Dissertations / Theses on the topic 'CD2BP2'

Le paludisme est l'une des maladies infectieuses les plus répandues, menaçant 40% de la population mondiale, provoquant environ 300 millions de cas et 450 000 décès chaque année, touchant principalement les enfants de moins de 5 ans. En l'absence d'un vaccin efficace et face à l'émergence de la résistance aux médicaments, il y a un besoin urgent pour mieux comprendre la biologie du parasite afin de proposer des traitements innovants. Le parasite du paludisme, Plasmodium, responsable de la maladie, présente un cycle de vie complexe et un processus de division cellulaire unique. Par rapport aux systèmes bien étudiés, la connaissance limitée de la biologie du Plasmodium empêche le développement thérapeutique. La phosphorylation des protéines, un mécanisme de régulation important, est moins comprise dans Plasmodium que dans les cellules mammifères ou de levure. Les kinases et les phosphatases impliquées dans la phosphorylation et la déphosphorylation respectivement sont des cibles potentielles de médicaments. La sous-unité catalytique de la protéine phosphatase de type 1 (PP1c) (PF3D7_1414400) opère en combinaison avec diverses protéines régulatrices pour diriger et contrôler spécifiquement son activité phosphatase. Cependant, peu d'informations sont disponibles sur cette phosphatase et ses régulateurs dans le parasite du paludisme humain, Plasmodium falciparum. Pour combler cette lacune de connaissances, nous avons mené une étude approfondie sur les caractéristiques structurelles et fonctionnelles d'un régulateur spécifique du Plasmodium appelé, Gametocyte EXported Protein 15, GEXP15 (PF3D7_1031600). Par analyses in silico, nous avons identifié trois régions d'intérêt significatives dans GEXP15 : une région N-terminale couvrant un motif RVxF interagissant avec PP1, un domaine conservé dont la fonction est inconnue, et un domaine de type GYF qui facilite potentiellement des interactions spécifiques protéine-protéine. Pour élucider davantage le rôle de GEXP15, nous avons réalisé des études d'interaction in vitro qui ont démontré une interaction directe entre GEXP15 et PP1 via le motif de liaison RVxF. Cette interaction avec PfGEXP15 a été montrée capable d'augmenter l'activité phosphatase de PP1 in vitro. De plus, en utilisant une lignée transgénique de P. falciparum exprimant la GEXP15-GFP, nous avons observé une forte expression de GEXP15 dans les stades asexués tardifs du parasite, avec une localisation principalement dans le noyau. Des expériences d'immunoprécipitation suivies d'analyses en spectrométrie de masse ont révélé l'interaction de GEXP15 avec des protéines de liaison aux ribosomes et à l'ARN. De plus, grâce à des analyses de capture de domaines fonctionnels recombinants de GEXP15 marqués avec un tag His, nous avons confirmé sa liaison avec PfPP1et au complexe ribosomal via le domaine GYF. Dans l'ensemble, notre étude éclaire l'interaction PfGEXP15-PP1-ribosome, qui joue un rôle crucial dans la traduction des protéines. Ces découvertes suggèrent que PfGEXP15 pourrait être une cible potentielle pour le développement de médicaments contre le paludisme
Malaria is one of the most prevalent vector-borne infectious diseases threatening 40% of the global population, causing around 300 million cases and 450,000 deaths annually, mostly affecting children under 5. With no effective vaccine and drug resistance emerging, there is an urgent need for innovative treatments. The malaria-causing Plasmodium parasite has a complex life cycle and unique cell division process. Compared to well-studied systems, limited knowledge of Plasmodium biology hampers therapeutic development. Protein phosphorylation, a key regulatory mechanism, is less understood in Plasmodium than in mammalian or yeast cells. Kinases and phosphatases involved in phosphorylation and dephosphorylation processes respectively are potential drug targets. The Protein Phosphatase type 1 catalytic subunit (PP1c) (PF3D7_1414400) operates in combination with various regulatory proteins to specifically direct and control its phosphatase activity. However, there is little information about this phosphatase and its regulators in the human malaria parasite, Plasmodium falciparum. To address this knowledge gap, we conducted a comprehensive investigation into the structural and functional characteristics of a conserved Plasmodium-specific regulator called Gametocyte EXported Protein 15, GEXP15 (PF3D7_1031600). Through in silico analysis, we identified three significant regions of interest in GEXP15: an N-terminal region hous-ing a PP1-interacting RVxF motif, a conserved domain whose function is unknown, and a GYF-like domain that potentially facilitates specific protein-protein interactions. To further elucidate the role of GEXP15, we conducted in vitro interaction studies that demonstrated a direct interaction between GEXP15 and PP1 via the RVxF-binding motif. This interaction was found to enhance the phosphatase activity of PP1. Additionally, utilizing a transgenic GEXP15-tagged line and live microscopy, we observed high expression of GEXP15 in late asexual stages of the parasite, with localization predominantly in the nucleus. Immunoprecipitation assays followed by mass spectrometry analyses revealed the interaction of GEXP15 with ribosomal- and RNA-binding proteins. Furthermore, through pull-down analyses of recombinant functional domains of His-tagged GEXP15, we confirmed its binding to PfPP1 and to the ribosomal complex via the GYF domain. Collectively, our study sheds light on the PfGEXP15-PP1-ribosome interaction, which plays a crucial role in protein translation. These findings suggest that PfGEXP15 could serve as a potential target for the development of malaria drugs

Orientador : Roy Edward Bruns
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica
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Doutorado

Genome wide association studies have identified genes associated with Late Onset Alzheimer’s disease (AD). Pathway analyses have used this data to implicate a number of biological mechanisms in AD pathogenesis. Endocytosis has been implicated in AD and is a critical biological mechanism involved in the production of Aβ. BIN1 and CD2AP are associated with AD and function in endocytosis. This thesis describes how depletion of BIN1 and CD2AP has contradicting effects on the processing of APP in a human brain cell line and affects endocytosis in different ways, suggesting multiple cellular trafficking mechanisms may be involved in Aβ production. As most genetic variants associated with complex diseases are located in the non-coding region of the genome, they may contribute to disease susceptibility by disrupting gene regulation. Variants at the BIN1 locus associated with AD are located approximately 30 Kb from the coding region, suggesting BIN1 regulation may be a risk mechanism in AD. BIN1 expression is influenced by cis-regulatory mechanisms in prefrontal cortex tissue. Differential allele expression implied that this cis-regulation was influenced by DNA variants. The variant responsible was not identified but there was suggestive evidence for an intronic variant associated with AD. ChIP-Seq and DNase-Seq data identified chromatin modifications and transcription factor binding in immune cell types at the BIN1 risk locus. Gene reporter assays showed that the BIN1 locus was capable of functioning as a genetic enhancer. Furthermore, assays used to investigate DNA protein interactions showed that SPI1, an AD associated transcription factor with a critical immune function, bound to the BIN1 locus. The BIN1 index SNP had no effect on gene expression or protein binding, but may have a greater impact on additional disease relevant immune cell types. Finally, gene-editing techniques were explored as a mechanism for generating isogenic cell models to investigate the effect of AD associated variants.

Little is known about the molecular biology of late onset Alzheimer’s disease (LOAD), the most common dementia in the elderly. Genetic loci associated with LOAD have been identified through genome-wide association studies (GWAS). However, the functional variants responsible for the observed GWAS association at each of the loci remain unknown. The aim of this project was to identify and assess potential functional rare variants at three associated loci, CD2AP, EPHA1 and CD33. Target enriched, pooled sequencing of 96 post-mortem confirmed LOAD patient samples was used to identify 1273 variants within the three GWAS loci. Variants were prioritised using a combination of in silico functional annotation and putative disease association. Disease association was assessed through comparison to an independent, imputed LOAD GWAS dataset (2067 cases, 7376 controls). 18 coding and untranslated region variants and 9 noncoding variants were prioritised for further investigation. Potential splicing variants in CD2AP (6:47544253A > G) and EPHA1 (rs6967117) were assessed using minigene assays, although neither were found to influence splicing products in vivo. Five untranslated variants from the three genes and a frameshift variant in CD33 (rs201074739) were assessed for potential cis-regulatory consequences using allelic expression imbalance in brain tissues and B-lymphoblast cell lines. Only the frameshift variant displayed significant allelic expression imbalance and was found to be targeted for nonsense-mediated decay. None of the prioritised variants investigated were both functional and significantly associated with LOAD. However, pooled next generation sequencing using target enrichment successfully identified potential functional rare variants in CD2AP, EPHA1 and CD33. Rare variants do have a role to play in late onset Alzheimer’s disease. With the development of additional functional databases and improvements imputing rare variants from GWAS datasets, the combined prioritisation strategy used in this thesis will be useful for similar studies investigating causal GWAS variants.

In questo progetto di ricerca, abbiamo descritto i meccanismi molecolari innescati dall'Inotuzumab Ozogamicina (CMC-544), un anticorpo anti-CD22 coniugato con la calichemicina, in linee immortalizzate ed in colture primarie di cellule linfoidi derivate da patologie linfoproliferative CD22-positive. Abbiamo utilizzato due linee cellulari derivate da linfoma di Burkitt (BL-2, RAJI, NAMALWA), una linea derivata da leucemia a precursori linfoidi B (SUP-B15) e una linea derivata da leucemia mieloide acuta (HL-60) come linea cellulare di controllo. Le cellule sono state trattate con il CMC-544 o il Gemtuzumab Ozogamicina (CMA-676), un anticorpo anti-CD33 coniugato con la calichemicina. Le linee cellulari BL-2 e SUP-B15 mostrano valori di IC50 per il CMC-544 significativamente piu' bassi rispetto a quelli ottenuti dopo trattamento con il CMA-676. Tuttavia, le RAJI e le NAMALWA presentano valori di IC50 simili per entrambi gli immunoconiugati, anche se presentano una bassa percentuale dell'antigene di superficie CD33. Inoltre, mentre le BL-2 e le SUP-B15 sopravvissute al trattamento per 48 ore con il CMC-544 sono arrestate in fase G2/M del ciclo cellulare, le RAJI e le NAMALWA - che presentano una mutazione nella sequenza amminoacidica della proteina p53 non solo non sono in grado di bloccarsi, ma l'esposizione all'immunoconiugato genera una popolazione poliploide. Nonostante cio', gli esperimenti di citofluorimetria, per valutare la distribuzione del ciclo cellulare, dimostrano che il trattamento con il CMC-544 per 12 ore induce un arresto in fase G2/M p53-indipendente in tutte le linee cellulari CD22-positive. Quando abbiamo ripetuto gli stessi esperimenti usando una combinazione sequenziale del CMC-544 e dellà à à à à ¢ UCN-01, un inibitore di ChK2, abbiamo osservato un significativo incremento della morte cellulare. Il trattamento per 48 ore con il CMC-544 determina un arresto in fase G2/M del ciclo cellulare solo nelle BL-2 e SUP-B15, un evento fortemente ridotto dopo esposizione all'UCN-01. Tuttavia, le RAJI e le NAMALWA à à à à à ¢ mancando del blocco in fase G2/M dopo 48 ore d'esposizione al CMC-544 - non rispondono al trattamento con l'UCN-01. Nonostante cio', l'espressione ectopica di p53 wild-type nelle NAMALWA le ha rese sensibili al trattamento con il CMC-544 ed ha generato un incremento della morte cellulare. Abbiamo, inoltre, confermato il valore predittivo dello status di p53 nel determinare la sensibilita' al trattamento con il CMC-544 anche in colture primarie derivate da pazienti affetti da patologie linfoproliferative CD22-positive. In conclusione, il CMC-544 mostra un alto potenziale terapeutico in cellule immortalizzate ed in linee primarie linfoidi CD22-positive. Lo status mutazionale della proteina p53 potrebbe rappresentare un marcatore molecolare di risposta al CMC-544 sia in vitro che in vivo. Inoltre, la combinazione con un inibitore della protein-chinasi ChK2 potrebbe ripristinare l'attivita' dell'immunoconiugato in pazienti con patologie linfoproliferative CD22-positive che presentano mutazioni nella proteina p53.

CD2AP is a member of the CD2AP/CIN85 family of adaptors and involved in several cellular processes, such as kidney podocyte development, actin mediated membrane trafficking and T cell activation. It contains three SH3 domains whose binding properties and interaction partners remain largely unexplored. The CD2AP SH3 interaction with the novel partner Rab5-activating GEF RIN3 was studied extensively by isothermal titration calorimetry (ITC), peptide scanning arrays, mutagenesis and X-ray crystallography. Mapping of the interaction regions showed that human RIN3 contains two binding sites for the CD2AP SH3 domains. From these studies, the CD2AP SH3 recognition motif P-x-P/A-x-x-R emerged. Two crystal structures (1.65 Å and 1.2 Å) of the SH3 1 and SH3-2 domains in complex with RIN3 epitopes 1 and 2 respectively revealed that these residues serve as anchoring points. With the aid of bioinformatics tools, this motif was used to conduct a peptide array-based screen for additional signalling partner candidates. One of the hits was the Arf-GAP ARAP1. ITC data indicate that the three SH3 domains differentially recognise three ARAP1 epitopes, with the first ARAP1 epitope binding to SH3-2 in the nanomolar range. A crystal structure (1.6 Å) of the SH3-2 domain in complex with the first ARAP1 epitope implicates two additional anchoring residues that extend beyond the PPII helical region of the canonical motif. The CD2AP/ARAP1 interaction was confirmed in podocytes and cancer cells at the endogenous protein level. Even though RIN3 and ARAP1 are involved in membrane trafficking, a direct link to CD2AP had not been reported before. Other candidates from the peptide array analyses were also investigated by ITC. In conclusion, this study led to the elucidation of the CD2AP SH3-1 and SH3-2 domain binding signatures and the identification of putative novel binding partners for all three SH3 domains. Lastly, insight was gained into the binding preferences of CD2AP SH3-3.

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A doença de Alzheimer (DA) é um dos tipos mais comuns de demência. Possui alterações patológicas como placas amilóides e emaranhados de neurofibrilas. A forma esporádica (DAE) representa 98% dos casos da doença de Alzheimer e apresenta etiologia multifatorial. A variante rs670139 do gene MS4A4E e rs9349407 do gene CD2AP foram consideradas, em estudos de Genome wide association studies, como associadas ao risco na DAE. O diagnóstico clínico da DA é difícil e apenas possível quando a doença já está em estágios avançados, sendo assim, estudos buscam encontrar biomarcadores para auxiliar no diagnóstico da doença em estágios precoces. Com isso, nos últimos anos, foram publicados vários estudos caso-controle investigando a associação de rs670139 do gene MS4A4E e rs9349407 do gene CD2AP com DAE em outras populações. Assim, este trabalho teve como objetivo investigar a associação entre o polimorfismo rs670139 MS4A4E e rs9349407 CD2AP com DAE em uma amostra de indivíduos da população da Grande Vitória, ES e realizar um estudo de meta-análise atualizado sobre a associação destas variantes com a doença. O estudo de associação foi realizado com uma amostra composta por 221 indivíduos não consanguíneos, sendo 139 indivíduos saudáveis e 82 pacientes com diagnóstico provável de DAE, pareados em relação à idade e gênero. As amostras foram genotipadas por Polymerase Chain Reaction Restriction Fragment Length Polymorphism. O Equilíbrio de Hardy-Weinberg foi calculado para cada grupo estudado. Os resultados de genotipagem foram validados por sequenciamento em 5% das amostras. Os dados foram analisados pelos testes estatísticos de Qui-quadrado, Odds ratio (Intervalo de confiança de 95%), Mann-Whitney e Regressão logística no programa SPSS (valores de p ≤ 0,05 foram considerados significativos). Na meta-análise a comparação alélica seguindo o modelo genético aditivo foi realizada pelo programa R. Os resultados obtidos neste estudo sugerem que o alelo A de rs670139 MS4A4E está associado ao risco na DAE. Este resultado apoia o papel de risco do rs670139, uma vez que, o polimorfismo, em estudos funcionais, foi correlacionado com o aumento das placas neuríticas. No entanto, não houve associação do polimorfismo rs670139 MS4A4E e rs9349407 CD2AP com DAE na população da Grande Vitória, ES e a meta-análise não encontrou associação para o rs9349407 CD2AP. Os resultados deste trabalho são importantes para ajudar a validar o papel das variantes genéticas sobre o risco de DAE.

Background: The treatment of B-cell acute lymphoblastic leukemia (B-ALL) has been enriched by novel agents targeting surface markers CD19 and CD22. Inotuzumab ozogamicin (INO) is a CD22-calicheamicin conjugated monoclonal antibody approved in the setting of relapse/refractory (R/R) B-ALL able to induce a high rate of deep responses, not durable over time. Aims: This study aims to identify predictive biomarkers to INO treatment in B- ALL by flow cytometric analysis of CD22 expression and gene expression profile. Materials and methods: Firstly, the impact on patient outcome in 30 R/R B-ALL patients of baseline CD22 expression in terms of CD22 blast percentage and CD22 fluorescent intensity (CD22-FI) was explored. Secondly, baseline gene expression profile of 18 R/R B-ALL patient samples was analyzed. For statistical analysis of differentially expressed genes (DEGs) patients were divided in non-responders (NR), defined as either INO-refractory or with duration of response (DoR) < 3 months, and responders (R). Gene expression results were analyzed with Ingenuity pathway analysis (IPA). Results: In our patient set higher CD22-FI, defined as higher quartiles (Q2-Q4), correlated with better patient outcome in terms of CR rate, OS and DoR, compared to lower CD22-FI (Q1). CD22 blast percentage was less able to discriminate patients’ outcome, although a trend for better outcome in patients with CD22 ≥ 90% could be appreciated. Concerning gene expression profile, 32 genes with corrected p value <0.05 and absolute FC ≥2 were differentially expressed in NR as compared to R. IPA upstream regulator and regulator effect analysis individuated the inhibition of tumor suppressor HIPK2 as causal upstream condition of the downregulation of 6 DEGs. Conclusions: CD22-FI integrates CD22-percentage on leukemic blasts for a more comprehensive target pre-treatment evaluation. Moreover, a unique pattern of gene expression signature based on HIPK2 downregulation was identified, providing important insights in mechanisms of resistance to INO.

La radioimmunothérapie (RIT) utilise des anticorps monoclonaux (MAbs) radiomarqués spécifiques d'antigènes tumoraux. Injectés par voie intraveineuse, les MAbs radiomarqués se fixent à la tumeur et produisent une irradiation locale permettant de faire régresser les tumeurs disséminées. La RIT anti-CD22 a démontré son efficacité dans des essais cliniques chez des patients atteints de lymphome B diffus à grandes cellules (DLBLC). L'optimisation de cette approche thérapeutique vers un traitement personnalisé impose de développer des modèles précliniques pertinents. Les modèles murins disponibles sont peu pertinents pour une optimisation de cette approche thérapeutique. Les chiens spontanément atteints de DLBCL sont en revanche représentatifs de la pathologie humaine d'un point de vue physiopathologique et constituent un modèle préclinique pertinent pour optimiser la RIT anti-CD22 chez l'Homme. Parmi les 7 MAbs murins anti-CD22 canin isolés au laboratoire, nous avons choisi le plus adapté au diagnostic par immunohistochimie et par imagerie fonctionnelle chez les chiens. La biodistribution de cet anticorps 10C6 radiomarqué à l' indium-11 1 a été déterminée chez des chiens sains et malades et la dosimétrie effectuée à partir des données recueillies en imagerie indique qu'une RIT peut-être effectuée de façon sécurisée chez les chiens atteints de DLBCL. L'influence de l'activité spécifique du MAb radiomarqué sur la biodistribution a été étudiée. Les variations importantes des doses déposées aux tumeurs et aux organes sains indiquent que l'activité spécifique est un paramètre à prendre en compte pour optimiser l'efficacité de la RIT dans une approche de personnalisation du traitement
Radioimmunotherapy (RIT) uses radiolabeled monoclonal antibodies (MAbs) specific for tumor antigens. After intravenous injection, radiolabeled MAbs bind to the tumor and produce local irradiation to reduce disseminated tumors. Anti-CD22 RIT has been shown to be effective in clinical trials in patients with diffuse large B-cell lymphoma (DLBCL). The optimization of this therapeutic approach towards personalized treatment requires the development of relevant preclinical models. The mouse models available are of little relevance for optimizing this therapeutic approach. Conversely, dogs with spontaneous DLBCL are representative of human pathology from a physiopathological point of view and constitute a relevant preclinical model for optimizing anti-CD22 RIT in humans. Among the 7 murine anti-canine CD22 Mabs isolated in the laboratory, we chose the most suitable for diagnosis in immunohistochemistry and functional imaging in dogs. The biodistribution of this indium-111 radiolabeled 10C6 antibody has been determinated in healthy and diseased dogs, and dosimetry from imaging data indicates that RIT can be safely performed in dogs with DLBCL. The influence of the specific activity of radiolabeled MAb on biodistribution has been studied. The large variations in the doses deposited to tumors and healthy organs indicate that the specific activity is a parameter to take into account to optimize the efficiency of the RIT in a personalization approach to treatment

Les immunoglobulines intraveineuses (IVIg) sont préparées à partir de pool de plasmas de milliers de donneurs sains et sont utilisées depuis les années 80 dans le traitement de certaines maladies autoimmunes. Leurs modes d’actions sont peu connus chez l’homme et nous avons donc souhaité étudier leurs effets sur les lymphocytes B (LB). Nous avons ainsi identifié CD22 comme étant un récepteur membranaire capable de lier les IVIg, d’empêcher la cellule de répondre à une stimulation de son BCR et d’entraîner un arrêt du cycle cellulaire et une entrée en apoptose. Nous avons également montré que les IVIg pouvaient inhiber l'activation des LB par les Toll-like receptors de l’immunité innée. Ce mécanisme fait intervenir une phosphatase et aboutit au blocage de la voie NF-kB, l’une des principales voies de signalisation de la cellule. Nos différentes observations nous ont montré qu’il existait de nombreuses similitudes entre le LB traité par IVIg et le LB en anergie qui est caractérisé par son absence de réponse face à la stimulation auto-antigénique. Ainsi, les IVIg perturbent le passage des BCR dans les radeaux lipidiques membranaires où sont émis les signaux activateurs et bloquent la libération de calcium, facteur déterminant dans l’activation de la cellule. Dans une dernière série d’expériences, nous avons vérifié notre hypothèse et montré que le traitement par IVIg prévenait la réponse à une immunisation chez la souris. Ainsi, les IVIg agissent sur les LB aussi bien au niveau des mécanismes de l’immunité adaptative que de l'immunité innée et cela dès les premiers évènements qui surviennent suite à la stimulation de la cellule. Tous ces phénomènes entraînent une non- réponse qui est suivie d’une élimination du LB
IVIg is prepared from pooled plasma of healthy donors and used as a therapeutic agent in autoimmunity since the 80’s. Few things are known about its mechanisms in human, we sought to better understand how IVIg interact with B-cells, which are key components of autoimmunity. Here, we find that CD22 can bind IVIg thereby blocking B-cell response to 8CR stimulation. IVIg also blocks ceil cycle and promotes apoptosis. Moreover we find that IVIg can inhibit TL. R-mediated activation by recruiting phosphatase resulting in NF-kB pathway inhibition. Our results show that (t exist a similarity between lVlg-treated B-cells and anergic B-cells whose response is inhibited. Thus, IVIg can block BCR translocation in lipid raft and intracellular calcium in flux which are both essential elements in B-cell activation. Finally, we con flan our hypothesis by treating mice with IVIg and showing that these mice can’t respond properly to immunization. This work shows that IVIg can act on adaptive as well as on innate immune system therefore resulting in a non-response followed by an elimination of B-cells

碩士
長庚大學
臨床行為科學研究所
96
Chapter 1 No abstract Chapter 2 Trunk Study of Distributed Constraint-Induced Therapy in Patients with Chronic Stroke Objective： Patients showed impairments of arm trunk coordination after stroke. Previous studies didn’t investigate the interventions’ effects on arm-trunk coordination after constraint-induced therapy (CIT). However, the trunk compensation often used by stroke patients may limit the improvement of the affected arm. Therefore, we tried to discuss the influence on arm trunk coordination and trunk compensation strategy after the intervention of distributed CIT. Methods： A total of 16 participants (at least 6 months post-onset) after stroke were randomly assigned to either distributed CIT group (practiced 2 hours every workday for 3 weeks and restrained the unaffected arm 6 hours per day) or control group (received traditional rehabilitation for equivalent intensity and duration). We used kinematics analysis (unilateral and bilateral tasks) to investigate the arm-trunk coordination and two clinical measures (Functional Independent Measure, FIM and Stroke Specific Quality of Life Scale, SSQOL) to assess functional ability and quality of life of the participants. All the outcomes were measured at the beginning and end of the 3-week intervention. Results： There were no differences between the groups at baseline (p > 0.05). The results showed that there were statistically different between the groups during the unilateral tasks on elbow extension angular change (p = 0.030), trunk flexion angular change (p = 0.045), elbow extension & trunk flexion correlation (p = 0.020), and early part, terminal part, and total of the trunk contribution slope (p < 0.05). There were also statistically different on elbow extension angular change (p = 0.017), trunk-arm delay (p = 0.030), and the total trunk contribution slope (p = 0.033) during the bilateral tasks. Statistically different on transfer domains of FIM (p = 0.046) and family role domain of SSQOL (p = 0.013) were noted as well. Conclusion： After 3-week distributed CIT, the participants reduced trunk compensation and performed normally as a result of gained better elbow joint control during both the unilateral or bilateral tasks. It showed that distributed CIT elicited better arm trunk coordination and improved greater motor control. Participants also had better performances in functional ability and quality of life after distributed CIT. Chapter 3 Effects of Bilateral Arm Training in Patients with Chronic Stroke Objective： To examine the effectiveness of bilateral arm training (BAT) on chronic stroke patients by measuring the performance of kinematics analysis, motor capacity and functional ability. Methods： A total of 33 patients (at least 6 months post-onset) after stroke were randomly assigned to either BAT or placebo-controlled traditional rehabilitation. All patients practiced 2 hours every workday for 3 weeks. The outcome measures were the performance of kinematics analysis (unilateral & bilateral tasks), motor capacity (the Fugl-Meyer Assessment, FMA) and functional ability (Functional Independent Measure, FIM), assessed at the beginning and end of treatment. Results： There were no significant differences between the groups at baseline (p > 0.05). We observed better performance in reaching kinematics of the affected arm after BAT as compared with the traditional rehabilitation by less nMT and nTD (p = 0.034; p = 0.039) during both unilateral and bilateral tasks. But a non-significant and small effect was found on PPV during the unilateral task (p = 0.396), rather than bilateral task (p = 0.001). Also, BAT showed a significant greater improvement in FMA than traditional rehabilitation (p = 0.041), but there were no differences in FIM (p > 0.05). Conclusion： BAT was associated with higher efficiency in the temporal and spatial aspects during the unilateral and bilateral tasks, also a greater preplanning control strategy during the bilateral tasks. Greater motor improvements were also observed by FMA. These findings provide some insight about the mechanisms that may be responsible for improved motor function of the affected arm after BAT. But we suggested that more studies were needed to examine the effect of BAT on functional ability.

碩士
國立臺灣藝術大學
書畫系造形藝術碩士班
105
This study is based on author’s childhood memory, to reflect on the way of life and meaning with subconscious and social values. Together with childhood memories and experience, combined with collage art, subconscious, flower language and other related literature, this report unites he concept of unity and practice creation. In the first chapter, the author introduces the research motive, purpose, and the research method. The second chapter explains the author's background to the creation of the relevant theoretical background. The third chapter analyzes author’s practice of creative techniques. The fourth chapter is the main creative theme, to explore the subconscious, memory and social value of the relationship between the three interactive, and the memory of childhood objects, summed up the impact of social value on life and analysis of the growth process of self-meaning and meaningless Life perception experience. The author of this research to the researchers from 2015 to 2016 works for the study, to the childhood memories of the object as the main creative thinking core. The content can be divided into four series: First, flower story series describes the growth process, with the time of the scheduled travel calendar, daily life, but neglect the relationship between life and self. Second, the fine series of flowers, the face of the world of flowers, the pursuit of life value, but easy to forget the value of self, and then explore the issue of social assimilation. Third, the flower park series, the main description of the value of the community packaging, life behind the busy, looking for selfless heart in the deep childhood pure childhood permanent memory. Fourth, the daily series of flowers, childhood growth process, mediocrity, did not stop. When we finally stopped, we learn to have a conversation with ourselves, their own and time alone on the daily imagination of life. I want to create ideas and experiments by the new order, in an attempt to personal memory and aesthetic inspiration, to reorganize the inner world.

In the first chapter, I measure the effects of street-level political advertising on voting behavior. I use a novel dataset on ad location in a major Spanish city during elections for the national parliament as well as granular socio-economic and voting data. This set-up, where more than two parties are running for office and elections are very competitive, allows me to explore the heterogeneous effects of ads across parties as well as how parties' ads affect other parties' vote shares. To identify the effects of parties' ads, I exploit a legally mandated randomized assignment of ad location to parties across multiple years. I find that a party's own ads have a positive effect on its vote share, although the effects are heterogeneous across parties. A one standard deviation increase in the number of ads increases a party's vote share by 0.79 percentage points on average. Ads of parties with ideologically distant platforms consistently have a negative effect on a party's vote share. In contrast, ads of parties that are close competitors may act either as complements or substitutes in different years. The second chapter analyses the effects of an economic shock on the emergence of new parties and other changes in voting parties by using regional variation in the exposure to the shock. I find that a worsening of economic conditions as measured by unemployment rate leads to an increase in electoral competition and volatility. In particular, the deeper the effects of the recession in a area, the larger the number of new parties emerge and become more successful and there is an increase in the changes in vote shares. On the other hand, the vote share of parties previously in government decreases and a decrease in vote share concentration. The third chapter is a co-authored works where we present experimental evidence establishing that the level of incentives affects both gameplay and beliefs. Holding fixed the actions of the other player, we find that, in the context of dominance-solvable games, higher incentives make subjects more likely to best-respond to their beliefs. Moreover, higher incentives result in more responsive beliefs but not necessarily less biased. We provide evidence that incentives affect effort and that it is effort, and not incentives directly, that accounts for the changes in belief formation. The results support models where, in addition to choice mistakes, players exhibit costly attention.

Gegenstand dieser Arbeit ist die Untersuchung von Aspekten der Funktion von CD22, einem B-Zell spezifischen Transmembran-Rezeptor der Siglec-Familie (Sialinsäure-bindende Immunglobulin-ähnliche Lectine). Mit der äußersten der 7 extrazellulären Ig-ähnlichen Domänen kann CD22 spezifisch mit a2,6-Sialinsäure interagieren. In der cytoplasmatischen Domäne von CD22 befinden sich 6 konservierte Tyrosine, 3 davon in ITIMs (Immunrezeptor tyrosinhaltige inhibitorischen Motiven). Nach Kreuzvernetzung des B-Zell Rezeptors wird CD22 tyrosinphosphoryliert. Die cytosolische Tyrosin-Phosphatase SHP-1 bindet in der Folge an die phosphorylierten ITIMs, wird aktiviert, und inhibiert das BCR Ca2+-Signal. Gleichzeitig binden jedoch auch positive Modulatoren des BCR-Signals (Lyn, Syk, PLCg PI3K, und Grb-2) an CD22, deren Rolle im Zusammenhang mit CD22 bislang ungeklärt ist. 1. In einem Hauptteil der Arbeit sollten zwei Knockin Mausmodelle generiert werden. Das eine Mausmodell (CD22-ITIM-KO) sollte zerstörte ITIM-Motive enthalten. Bei dem anderen (CD22-Tailless) sollte die gesamte cytoplasmatische Domäne von CD22 fehlen. Beide Modelle sollten der Untersuchung der Rolle der an CD22 bindenden positiven Modulatoren des BCR-Signals, und des Zusammenhangs zwischen Signaltransduktion und Ligandenbindung in vivo dienen. Die Klonierung der Targeting-Vektoren für CD22-ITIM-KO (pCD22-ITIM-KO) und CD22-Tailless (pCD22-Tailless) wurde abgeschlossen. Mit Hilfe ebenfalls klonierter Kontrollvektoren wurden PCRs zur Identifizierung homolog rekombinanter ES-Zell Klone etabliert. Für beide Targeting-Konstrukte wurden nach Transfektion von C57BL/6 ES-Zellen homolog rekombinante Klone erhalten, und mittels Southern Blot und Sequenzierung der eingeführten Mutationen vollständig charakterisiert. Nach Cre/lox-vermittelter Deletion der Selektionskassette des Targeting-Konstrukts folgte Injektion voll charakterisierter CD22-ITIM-KO Klone in BALB/c-Blastozysten. Es wurden 5 chimäre Tiere erhalten, von denen keines die Mutationen durch die Keimbahn weitergab. Transfektionen der C57BL/6 Targeting-Konstrukte in anderen, nicht-isogenen ES-Zell Linien ergaben keine homologen Rekombinanten. Das Auffinden der genomischen Sequenz von CD22 in einer Internet-Datenbank ermöglichte die Verlängerung von pCD22-ITIM-KO um ca. 4 kb mit 129/ola-DNA. Eine Transfektion dieses neuen Konstruktes in eine 129/ola ES-Zelllinie ergab keine homologen Rekombinanten. Jedoch öffnet die nun bekannte genomische CD22-Sequenz den Weg zu einfacher Neukonstruktion von pCD22-ITIM-KO mit 129/ola-DNA, oder zu einer Veränderung und Verbesserung der vorhandenen C57BL/6-Vektoren. 2. Zur Untersuchung der Auswirkung der zerstörten ITIMs auf Tyrosinphosphorylierung und SHP-1 Assoziation von CD22 in vitro in einer Zelllinie wurde ein CD22-ITIM-KO-Expressionsvektor konstruiert, und Sialyltransferase/CD22-ITIM-KO Doppeltransfektanten der Plasmozytom-Zelllinie J558L gewonnen. CD22-ITIM-KO wurde nach BCR-Stimulation nicht tyrosinphosphoryliert, SHP-1 konnte entsprechend nicht mit CD22-ITIM-KO assoziieren. Die Ergebnisse zeigen die Funktionalität des CD22-ITIM-KO Konstrukts hinsichtlich ITIM-Phosphorylierung und SHP-1 Bindung. Weiterhin zeigten die Ergebnisse, daß die ITIM-Tyrosine wichtig für die Phosphorylierung der nicht-ITIM-Tyrosine sind. 3. Interaktion von CD22 mit a2,6-Sialinsäure auf der selben Zelloberfläche (in Cis) spielt eine wichtige Rolle bei der Zell-Zell-Interaktion und bei der intrazellulären Signaltransduktion. In dieser Arbeit wurden erstmals mittels Durchflußcytometrie B-Zellen mit CD22, dessen Liganden-Bindungsstelle nicht durch endogene a2,6-Sialinsäure besetzt ist (demaskiertes CD22), identifiziert. Ca. 10,5% aller B220+ Milzzellen von Wildtyp-Mäusen, aber nur ca. 4,5% der B220+ Milzzellen aus CD22-/- Mäusen waren in der Lage, exogene a2,6-Sialinsäure zu binden. Dieser Effekt ist zum Großteil auf CD22 zurückzuführen. Genauere FACS-Analyse zeigte, daß Zellen mit demaskiertem CD22 in der Fraktion der Transitionalen B-Zellen Typ 2 (T2-Zellen) angereichert sind, und Zeichen von Aktivierung (B7.2, CD25, CD69) zeigen. In Übereinstimmung damit führte in vitro Aktivierung von B-Zellen mit LPS oder IL4 zu CD22-abhängiger Demaskierung. 4. FACS-Färbungen zeigten, daß das Marginalzonen (MZ) B-Zell Kompartiment in CD22-/- Mäusen um ca. 70-80% gegenüber wt-Mäusen verkleinert ist. In Bestätigung früherer Arbeiten war die Immunantwort gegen i.p.-injizierte Thymusunabhängige Antigene Typ 2 (TI2-Antigene) in CD22-/- Mäusen 2-fach reduziert. Die Antwort war jedoch signifikant stärker reduziert (3-4-fach), wenn die gleiche Antigen-Menge i.v.-injiziert wurde, eine Situation, in der bevorzugt die MZ B-Zellen der Milz in Kontakt mit im Blutstrom transportierten Antigenen kommen. Es ist wahrscheinlich, daß die bekannte Defizienz in TI2-Immunantworten in CD22-/- Mäusen auf die verringerte MZ B-Zell Anzahl zurückzuführen ist
Aim of this thesis was to investigate aspects of the function of CD22, a B-cell specific member of the Siglec-family (sialic acid binding immunoglobulin–like lectins). CD22 can specifically bind a2,6-sialic acid with the outermost of its 7 extracellular Ig-domains. There are 6 conserved tyrosine residues in the cytoplasmic domain, 3 of which lie within ITIMs (immunoreceptor tyrosine-based inhibitory motifs). Following BCR engagement, CD22 becomes tyrosine-phosphorylated. Consecutively, the cytosolic tyrosin-phosphatase SHP-1 binds to the phosphorylated ITIMs, becomes activated and inhibits the BCR-signal. There are also some positive modulators of the BCR-signal which bind to CD22 (Lyn, Syk, PLCg PI3K, und Grb-2). The role of these molecules in the context of CD22 remains to be elucidated. 1. One main goal of this work was the generation of two new knockin mouse lines. In the first line (CD22-ITIM-KO), the ITIMs of CD22 were to be destroyed. A second line in which CD22 lacks its cytoplasmic tail was to be generated (CD22-Tailless). Both models were supposed to serve the in vivo investigation of the role of the positive modulators of the BCR-signal and the interrelation of CD22 signaling and ligand binding. Cloning of the targeting vectors pCD22-ITIM-KO and pCD22-tailless was completed. Using control vectors, which were also cloned, screening-PCRs for the identification of homologous recombined ES-cell clones were established. C57BL/6 ES-cells were transfected with the targeting constructs, and homologous recombinants were identified with PCR and Southern blot. The introduced mutations were confirmed by sequencing. Following cre/lox-mediated deletion of the selection cassette, fully characterised CD22-ITIM-KO clones were injected into BALB/c-blastocysts. Five chimaeras were obtained, none of which transmitted the mutations through the germline. No homologous recombinants were obtained upon injection of the C57BL/6 targeting constructs into other, non-isogenic ES-cell lines. Finding of the genomic sequence of murine CD22 in a database made it possible to extend pCD22-ITIM-KO by 4 kb with DNA from a 129/ola mouse strain. The new construct was used to transfect 129/ola ES-cells, which are generally known to be kariotypically more stable than C57BL/6 ES-cells. No homologous recombinants were obtained. The now known sequence of the CD22 gene will make it possible to reconstruct pCD22-ITIM-KO based on 129-DNA or to modify and improve the already existing C57BL/6-constructs. 2. To investigate the consequences of the destroyed ITIMs on tyrosine-phosphorylation and SHP-1 association of CD22 in vitro, a CD22-ITIM-KO expression-vector was constructed and sialyl-transferase/CD22-ITIM-KO double-transfectants of the murine plasmocytoma-line J558L were generated. CD22 tyrosine-phosphorylation after BCR-stimulation was completely abolished in CD22-ITIM-KO transfectants. Accordingly, SHP-1 couldnt associate with CD22-ITIM-KO. The results prove the funcionality of the CD22-ITIM-KO construct with respect to ITIM-phosphorylation and binding of SHP-1. Furthermore the results show that the ITIM-tyrosines are also important for the phosphorylation of the non-ITIM-tyrosines. 3. Interaction of CD22 with its ligand a2,6-sialic acid on the surface of the same cell (in cis) plays an important role in cell-cell-interaction and intracellular signal transduction. In this work B-cells on which the ligand binding site of CD22 is not occupied by endogenous a2,6-sialic acid (demasked CD22) were identified by FACS for the first time. 10,5% of all B220+ spleen cells from wild type mice, in contrast to only 4,5% B220+ spleen cells from CD22-/- mice were able to bind exogenous a2,6-sialic acid. This effect is mainly due to the presence or absence of CD22, respectively. Detailed FACS-analyses revealed that cells with demasked CD22 are enriched in the fraction of the transitional B-cells type 2 (T2 cells) and show signs of activation. Accordingly, in vitro activation of B-cells with LPS or IL4 resulted in CD22-dependent demasking. 4. FACS-analyses showed that the marginal zone (MZ) B-cell compartment in CD22-/- mice is strongly reduced (by about 70-80%). Earlier results showing that the immune response against i.p.-injected TI2-antigens is about 2-fold reduced in CD22-/- mice could be confirmed. However, the response was significantly more impaired (3-4-fold) when the same dose of antigen was applied i.v., a situation where preferentially the MZ B-cells of the spleen come in contact with antigen carried along with the blood flow. The known deficiency in TI2-responses in CD22-/- mice is likely due to non-sufficient MZ B-cell numbers in these animals

Das Hauptthema der hier vorliegenden Arbeit befaßt sich mit dem B-Zell spezifischen Oberflächenprotein CD22, einem Mitglied der Siglec (Sialinsäure bindende Igähnliche Lektine) Proteinfamilie. Dieses Transmembranprotein besitzt sieben extrazelluläre Immunoglobulin-ähnliche Domänen und kann über die äußerste V-set Domäne seine Liganden: α2,6 verknüpfte Sialinsäuren binden. CD22 hat eine Transmembrandomäne und eine cytoplasmatische Domäne mit sechs potentiellen Tyrosin Phosphorylierungsstellen, von denen drei eine ITIM-Sequenz (engl. immunoreceptor tyrosine-based inhibitory motif) aufweisen. CD22 defiziente Mäuse zeigten eindeutig, daß das Siglec CD22 ein negativer Regulator des BCR-Signals ist. Durch BCR-Kreuzvernetzung wird CD22 tyrosinphosphoryliert, die inhibitorische Tyrosinphosphatase SHP-1 gebunden, aktiviert, und ist nun in der Lage das BCR Ca2+ Signal zu inhibieren. Um die Rolle der CD22Ligandenbindungsdomäne, in vivo zu untersuchen, sollte in dieser Arbeit eine CD22 knock -in Maus erzeugt werden (CD22R130E Maus), in der die Ligandenbindungsdomäne von CD22 durch eine Punktmutation funktionell ausgeschaltet ist. In der hieraus resultierenden Mauslinie sollte dann die BZellentwicklung, Signaltransduktion und der Immunstatus analysiert werden. Der Vergleich des Phänotyps der CD22R130E Maus und der CD22 defizienten Maus sollte dann zeigen, wie die Adhäsions- und Signalleitungseigenschaften von CD22 zusammenhängen. Der „Targeting“ Vektor für die „Gene Targeting“ Experimente wurde von der Arbeitsgruppe Dr. Anton van der Merwe (von Christina Piperi) angefertigt. Ursprünglich wurde ein „Targeting“ Vektor aus genomischer C57BL/6-DNA verwendet, um den genetischen Hintergrund der CD22-defizienten Maus beizubehalten. Dieser Vektor wurde von mir für ES-Zell Transfektionen in der C57Bl/6 ES-Zellline verwendet. Aus den Gene Targeting Experimenten mit der C57Bl/6-III ES-Zelllinie konnten zwei ES-Zellklone isoliert werden, die eine korrekte homologe Integration des Targetvektors trugen. Aus einem Blastozysteninjektions- Experiment mit einem Cre-deletierten C57BL/6-III Subklon wurden sechs hochchimäre Mäuse erhalten, mit denen allerdings keine Keimbahntransmission erzielt werden konnte. Nach Problemen mit Keimbahntransmission von Klonen aus der C57BL/6-III ESZelllinie, wurden noch die BALB/c und die E14Tg2a ES-Zelllinie für neue Gene Targeting Experimente verwendet. Die Experimente mit der BALB/c ES-Zelllinie ergaben keine ES-Zellklone mit korrekter homologer Integration, dies beruhte wahrscheinlich auf dem nicht isogenen Hintergrund. Alle folgenden Experimente mit der E14Tg2a ES-Zelllinie (genetischer Hintergrund: 129/ola) wurden mit dem verlängerten R130E-Targetvektor (Targetvektor 2), der mit 129/ola DNA um 2,3 Kb in 5’-Richtung verlängert wurde, um den isogenetischen Anteil des Targetvektors zu erhöhen, durchgeführt. Aus diesen Experimenten resultierten wiederum zwei ESZellklone, deren korrekte homologen Rekombination durch Southern Blot bestätigt werden konnten. Bei den darauffolgenden Blastozysten-Injektionsexperimenten mit diesen zwei E14Tg2a Klonen konnten fünf chimäre Tiere gewonnen werden. Ein 80 %ig chimäres Männchen erzeugte eine hohe Anzahl von Nachkommen mit Keimbahntransmission. Bei der Analyse dieser Tiere trat das Resultat zutage, daß alle diese Tiere mit Keimbahntransmission einen wildtypischen Genotyp besaßen. Ein weiteres Mitglied der Siglecproteinfamilie, das murine SiglecG (ein Ortholog zu humanem Siglec10), wurde in dieser Arbeit untersucht. In Zusammenarbeit mit dem Labor von Dr. Paul Crocker sollte eine SiglecG knock out Maus hergestellt werden. Die Strategie für die Gene Targeting Experimente für einen SiglecG knock out basierten auf der Verwendung der BalbI ES-Zelllinie (aus BALB/c Mäusen), da hiermit sehr gute Erfahrungen vorlagen, was die Stabilität ihrer Pluripotenz und des Keimbahntransmissionspotenzials angeht. Daher wurde im Labor von Paul Crocker (von Sheena Kerr) ein Kontroll- und ein Targetvektor kloniert, mit dem große Teile der ersten und zweiten Ig-Domäne von SiglecG ausgeschaltet werden sollte. Mit diesem Vektor führte ich mehrere ES-Zell Transfektionsexperimente durch. Innerhalb der Zeitspanne meiner Doktorarbeit konnten keine ES-Zellklone mit einem korrekten homologen Integrationsereignis gewonnen werden. Mittels der ursprünglichen Strategie konnte die mir nachfolgende Doktorandin jedoch ES-Zell Klone isolieren, nach Blastozysteninjektion Keimbahntransmission erzielen und somit eine SiglecGdefiziente Maus generieren. Eine andere Zusammenarbeit kam mit Dr. Burkhard Kneitz (Physiologisches Chemie I, Biozentrum, Universität Würzburg) zustande. Seine Intention war es, die Rolle des TGF-β Signalmediators SMAD2 auf B-Zellebene näher zu untersuchen. Von Erwin Böttinger bekamen wir eine Mauslinie, in der das Smad2-Gen gefloxt ist, die mit der CD19-Cre Maus gekreuzt wurde. So wurde eine B-Zell spezifische SMAD2 knock out Maus (bSmad2-/-) erzeugt. Meine Aufgabe bestand darin, die B-Zellkompartmente und die Immunantworten der B-Zell spezifischen Smad2-defizienten Maus zu analysieren. Faßt man alle gewonnenen Daten aus den hier generierten B-Zell spezifischen Smad2 knock out Tieren zusammen, so kann man zu dem klaren Ergebnis kommen, daß der TGF-β Signalmediator Smad2 eine entscheidende Rolle bei der Weiterleitung von TGF-β Signalen in das Zellinnere von B-Zellen spielt. Hierbei zeigten sich klare Veränderungen, im Vergleich zu Kontrolltieren, eine Erhöhung der Zellzahl in den Peyerschen Plaques (PP), und der B1-Zellen im Peritoneum. Die IgA-Immunantwort war in bSmad-/- Tieren stark erniedrigt. Der für TGF-β beschriebene Effekt der Proliferationshemmung von aktivierten B-Zellen war bei aktivierten B-Zellen der bSmad2-/- Mäuse hingegen nicht beeinträchtigt
The main topic of this thesis dealt with the B cell-specific transmembrane protein CD22, a member of the Siglec (Sialic-acid binding Ig-like lectin) protein family. This transmembrane protein posseses seven extracellular domains and is capable to bind α2,6 sialic acids via its most outer V-set domain. Furthermore there are one transmembrane domain and six potential tyrosine-based phosphorylation motifs, three of which match ITIM (immunoreceptor tyrosine-based inhibitory motif) consensus sequences. CD22 deficient mice clearly showed that the Siglec CD22 is a negativ modulator of BCR signalling. BCR engagement causes tyrosine phosphorylation of CD22, now the inhibitory tyrosine phosphatase SHP-1 is able to bind, is then getting activated and is thus inhibiting the BCR Ca2+ signal. To elucidate the function of the CD22 adhesion domain in vivo, especially concerning the connection with CD22 signalling, one main topic of this work was to generate a CD22 knock in mouse (CD22R130E), in order to functionally eliminate the CD22 adhesion domain through a point mutation. The resulting new mouse line should give the opportunity to investigate B-cell development, signal transduction and the immune status ot the CD22R130E mouse. The comparison between the phenotypes of the CD22R130E mouse and the CD22 deficient mouse should resolve the interplay of adhesion and signalling of CD22. The cloning of the targeting vector for the gene targeting experiments was done in the laboratory of Dr. Anton van der Merwe (by Christina Piperi). Basically, the idea was to keep the C57BL/6 genetic background, which was already used to generate the CD22 deficient mouse by Dr. Lars Nitschke (Nitschke et al. 1997). This vector was used by me for the ES cell transfection experiments with the C57Bl/6-III ES cell line. Finally, two ES-cell clones could be identified from gene targeting experiments with the C57BL/6 ES-cell line, which carried a correct homologous integrated target vector. With one Cre-deleted C57BL/6 subclone it was possible to generate six chimaeric animals from one injection experiment, although none of these animals could give rise to germline transmission. Since occurence of crucial problems with the germline transmission of C57BL/6-III ES-cell clones, the BALB/c and E14Tg2a ES-cell lines were used for new gene targeting experiments. With the following gene targeting experiments using the BALB/c ES-cell line no homologous recombinants were obtained. This was probably due to the non-isogenic background. All following experiments performed with the E14Tg2a ES-cell line (genetic background: 129/ola) were carried out with the elongated R130E-targeting vector (targeting vector 2). This vector was created by using a genomic 129/ola template, in order to gain a new isogenetic 2.3 kb 5’- fragment. These experiments gave rise to two ES-cell clones with a correct homologous recombination event confirmed by southern blot. It was now possible to generate five chimaeric animals out of three injection experiments with these two EScell clones. One male animal, with 80 % chimaerism, produced offspring with germline transmission. The analysis of these animals with germline transmission showed that all of them possessed a wildtype like genotype. This thesis dealt with a further member of the Siglec protein family, the murine SiglecG (an ortholog to human Siglec10). In collaboration with the laboratory of Dr. Paul Crocker a SiglecG knock out mouse was to be generated. The strategy to do the gene targeting experiments was based on the usage of the BalbI ES-cell line (from BALB/c mice), since it possesses well known stability concerning pluripotential and germline transmission potential. In the laboratory of Paul Crocker (by Sheena Kerr) a control and target vector was cloned, which should eliminate a large part of the first and second Ig-domain of SiglecG. I performed different ES-cell transfection experiments with this vector. Within the timecourse of my work it was not possible to gain any ES-cell clones with correct homologous integration events. Later on ES-cell clones, germline transmission and generation of the SiglecG deficient mouse was achieved with the original strategy by the following Phd student. Another collaboration was evolved by Dr. Burkhard Kneitz (Department of Physiological Chemistry I, Würzburg). His intention was to investigate the meaning of the TGF-β signalmediator SMAD2 in a B-cell specific manner. From Erwin Böttinger we received a mouse line with a floxed Smad2 gene, which was crossed with a CD19-Cre mouse line (Rickert et al. 1997). Thus a B-cell specific SMAD2 knock out mouse (bSmad2-/-) was generated. I had to analyse the B-cell compartments and the immune responses of the B-cell specific SMAD2 knock out mouse. Taking together all data gained with the newly generated B-cell specific SMAD2 knock out mouse showed that the signalmediator SMAD2 is a crucial downstream component of TGF-β signalling in B-cell biology. The crucial differences of bSmad2-/- animals in comparison to control animals were given in an increase of cells of Payers Patches (PP) and B1 cells of peritoneal lavages. The IgA immune response was strongly reduced in bSmad2-/- animals. The well known effect of TGF-β concerning inhibition of proliferation with activated B-cells (Kehrl et al. 1986; 1989; 1991) was not impaired with activated B-cells of bSmad2-/- animals

CD22 ist ein B-zellspezifisches Transmembranprotein der Immunglobulin (Ig)-Superfamilie. Es übernimmt zwei unterschiedliche Aufgaben. Zum einen hat CD22 eine inhibitorische Wirkung auf das BZR-Signal. Nach BZR-Ligation lagert sich die Tyrosin-Phosphatase SHP-1 an die cytoplasmatische Domäne von CD22 an, wodurch SHP-1 aktiviert wird. Durch die dephosphorylierende Aktivität der Phosphatase moduliert sie das BZR-Signal. Zum anderen ist CD22 ein Adhäsionsmolekül, das in die Gruppe der Siglecs (Sialic acid-binding Ig-like lectin) gehört. Die N-terminale Ig-Domäne von CD22 weist die Bindungseigenschaften eines Lectins auf und bindet spezifisch 2,6-gekoppelte Sialinsäure. Das Ziel der vorliegenden Doktorarbeit war es, die inhibitorische Wirkung von CD22 auf das BZR-Signal molekular zu definieren. Daher wurde das Substrat der an CD22 rekrutierten und aktivierten Phosphatase SHP-1 in vergleichenden Analysen von CD22-defizienten und Kontroll-B-Zellen nach BZR-Stimulation gesucht. Wir konnten zeigen, dass in CD22-defizienten B-Zellen nach BZR-Stimulation das Adaptermolekül SLP-65, auch BLNK oder BASH genannt, früher und stärker Tyrosin-phosphoryliert vorliegt, als in Kontroll-B-Zellen. Transfektionsexperimente mit der CD22-defizienten Plasmazytom-Zelllinie J558Lm3 wurden begonnen, um den molekularen Zusammenhang zwischen CD22, SHP-1 und SLP-65/BLNK zu bestätigen. Das in J558Lm3 Zellen ektopisch exprimierte CD22 wurde Tyrosin-phosphoryliert, und SHP-1 konnte mit CD22 co-präzipitiert werden. Jedoch war in den CD22-Transfektanten keine Reduktion der Tyrosin-Phosphorylierung von SLP-65/BLNK nach BZR-Stimulation im Vergleich zu untransfizierten Zellen nachweisbar. Da in unabhängigen Experimenten die Liganden-Bindung von CD22 als Voraussetzung für die inhibitorische Wirkung von CD22 deutlich wurde, etablierten wir 2,6 Sialinsäure- und CD22-positive J558Lm3 Doppeltransfektanten. Auf diesen konnte die cis-Maskierung von CD22 nachgewiesen werden. In Stimulationsexperimenten der Doppeltransfektanten wurde die reduzierte Tyrosin-Phosphorylierung von SLP-65/BLNK in Abhängigkeit von CD22 in der Mehrzahl der Klone bestätigt. Allerdings mussten die Resultate in Frage gestellt werden, als in den meisten Klonen einer Kontrolltransfektion ebenfalls eine Reduktion der Tyrosin-Phosphorylierung von SLP-65/BLNK festgestellt wurde. Um den Einfluss von CD22 und SLP-65 auf das BZR-Signal zu klären, wurden CD22- und SLP-65-defiziente Mäuse gekreuzt. Durch die zusätzliche Deletion von CD22 in SLP-65-/- Mäusen konnte das Ca2+-Signal nach BZR-Stimulation wieder hergestellt werden. Jedoch zeigte die weitere Analyse der doppel-defizienten Mäuse, dass in der Regel der Phänotyp der SLP-65-defizienten Mäuse dominiert. Diese Ergebnisse verdeutlichten, dass das Adaptermolekül SLP-65/BLNK zwar ein Substrat des CD22/SHP-1 Signalweges ist, aber keine essentielle Rolle in der inhibitorischen Wirkung von CD22 übernimmt. Weiterhin wurde der Einfluss der Ligandenbindung von CD22 auf dessen intrazelluläre, inhibitorische Wirkung auf das BZR-Signal untersucht. Ein synthetisches Sialoside stand zur Verfügung, das hochspezifisch die Interaktion von CD22 mit dessen Liganden stört. Wurden die Zellen einer humanen B-Zelllinie in Gegenwart des Sialosids über den BZR stimuliert, konnte ein erhöhtes Ca2+-Signal gemessen werden. Dieses Resultat erinnerte an die stärkere Ca2+-Mobilisierung in CD22-defizienten B-Zellen. Entsprechend war die Tyrosin-Phosphorylierung von CD22 nach Vorbehandlung mit dem Sialosid in den humanen B-Zellen verringert, und weniger SHP-1 konnte mit CD22 co-präzipitiert werden. Mit diesen Ergebnissen wurde deutlich, dass die Adhäsionsdomäne von CD22 einen direkten, positiven Einfluss auf die intrazelluläre, inhibitorische Domäne von CD22 hat. Als Nebenprojekt wurde die Rolle von CD22 in knock-out Mäusen des transkriptionellen Co-Aktivators BOB.1/OBF.1 untersucht. Ein Entwicklungsblock im transitionalen B-Zellstadium im Knochenmark der BOB.1/OBF.1-defizienten Mäuse verursacht eine deutliche Reduktion reifer B-Zellen in der Milz. Die Analyse des Knochenmarks der BOB.1/OBF.1-defizienten Mäuse zeigte, dass ausschließlich die Expression von CD22 auf den B-Vorläufer Zellen erhöht war. Nach zusätzlicher Deletion von CD22 in BOB.1/OBF.1-/- Mäusen war nach BZR-Stimulation ein deutliches Ca2+-Signal in den doppel-defizienten B-Zellen messbar. Dieses könnte die normalisierte Anzahl transitionaler B-Zellen im Knochenmark und die gestiegene Anzahl reifer B-Zellen in der Milz der doppel-defizienten Mäuse bewirken. Allerdings waren die doppel-defizienten Mäuse, entsprechend den BOB.1/OBF.1-/- Mäusen, nicht in der Lage, eine humorale Immunantwort einzuleiten oder Keimzentren zu bilden. Die Proliferation von CD22-defizienten B-Zellen nach LPS-Stimulation verlief unabhängig von der An- oder Abwesenheit von BOB.1/OBF.1. Mit den Untersuchungen konnten wir zeigen, dass die Differenzierungsschwierigkeiten der BOB.1/OBF.1-defizienten B-Zellen vom BZR-Signal abhängen. Allerdings muss das Ausbleiben der Keimzentrenbildung auf einen anderen Mechanismus zurückgeführt werden
CD22 is a B cell-specific transmembran protein of the Immunoglobulin (Ig) superfamily, which has two distinct functions. On the one hand, CD22 acts as a negative regulator of the BCR-signal. Upon BCR-ligation the tyrosine phosphatase SHP-1 associates with the cytoplasmatic tail of CD22 leading to the activation of SHP-1. The activated tyrosine phosphatase dephosphorylates signaling molecules within the BCR-signaling cascade, thereby inhibiting the BCR-signal. On the other hand, CD22 is an adhesion molecule belonging to the Siglec family (Sialic acid-binding Ig-like lectin). The N-terminal Ig-domain confers lectin function by specifically binding to 2,6-linked sialic acid. The main focus of this thesis was to molecularly define the inhibitory role of CD22 on the BCR-signal. Therefore, we looked for possible substrates of the tyrosine phosphatase SHP-1 by comparing the tyrosine phosphorylation level of proximal signaling molecules upon BCR-stimulation in CD22-deficient and control-B-cells. The adaptor protein SLP-65, also called BLNK or BASH, was earlier and stronger tyrosine-phosphorylated in CD22-deficient B-cells, compared to control-B-cells upon BCR-stimulation. Reconstitution experiments were started with the CD22-deficient plasmacytoma cell-line J558Lm3 to demonstrate the molecular correlation between CD22, SHP-1, and SLP-65/BLNK. Ectopically expressed CD22 was tyrosine-phosphorylated in transfected J558Lm3 cells and SHP-1 could be co-precipitated with CD22. However, the tyrosine-phosphorylation level of SLP-65/BLNK was unaffected or even increased in CD22-positive J558Lm3 transfectants upon stimulation. In the meantime we were able to show that the adhesion domain of CD22 has a direct influence on the inhibitory role of CD22. Therefore, plasmacytoma cells were established stably expressing 2,6 sialic acid and CD22. On those cis-masking of CD22 could be detected. The tyrosine-phosphorylation level of the adaptor molecule SLP-65/BLNK was decreased in most of the double-transfected J558Lm3 clones depending on the expression of CD22. But the obtained results had to be questioned when the tyrosine-phosphorylation of SLP-65/BLNK was also decreased in most of the clones of a control-transfection. To further analyze the mechanism of BCR-signal regulation by CD22 and SLP-65, CD22- and SLP-65-deficient mice were crossed. The additional deletion of CD22 in SLP-65-/- mice restored the Ca2+-signal in double-deficient B-cells upon BCR-stimulation. Further analysis of SLP-65xCD22-double-deficient mice revealed that the phenotype of the SLP-65-/- mice was dominant in most of the explored aspects. Therefore, we concluded that the adaptor protein SLP-65/BLNK is a substrate of the CD22/SHP-1 pathway but not crucial for mediating the inhibitory function of CD22. So far it was not known whether the ligand-binding of CD22 influences its intracellular signaling domain. To investigate this question we used a synthetic sialoside, which specifically binds to the lectin domain of CD22 and thereby interferes with the ligand-binding. When cells of a human B cell-line were stimulated with anti-IgM in the presence of this sialoside, a higher Ca2+-signal was observed, similar to the one measured in CD22-deficient B-cells. Accordingly, a lower tyrosine-phosphorylation of CD22 and less SHP-1 recruitment was demonstrated in the presence of this sialoside in those human B-cells. Thus, by interfering with the ligand binding of CD22 on the B-cell surface, we have shown that the lectin domain of CD22 has a direct, positive influence on its intracellular inhibitory domain. As a side project we investigated the role of CD22 in mice lacking the transcriptional co-activator BOB.1/OBF.1. A developmental block at the transitional B cell stage in the bone marrow of BOB.1/OBF.1-deficient mice causes a reduced number of splenic B cells. By analyzing the bone marrow of BOB.1/OBF.1-/- mice, we found that the expression of CD22 is selectively increased on B-lineage cells. Furthermore, the Ca2+-signal in BOB.1/OBF.1-deficient B-cells was restored upon BCR-stimulation, when CD22 was additionally deleted. The increased Ca2+-signal could cause the normal number of transitional B cells in the bone marrow and the increased number of mature B-cells in the spleen of BOB.1/OBF.1xCD22-double-deficient mice. Nevertheless, double-deficient animals were unable to mount humoral immune response and to form germinal centers as has been described for BOB.1/OBF.1-deficient mice. Finally, CD22-deficient B-cells proliferate independently of BOB.1/OBF.1 upon stimulation with LPS. These studies suggest that the differentiation defect of B-cells observed in BOB.1/OBF.1-/- mice is BCR-signal dependent. However, the impairment of BOB.1/OBF.1-/- B-cells to form germinal centers is caused by a different mechanism

Tese de mestrado. Biologia (Biologia Molecular e Genética). Universidade de Lisboa, Faculdade de Ciências,2014
A doença de Alzheimer (AD) foi identificada em 1907 pelo Dr. Alois Alzheimer. Trata-se de uma doença neurodegenerativa bastante complexa, sendo a maior causa de demência a nível mundial. Após mais de 100 anos de investigação na AD os únicos tratamentos alcançados apenas conseguem retardar a doença, e melhorar a qualidade de vida do paciente, por mais algum tempo. A neuropatologia é caracterizada pela presença no cérebro de placas de péptidos β-amilóide (Aβ) e aglomerados intraneuronais de formas hiperfosforiladas da proteína tau associada aos microtúbulos. Numa fase inícial da AD inicia-se a acumulação e oligomerização do péptido Aβ, tanto a nível extra como intracelular que causa disfunção das sinapses levando à perda de sinapses e neurónios. Os principais sinais e sintomas clínicos da AD são a perda de memória, de habilidades intelectuais, racionais e sociais gerando a perda de qualidade de vida do paciente, acabando por num estado avançado levar á perda total da autonomia e da própria identidade do paciente, até à morte. O péptido Aβ é um péptido de 39 a 43 aminoácidos produto do processamento sequencial da proteína precursora amilóide (APP) pelas enzimas β-secretase (BACE1) e posteriormente γ-secretase (via de processamento amilodoigénica). O processamento do APP origina dois péptidos Aβ40 ou Aβ42, sendo o último o responsável pelo efeito de toxicidade nos neurónios. A anormal acumulação de Aβ42 nos neurónios resulta de um desequilíbrio entre os níveis de produção e degradação do Aβ42. A produção do Aβ42 via o processamento de APP está dependende do encontro do APP e seus derivados com as secretases específicas do seu processamento nos endossomas. Sendo a desregulação do tráfego neuronal para os endosomas uma das causas possíveis para o desenvolvimento da AD. A AD está fortemente associada à idade sendo o envelhecimento celular o principal factor de risco para o seu desenvolvimento. A AD pode ser classificada em dois tipos, a AD de aparecimento precoce ou a AD de aparecimento tardio (também conhecida por tipo familiar ou esporádico respectivamente). Contudo, estão identificados outros factores de risco para a doença como factores genéticos; predisposição clínica, ou comportamentos de risco. Estudos de associação genética com a AD em populações de diversas regiões do globo, identificaram um conjunto de genes com forte associação à AD de aparecimento tardio. O polimorfismo rs9349407 do CD2AP foi um dos genes identificados como factor de risco para AD, p = 8.78E-07, odds racio (OR) = 1.08, 95 % intervalo de confiança. Cluster of Differentiation 2-Associated Protein (CD2AP) é uma proteína citoplasmática reconhecida pela sua função em células T, podócitos, ou pela sua interacção com actina. É referida como sendo uma proteína efectora da Rab4 na maturação dos endossomas. A função desempenhada nos neurónios e no desenvolvimento da AD é completamente desconhecida. O objectivo deste trabalho de dissertação é estudar o papel da proteína CD2AP nos mecanismos celulares e moleculares, que podem levar ao desenvolvimento da AD. A nossa hipótese biológica é que o CD2AP como regulador da via endocítica neuronal tem acção na acumulação de Aβ42 nos neurónios levando ao desenvolvimento da AD. Para estudar o CD2AP utilizamos uma linha celular de neuroblastoma de rato (N2a) e culturas primárias de neurónios (PN) de murganho (Balb-C). Iniciamos o estudo do CD2AP comprovando a expressão endógena do CD2AP nos neurónios primários e células N2a. Observamos uma distribuição homogénea do CD2AP no corpo celular e dendrites dos primários neurónios, e um enriquecimento junto á membrana plasmática e espiculas. Nos axónios a concentração de CD2AP era menor. Nas células N2a verificamos por imunofluorescência que o CD2AP se distribui por toda a célula com uma maior concentração na região perinuclear (PR). Para a subexpressão do CD2AP nas células utilizamos uma construção do CD2AP marcada por uma proteína de fluorescência verde (GFP-CD2AP), e uma segunda construção que possuí um codão stop após o segundo domínio SH3 (GFP-CD2AP(SH3)). Verificamos que o GFP-CD2AP nas células tem tendência a acumular-se junto á membrana plasmática e processos celulares, e que ao contrário da proteína endógena o GFP-CD2AP aglomera e forma grandes e brilhantes vesiculas. Verificamos também que os dois primeiros domínios do GFP-CD2AP(SH3) alteraram a dispersão do CD2AP pelas células. Tendo em conta a hipotese colocada utilizámos a técnica de transfecção de DNA e RNA de interferência alteramos a expressão do CD2AP nas células e analisámos os níveis de Aβ42 em células a sobrexpressar o CD2AP ou tratadas com siRNA contra CD2AP (subexpressando CD2AP). Detectámos um aumento significativo dos níveis de Aβ42 nas células a sobrexpressar CD2AP, de igual forma se registou o aumento de Aβ42 nas células a subexpressar CD2AP. Este resultado sugere que para CD2AP funcionar normalmente os seus níveis são regulados com precisão pelos neurónios sendo que excesso ou falta de CD2AP induz um aumento de Aβ42. Seguidamente, investigámos se o CD2AP regula o processamento do APP em PN e em células N2a, em células a sobrexpressar ou a subexpressar o CD2AP. Comprovámos por western blot que o CD2AP tem tendência a diminuir o processamento do APP. Sugerindo que o CD2AP regula o processamento do APP pela γ-secretase, reacção enzimática que forma o péptido Aβ. Por imunofluorescência analisámos o efeito do CD2AP na localização endossomal do APP e da BACE1 nas células N2a. Destacamos que em células N2a tratadas com siRNA-CD2AP ocorre um aumento do APP/CTFs transportado por endossomas positivos para Lamp1. Sugerimos que nas células que subexpressam CD2AP ocorre um aumento do encontro do APP/CTFs com a γ-secretase, levando ao aumento de produção do Aβ40/42. Porque o CD2AP esta descrito como tendo uma função reguladora da estrutura F-actina e que a F-actina participa na dinamica dos endosomas.Verificamos por imunofluorescência o efeito do CD2AP na regulação da F-actina nas células N2a e PN, comprovamos que as células tratadas com siRNA-CD2AP assumem uma morfologia differente com a presença de longas extensões da membrana plasmática, que poderam ser enormes filopodia ricas em F-actina, semelhantes às que se observam em zonas de crescimento de processos neuronais. Verificàmos também que os níveis de F-actina estão aumentados nas células que subexpressam siRNA-CD2AP. Em suma, comprovamos que o CD2AP é uma proteína com os níveis de expressão altamente regulados pela célula, que uma alteração dos níveis de expressão do CD2AP provoca um aumento da acumulação do Aβ42 nas células, possivelmente associado ao efeito do CD2AP na maturação dos endossomas e na regulação do citosqueleto de actina.
CD2AP is a cytoplasmatic protein known to be an effector of maturation of endosomes. Studies of genetic associations recognized CD2AP as a risk factor of Alzheimer’s disease. The intraneuronal accumulation of beta-amyloid 42 (Aβ42) peptide is a hallmark of early stage of Alzheimer’s disease. In this dissertation we studied the effect of CD2AP on the accumulation of cell associated Aβ42, in primary mouse neuronal culture (PN) from cerebral cortices of BALB/c mice with 16 days of gestation and a cell line of mouse neuroblastoma cells (N2a). Using cells, transiently transfected with CD2AP or treated with siRNA against CD2AP, we describe CD2AP effects on cells that may lead to development of Alzheimer’s disease. We found that the deregulation of CD2AP expression level lead to increase of Aβ42 in the cells. The Aβ42 is product from sequential processing of the amyloid precursor protein (APP) by β-secretase enzyme (BACE1) and next γ-secretase. Cells overexpressing or depleted for CD2AP have tendency to decrease mainly the levels of the C-terminal fragments (CTFs) of APP. We described in cells depleted for CD2AP an increase of APP/CTFs in late endosomes, suggesting that an increase in the meeting of APP with the γ-secretase. We analyzed the BACE1 endocytosis pathway in cells overexpressing CD2AP, we did not find significant differences in the distribution of BACE1. Knowing that CD2AP regulates actin polimerization, we studied the effect of CD2AP depletion in cells. CD2AP knockdown caused an increase in F-actin level and altered the morphology of cells. I suggest that the level of expression of CD2AP is highly regulated in the cell, and that the interaction of CD2AP in the maturation of cells leads to increase on cell associated Aβ42. Justifying CD2AP as risk factor for the AD.