Teoria dos números e o processo de ensino-aprendizagem

Com a corrente da Matemática “Moderna”, tanto a Geometria como a Teoria dos Números ficaram relegadas a segundo plano nos currículos da Matemática no Brasil, nos últimos anos a Geometria voltou a recuperar sua força e importância, porém não ocorreu o mesmo com a Teoria dos Números, talvez por não ter se encontrado um meio termo para sua apresentação como simples receituário ou, porque, seu ensino mais profundo apresenta muitas dificuldades de compreensão, tanto para os professores como para os alunos. O trabalho aqui proposto objetiva analisar o processo de ensino e aprendizagem dos conceitos elementares da Teoria dos Números

Das Protein La/SS-B: Vom Autoantigen zur Zielstruktur für die Immuntherapie

Das La-Protein wurde als Autoantigen bei Autoimmunpatienten, die an SLE oder Sjögren-Syndrom erkrankt sind, entdeckt. Es kommt in phosphorylierter Form im Zellkern aller Eukaryonten vor und nimmt Aufgaben bei der Faltung, Prozessierung und nukleären Retention von RNA-Polymerase III-Transkripten wahr. Unter normalen zellulären Bedingungen ist das La-Protein außerdem in der Lage, zwischen Zellkern und Zytoplasma zu pendeln. Bei Zellstress, der nach UV-Exposition oder während einer viralen Infektion entsteht, wird das Protein verstärkt im Zytoplasma beobachtet, wo es an der Cap-unabhängigen Translation zellulärer und ggf. viraler Proteine beteiligt ist. Wird in der Zelle daraufhin Apoptose induziert, so ist das La-Protein auf der Zellmembran bzw. in apoptotischen Körperchen nachweisbar. Ein wesentlicher Bestandteil dieser Arbeit war die Untersuchung verschiedener monoklonaler anti-La-Antikörper. Einige wenige konnten durch wiederholte Immunisierung von Mäusen mit rhLa-Protein generiert werden. Im Gegensatz dazu resultierte die einmalige Übertragung von gegen das hLa-Protein aktivierten CD4+ T-Zellen auf eine hLaTg-Maus in der Gewinnung mehrerer La-spezifischer Antikörper. Die Sequenzanalyse der Gene, die für die variablen Antikörperdomänen codieren, bestätigte, dass es sich um individuell rekombinierte und hypermutierte Immunglobuline handelt. Die Antikörper zeichneten sich außerdem durch unterschiedliche Eigenschaften bei der Bindung von humanem und murinem La-Protein in der Immunfluoreszenz, im Immunoblot oder während der Immunpräzipitation aus. Für die IgG-Antikörper konnten die Epitopbereiche innerhalb des La-Proteins eingegrenzt werden. Auffällig waren die kurzen linearen Peptidepitope, die von den auf konventionelle Art erzeugten Antikörpern gebunden wurden. Hingegen erkannten alle Antikörper, die aus dem adoptiven T-Zell-Transfer hervorgegangen waren, Konformationsepitope. Darüber hinaus wurde gezeigt, dass einige mAks aber auch anti-La-Patientenseren die reduzierte von der oxidierten Form des La-Proteins unterscheiden können. Unerwartet ist die Erkenntnis, dass sich offensichtlich zahlreiche B-Zellen mit anti-La-Spezifität von wenigen variablen Ketten ableiten und dass diese bei einer herkömmlichen Immunisierung entweder nicht aktiviert werden (und deshalb nicht in der Milz zu finden sind) oder sogar eliminiert werden. Der Import des La-Proteins in den Zellkern wird durch die klassischen Transportmoleküle Karyopherin-α und Karyopherin–β vermittelt. Für den Shuttlingprozess muss das Protein auch wieder aus dem Kern exportiert werden. Da es kontroverse Daten bezüglich eines Crm1-abhängigen Kernexports gab, wurde das Shuttlingverhalten von GFP-La-Fusionsproteinen in dieser Arbeit genauer analysiert. Mit Hilfe von Heterokaryonexperimenten konnte bestätigt werden, dass sowohl das hLa- als auch das mLa-Protein zwischen humanen und murinen Zellkernen pendeln kann und dass der Export unabhängig von Crm1 stattfindet. Aufgrund der kurzen Verweildauer im Zytoplasma schienen die Proteine quantitativ im Zellkern vorzuliegen, doch ein Teil konnte stets in den im Heterokaryon enthaltenen Nachbarzellkernen detektiert werden. Die Verwendung von N-terminal deletierten La-Fragmenten, die alle über das C-terminale NLS verfügten, gab Aufschluss über die Regulation des Shuttlings. Es zeigte sich, dass die Menge des exportierten Proteins von einem nukleären Retentionspartner festgelegt wird, der das La-Protein bindet und dadurch im Zellkern festhält. Wird diese Assoziation aufgehoben, gelangt das La-Protein in das Zytoplasma. Dort ist es allerdings nicht detektierbar, da das NLS einen umgehenden Import zurück in den Zellkern hervorruft. Zusätzlich wurde die Auswirkung von zellulärem Stress (z. B. durch ROS) auf die intrazelluläre Lokalisation des Proteins untersucht. Unter oxidativen zellulären Bedingungen wird einerseits die Wechselwirkung mit dem nukleären Retentionspartner aufgehoben und andererseits findet kein Kernimport über Karyopherin-α mehr statt. Aus diesem Grund reichert sich das La-Protein nun verstärkt im Zytoplasma an. Darüber hinaus wurde nachgewiesen, dass das La-Protein von apoptotischen Zellen freigesetzt wird und daraufhin auf die Membran von Nachbarzellen binden kann. Die Bindungs-eigenschaften wurden mit rhLa-Protein genauer untersucht. Das La-Protein war auf Endothel- und Epithelzellen nachweisbar und die Bindung fand sowohl bei Inkubation auf Eis als auch bei 37 °C statt. Da das La-Protein auch über DNA-Bindungseigenschaften verfügt, war es in der Lage, DNA auf der Zelloberfläche zu immobilisieren. Innerhalb von PBMCs wurde es selektiv auf Antigen-präsentierenden Zellen nachgewiesen. Diese Eigenschaften lassen eine Beteiligung des Proteins bei der Induktion von anti-dsDNA-Antikörpern in Autoimmunpatienten vermuten. Es ist bekannt, dass die Bedingungen (Virusinfektion, UV-Exposition), die zur Translokation des La-Proteins auf die Zelloberfläche führen, bei SLE-Patienten Krankheitsschübe auslösen können. Bisher wurden anti-La-Autoantikörper aber eher nicht als pathophysiologisch erachtet, da sie bei der Bindung an bereits apoptotische Zellen keine weiteren Schäden verursachen können. Jedoch wurde in dieser Arbeit gezeigt, dass das La-Protein apoptotischer Zellen auf der Oberfläche von lebenden Zellen in der Umgebung nachgewiesen werden kann. Daran könnten anti-La-Autoantikörper binden und eine Komplement- oder NK-Zell-vermittelte Zerstörung der Nachbarzellen hervorrufen. Dadurch entstehen zusätzliche Gewebeschäden. Im Chromfreisetzungstest waren NK-Zellen tatsächlich in der Lage, La-dekorierte Zielzellen Antikörper-abhängig zu lysieren, sofern zusätzliche in vitro Stimuli präsent waren, die z. B. eine virale Infektion simulierten. Die Immuntherapie von Tumoren ist auf bestimmte Zielstrukturen auf den Tumorzellen angewiesen, über welche die Wirkstoffe spezifisch zu den maligne transformierten Zellen gebracht werden. Die Therapeutika, die sich oft von mAks gegen diese Zielstrukturen ableiten, müssen für verschiedene Tumorarten individuell entwickelt werden. Da das La-Protein von apoptotischen Zellen freigesetzt wird und auf die Membran benachbarter (bestrahlungsresistenter) Zellen binden kann, ist es in Kombination mit einer vorangegangenen Bestrahlung als universelle Zielstruktur für die Immuntherapie nutzbar. Aus diesem Grund wurde unter Verwendung eines ausführlich in dieser Arbeit charakterisierten anti-La-Antikörpers ein rekombinantes bispezifisches Antikörperderivat entwickelt. Es ist in der Lage, das La-Protein auf der Oberfläche von Tumorzellen zu binden und auf diesen zytotoxische T-Lymphozyten zu immobilisieren. Durch die Quervernetzung werden die T-Lymphozyten aktiviert und induzieren in den Zielzellen Apoptose. Das neue Antikörperderivat verspricht eine vielseitige Anwendung in Kombination mit Strahlentherapie oder auch mit rekombinanten Antikörpermolekülen, die gegen spezifische Zielstrukturen auf den Tumorzellen gerichtet sind

Larval migration in PERL chambers as an in vitro model for percutaneous infection stimulates feeding in the canine hookworm Ancylostoma caninum

<p>Abstract</p> <p>Background</p> <p><it>Ancylostoma caninum </it>third-stage larvae are the non-feeding infective stage of this parasite and are able to infect potential hosts via different infection routes. Since percutaneous infection is one of the most important routes and skin penetration is the first step into parasitic life, an existing <it>in vitro </it>model for percutaneous migration was modified and evaluated. The main parameter used to evaluate migration was the migration ratio (migrated larvae as a percentage of total number of larvae recovered). Additionally, the skin lag was calculated, expressing the percentage of larvae remaining in the skin and therefore not being recovered. Since initiation of feeding is proposed to be an important step in the transition from free-living to parasitic <it>A. caninum </it>larvae, feeding assays were performed with <it>in vitro </it>percutaneously migrated larvae. Additionally, infective larvae of <it>A. caninum </it>were activated via serum-stimulation and feeding behaviour was analysed and compared between percutaneously migrated and serum-stimulated larvae.</p> <p>Results</p> <p>Maximum skin migration levels of infective larvae were observed at temperatures above 32°C when larvae were placed on the epidermal side of skin for more than 12 hours. The medium beneath the skin had no effect on migration ratio, and no significant difference between the migration ratios through fresh and frozen/thawed skin was observed.</p> <p>Maximum feeding levels of 93.2% were observed for percutaneously migrated larvae after 48 h incubation, whereas serum-stimulated larvae reached the maximum of 91.0% feeding larvae after 24 h.</p> <p>Conclusions</p> <p>The PERL chamber system was optimised and standardised as an <it>in vitro </it>model for percutaneous migration. The larvae recovered after percutaneous migration showed characteristic signs of activation similar to that of serum-stimulated larvae. The observed difference in time course of resumption of feeding indicates that percutaneously migrated larvae are not identical to serum-stimulated larvae, which are currently representing the model for early parasitic stages.</p

Environmental Effects on Temperature Stress Resistance in the Tropical Butterfly Bicyclus Anynana

BACKGROUND: The ability to withstand thermal stress is considered to be of crucial importance for individual fitness and species' survival. Thus, organisms need to employ effective mechanisms to ensure survival under stressful thermal conditions, among which phenotypic plasticity is considered a particularly quick and effective one. METHODOLOGY/PRINCIPAL FINDINGS: In a series of experiments we here investigate phenotypic adjustment in temperature stress resistance following environmental manipulations in the butterfly Bicyclus anynana. Cooler compared to warmer acclimation temperatures generally increased cold but decreased heat stress resistance and vice versa. In contrast, short-time hardening responses revealed more complex patterns, with, e.g., cold stress resistance being highest at intermediate hardening temperatures. Adult food stress had a negative effect on heat but not on cold stress resistance. Additionally, larval feeding treatment showed interactive effects with adult feeding for heat but not for cold stress resistance, indicating that nitrogenous larval resources may set an upper limit to performance under heat stress. In contrast to expectations, cold resistance slightly increased during the first eight days of adult life. Light cycle had marginal effects on temperature stress resistance only, with cold resistance tending to be higher during daytime and thus active periods. CONCLUSIONS/SIGNIFICANCE: Our results highlight that temperature-induced plasticity provides an effective tool to quickly and strongly modulate temperature stress resistance, and that such responses are readily reversible. However, resistance traits are not only affected by ambient temperature, but also by, e.g., food availability and age, making their measurement challenging. The latter effects are largely underexplored and deserve more future attention. Owing to their magnitude, plastic responses in thermal tolerance should be incorporated into models trying to forecast effects of global change on extant biodiversity

Bayesian test for colocalisation between pairs of genetic association studies using summary statistics.

Genetic association studies, in particular the genome-wide association study (GWAS) design, have provided a wealth of novel insights into the aetiology of a wide range of human diseases and traits, in particular cardiovascular diseases and lipid biomarkers. The next challenge consists of understanding the molecular basis of these associations. The integration of multiple association datasets, including gene expression datasets, can contribute to this goal. We have developed a novel statistical methodology to assess whether two association signals are consistent with a shared causal variant. An application is the integration of disease scans with expression quantitative trait locus (eQTL) studies, but any pair of GWAS datasets can be integrated in this framework. We demonstrate the value of the approach by re-analysing a gene expression dataset in 966 liver samples with a published meta-analysis of lipid traits including >100,000 individuals of European ancestry. Combining all lipid biomarkers, our re-analysis supported 26 out of 38 reported colocalisation results with eQTLs and identified 14 new colocalisation results, hence highlighting the value of a formal statistical test. In three cases of reported eQTL-lipid pairs (SYPL2, IFT172, TBKBP1) for which our analysis suggests that the eQTL pattern is not consistent with the lipid association, we identify alternative colocalisation results with SORT1, GCKR, and KPNB1, indicating that these genes are more likely to be causal in these genomic intervals. A key feature of the method is the ability to derive the output statistics from single SNP summary statistics, hence making it possible to perform systematic meta-analysis type comparisons across multiple GWAS datasets (implemented online at http://coloc.cs.ucl.ac.uk/coloc/). Our methodology provides information about candidate causal genes in associated intervals and has direct implications for the understanding of complex diseases as well as the design of drugs to target disease pathways

Compositional and functional stability of aerobic methane consuming communities in drained and rewetted peat meadows

The restoration of peatlands is an important strategy to counteract subsidence and loss of biodiversity. However, responses of important microbial soil processes are poorly understood. We assessed functioning, diversity and spatial organization of methanotrophic communities in drained and rewetted peat meadows with different water table management and agricultural practice. Results show that the methanotrophic diversity was similar between drained and rewetted sites with a remarkable dominance of the genus Methylocystis. Enzyme kinetics depicted no major differences, indicating flexibility in the methane (CH4) concentrations that can be used by the methanotrophic community. Short-term flooding led to temporary elevated CH4 emission but to neither major changes in abundances of methane-oxidizing bacteria (MOB) nor major changes in CH4 consumption kinetics in drained agriculturally used peat meadows. Radiolabeling and autoradiographic imaging of intact soil cores revealed a markedly different spatial arrangement of the CH4 consuming zone in cores exposed to near-atmospheric and elevated CH4. The observed spatial patterns of CH4 consumption in drained peat meadows with and without short-term flooding highlighted the spatial complexity and responsiveness of the CH4 consuming zone upon environmental change. The methanotrophic microbial community is not generally altered and harbors MOB that can cover a large range of CH4 concentrations offered due to water-table fluctuations, effectively mitigating CH4 emission

Involvement of GPR17 in Neuronal Fibre Outgrowth

Characterization of new pharmacological targets is a promising approach in research of neurorepair mechanisms. The G protein-coupled receptor 17 (GPR17) has recently been proposed as an interesting pharmacological target, e.g., in neuroregenerative processes. Using the well-established ex vivo model of organotypic slice co-cultures of the mesocortical dopaminergic system (prefrontal cortex (PFC) and substantia nigra/ventral tegmental area (SN/VTA) complex), the influence of GPR17 ligands on neurite outgrowth from SN/VTA to the PFC was investigated. The growthpromoting effects of Montelukast (MTK; GPR17- and cysteinyl-leukotriene receptor antagonist), the glial cell line-derived neurotrophic factor (GDNF) and of two potent, selective GPR17 agonists (PSB-16484 and PSB-16282) were characterized. Treatment with MTK resulted in a significant increase in mean neurite density, comparable with the effects of GDNF. The combination of MTK and GPR17 agonist PSB-16484 significantly inhibited neuronal growth. qPCR studies revealed an MTK-induced elevated mRNA-expression of genes relevant for neuronal growth. Immunofluorescence labelling showed a marked expression of GPR17 on NG2-positive glia. Western blot and RT-qPCR analysis of untreated cultures suggest a time-dependent, injury-induced stimulation of GPR17. In conclusion, MTK was identified as a stimulator of neurite fibre outgrowth, mediating its effects through GPR17, highlighting GPR17 as an interesting therapeutic target in neuronal regeneration

Growth and differentiation of primary and passaged equine bronchial epithelial cells under conventional and air-liquid-interface culture conditions

<p>Abstract</p> <p>Background</p> <p>Horses develop recurrent airway obstruction (RAO) that resembles human bronchial asthma. Differentiated primary equine bronchial epithelial cells (EBEC) in culture that closely mimic the airway cells <it>in vivo </it>would be useful to investigate the contribution of bronchial epithelium in inflammation of airway diseases. However, because isolation and characterization of EBEC cultures has been limited, we modified and optimized techniques of generating and culturing EBECs from healthy horses to mimic <it>in vivo </it>conditions.</p> <p>Results</p> <p>Large numbers of EBEC were obtained by trypsin digestion and successfully grown for up to 2 passages with or without serum. However, serum or ultroser G proved to be essential for EBEC differentiation on membrane inserts at ALI. A pseudo-stratified muco-ciliary epithelium with basal cells was observed at differentiation. Further, transepithelial resistance (TEER) was more consistent and higher in P₁cultures compared to P₀cultures while ciliation was delayed in P₁cultures.</p> <p>Conclusions</p> <p>This study provides an efficient method for obtaining a high-yield of EBECs and for generating highly differentiated cultures. These EBEC cultures can be used to study the formation of tight junction or to identify epithelial-derived inflammatory factors that contribute to lung diseases such as asthma.</p

Case Report: Graft Versus Tumor Effect After Non-Myeloablative Allogeneic Stem-Cell Transplantation in a Patient With Brentuximab-Vedotin Refractory Sezary Syndrome

Sezary Syndrome (SS) is a rare leukemic variant of primary cutaneous T-cell lymphoma. Relapsed or refractory disease is generally considered incurable by conventional therapeutic approaches, although durable responses can be achieved with novel monoclonal antibodies. Allogeneic hematopoietic stem cell transplantation (alloHSCT) may have potential value by inducing graft vs-lymphoma (GvL) effects, but there is currently no consensus regarding the timing of alloHSCT or type of conditioning regimen. Here we present the case of a male patient who achieved a complete remission (CR) of primary refractory SS after non-myeloablative alloHSCT. Patient: Two years prior to HSCT, the patient had been refractory to CHOEP-based chemotherapy, interferon, extracorporeal photopheresis (ECP), and bexarotene. Directly prior to alloHSCT brentuximab-vedotin (BV) was applied resulting in a partial remission of the skin compartment and overall in a stable disease. Prior to HSCT, flow cytometry of the bone marrow and peripheral blood showed an infiltration with T-cells positive for CD5, CD4, low CD3, low CD2 and negative for CD7, CD38, HLA-DR and CD8. The trephine biopsy showed a 7% infiltration of SS cells. The CD4:CD8 ratio in peripheral blood (pb) was massively increased at 76.67, with 63.5% of white blood cells expressing a SS immune phenotype. The conditioning regimen included 30 mg/m2 fludarabine on days -5, -4 and -3 and total body irradiation with 2 Gy on day -1. Immunosuppression consisted of cyclosporine A from day-1 and mycophenolate mofetil from day 0. The patient received 6.55x106 CD34+ cells and 1.11x108 CD3+ cells/kg body weight. Bone marrow evaluation on day 28 still showed persistent SS cells by flow cytometry. After tapering immunosuppression until day 169, the CD4:CD8 ratio in pb normalized. CR was documented on day 169 after alloHSCT and is now ongoing for almost 3 years after alloHSCT. Conclusions: We confirm that an alloHSCT can be a curative option for refractory patients with SS. The achievement of a CR after tapering the immunosuppressive therapy indicates a significant role of the GvL effect. In present treatment algorithms for patients with SS, the timing of an alloHSCT and the intensity of conditioning should be further explored