<p>T lymphocytes regulate the initiation, duration, and magnitude of adaptive immune responses and function as effector cells in cell mediated immunity. To become immunologically competent they must generate functional antigen receptors. This process takes place in the thymus and requires somatic recombination of T cell receptor (TCR) genes. It is mediated by the endonucleases recombination activating gene-1 (RAG1) and RAG2. Although the thymus regresses at puberty, T cells are present throughout life implying that other tissues must provide the proper milieu for T cell development. This thesis describes extrathymic T cell maturation in man. RAG1, RAG2, and the preTα-chain (pTα), which is exclusively utilized in developing T cells, were used as markers for TCR gene rearrangement. Two new exons (1A and 1B) encoding sequences in the 5’ untranslated region (5’UTR) of mRNA were discovered in the human RAG1 gene. The previously described 5’UTR exon (renamed 1C) was located between the new exons and exon 2, the latter containing the entire coding sequence. We found that small intestinal lymphocytes of the T cell lineage expressed the new exons in three different splice forms. RAG1 mRNA containing the 1C exon was not expressed in small intestinal lymphocytes. In contrast, splice forms containing the 1A exon were not expressed in thymocytes. RAG1 and pTα mRNA expressing lymphocytes were seen both within the epithelium and in lamina propria. Thymocyte-like CD2⁺CD7⁺CD3^-, CD4⁺CD8⁺, CD1a⁺, and IL7-R+ lymphocytes were identified in the small intestinal mucosa. CD2⁺CD7⁺CD3^- cells had the highest expression levels of mRNA for RAG1 and pTα, suggesting that the small intestinal mucosa is indeed a site for T cell maturation. Small intestinal T lymphocytes were also shown to kill via the Fas/FasL pathway in a TCR/CD3 independent manner and via the perforin/granzyme pathway in a TCR/CD3 dependent manner. The Fas/FasL-mediated cytotoxicity may reflect an ongoing selection process of extrathymically maturated T cells. </p><p>The nasopharyngeal tonsil is the major inductive site for immune reactions against inhaled antigens. Previous demonstration of RAG1 expression in tonsillar B cells was interpreted as antigen driven receptor revision. The present study confirms the expression of RAG1 in B cells. We also found that RAG1, RAG2, and pTa mRNAs were expressed in lymphocytes of the T cell lineage. A small population of cells with the immature phenotype CD2+CD7+CD3- was demonstrated. This population had the highest expression levels of mRNA for RAG1, RAG2, pTα and terminal deoxynucleotidyl transferase. All four splice-forms of RAG1 mRNA were expressed. RAG1 and pTα mRNA expressing cells were mainly located in the proximity of the surface epithelium and in the outer rim of the follicles. These results suggest that the nasopharyngeal tonsil is a site where extrathymic T cell development and antigen driven TCR revision are occurring in parallel. </p><p>Celiac disease (CD) is a small intestinal enteropathy characterized by permanent intolerance to gluten. Gluten reactive intestinal T cells are central in the pathogenesis and CD can be regarded as a failure to maintain tolerance to this food antigen. Expression of the RAG1 1A/2 splice form was significantly decreased in small intestinal T cell subsets of CD patients suggesting that impaired TCR gene rearrangement could contribute to failure of maintain tolerance in CD. </p><p>Together, these findings show that both small intestinal and nasopharyngeal tonsillar lymphocytes of T cell lineage have the molecular machinery for antigen receptor rearrangement and that thymocyte-like lymphocytes are present in both tissues. Thus these organs are likely sites of T lymphocyte ontogeny as well as for secondary T cell receptor rearrangement in man. </p>

T lymphocytes regulate the initiation, duration, and magnitude of adaptive immune responses and function as effector cells in cell mediated immunity. To become immunologically competent they must generate functional antigen receptors. This process takes place in the thymus and requires somatic recombination of T cell receptor (TCR) genes. It is mediated by the endonucleases recombination activating gene-1 (RAG1) and RAG2. Although the thymus regresses at puberty, T cells are present throughout life implying that other tissues must provide the proper milieu for T cell development. This thesis describes extrathymic T cell maturation in man. RAG1, RAG2, and the preTα-chain (pTα), which is exclusively utilized in developing T cells, were used as markers for TCR gene rearrangement. Two new exons (1A and 1B) encoding sequences in the 5’ untranslated region (5’UTR) of mRNA were discovered in the human RAG1 gene. The previously described 5’UTR exon (renamed 1C) was located between the new exons and exon 2, the latter containing the entire coding sequence. We found that small intestinal lymphocytes of the T cell lineage expressed the new exons in three different splice forms. RAG1 mRNA containing the 1C exon was not expressed in small intestinal lymphocytes. In contrast, splice forms containing the 1A exon were not expressed in thymocytes. RAG1 and pTα mRNA expressing lymphocytes were seen both within the epithelium and in lamina propria. Thymocyte-like CD2+CD7+CD3-, CD4+CD8+, CD1a+, and IL7-R+ lymphocytes were identified in the small intestinal mucosa. CD2+CD7+CD3- cells had the highest expression levels of mRNA for RAG1 and pTα, suggesting that the small intestinal mucosa is indeed a site for T cell maturation. Small intestinal T lymphocytes were also shown to kill via the Fas/FasL pathway in a TCR/CD3 independent manner and via the perforin/granzyme pathway in a TCR/CD3 dependent manner. The Fas/FasL-mediated cytotoxicity may reflect an ongoing selection process of extrathymically maturated T cells. The nasopharyngeal tonsil is the major inductive site for immune reactions against inhaled antigens. Previous demonstration of RAG1 expression in tonsillar B cells was interpreted as antigen driven receptor revision. The present study confirms the expression of RAG1 in B cells. We also found that RAG1, RAG2, and pTa mRNAs were expressed in lymphocytes of the T cell lineage. A small population of cells with the immature phenotype CD2+CD7+CD3- was demonstrated. This population had the highest expression levels of mRNA for RAG1, RAG2, pTα and terminal deoxynucleotidyl transferase. All four splice-forms of RAG1 mRNA were expressed. RAG1 and pTα mRNA expressing cells were mainly located in the proximity of the surface epithelium and in the outer rim of the follicles. These results suggest that the nasopharyngeal tonsil is a site where extrathymic T cell development and antigen driven TCR revision are occurring in parallel. Celiac disease (CD) is a small intestinal enteropathy characterized by permanent intolerance to gluten. Gluten reactive intestinal T cells are central in the pathogenesis and CD can be regarded as a failure to maintain tolerance to this food antigen. Expression of the RAG1 1A/2 splice form was significantly decreased in small intestinal T cell subsets of CD patients suggesting that impaired TCR gene rearrangement could contribute to failure of maintain tolerance in CD. Together, these findings show that both small intestinal and nasopharyngeal tonsillar lymphocytes of T cell lineage have the molecular machinery for antigen receptor rearrangement and that thymocyte-like lymphocytes are present in both tissues. Thus these organs are likely sites of T lymphocyte ontogeny as well as for secondary T cell receptor rearrangement in man.

ADP Ribosylation Factor 1 (ARF1) is a universal small GTP binding protein which has an important role in vesicular trafficking between endoplasmic reticulum and Golgi. ARF1 is a basic component of Coat Protein I (COPI) vesicles which have functions in both formation of coatomer complex and recruitment of cargo proteins. In this study, the expression ARF1 was analyzed in pea (P. sativum L. cv. Araka) grown at different developmental stages. Because of the differential hormonal levels at corresponding stages, the effects of hormones on ARF1 expression were also studied. The results of present research show that ARF1 expression in embryos and 2 days grown plants after germination is lower when compared to 6 days grown plants. In order to see the hormonal effect, 3 weeks old plants were supplied with 50&micro / M of each hormone for 3 times on alternate days. Protein extraction, cell fractionation,Western blot was carried out and immunoblot analysis was conducted with AtARF1 polyclonal antibodies. It was shown that, in pea shoots, abscisic acid and gibberellin increases the inactive GDP bound ARF1 by hydrolyzing ARF-GTP through activating ARFGTPase activating protein (ARF-GAP) or partially inhibiting ARF-Guanine Nucleotide Exchange Factor (ARF-GEF). In roots, ARF-GDP (cytosolic fraction), ARF-GTP (microsomal fraction) and total amount of ARF1 (13.000 x g supernatant fraction) were down regulated by ~11, ~19 and ~11 fold respectively with the application of gibberellin / and by ~11, ~7 and ~3 fold respectively with the application of abscisic acid / when compared to control plants. These results indicate the importance of plant hormones in the regulation of ARF1 in pea.

Gαq signaling is implicated in a number of physiological processes that include platelet activation, cardiovascular development and smooth muscle function. Historically, Gαq is known to function by activating its effector, phospholipase Cβ. Desensitization of Gαq signaling is mediated by G-protein coupled receptor kinases (GRK) such as GRK2 that phosphorylates the activated receptor and also sequesters activated Gαq and Gβγ subunits. Our crystal structure of Gαq-GRK2-Gβγ complex shows that Gαq forms effector-like interactions with the regulator of G-protein signaling (RGS) homology domain of GRK2 involving the classic effector-binding site of Gα subunits, raising the question if GRK2 can itself be a Gáq effector and initiate its own signaling cascade. In the structure, Gα and Gβγ subunits are completely dissociated from one another and the orientation of activated Gαq with respect to the predicted cell membrane is drastically different from its position in the inactive Gαβγ heterotrimer. Recent studies have identified a novel Gαq effector, p63RhoGEF that activates RhoA. Our crystal structure of the Gαq-p63RhoGEF-RhoA complex reveals that Gαq interacts with both the Dbl homology (DH) and pleckstrin homology (PH) domains of p63RhoGEF with its C-terminal helix and its effector-binding site, respectively. The structure predicts that Gαq relieves auto-inhibition of the catalytic DH domain by the PH domain. We show that Gαq activates p63RhoGEF-related family members, Trio and Kalirin, revealing several conduits by which RhoA is activated in response to Gq-coupled receptors. The Gαq effector-site interaction with p63RhoGEF/GRK2 does not overlap with the Gαq-binding site of RGS2/RGS4 that function as GTPase activating proteins (GAPs). This suggests that activated G proteins, effectors, RGS proteins, and activated receptors can form high-order complexes at the cell membrane. We confirmed the formation of RGS-Gαq-effector complexes and our results suggest that signaling pathways initiated by GRK2 and p63RhoGEF are regulated by RGS proteins via both allosteric and GAP mechanisms. Our structural studies of Gαq signaling provide insight into protein-protein interactions that induce profound physiological changes. Understanding such protein interfaces is a key step towards structure-based drug design that can be targeted to treat diseases concerned with impaired Gαq signaling. / text

Activated receptor tyrosine kinases recruit many signalling proteins to initiate downstream cell proliferation and survival pathways, including phosphatidylinositol 3-kinase (PI3K), a heterodimer consisting of a p85 regulatory protein and a p110 catalytic protein. Our laboratory has previously shown the p85α protein also has in vitro GTPase activating protein (GAP) activity towards Rab5 and Rab4, small GTPases that regulate vesicle trafficking events for activated receptors. Expression of a p85α protein containing an arginine to alanine substitution at position 274 (p85R274A) that affects its GAP activity, caused sustained levels of activated platelet-derived growth factor receptors (PDGFRs), enhanced downstream signalling, and resulted in cellular transformation. Together with other data, this suggested that in p85R274A-expressing cells, PDGFRs are more rapidly trafficked through the endocytic pathway, which reduces opportunities for sorting events necessary for receptor degradation. Our laboratory has observed previously that p85 was capable of binding to both Rab5-GDP, as well as Rab5-GTP, which is an atypical characteristic of GAP proteins, whereas p110β had previously been reported to bind Rab5-GTP selectively. Based on these observations, this thesis project was designed to test the hypothesis that both proteins contributed GAP activity towards Rab5, with p85 providing a catalytic arginine residue (R274) and p110β providing switch stabilization functions specific to the GTP-bound state. To accomplish the thesis objective, cells expressing individual p85 defects (lacking GAP activity, R274A; or lacking p110-binding ability through deletion of residues 478-513, Δ110) were compared to cells expressing a double mutant missing both functions. Stable clonal NIH 3T3 cell lines were generated and selected in G418 and clones expressing similar levels of FLAG-tagged p85 wild type or mutants compared to the control cell lines (NIH 3T3, FLAG-vector control, p85 wild type, and p85R274A) were chosen for analysis. A time-course of PDGF stimulation showed that cells expressing p85R274A or p85Δ110+R274A have sustained phosphorylation levels of the PDGFR, reduced rates of PDGFR degradation and sustained MAPK/Erk signalling. Contrary to the cellular transformation previously reported for p85R274A-expressing cells, expression of p85Δ110+R274A did not lead to cellular transformation. These divergent results suggest that p85-associated p110 serves two functions. As the catalytic subunit of PI3K, one function is the localized generation of PI3,4,5P3 lipids at the plasma membrane for Akt activation, and possibly during receptor endocytosis where it could impact MAPK/Erk activation/deactivation kinetics and cell transformation. These results support a second function for p110 in the regulation of PDGFR activation/deactivation kinetics and PDGFR half-life, both strongly influenced by alterations in PDGFR trafficking. This suggests that p110β may regulate PDGFR trafficking by providing Rab5-GTP switch stabilization that complements the catalytic arginine residue (R274) within p85, and that p85α and p110β work together as a Rab5 GAP. The role of PDGFR in the localization of the RabGAP function of p85 to specific subcellular compartments was also examined. It was hypothesized that PDGFR may help localize the RabGAP function of p85 to vesicles containing Rab5 or Rab4 through the binding of p85 to phosphorylated tyrosine residues on activated PDGFR. Stable cell lines expressing individual p85 defects (lacking GAP activity, R274A; or lacking PDGFR-binding ability through site-directed mutation of residues 358 and 649 from arginine to alanine, ΔR; or a double mutant missing both functions) demonstrated that p85R274A or p85ΔR+R274A expression leads to sustained PDGFR activation and signalling, and to delayed PDGFR degradation in response to PDGF stimulation. The sustained signalling observed resulted in cellular transformation in cells expressing p85R274A or p85ΔR+R274A. The data suggests that PDGFR does not play a role in the localization of the RabGAP activity of p85. The findings of this study elucidates important non-canonical functions of the PI3K heterodimer and contributes to our understanding of how specific mutations in both p85 and p110β within regions implicated in the regulation of RabGAP activity can alter signalling events and lead to enhancement of tumour-associated phenotypes.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 3 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2004. / Vita. Bibliography: 106-117.

On-line experiments by physics education at basic school The aim of this diploma thesis is to prove the usefulness of on-line experiments during physics teaching. The work is divided into two parts: theoretical one and practical one. The theoretical part deals with the teaching in primary schools, the possibility of the usage of informational and communication technologies, the increase of student´s motivation through new teaching methods and the student´s life in a school surrounding. The practical part consists of the experiment complex created by the set PASCO. The working sheets, which are given to some of the experiments, enable to attest the effectiveness of the chosen experiments in practice. The results of the didactic samplers to the particular classes are enclosed and described.

The diploma thesis explores activating methods in music education at elementary music schools. The theoretical part of the thesis deals with the description and the issue of activating methods and also focuses on methods suitable for teaching music subjects at elementary music school in the classes of collective and individual lessons. The practical part deals with the purposefulness of the use of activating methods with emphasis to the support of creativity and coordination in lessons of music education. The diploma thesis uses the questionnaire method in various modifications and the method of observation. The aim of the presented diploma work is to describe the activating teaching methods and to focus on these suitable for teaching music subjects at elementary music schools in the classes of collective and individual lessons. The aim of the practical part of the diploma thesis is to find out whether the activating teaching methods are used in education at elementary music schools and how they are evaluated and accepted by teachers and pupils.