Background: Platelet activating factor (PAF) is a phospholipid synthesized by the action of phospholipase A2 and acetyl transferase. PAF possesses a wide range of biological activities. In the lung of the fetus and newborn, PAF binds to its G protein couple receptor to evoke its biological activities via a well-defined signaling pathway. High levels of PAF receptor (PAFr) activity in fetal ovine lung vascular smooth muscle cells (PVSMC) at baseline has previously been demonstrated, a finding that is further perpetuated by conditions of hypoxia similar to fetal lung environment. Additionally in fetal ovine PVSMC, a cross-talk between PAFr-mediated cell signaling and activity of the vasodilator cyclic nucleotides cGMP and cAMP acting via their respective receptors protein kinase (PK) G and PKA has been shown. The interaction of PAF with its receptor has been implicated in the pathogenesis of persistent pulmonary hypertension in the newborn (PPHN) which has a high incidence of hospitalization and death of newborn infants. Successful transition of fetus to newborn life entails a mechanism whereby vasoconstrictors necessary for fetal existence are abrogated in the immediate newborn. Hypothesis: We hypothesize that PPHN results from the failure to down regulate PAFr- mediated activity and /or failure to up-regulate activity of the vasodilators cGMP and cAMP. PPHN is triggered by chronic intrauterine or postnatal hypoxia. Then newborn PVSMC undergo hyperplasia and hypertrophy, which over time, results in irreversible vascular remodeling. Methods: My study aims to employ in vitro models to delineate the consequences of PAF-PAFr mediated pathway in the pharmacological effects of the cAMP-PKA and cGMP-PKG signaling and the involvement of this cross-talk in the pathogenesis of PPHN. I modeled my cell culture studies to mimic the low oxygen environment of fetal lungs (hypoxia), the normal oxygen environment of newborn lungs (normoxia) and high oxygen environment (hyperoxia) to which the newborn lung may be exposed in incidental clinical condition of PPHN. I studied the effect of PAF, a vasoconstrictor, cAMP/cGMP, vasodilators, and other inhibitors of the PAFr pathway on growth of newborn PVSMC, by DNA synthesis, and measured their effects on expression of mitogenic and non-mitogenic proteins. Results: We found that both hypoxia and hyperoxia decreased cell growth even in the presence of PAF which up-regulates cell growth in fetal PVSMC. Also PAF treatment of cells resulted in down regulation of the vasodilator proteins, PKA and PKG. Conclusion: Our data suggests that in the lung of the newborn a high activity of PAF-PAFr mediated activities will worsen the condition of PPHN imposed on the newborn lung by environmental or therapeutic conditions. We can speculate that, in the long run, these findings may translate into the establishment of less toxic protein-based management of PPHN.

Muscle aging is associated with a decrease in the number of satellite cells and their progeny, muscle progenitor cells (MPCs) that are available for muscle repair and regeneration. However, there is an increase in non-immuno-hematopoietic cells (CD45 negative) in regenerating muscle from aged mice characterized by high stem cell antigen -1(Sca-1) expression. In aged regenerating muscle, 14.2% of cells are CD45neg Sca-1pos while 7.2% of cells are CD45neg Sca-1pos in young adult muscle. In vitro, CD45neg Sca-1pos cells over express genes associated with fibrosis, potentially controlled by Wnt2. These cells are proliferative, non-myogenic and non-adipogenic, and arise in clonally-derived MPCs cultures from aged mice. Both in vitro and in vivo studies suggest that CD45neg Sca-1pos cells from aged muscle are more susceptible to apoptosis than their MPCs, which may contribute to depletion of the satellite cell pool. Therefore, with age, a subset of MPCs takes on an altered phenotype, which is marked by high Sca-1 expression. This altered phenotype prevents these cells from participating in muscle regeneration or replenishing the satellite cell pool, and instead may contribute to fibrosis in aged muscle.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed June 10, 2008). Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 92-104).

The submitted diploma thesis deals with the use of activating methods in foreign language tuition. The thesis aims at finding out which teaching and activating methods are used by teachers in foreign language tuition most often, and what the advantages and disadvantages of activating methods are. The theoretical part of the thesis deals with activity and creativity in lessons and with teaching methods. The research part of the thesis is based on questionnaire survey that foreign language teachers took part in. The survey results contribute to the finding which teaching methods are employed to teach foreign languages at primary and secondary schools at present.

The KIL gene of vaccinia virus encodes for a host range protein; in the absence of which, the virus is unable to grow in certain cell lines (RK-13 and some human cell lines). KIL function can be complemented in RK-13 cells by the cowpox host range gene product CP77 despite a lack of homology between the two proteins except for ankyrin repeats. We investigated the role of ankyrin repeats ofthe K1L gene in the host-range restriction of growth in RK-13 cells. The growth of a recombinant vaccinia virus, with the K1L gene mutated in the most conserved ankyrin repeat, was severely impaired as evidenced by lack of plaque formation and reduction in viral titers. Infection of RK-I3 cells with the mutant recombinant vaccinia virus resulted in total shutdown of both cellular and viral protein synthesis early in infection, indicating that the host restriction mediated by the ankyrin repeat is due to a translational block. A comparison of the cellular localization of the K1L wild type and mutated forms showed no difference, as both localized exclusively in the cytoplasm of RK-I3 cells. We also investigated the interaction of the vaccinia virus K1L protein with cellular proteins in RK-13 cells and co-immunoprecipitated a 90 kDa protein identified as the rabbit homologue of human ACAP2, a GTPase-activating protein with ankyrin repeats. Our result suggests the importance of ankyrin repeat for host-range function of K1L in RK-13 cells and identifies ACAP2 as a cellular protein which may be interacting with K1L.

The presented master's thesis is concerned with the topic of the revised version of Bloom's taxonomy and its implementation to second language teaching with regards to its higher levels through the use of different activating methods of teaching. The aim of this thesis is to, with the support of academic literature, consider the role of the activating methods in language teaching and their potential relationship to the revised taxonomy. Based on these findings it is later aimed to present suggestions applicable in practice about how to combine these two instruments with regards to Spanish language teaching. The thesis includes a research done through a questionnaire which aims to map the teachers' knowledge of the activating teaching methods and the potential means of using them while teaching.

Submitted by Ana Hilda Fonseca (anahilda@ufba.br) on 2014-09-22T13:52:31Z No. of bitstreams: 1 TESE FINAL.pdf: 3692054 bytes, checksum: b5b71f5f7c08c260cdbbe30893d2de24 (MD5) / Approved for entry into archive by Fatima Cleômenis Botelho Maria (botelho@ufba.br) on 2014-09-23T13:31:21Z (GMT) No. of bitstreams: 1 TESE FINAL.pdf: 3692054 bytes, checksum: b5b71f5f7c08c260cdbbe30893d2de24 (MD5) / Made available in DSpace on 2014-09-23T13:31:21Z (GMT). No. of bitstreams: 1 TESE FINAL.pdf: 3692054 bytes, checksum: b5b71f5f7c08c260cdbbe30893d2de24 (MD5) / CNPq / Nos últimos tempos, a intensa atividade humana levou a um aumento da disposição de resíduos agrícolas no meio ambiente. Visando encontrar novas aplicações para eles estudou- se, neste trabalho, o efeito do agente ativador (cloreto de cálcio, magnésio, cobre e ferro) e da temperatura de carbonização/ativação sobre as propriedades físico-químicas de carvões ativados preparados a partir do mesocarpo de coco verde. Essas propriedades dependem amplamente dos métodos de preparação e podem ser ajustadas durante essa etapa. As amostras foram obtidas usando uma razão biomassa/agente ativador de 2:1 e carbonizados/ativados a 600 e 800 °C, sob fluxo de nitrogênio, por 1 h. Os sólidos foram caracterizados por thermogravimetria, difração de raios X, medida da área superficial específica e porosidade, espectroscopia no infravermelho com transformada de Fourier, espectroscopia Ramam, microscopia eletrônica de varredura, dessorção à temperatura programada, espectroscopia fotoeletrônica de raios X e determinação do ponto de carga zero. Foram obtidos carvões ativados com diferentes propriedades texturais, que variaram com o agente ativador e com a temperatura de carbonização/ativação. Observou-se a formação de carbonato de cálcio, óxido de magnésio, atacamita, paratacamita e óxido de ferro, dependendo do agente ativador, suportados em carvões ativados com baixa organização estrutural. Todos os sólidos apresentaram isotermas do tipo II, típicas de materiais macroporosos e mesoporosos com microporos associados. As isotermas apresentaram laços de histerese H1, característicos de sólidos com distribuição de poros uniformes e com formatos cilíndricos e/ou poliédrico, com as extremidades abertas. Observou-se a presença de grupos fenólicos, carboxílicos, quinônicos, lactônicos, anidridos e carbonílicos na superfície de todos os sólidos. os cloretos de ferro III e cobre II foram os agentes ativadores que promoveram uma maior quantidade de grupos superficiais nos carvões ativados, independente da temperatura de preparação. As amostras ativadas com cloreto de magnésio, cloreto de ferro III e cloreto de cobre II a 600 °C apresentaram as áreas superficiais específicas mais elevadas. Entretanto, elas decresceram com o aumento da temperatura, com exceção da amostra preparada com cloreto de ferro III, que apresentou uma área superficial específica de 471 m2.g-1 a 800 oC, indicando que esse é o agente ativador mais eficiente na preparação dos carvões ativados, para serem utilizado como suportes catalíticos. / In recent times, the intense man activity has led to an increase disposal of agricultural wastes into the environment. Aiming to find new applications for them, such as catalyst supports, the effect of the activating agent (calcium, magnesium, copper and iron chloride) and of the carbonization/activation temperature on the physicochemical properties of activated carbons, prepared from coconut mesocarp, was studied in this work. These properties largely depend on the preparation methods and can be tailored during this step. Samples were obtained by using biomass to activating agent ratio of 2:1 and carbonized/activated at 600 or 800 °C under nitrogen flow, for 1 h. The solids were characterized by thermogravimetry, X-ray diffraction, specific surface area and porosity measurements, Fourier transform infrared spectroscopy, Raman spectroscopy, scanning electron microscopy, temperature programmed desorption, X- ray photoelectron spectroscopy and point of zero charge determination. Activated carbons with different textural properties were obtained, depending on the activating agent and of the carbonization/activation temperature. It was obtained calcium carbonate, magnesium oxide, atacamite, paratacamite, or iron oxide supported on poorly organized activated carbons, depending on the activating agent. All solids showed type II isotherm, which is typical of mesoporous and macroporous materials with associated micropores. The isotherms displayed H1 hysteresis loop, characteristic of solids with uniform pore distribution and with cylindrical and/or polyhedral pores with open ends. The presence of phenolic, carboxylic, quinone, lactones, anhydrides and carbonyl groups on the surface was found for all solids. The samples activated with magnesium chloride, iron III chloride and copper II chloride at 600 °C showed the largest specific surface areas. However, they decreased with temperature increase, for the sample prepared with iron chloride, which showed a specific surface area of 471 m2.g-1 at 800 oC, indicating that this is the most effective activating agent for the preparation of activated carbons to be used as catalyst support.

SYNGAP1 haploinsufficiency is one of the most common monogenic causes of nonsyndromic moderate to severe intellectual disability (NSID) and autism (Hamdan et al., 2009; Pinto et al., 2010). De novo truncating or frameshift mutations in the SYNGAP1 gene lead to the loss of the encoded protein Synaptic GTPase activating protein (SynGAP), one of the most abundant of postsynaptic proteins (Hamdan et al., 2011). SynGAP, present at excitatory and inhibitory synapses (Kim et al., 1998), acts as a key regulator of highly conserved signaling pathways linked to AMPA- and NMDA-receptor dependent plasticity at the post synaptic density (Krapivisky et al., 2004; Vazquez et al., 2004). The Syngap mouse model has been extensively used to understand the pathophysiology underlying abnormal SynGAP-mediated signaling. Syngap heterozygous (het) mice demonstrate a range of physiological and behavioural abnormalities from development to adulthood (Komiyama et al., 2002; Muhia et al., 2010). However, recent advances in techniques for genome manipulation have allowed for the generation of rat models of neurodevelopmental disorders, including Syngap; enabling phenotypes to be validated across species and to address cognitive and social dysfunction, using paradigms that are more difficult to assess in mice. In this study, we examined the pathophysiology associated with a heterozygous deletion of the C2 and catalytic GAP domain of the protein, in Long-Evans rats (het). In contrast with het mice, het rats do not present with hyperactivity and can be habituated to an open field environment. To examine associative recognition memory, we tested the rats in five spontaneous exploration tasks for short-term and long-term memory, object-recognition (OR), object-location (OL), object-place (OP), object-context (OC) and object-place-context (OPC). Both groups were able to perform short-term memory tasks, but only wild type rats performed above chance in OL with a 24hour delay, suggesting deficits in long- term spatial memory. We also tested if partial loss of the GAP domain in SynGAP affects social behaviour in rats and we found that het rats exhibited impaired short- term social memory, with no signs of social isolation. These findings do not fully recapitulate previous abnormalities reported in the mouse model of SYNGAP1 haploinsufficiency, suggesting that some key behavioural phenotypes may be species-specific. Furthermore, based on physiological deficits that Syngap het mice exhibit, such as alterations in mEPSC/mIPSC amplitude and frequency and evoked cortical hyperexcitability in vitro (Guo et al., 2009; Ozkan et al., 2014), we also aimed to test if in vivo neuronal activity and circuit properties are altered. Using two-photon calcium imaging in awake mice, we focused on two areas of the cortex; a primary sensory area, the binocular region of the visual cortex (V1), and an association area, the medial posterior parietal cortex (PPC). Both areas have been found to maintain activity during visual discrimination tasks but to present with divergent activity trajectories (Harvey et al., 2012; Goard et al., 2016). We found preliminary evidence that neurons in layer 2-3 of the PPC of Syngap mice are hypoactive in basal conditions when animals are still in the dark, compared to wild type controls. When we assessed whether that changes when animals are running, we found that during locomotion neurons of both genotypes increase their activity, consistent with previous findings in wild type mice (McGinley et al., 2015; Pakan et al., 2016). However, this response gain is exaggerated in Syngap het neurons of the PPC. In contrast to above findings in PPC, results in V1 show that layer 2-3 neurons are hyperactive during both behavioural states, suggesting seemingly different computations of these two cortical areas. This work provides the first evidence for a dysregulated neuronal circuit in vivo in both visual and parietal cortex of Syngap mice, two areas critical for sensory processing that has been found to be affected in individuals with NSID and autism (Joosten and Bundy, 2010). We also provide first evidence of the effect of loss of SynGAP activity in behaviour of rats, complimenting existing data in the literature in a species-specific manner and providing greater insight into sensory and cognitive dysfunction associated with dysregulation in SynGAP-mediated signaling.

The lymphoid tissues consist of distinct cell subpopulations of B and T cell lineages and possess complex signaling pathways that are controlled by a myriad of molecular interactions. During the fine-tuned developmental process of the lymphoid system, inappropriate activation of oncogenes and loss of tumor suppressor gene activity can push lymphocytes into uncontrolled clonal expansion, causing several lymphoid malignancies. V(D)J recombination is one such essential process, important for the proper development of the mammalian immune system. However, mistakes in normal V(D)J recombination can lead to deletion of tumor suppressor genes or activation of proto-oncogenes. In the first part of the study, the physiological and pathological roles of DNA binding domain of RAG1 have been characterized. RAG (Recombination Activating Gene) complex consisting of RAG1 and RAG2, is a site specific endonuclease responsible for the generation of antigen receptor diversity. It cleaves a specific DNA sequence termed as recombination signal sequence (RSS), comprising of a conserved heptamer and nonamer. Recent studies have shown that RAGs can also act as a structure-specific nuclease by cleaving flaps, heterologous loops, bubbles, hairpins etc. Nonamer binding domain (NBD) of RAG1 plays a central role in the recognition of RSS during its sequence specific activity. To investigate its DNA binding properties, NBD of murine RAG1 was cloned, overexpressed and purified from E. coli. Electrophoretic mobility shift assays showed that NBD binds with high affinity to nonamer in the context of 12/23 RSS. However, it did not bind to heteroduplex DNA, irrespective of the sequence of the single-stranded region. Interestingly, when a nonamer was present next to a heteroduplex DNA, NBD exhibited robust binding. NBD binding was specific to thymines when single stranded DNA containing poly A, C, G and T were used. Biolayer interferometry studies showed that the observed poly T binding to NBD was robust with a binding constant of 0.45±0.16 µM. >23 nt was essential for NBD binding at homothymidine stretches. On a double-stranded DNA, NBD could bind to A:T stretches, but not G:C stretches or random sequences. Although NBD is indispensable for sequence-specific activity of RAGs, external supplementation of purified nonamer binding domain to NBD deleted cRAG1/cRAG2 did not restore the sequence specific activity, suggesting that the overall domain architecture of RAG1 is important for maintaining its properties. Therefore, we define the sequence requirements of NBD binding to double- and single-stranded DNA, which will have implications in generation of chromosomal rearrangement and genomic instability in lymphoid cells. Genetic alterations are one of the hallmarks of lymphoid malignancies. Many genes involved in chromosomal abnormalities are known to play central roles in the development of normal lymphocytes. In the second part of the study, molecular mechanism associated with fragility of the transcription factor, B cell leukemia 11B (BCL11B) that drives malignant transformation of T-cells has been studied. BCL11B is a zinc finger protein transcription factor with multiple functions. It plays a key role in both development and subsequent maintenance of T-cells. BCL11B gene alterations are implicated in a number of diseases including T-cell malignancies. It acts as a haplo-insufficient tumor suppressor and loss of BCL11B allele leads to susceptibility to mouse thymic lymphoma and human T-ALL. Recent studies reveal heterozygous BCL11B mutations and deletions across each of the major molecular subtypes of T-ALL (15% of patients). Most of the BCL11B missense mutations identified so far affected the residues within BCL11B zinc finger domains of the exon 4. However, mechanism of generation of such specific mutations leading to altered functions of BCL11B remains to be explored. In the present study, we address the potential mechanism of fragility of BCL11B gene during leukemia genesis. Firstly, we have evaluated different regions of BCL11B gene for presence of non-B DNA sequence motifs. Studies using non-B DB database reveal clustering of several non-B DNA forming motifs at the region spanning exon 4 of BCL11B gene. In order to biochemically evaluate the potential of non-B DNA structure formation, two different regions of exon 4 were PCR amplified and cloned. Using bisulfite modification assay we demonstrate that, single strandedness exists at both region I and II of BCL11B exon 4, when the region is present on a plasmid DNA. Bisulfite reactivity on chromosomal DNA confirmed existence of such altered DNA structures in the context of human genome. In vitro gel shift assays showed formation of both intra and intermolecular G-quadruplexes. Primer extension studies revealed that non-B DNA structures could block polymerization during replication on a plasmid, leading to DNA replication arrest. Extrachromosomal assays showed that non-B DNA structure motifs, in contrast to its mutants, blocked transcription leading to reduced expression of green fluorescent protein (GFP) within cells. Many non-B DNA-forming sequences have been mapped to regions of common chromosomal breakpoints in human tumors, known as “hotspots”, which are associated with leukemia, lymphomas and genomic disorders. Thus, alternative DNA conformations are believed to contribute to mutations, deletions and other genetic instability, leading to the deregulation of cancer-related genes in malignant diseases such as leukemia and lymphoma. Activation induced cytidine deaminase (AID), is an essential enzyme involved in antibody diversification of immunoglobulin genes. However, aberrant AID expression in B- cell and non-B cell background is reported in various cancers including leukemia and lymphoma. AID activity requires single stranded DNA (ssDNA) as a substrate. Since activation induced cytidine deaminase (AID) deaminates cytosines when present on a single stranded DNA and its expression is deregulated in many cancers, we investigated the role of AID in BCL11B gene mutagenesis. We observed substantial AID expression in many T-cell leukemic cell lines. Thus, we hypothesize that AID might be targeted to single stranded DNA present at BCL11B exon 4 due to formation of non-B DNA structures such as G-quadruplexes causing AID mediated deamination, further leading to nucleotide alterations and the mutational signature observed at BCL11B exon 4 resulting in T-ALL. Based on our findings, we propose that single strandedness resulted due to formation of non-B DNA structures such as G-quadruplex DNA, triplex DNA or cruciform DNA during physiological processes like DNA replication and transcription at exon 4 of BCL11B, can act as the target for AID. Thus, our findings uncover a new possible link between non-B DNA structure motifs and AID expression in causing mutations at BCL11B exon 4 which could lead to T cell leukemia genesis. BCL11B is a bifunctional transcriptional regulator that can act as a repressor and transactivator, and is known to differentially control the expression of specific genes in a context-dependent manner. In order to understand the transcriptional network involving BCL11B, it was cloned, overexpressed and purified from E. coli. To investigate the DNA binding properties of BCL11B protein, electrophoretic mobility shift assays were performed. Our results lead to identification of a specific sequence motif that is responsible for DNA binding. Competition experiments in presence of specific and nonspecific oligomers further confirmed the binding specificity. Thus, in the present study, we have characterized the binding properties of nonamer binding domain of RAG1, emphasizing its pathological relevance in causing genomic instability in lymphoid cells. The study may help in better understanding of RAG induced genomic instability in lymphoid tissues and role of aberrant AID expression in inducing mutations at BCL11B Zinc finger domain, leading to its deregulation and culminating into T-cell leukemia