coupled to capillary zone electrophoresis (CE) in a comprehensive manner was used to separate mixture components in both insufficient and sufficient concentrations without heart-cutting or splitting. Examples of comprehensive ITP-CE involving multiple CE injections of preconcentrated ITP zones are demonstrated. In the comprehensive arrangement, all of the sample in the first dimension (ITP) is subjected to analysis in the second dimension (CE), without significant sample loss or decrease in sample detectability resulting from removal of a portion of the sample. This is especially important for analytes at low concentrations which may form a single mixed zone instead of individual ITP zones. Direct online coupling of ITP to CE in this comprehensive arrangement involved the use of columns having different diameters with one directly inserted inside the other. A counterflow was applied when the isotachophoretic sample stack reached the bifurcation point. Large volume (10 µL) injections were made using an electrically-insulated commercial polymeric rotary valve injector for increased reproducibility compared to previous comprehensive ITP-CE studies, with ITP and CE retention time RSD values ranging from 2-5%. An ultraviolet (UV) detector positioned at the bifurcation point was used to determine the beginning of CE injection. Application of a splitting voltage at the bifurcation point showed no affect on analyte transfer into the CE column. By using multiple injections of the ITP band(s), CE column overloading was not observed. Online capillary isotachophoresis (ITP) and comprehensive isotachophoresis-capillary electrophoresis (ITP-CE) were also coupled with electrospray ionization (ESI)-orthogonal acceleration time-of-flight mass spectrometry (TOFMS). Separations were performed using 200 µm I.D. and 50 µM I.D. polyvinylalcohol (PVA)-coated fused silica capillaries for ITP and CE, respectively. Both ITP and ITP-CE were coupled to TOFMS for analysis of sufficient (10-5 M) and insufficient (10-6-10-7 M) concentrations of angiotensins in mixtures. ITP-TOFMS of a single mixed zone of five angiotensins (3 x 10-7 M) showed that ion suppression due to the co-elution of angiotensin III in the electrospray significantly reduced the ionization of other analytes. A practical solution to the detection difficulties for ITP mixed zones involved the insertion of a CE separation between the ITP and TOFMS for online preconcentration, separation, and identification in one system.