published_or_final_version / Zoology / Master / Master of Science

The NF kappa B family of heterodimeric transcription factors is activated by many stimuli, and lead to the upregulation of countless genes. Not surprisingly, NF kappa B plays a critical role in many aspects of cellular function. In T and B lymphocytes, antigen receptor stimulation leads to the activation of NF kappa B through a signal transduction cascade involving the Bcl10-MALT1 complex. We hypothesized that this complex may be critical to signalling cascades other than those emanating from antigen receptors. B cell activation factor of the TNF family (BAFF) activates non-canonical NF kappa B heterodimers that promote B cell survival. Here, we show that MALT1 is required for BAFF-induced phosphorylation of NF kappa B2 (p100), p100 degradation and RelB nuclear translocation in B220+ B cells. TRAF3, a known negative regulator of BAFF-R mediated signaling, interacts with MALT1 in a manner which is negatively regulated by BAFF, and TRAF3 levels are enhanced in MALT1-/- B cells. MALT1-/- CD21highCD23low (MZ) B cells show a defect in BAFF-induced survival and MALT1-/- x BAFF-transgenic (Tg) mice have decreased MZ and B1 B cell levels compared to BAFF-Tg mice. In agreement with this in vitro data, phenotypes associated with over-expression of BAFF including increased serum immunoglobulin titres, spontaneous germinal center (GC) formation, and immune complex deposition in the kidney were found to be dependent on B cell-intrinsic MALT1 expression. Our results demonstrate a novel role for MALT1 in biological outcomes induced by BAFF-mediated signal transduction. The mechanism by which the Bcl10-MALT1 complex regulates antigen induced NF kappa B activation in T cells remains controversial. To shed light on this regulatory network, we conducted biochemical purification of Bcl10, and identified Uev1a, a known regulator of antigen receptor mediated NF kappa B activation. We hypothesized that mms2, and structurally similar molecule to Uev1a, may also impinge on NF kappa B activation. Mms2 overexpression in 293T cells inhibited the Bcl10-induced activation of an NF kappa B sensitive luciferase. Lymphocyte development and antigen receptor induced activation occurs normally mms2-/- mice. However, class switched serum immunoglobulins, and survival responses to DNA damage inducing gamma-irradiation, are decreased in mms2-/- mice. Therefore, mms2 is dispensible in vivo for lymphocyte function and development, but is required for DNA damage responses.

Las pruebas serológicas de Inhibición de la Hemaglutinación (HI) y la Prueba de Inmunoensayo con enzimas asociadas (ELISA) son usadas rutinariamente para la detección de anticuerpos contra el virus de la Enfermedad de Newcastle (vENC). El antígeno de la prueba de HI puede ser una cepa de tropismo respiratorio o de tropismo entérico. En el presente estudio fue analizada la concordancia entre las dos pruebas de HI y la prueba de ELISA. Con tal fin fueron analizados los resultados serológicos obtenidos en 196 sueros colectados en pollos de engorde de 26 y 42 días, antes y después de ser desafiados con una cepa velogénica viscerotrópica del virus de la enfermedad de Newcastle. Los anticuerpos detectados por ambas pruebas fueron agrupados en cuatro escalas según su nivel de detección (negativo, bajo, medio y alto) para posteriormente ser analizados por la prueba estadística de Kappa de Cohen. Se analizó la concordancia entre las dos pruebas de HI con antígeno respiratorio o entérico y, entre cada una de estos últimas (HI respiratoria y HI entérica) con la prueba de ELISA. Se determinó que la concordancia entre las prueba de HI y ELISA, tanto usando antígeno entérico como antígeno respiratorio fue muy pobre, mientras que la concordancia entre las dos pruebas de HI (con antígeno respiratorio o entérico) fue moderada. Palabras claves: virus de la enfermedad de Newcastle (ENC), ELISA, HI, índice de kappa, concordancia. / --- Hemagglutination Inhibition (IH) and Enzyme-linked Immunosorbent Assay (ELISA) Serological tests are routinely used for antibodies detection against the virus of Newcastle disease (NDV). Antigen for IH test may be a strain of respiratory tropism or enteric tropism. This study analyzed the concordance between the HI and ELISA tests. For this purpose, the serological results obtained on 196 sera collected in broilers of 26 and 42 days old were analyzed, before and after being challenged with a velogenic viscerotropic strain of newcastle disease virus. The antibodies detected by both tests were grouped into four scales according to their detection level (negative, low, medium and high) in order to later be analyzed by the Kappa Cohen statistic test. Concordance between the two tests of IH with respiratory or enteric antigen was analyzed, and concordance between each these (IH respiratory and IH enteric) with the ELISA test was analyzed. It was found that the concordance between IH test and ELISA, using both enteric antigen and respiratory antigen was very poor, while the concordance between the two IH tests (respiratory or enteric antigen) was moderate. Keywords: virus Newcastle disease (NDV), ELISA, HI, kappa index, concordance.

There has been much evidence in recent years to suggest that the kappa opioid receptor plays a significant role in mediating a number of behavioural disorders including drug abuse and depression. Previous in vitro evaluation within the group of secondary and tertiary amines derived from 2-amino-1,1-dimethyl-1,2,3,4-tetrahydronaphthalen-7-ol has suggested that all behave as pure opioid antagonists. These findings prompted further synthetic and pharmacological investigations of this scaffold, with the eventual aim of developing a short-acting selective kappa opioid antagonist to further probe the receptor.

Thesis (M. Sc.)--University of Hong Kong, 1972. / Typewritten.

The NF kappa B family of heterodimeric transcription factors is activated by many stimuli, and lead to the upregulation of countless genes. Not surprisingly, NF kappa B plays a critical role in many aspects of cellular function. In T and B lymphocytes, antigen receptor stimulation leads to the activation of NF kappa B through a signal transduction cascade involving the Bcl10-MALT1 complex. We hypothesized that this complex may be critical to signalling cascades other than those emanating from antigen receptors. B cell activation factor of the TNF family (BAFF) activates non-canonical NF kappa B heterodimers that promote B cell survival. Here, we show that MALT1 is required for BAFF-induced phosphorylation of NF kappa B2 (p100), p100 degradation and RelB nuclear translocation in B220+ B cells. TRAF3, a known negative regulator of BAFF-R mediated signaling, interacts with MALT1 in a manner which is negatively regulated by BAFF, and TRAF3 levels are enhanced in MALT1-/- B cells. MALT1-/- CD21highCD23low (MZ) B cells show a defect in BAFF-induced survival and MALT1-/- x BAFF-transgenic (Tg) mice have decreased MZ and B1 B cell levels compared to BAFF-Tg mice. In agreement with this in vitro data, phenotypes associated with over-expression of BAFF including increased serum immunoglobulin titres, spontaneous germinal center (GC) formation, and immune complex deposition in the kidney were found to be dependent on B cell-intrinsic MALT1 expression. Our results demonstrate a novel role for MALT1 in biological outcomes induced by BAFF-mediated signal transduction. The mechanism by which the Bcl10-MALT1 complex regulates antigen induced NF kappa B activation in T cells remains controversial. To shed light on this regulatory network, we conducted biochemical purification of Bcl10, and identified Uev1a, a known regulator of antigen receptor mediated NF kappa B activation. We hypothesized that mms2, and structurally similar molecule to Uev1a, may also impinge on NF kappa B activation. Mms2 overexpression in 293T cells inhibited the Bcl10-induced activation of an NF kappa B sensitive luciferase. Lymphocyte development and antigen receptor induced activation occurs normally mms2-/- mice. However, class switched serum immunoglobulins, and survival responses to DNA damage inducing gamma-irradiation, are decreased in mms2-/- mice. Therefore, mms2 is dispensible in vivo for lymphocyte function and development, but is required for DNA damage responses.

Ulm, Univ., Diss., 2008.

Ulm, Univ., Diss., 2007.

Thesis (Ph. D.)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Medicinal Chemistry. Title from title-page of electronic thesis. Bibliography: leaves 126-138.

Memoria para optar al título de Químico Farmacéutico / Las vacunas de ADN son una terapia alternativa altamente atractiva para combatir el cáncer. Esta estrategia consiste en la entrega in vivo de ADN plasmidial codificante de antígenos tumorales que son expresados y presentados por células dendríticas (DC) con el fin de activar linfocitos T capaces de eliminar específicamente células tumorales. Un importante paso hacia la aplicación clínica de las vacunas de ADN ha sido el uso de la electroporación in vivo, un método de entrega altamente eficiente y clínicamente aplicable, que aumenta considerablemente el ingreso de plásmidos al interior de las células. Sin embargo, la mayoría de los antígenos tumorales son antígenos propios expresados en células normales, por lo que el sistema inmune es incapaz de discriminar a los tumores como agentes potencialmente peligrosos. Es así como los esfuerzos para resolver estos inconvenientes se han enfocado, en parte, en la búsqueda y elaboración de nuevas estrategias que sean capaces de promover eficientemente la inducción de linfocitos T específicos contra estos antígenos tumorales poco inmunogénicos. El ADN usado en las vacunas puede ser reconocido por numerosos receptores de la inmunidad innata, los cuales al ser activados, señalizan a través del factor de transcripción NF-κB, induciendo la expresión de citoquinas proinflamatorias e interferones de tipo I. NF-κB es un regulador maestro de la función de las DC y de la inducción de linfocitos T mediada por vacunas de ADN, por lo tanto, el desarrollo de adyuvantes que modulen su actividad representa una estrategia interesante para promover la inducción eficiente de linfocitos T antitumorales. IκBα es uno de los principales inhibidores de NF-κB, el cual además es un gen blanco de NF-κB, por lo que actúa en un sistema de retroalimentación negativa para detener la activación de esta vía. Por otro lado, el uso de shARN que silencian la expresión de reguladores negativos de las DC ha demostrado su capacidad como adyuvantes génicos en vacunas de ADN, al inducir una respuesta inmune antitumoral más potente. Por tanto, nuestra hipótesis plantea que un shARN contra IκBα es capaz de potenciar la activación de la inmunidad innata mediada por NF-κB y, como consecuencia, la magnitud de la respuesta inmune adaptativa específica contra el antígeno tumoral codificado. En este proyecto se utilizaron plásmidos codificantes de un shARN contra IκBα como adyuvantes génicos para ser administrado junto a una vacuna de ADN que codifica para el antígeno tumoral TRP2. Se evaluó el efecto del silenciamiento de IκBα sobre la migración de las células dendríticas de la piel, particularmente sobre las células de Langerhans y sobre las células dendríticas dermales CD103+. La generación de linfocitos T específicos contra TRP2 se analizó en ratones vacunados mediante citometría de flujo y el efecto antitumoral in vivo se evaluó usando un modelo de profiláctico de melanoma metastásico y un modelo terapéutico de melanoma subcutáneo. Los resultados de este trabajo indican que la administración de ADN plásmidial en la piel incrementa la migración de células dendríticas hacia los nódulos linfáticos drenantes y que la inhibición de la expresión de IκBα con un shARN en el sitio de vacunación incrementa la migración de células dendríticas dermales CD103+ hacia el nódulo linfático drenante. Además se observó que la coadministración del shARN contra IκBα en conjunto con una vacuna de ADN dirigida contra el antígeno tumoral TRP2 potencia la generación de linfocitos T CD8 específicos e incrementa la respuesta inmune antitumoral en ensayos de metástasis pulmonar y crecimiento tumoral utilizando el modelo de melanoma murino B16F10. Dada la gran capacidad de las células dendríticas dermales CD103+ para activar linfocitos T CD8 es posible correlacionar su incremento en los nódulos linfáticos drenantes del sitio de vacunación con una mayor generación de linfocitos T CD8 específicos contra TRP2 y con un incremento en la respuesta inmune antitumoral contra melanoma cuando se coadministra el shARN contra IκBα junto con una vacuna de ADN dirigida contra el antígeno tumoral TRP2 / DNA vaccines are a highly attractive therapeutic alternative to treat cancer. This strategy consists of in vivo delivering plasmid vectors encoding tumor antigens, which are expressed and presented by dendritic cells (DC) in order to activate T cells capable of specifically eliminating tumor cells. An important step towards the clinical application of DNA vaccines has been the use of in vivo electroporation, a highly efficient delivery method that is clinically applicable and considerably increases plasmid uptake in cells. However, most of tumor antigens are self-antigens expressed by normal cells, so that the immune system is unable to recognize tumors as potentially dangerous agents. Thus, the efforts to solve these inconvenient have been focused on developing new strategies to promote the induction of T cells responses specific against such poorly immunogenic tumor antigens. DNA used for vaccines can be recognized by several innate immune receptors that, upon activation, signal through the transcription factor NF-κB which induces the expression of proinflamatory cytokines and type I interferons. NF-κB is a master regulator of both DC function and DNA vaccine-mediated induction of T cell responses, therefore the development of adjuvants that modulate NF-κB activity represents an interesting strategy to promote the efficient induction of antitumor T cell responses. IκBα is one of the major inhibitors and downstream target genes of NF-κB, therefore acting in a negative feedback loop to stop NF- κB activation. The use of shRNA silencing negative regulators of DC function has demonstrated its potential as genetic adjuvants for DNA vaccines by inducing enhanced antitumor immune responses. Hence, our hypothesis states that a shRNA targeting IκBα is able to promote the NF-κB innate immune activation and, as a consequence, the magnitude of tumor antigen-specific adaptive immune responses. In this project, plasmid-encoded shRNA targeting IκBα were generated as genetic adjuvants to be coadministrated with a DNA vaccine encoding the tumor antigen TRP2.The effect of IκBα silencing was analyzed over the migration of skin dendritic cells, particularly Langerhans cells and CD103+ dermal dendritic cells. Generation of TRP2-specific T cells was studied in vaccinated mice by flow cytometry and the in vivo antitumor effect was evaluated using prophylactic model of metastatic melanoma and a subcutaneous model of melanoma. The results obtained in this work shows that the inhibition of IκBα using a shRNA increases the migration of CD103+ dermal dendritic cells to the skin draining lymph nodes. Also the coadministration of the shRNA against IκBα and a DNA vaccine against TRP2 enhance the generation of TRP2 specific CD8 T cells increasing the antitumor immune response against melanoma. Finally due to the high ability of CD103+ dermal dendritic cells to cross-present antigens and activate CD8 T cell is possible to correlate their increase in the migration with higher levels of TRP2 specific CD8 T cells and higher antitumor immune response when the shRNA against IκBα is coadministered with a DNA vaccine against TRP2