No description available.

Optimization in immunohistochemistry is often a time consuming and complex process. There are a varying array of moving parts to consider all while preserving the sensitivity and specificity of the test. When optimizing an antibody it is important to consider the fixation of the tissue and the type of epitope retrieval that would be best suited for the test. The dilution of the primary antibody is a key marker for the efficiency and effectiveness of the laboratory protocol. The purpose of this study was to produce an optimized antibody for the nuclear protein in testis to detect NUT midline carcinoma that provides a sensitive and specific test but is also efficient and can be useful for everyday pathological dedications. The midline carcinoma defined by the translocation of the NUT gene on chromosome 15q14 that bonds with BRD4 or BRD3 commonly known as NUT midline carcinoma (NMC) is a rapidly aggressive and fatal disease. Commonly a fluorescent in situ hybridization (FISH) test is used to diagnosis this carcinoma. This test takes longer than traditional IHC and can delay the treatment of the patient. Therefore this is why, irrespective of the levels of tumor markers, immunohistochemistry for the NUT marker should be performed in any case where there is poorly differentiated carcinomas that do not have glandular differentiation that come from midline structures.

No description available.

A transição da adolescência (ADOLESC) para a idade adulta (ADULTO) é caracterizada por uma maturação de comportamentos, assim como de estruturas e sistemas do SNC. Este estudo investigou os efeitos da administração aguda e repetida de baixas doses de etanol (2,0 g/kg) em camundongos ADULTO (60 dias) e ADOLESC (28 dias), bem como a expressão de c-Fos e Egr-1 em distintas regiões cerebrais. Camundongos ADULTO e ADOLESC receberam agudamente salina (SAL) ou EtOH e sua atividade locomotora foi quantificada em campo aberto (CA). Uma hora após a injeção, a expressão de c-Fos e Egr-1 foi avaliada por imuno-histoquímica. Foram constatados: aumento na atividade locomotora e na expressão de c-Fos e Egr-1 nos animais tratados com EtOH. Outros animais ADULTO e ADOLESC foram tratados com EtOH ou SAL repetidamente (15 dias). Uma semana após o pré-tratamento, todos animais receberam etanol. A atividade locomotora foi quantificada e a expressão de c-Fos e Egr-1 foi avaliada. Os ADOLESC desenvolveram tolerância, enquanto que os ADULTO apresentaram sensibilização comportamental locomotora. A administração prolongada de etanol induziu alterações adaptativas diferenciadas, dependentes da idade, na expressão de c-Fos e Egr-1 em várias regiões encefálicas. / The transition from adolescence (ADOLESC) into adulthood (ADULT) is characterized by behavioral maturation, as well as of structures and systems of CNS. This study investigated the behavioral effects of acute and repeated administration of EtOH (2,0 g/kg) in ADULT and ADOLESC mice, as well as the c-Fos and Egr-1 expression induced by EtOH in distinctive brain structures. Locomotor activity from ADULT and ADOLESC mice acutely treated with either saline (SAL) or EtOH was measured in open field (OF). One hour after injections, c-Fos and Egr-1 expressions were evaluated by immunohistochemistry. The results demonstrated increased locomotor activity and increased c-Fos and Egr-1 expression in EtOH-treated mice. Other groups of ADULT and ADOLESC mice were treated repeatedly with SAL or EtOH during 15 days. One week after this pretreatment, all animals received an injection of EtOH. The locomotor activity was quantified and c-Fos and Egr-1 expressions were evaluated. While ADULT mice presented behavioral sensitization, ADOLESC mice developed tolerance. The repeated administration of EtOH induced an age-dependent differential expression of c-Fos and Egr-1 in several brain regions.

A transição da adolescência (ADOLESC) para a idade adulta (ADULTO) é caracterizada por uma maturação de comportamentos, assim como de estruturas e sistemas do SNC. Este estudo investigou os efeitos da administração aguda e repetida de baixas doses de etanol (2,0 g/kg) em camundongos ADULTO (60 dias) e ADOLESC (28 dias), bem como a expressão de c-Fos e Egr-1 em distintas regiões cerebrais. Camundongos ADULTO e ADOLESC receberam agudamente salina (SAL) ou EtOH e sua atividade locomotora foi quantificada em campo aberto (CA). Uma hora após a injeção, a expressão de c-Fos e Egr-1 foi avaliada por imuno-histoquímica. Foram constatados: aumento na atividade locomotora e na expressão de c-Fos e Egr-1 nos animais tratados com EtOH. Outros animais ADULTO e ADOLESC foram tratados com EtOH ou SAL repetidamente (15 dias). Uma semana após o pré-tratamento, todos animais receberam etanol. A atividade locomotora foi quantificada e a expressão de c-Fos e Egr-1 foi avaliada. Os ADOLESC desenvolveram tolerância, enquanto que os ADULTO apresentaram sensibilização comportamental locomotora. A administração prolongada de etanol induziu alterações adaptativas diferenciadas, dependentes da idade, na expressão de c-Fos e Egr-1 em várias regiões encefálicas. / The transition from adolescence (ADOLESC) into adulthood (ADULT) is characterized by behavioral maturation, as well as of structures and systems of CNS. This study investigated the behavioral effects of acute and repeated administration of EtOH (2,0 g/kg) in ADULT and ADOLESC mice, as well as the c-Fos and Egr-1 expression induced by EtOH in distinctive brain structures. Locomotor activity from ADULT and ADOLESC mice acutely treated with either saline (SAL) or EtOH was measured in open field (OF). One hour after injections, c-Fos and Egr-1 expressions were evaluated by immunohistochemistry. The results demonstrated increased locomotor activity and increased c-Fos and Egr-1 expression in EtOH-treated mice. Other groups of ADULT and ADOLESC mice were treated repeatedly with SAL or EtOH during 15 days. One week after this pretreatment, all animals received an injection of EtOH. The locomotor activity was quantified and c-Fos and Egr-1 expressions were evaluated. While ADULT mice presented behavioral sensitization, ADOLESC mice developed tolerance. The repeated administration of EtOH induced an age-dependent differential expression of c-Fos and Egr-1 in several brain regions.

Made available in DSpace on 2015-03-26T13:36:45Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2928675 bytes, checksum: b8457e10b48f36e111d09ef311ae3d5a (MD5) Previous issue date: 2010-02-18 / The begomovirus NSP (nuclear shuttle protein) facilitates the transport of viral DNA from the nucleus to the cytoplasm and cooperates with the movement protein MP to promote the translocation of viral DNA to the adjacent, uninfected cells through plasmodesmata. NSP interacts with members of the LRR-RLK ( leucine-rich repeat receptor like kinase ) family, designated NIKs ( NSP-Interacting Kinase ). Binding of NSP to the activation loop of NIK inhibits kinase activity and hence the viral protein suppresses receptor autophosphorylation and defense responses. Mutagenesis assays in the activation loop of NIK have demonstrated that the threonine 474 residue is phosphorylated in vitro and plays a crucial role in the kinase activity that is required for signaling. Replacement of Thr-474 with aspartate produces the T474 mutant, which exhibits constitutive activation, enhanced substrate phosphorylation activity and less inhibitory effect by NSP binding. The goal of this investigation was to analyse the NIK kinase domain in defense responses against begomovirus in tomato. The NIK mutant T474D cDNA was placed under the control of 35S promoter into a binary vector for plant transformation (35S-AtNIK-T474D). Primary transformants were selected by PCR and the expression of the transgene was confirmed by normal and quantitative RT- PCR in independently transformed lines. NIK and NIK-T474D overexpression in tomato plants affected the overall developmental performance of transgenic lines, which display elongated stems and a root system less developed. These phenotypes were consistent with a cross-communication between the NIK-mediated antiviral signaling and developmental signaling pathways. Infectivity assays were carried out in AtNIK- and AtNIK-T474D-overexpressing lines, with the virus ToYSV-[MG-Bi2]. Overexpression of super active AtNIK-T474D altered the infection rate by ToYSV, and interfered in symptom development. As compared to untransformed plants and NIK- overexpressing 35S-AtNIK1-6 transgenic lines, independent transgenic AtNIK-T474D lines displayed lower infection rate and attenuated symptoms. These results confirmed in planta the essential role for phosphorylation of the Thr-474 residue for NIK function and underlined the possibility for the development of more efficient tolerance strategies against geminiviruses. / A proteína NSP de begomovírus facilita o transporte do DNA viral do núcleo para o citoplasma e coopera com a proteína de movimento MP para promover o transporte do DNA viral às células adjacentes não infectadas através dos plasmodesmas. A proteína NSP interage com membros da família LRR-RLK ( leucine- rich repeat receptor like kinase ), designados NIK ( NSP-Interacting Kinase ). A ligação de NSP na alça de ativação de NIK inibe a atividade quinase, e conseqüentemente, a proteína viral inibe a atividade de autofosforilação desses receptores e sua atividade de defesa antiviral. Estudos de mutagênese na alça de ativação de NIK demonstraram que o resíduo Treonina 474 é fosforilado in vitro e exerce uma função crucial para atividade de quinase que é requerida para sinalização antiviral. Mutação no resíduo de Thr-474 para aspartato resulta no mutante T474D que exibe ativação constitutiva, atividade de fosforilaçao do substrato aumentada e menor efeito inibidor de NSP. Este trabalho teve como objetivo caracterizar o domínio quinase de NIK na resposta de defesa antiviral em tomateiros. Tomateiros foram transformados com a construção que codifica para NIK super ativa (35S-AtNIK-T474D). Os transformantes primários foram selecionados por PCR e a expressão do transgene em linhagens independentes foi confirmada por RT-PCR normal e em tempo real. A super expressão da NIK1 e NIK- T474D super ativa em tomateiros promoveu um alongamento de entrenós, mas afetou negativamente o desenvolvimento do sistema radicular, demonstrando uma possível comunicação cruzada entre a via de sinalização antiviral mediada por NIK e vias de sinalização de desenvolvimento. Experimentos de infectividade foram conduzidos em linhagens transgênicas superexpressando AtNIK ou AtNIK-T474D, utilizando o vírus ToYSV-[MG-Bi2]. Super expressão de NIK super ativa alterou a taxa de infecção por ToYSV e interferiu no desenvolvimento dos sintomas. Comparado com as plantas não transformadas e a linhagem transgênica 35S-AtNIK1-6 superexpressando NIK normal, a taxa de infecção foi inferior e os sintomas mais atenuados em linhagens transgênicas independentes superexpressando AtNIK-T474D. Estes resultados confirmam in planta o papel essencial da fosforilação do resíduo de Treonina 474 de NIK e indicam a possibilidade de se desenvolverem estratégias de tolerância a geminivirus mais eficientes.

L’augmentation des rendements est un enjeu majeur chez les céréales. Dans cet objectif, il est nécessaire de maintenir la qualité du grain de blé, qui est principalement déterminée par sa teneur et sa composition en protéines de réserve. En effet, une forte relation négative existe entre le rendement et la teneur en protéines. Par ailleurs, la qualité du grain est fortement influencée par la disponibilité en azote et en soufre dans le sol. La limitation des apports d’intrants azotés à la culture et la carence en soufre récemment observée dans les sols représentent ainsi des difficultés supplémentaires pour maitriser cette qualité. Une meilleure connaissance des mécanismes moléculaires impliqués dans le contrôle du développement du grain et la mise en place de ses réserves protéiques en réponse à la nutrition azotée et soufrée est donc primordiale. L’objectif de cette thèse a ainsi été d’apporter de nouveaux éléments à la compréhension de ces processus de régulation, aujourd’hui peu connus. Pour cela, les approches -omiques sont apparues comme une stratégie de choix pour identifier les acteurs moléculaires mis en jeu. Le protéome nucléaire a été une cible importante dans les travaux menés. L’étude de ces protéines nucléaires a révélé certains régulateurs transcriptionnels qui pourraient être impliqués dans le contrôle de la mise en place des réserves du grain. Dans une approche combinant des données de protéomique, transcriptomique et métabolomique, une vision intégrative de la réponse du grain à la nutrition azotée et soufrée a été obtenue. L’importance d’un apport de soufre dans le contrôle de la balance azote/soufre du grain, déterminante pour la composition du grain en protéines de réserve, a été clairement vérifiée. Parmi les changements observés au niveau du métabolisme cellulaire, certains des gènes affectés par la modification de cette balance pourraient orchestrer l’ajustement de la composition du grain face à des situations de carences nutritionnelles. Ces nouvelles connaissances devraient permettre de mieux maitriser la qualité du grain de blé dans un contexte d’agriculture durable. / Improving the yield potential of cereals represents a major challenge. In this context, wheat grain quality has to be maintained. Indeed, grain quality is mainly determined by the content and the composition of storage proteins, but there is a strongly negative correlation between yield and grain protein concentration. In addition, grain quality is strongly influenced by the availability of nitrogen and sulfur in soils. Nowadays, the limitation of nitrogen inputs, and also the sulfur deficiency recently observed in soils represent major difficulties to control the quality. Therefore, understanding of molecular mechanisms controlling grain development and accumulation of storage proteins in response to nitrogen and sulfur supply is a major issue. The objective of this thesis was to create knowledge on the comprehension of these regulatory mechanisms. For this purpose, the best strategy to identify molecular actors involved in these processes consisted of -omics approaches. In our studies, the nuclear proteome was an important target. Among these proteins, we revealed some transcriptional regulators likely to be involved in the control of the accumulation of grain storage compounds. Using an approach combining proteomic, transcriptomic and metabolomic data, the characterization of the integrative grain response to the nitrogen and sulfur supply was obtained. Besides, our studies clearly confirmed the major influence of sulfur in the control of the nitrogen/sulfur balance that determines the grain storage protein composition. Among the changes observed in the cell metabolism, some genes were disturbed by the modification of this balance. Thus these genes could coordinate the adjustment of grain composition in response to nutritional deficiencies. These new results contribute in facing the challenge of maintaining wheat grain quality with sustainable agriculture.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

No description available.

In bacteria, protein synthesis can occur tightly coupled to transcription. In eukaryotes, it is believed that translation occurs solely in the cytoplasm; I test whether some occurs in nuclei and find: (1) L-azidohomoalanine (Aha) – a methionine analogue (detected by microscopy after attaching a fluorescent tag using ‘click’ chemistry) – is incorporated within 5 s into nuclei in a process sensitive to the translation inhibitor, anisomycin. (2) Puromycin – another inhibitor that end-labels nascent peptides (detected by immuno-fluorescence) – is similarly incorporated in a manner sensitive to a transcriptional inhibitor. (3) CD2 – a non-nuclear protein – is found in nuclei close to the nascent RNA that encodes it (detected by combining indirect immuno-labelling with RNA fluorescence in situ hybridization using intronic probes); faulty (nascent) RNA is destroyed by a quality-control mechanism sensitive to translational inhibitors. I conclude that substantial translation occurs in the nucleus, with some being closely coupled to transcription and the associated proof-reading. Moreover, most peptides made in both the nucleus and cytoplasm are degraded soon after they are made with half-lives of about one minute. I also collaborated on two additional projects: the purification of mega-complexes (transcription ‘factories’) containing RNA polymerases I, II, or III (I used immuno-fluorescence to confirm that each contained the expected constituents), and the demonstration that some ‘factories’ specialize in transcribing genes responding to tumour necrosis factor α – a cytokine that signals through NFκB (I used RNA fluorescence in situ hybridization coupled with immuno-labelling to show active NFκB is found in factories transcribing responsive genes).