The aim of the thesis is to compare the efficiency of innovative and traditional teaching methods. In the theoretical part the particular traditional and innovative methods and procedures are described which can be applied in the educational process. In the practical part the goal was to find out which teaching methods are used by the primary school teachers and which of them are most effective. Further, to compare the efficiency of teaching applying innovatice and traditional methods in natural history lessons in grade four. A questionnare was used to establish the most effective and mostly used teaching methods. It follows from the results that teachers most often use the methods of explanation, texts, pictures, educational games, group and co-operative forms of work and discussion. Most teachers respond that for pupils´ learning the innovative methods are more effective. An experiment was carried out during which both traditional methods (narration, explanation, work with a text) and innovative methods (group work and brainstorming) were applied. The results showed that for pupils´ learning the traditional methods were more effective. This result was caused by the fact that the innovative methods were more time-consuming.

This diploma thesis deals with the activation of activities teachers, which has a stimulating effect on the activities of students in learning the German language at secondary schools in Jihocesky county. German language was chosen purposely, because the most important thing while teaching foreign languages is to involve the student into a mutual communication as much as possible. The student has to be very active to be able to assume the foreign language. This thesis is divided into theoretical and research. The theme of this thesis is to release the ways how teachers stimulate the activity of pupils in the German language teaching. It is also important to find out if there is any interaction between the activation of activities teachers and a student?s activity during the lesson. We can get to know this interaction by statistical calculations. Among one of the methods of quantitatively directed analyses belongs standardized method of observation, which was used right for this research. To be specific it was an interactive analysis. There was applied Flanders observation system for an activity of a teacher and Tabers observation system for an activity of a student. There are described various ways and methods how to stimulate a student to be more active.

Transfusion of platelet concentrates (PC) can be necessary for patients to maintain coagulability. It is vital that the platelets maintain viability and function during processing and storage to obtain enhanced coagulability in the transfused patient. Today, no test is used to verify platelet function in either donors or in PC’s. Observing swirling effect is the only test applied to control platelets before transfusion but the method is based on platelet morphology and does not directly evaluate platelet function.Impedance aggregometry (IA) (Multiplate analyzer, Roche Diagnostic) is a promising method for measuring platelet function, measuring changes in impedance over time when platelets adhere to electrodes. IA has been well evaluated for the purpose of analyzing whole blood but analyzing PC’s is a relatively new application of the method.Samples from platelet donors and PC’s were analyzed with IA to evaluate correlation in function between donors and PC’s, in the hope of being able to predict function in PC. Different platelet concentrations were also analyzed to evaluate the impact of varying concentration on impedance. Adenosine diphosphate (ADP), collagen and thrombin receptor activating peptide 6 (TRAP-6) were used to induce aggregation. Platelet function was measured in PC’s on day 1 and 4 after donation.A significant correlation was observed between platelet function in donors and in PCs on day 1, measured with ADP. An important finding was also that platelet concentration does affect impedance, in collagen-induced aggregation more than ADP-induced. It is therefore possible that a correlation would also have been found between donors and PC’s analyzed with collagen if the platelet concentration would have been standardized.

Le choc anaphylactique est une réaction allergique systémique qui survient en quelques minutes et pouvant être fatale. Mon travail de thèse s’articule autour de deux projets dont la finalité est de mieux comprendre le mécanisme physiopathologique. La première partie de ce travail consiste à étudier in vivo chez la souris les contributions des récepteurs Fc aux IgG (FcγRs), des cellules effectrices et des médiateurs contribuant dans un modèle d’anaphylaxie systémique passive induit par une sous-classe particulière d’IgG : des IgG1, des IgG2a ou des IgG2b monoclonales dirigées contre un même antigène. Cette étude a permis de démontrer que le FcγRIII, les neutrophiles et les monocytes/macrophages sont les acteurs majoritaires quelque soit la sous-classe d’IgG de souris ; en revanche, la participation des basophiles ainsi que la contribution relative des médiateurs histamine et PAF sont dépendantes de la sous-classe d’IgG utilisée. La deuxième partie de ce travail consiste à étudier plus particulièrement la population plaquettaire dans un modèle de souris humanisées. Contrairement à la souris, les plaquettes humaines expriment un FcγR, le FcγRIIA déjà identifié comme acteur clé de l’anaphylaxie. Un modèle de choc allergique induit par des IgG humaines dans des souris transgéniques pour le FcγRIIA m’a permis de tester l’hypothèse suivant laquelle les plaquettes participent à l’initiation et/ou à la propagation de la réaction. Ce modèle a permis de mettre en évidence une thrombocytopénie sévère, des complexes plaquettes-leucocytes circulants et de montrer que le transfert de plaquettes ou de leur surnageant restaure les signes cliniques du choc allergique. / Anaphylaxis is a systemic hyperacute allergic reaction that occurs within minutes and can be fatal. The aim of my PhD project is to investigate the physiopathological mechanisms underlying anaphylaxis induction. The first part of my work focused on the contribution of FcγRs, effector cells and mediators in passive murine models of systemic anaphylaxis induced by the different subclasses of mouse specific IgG ; directed against the same antigen: IgG1, IgG2a or IgG2b. This study demonstrated that FcγRIII, neutrophils and monocytes/macrophages are the key players of anaphylaxis induction whatever the mouse IgG subclasses used. On the contrary, basophil participation and the relative contribution of histamine and PAF are IgG subclass dependent. The second part of this work examined the role of platelets in anaphylaxis using a humanized mouse model. Opposing the murine situation, human platelets express an IgG receptor, FcγRIIA. This receptor has already been identified as a key player in anaphylaxis. Using aggregated human IgG to induce anaphylaxis in mice transgenic for FcγRIIA, we tested our hypothesis that platelets contribute to the initiation and/or the propagation of this reaction. Anaphylaxis in this model was accompanied by a severe thrombocytopenia, the presence of circulating platelet-leukocyte complexes and activated platelets. I further demonstrated that the transfer of platelets or their activated supernatent into resistant mice restored features of anaphylactic shock.

Recombination Activating Genes (RAGs) orchestrate the process called V (D) J recombination, which enables the vertebrate adaptive immune system to specifically recognize millions of antigens. During this recombination process, V (variable), D (diversity) and J (joining) gene segments of antibody (B cell receptor) and TCR (T cell receptor) join by different possible combinations to generate antigen receptor diversity. This unique site specific recombination process is actuated by lymphoid specific proteins called RAG1 and RAG2 (RAGs or RAG complex). RAGs recognize a conserved sequence motif flanking the above subexons called Recombination Signal Sequence (RSS). There are two types of RSS known as 12-RSS and 23-RSS, where a conserved heptamer sequence and nonameric sequence is separated by 12 or 23 bp, respectively. RAGs specifically bind to RSS by RAG1 Nonamer Binding Domain (NBD) and generate nicks which are converted to DSBs via a hairpin intermediate and finally repaired by Non-Homologous DNA End Joining (NHEJ), a major DSB repair pathway in eukaryotes. Thus, RAGs act as a sequence specific endonuclease, and is unique to higher eukaryotes. Therefore, reduced or loss of RAG activity could result in immune deficiency syndromes like Omenn Syndrome (OS) and Severe Combined Immunodeficiency (SCID). Apart from acting as a sequence specific nuclease, RAGs have been shown to cleave on altered DNA structures like mismatches (bubbles), hairpins, flaps, gaps, triplexes and 3’ overhangs. This structure specific nuclease activity is implicated in causing genomic instability in B and T cells, particularly leading to generation of chromosomal translocations in certain lymphoid cancers. However, unlike the sequence- specific cleavage activity, this novel property of RAGs is poorly understood. Structure-specific nuclease activity of RAGs was characterized by using heteroduplex DNA substrate containing bubble region. RAG proteins were overexpressed and purified from human cell line and used for the assay. Results showed that RAGs cleave different bubble substrates with different efficiency. The role of DNA sequence at single-stranded region of heteroduplex DNA on RAG cleavage was investigated by synthesizing the substrate DNA having either adenineguanine/ thymine/ cytosine in the bubble sequence. Interestingly, efficient RAG cleavage was observed only when cytosines were present at single-stranded region, while thymine bubbles were cleaved with much lower efficiency. Adenine and guanine containing bubbles were not cleaved by RAGs. This was the first observation showing sequence specificity during structure-specific nuclease activity of RAGs. Besides, it was observed that RAG cleavage on bubbles with cytosines resulted in DSB formation, which is essential for generation of chromosomal translocations. Further, such specificity and cytosine preference was observed, even when RAGs acted on other altered DNA substrates like hairpin loops, 3’ overhangs and gaps. When the role of flanking duplex region on RAG cleavage was tested, only the 5’ duplex nucleotide was critical for RAG cleavage reaction and cytosine was the most preferred nucleotide. By systematic mutation of bubble region, it was observed that the two cytosines present at the double strand-single strand junction are critical for RAG cleavage. A single nucleotide bubble (mismatch) with cytosines was cleaved by RAGs with low but detectable efficiency. A bubble with at least 2 nt length possessing cytosine was cleaved with higher efficiency resulting in both single-stranded nicks and DSBs. Based on these studies, “C(d)C(s)C(s)” was proposed as a novel recognition motif for RAG cleavage, on altered DNA structures, where“d” and “s” represent double- and single-strand region, respectively. To be targeted by RAGs in vivo, the altered DNA substrates have to compete with RSS, the physiological substrate. It is not known whether such structures will be cleaved by RAGs, when present along with RSS. Besides, the regulation of the both structure and sequence specific nuclease activities are not studied. To address the above questions, RAG cleavage on bubble substrates was performed in presence of RSS either in cis or trans configuration. Results showed that both bubble substrates and RSS were cleaved with similar efficiency by RAGs. In fact, they can compete out each other in a concentration dependent manner. When kinetics of RSS and bubble cleavage were performed, RSS cleavage reaction was faster and saturated within 10-15 min, while bubbles cleavage started slow and went on increasing with time. This difference in kinetics persisted when both substrates were present together. This could be a regulatory mechanism for targeting RAGs to RSS sites and limiting bubble cleavage which can be deleterious to cells. HMGB1, a DNA binding protein which is shown to enhance RSS binding and synapsis, does not affect RAG action on bubble substrates. RAG postcleavage complex is formed during V(D)J recombination process where RAGs remain bound to cleaved RSS after cleavage, which limits further RAG action on other sites. Such cleavage complex was not detected on bubble substrates, which suggests that after cleavage RAGs were not associated to DSBs of bubble cleavage. Finally, the nonamer binding domain of RAG1 involved in RSS binding in V(D)J recombination, was found to be dispensable for the structure-specific nuclease activity and it appears that RAGs bind to bubble substrates using a different domain. In summary, in this study, the structure-specific nuclease activity of RAGs was characterized. A novel sequence motif that can regulate this activity of RAGs was identified. Though altered structures can be equally favored substrates as RSS, differences in reaction kinetics, cleavage complex formation and separate DNA binding domains regulate RAG cleavage, when it acts as a structure-specific nuclease. Thus, this study may help in the better understanding of RAG induced genomic instabilities in lymphoid tissues.

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

Human immunodeficiency virus type 1 (HIV-1) is a member of the lentivirus subfamily of retroviruses. HIV-1 expresses multiple genes from a single provirus by alternative splicing. Early in viral expression, fully spliced 2-kb viral RNA is exported from the nucleus and encodes the viral regulatory protein, Rev, which is essential for nuclear transport of partially spliced and unspliced genomic-length RNA. Rev binds to an RNA structural element called the Rev response element (RRE) and mediates nuclear export through the leucine-rich nuclear export signal (NES) pathway. The human Rev Interacting Protein (hRIP) interacts specifically with the Rev NES. Rev NES mutants that are unable to export Rev-dependent RNAs are also unable to bind to hRIP. The hRIP cDNA encodes a 562 amino acid protein containing an N-terminal zinc finger with homology to Arf GAP domains, a central serine and threonine rich region, and C-terminal phenylalanine-glycine (FG) repeats characteristic of nucleoporins. To identify an hRIP ortholog in a genetically tractable organism, we performed database searches using the N-terminal zinc finger of hRIP. Using this approach, we identified a novel gene in Schizosaccharomyces pombe. Alignment of the entire reading frame of the putative ortholog with hRIP indicates similarity with the serine/threonine rich region and with the FG repeats, suggesting that S. pombecould be a good model system to study the cellular function of hRIP. We find that the S. pombe ORF is an essential gene, which encodes a 483 amino acid protein that is also able to interact with the NES of HIV-1 Rev. Based on being an essential gene, and the presence of a putative Arf GAP domain, the ORF was named an Arf GAP essential for viability, agv1+. We show that Agv1 is not directly involved in the nuclear export of poly(A+) RNA or 5S rRNA, nuclear export of leucine-rich NES-containing proteins, or nuclear import of nuclear localization signal (NLS)-containing proteins. However, Agv1 does appear to play a role in the cytoplasmic localization of 5S rRNA. We demonstrate that loss of Agv1 alters the localization of endoplasmic reticulum (ER) membrane and Golgi membrane resident proteins, accumulates intracellular membrane, and blocks processing of carboxypeptidase Y. Furthermore, the S. cerevisiae ADP-ribosylation factor (Arf) GTPase activating protein (GAP) Glo3, but not a catalytically inactive Glo3 mutant [R59K], is able to partially compensate for the loss of Agv1 function in temperature sensitive strains, indicating that Agv1 is an S. pombe Arf GAP with some functional features similar to S. cerevisiae Glo3.

No description available.

This study deals with social-activating programs for seniors. It tries to answer the question how seniors evaluate social-activating programs in Klub REMEDIUM. The main task is then to find reviews of the social services provided at Klub REMEDIUM its visitors and draw cocnclusions along with a concept of posible improvement of service delivery. As a target then I'm also interested in the contribution that seniors are seeing from the use of these services. To achieve the goals I have used a quantitative method of data collection survey, which I also completed by the qualitative method of personal interviews and my own observation for deeper understanding of the topic. I have found that seniors evaluate these programs strongly positive. But I also encountered the problem areas that have become an incentive to improve service delivery.

Chronic pressure overload (PO) is associated with cardiac hypertrophy and altered autonomic control of cardiac function, in which the latter may involve adaptations in central and/or peripheral cardiac neural control mechanisms. To evaluate the specific remodeling of the intrinsic cardiac nervous system following pressure overload, the descending thoracic aorta artery of the guinea pig was constricted ∼20%, and the animals recovered for 9 wk. Thereafter, atrial neurons of the intrinsic cardiac plexus were isolated for electrophysiological and immunohistochemical analyses. Intracellular voltage recordings from intrinsic cardiac neurons demonstrated no significant changes in passive membrane properties or action potential depolarization compared with age-matched controls and sham-operated animals, but afterhyperpolarization duration was increased in PO animals. Neuronal excitability, as determined by the number of action potentials produced with depolarizing stimuli, was differentially increased in phasic neurons derived from PO animals in response to exogenously applied histamine compared with sham and age-matched controls. Conversely, pituitary adenylate cyclase-activating polypeptide-induced increases in intrinsic cardiac neuron evoked AP frequency were similar between control and PO animals. Immunohistochemical analysis demonstrated a two-fold increase in the percentage of neurons immunoreactive for neuronal nitric oxide synthase in PO animals compared with control. The density of mast cells within the intrinsic cardiac plexus from PO animals was also increased twofold compared with preparations from control animals. These results indicate that congestive heart failure associated with chronic pressure overload induces a differential remodeling of intrinsic cardiac neurons and upregulation of neuronal responsiveness to specific neuromodulators.