Microfluidic platforms for the characterization of in meso membrane protein crystallization

Membrane proteins, which reside in the membranes of cells, play a critical role in many important biological processes including cellular signaling, immune response, and material and energy transduction. Because of their key role in maintaining the environment within cells and facilitating intercellular interactions, understanding the function of these proteins is of tremendous medical and biochemical significance. Indeed, the malfunction of membrane proteins has been linked to numerous diseases including diabetes, cirrhosis of the liver, cystic fibrosis, cancer, Alzheimer's disease, hypertension, epilepsy, cataracts, tubulopathy, leukodystrophy, Leigh syndrome, anemia, sensorineural deafness, and hypertrophic cardiomyopathy.1-3 However, the structure of many of these proteins and the changes in their structure that lead to disease-related malfunctions are not well understood. Additionally, at least 60% of the pharmaceuticals currently available are thought to target membrane proteins, despite the fact that their exact mode of operation is not known.4-6 Developing a detailed understanding of the function of a protein is achieved by coupling biochemical experiments with knowledge of the structure of the protein. Currently the most common method for obtaining three-dimensional structure information is X-ray crystallography. However, no a priori methods are currently available to predict crystallization conditions for a given protein.7-14 This limitation is currently overcome by screening a large number of possible combinations of precipitants, buffer, salt, and pH conditions to identify conditions that are conducive to crystal nucleation and growth.7,9,11,15-24 Unfortunately, these screening efforts are often limited by difficulties associated with quantity and purity of available protein samples. While the two most significant bottlenecks for protein structure determination in general are the (i) obtaining sufficient quantities of high quality protein samples and (ii) growing high quality protein crystals that are suitable for X-ray structure determination,7,20,21,23,25-47 membrane proteins present additional challenges. For crystallization it is necessary to extract the membrane proteins from the cellular membrane. However, this process often leads to denaturation. In fact, membrane proteins have proven to be so difficult to crystallize that of the more than 66,000 structures deposited in the Protein Data Bank,48 less than 1% are for membrane proteins, with even fewer present at high resolution (< 2Å)4,6,49 and only a handful are human membrane proteins.49 A variety of strategies including detergent solubilization50-53 and the use of artificial membrane-like environments have been developed to circumvent this challenge.43,53-55 In recent years, the use of a lipidic mesophase as a medium for crystallizing membrane proteins has been demonstrated to increase success for a wide range of membrane proteins, including human receptor proteins.54,56-62 This in meso method for membrane protein crystallization, however, is still by no means routine due to challenges related to sample preparation at sub-microliter volumes and to crystal harvesting and X-ray data collection. This dissertation presents various aspects of the development of a microfluidic platform to enable high throughput in meso membrane protein crystallization at a level beyond the capabilities of current technologies. Microfluidic platforms for protein crystallization and other lab-on-a-chip applications have been well demonstrated.9,63-66 These integrated chips provide fine control over transport phenomena and the ability to perform high throughput analyses via highly integrated fluid networks. However, the development of microfluidic platforms for in meso protein crystallization required the development of strategies to cope with extremely viscous and non-Newtonian fluids. A theoretical treatment of highly viscous fluids in microfluidic devices is presented in Chapter 3, followed by the application of these strategies for the development of a microfluidic mixer capable of preparing a mesophase sample for in meso crystallization at a scale of less than 20 nL in Chapter 4. This approach was validated with the successful on chip in meso crystallization of the membrane protein bacteriorhodopsin. In summary, this is the first report of a microfluidic platform capable of performing in meso crystallization on-chip, representing a 1000x reduction in the scale at which mesophase trials can be prepared. Once protein crystals have formed, they are typically harvested from the droplet they were grown in and mounted for crystallographic analysis. Despite the high throughput automation present in nearly all other aspects of protein structure determination, the harvesting and mounting of crystals is still largely a manual process. Furthermore, during mounting the fragile protein crystals can potentially be damaged, both from physical and environmental shock. To circumvent these challenges an X-ray transparent microfluidic device architecture was developed to couple the benefits of scale, integration, and precise fluid control with the ability to perform in situ X-ray analysis (Chapter 5). This approach was validated successfully by crystallization and subsequent on-chip analysis of the soluble proteins lysozyme, thaumatin, and ribonuclease A and will be extended to microfluidic platforms for in meso membrane protein crystallization. The ability to perform in situ X-ray analysis was shown to provide extremely high quality diffraction data, in part as a result of not being affected by damage due to physical handling of the crystals. As part of the work described in this thesis, a variety of data collection strategies for in situ data analysis were also tested, including merging of small slices of data from a large number of crystals grown on a single chip, to allow for diffraction analysis at biologically relevant temperatures. While such strategies have been applied previously,57,59,61,67 they are potentially challenging when applied via traditional methods due to the need to grow and then mount a large number of crystals with minimal crystal-to-crystal variability. The integrated nature of microfluidic platforms easily enables the generation of a large number of reproducible crystallization trials. This, coupled with in situ analysis capabilities has the potential of being able to acquire high resolution structural data of proteins at biologically relevant conditions for which only small crystals, or crystals which are adversely affected by standard cryocooling techniques, could be obtained (Chapters 5 and 6). While the main focus of protein crystallography is to obtain three-dimensional protein structures, the results of typical experiments provide only a static picture of the protein. The use of polychromatic or Laue X-ray diffraction methods enables the collection of time resolved structural information. These experiments are very sensitive to crystal quality, however, and often suffer from severe radiation damage due to the intense polychromatic X-ray beams. Here, as before, the ability to perform in situ X-ray analysis on many small protein crystals within a microfluidic crystallization platform has the potential to overcome these challenges. An automated method for collecting a "single-shot" of data from a large number of crystals was developed in collaboration with the BioCARS team at the Advanced Photon Source at Argonne National Laboratory (Chapter 6). The work described in this thesis shows that, even more so than for traditional structure determination efforts, the ability to grow and analyze a large number of high quality crystals is critical to enable time resolved structural studies of novel proteins. In addition to enabling X-ray crystallography experiments, the development of X-ray transparent microfluidic platforms also has tremendous potential to answer other scientific questions, such as unraveling the mechanism of in meso crystallization. For instance, the lipidic mesophases utilized during in meso membrane protein crystallization can be characterized by small angle X-ray diffraction analysis. Coupling in situ analysis with microfluidic platforms capable of preparing these difficult mesophase samples at very small volumes has tremendous potential to enable the high throughput analysis of these systems on a scale that is not reasonably achievable using conventional sample preparation strategies (Chapter 7). In collaboration with the LS-CAT team at the Advanced Photon Source, an experimental station for small angle X-ray analysis coupled with the high quality visualization capabilities needed to target specific microfluidic samples on a highly integrated chip is under development. Characterizing the phase behavior of these mesophase systems and the effects of various additives present in crystallization trials is key for developing an understanding of how in meso crystallization occurs. A long term goal of these studies is to enable the rational design of in meso crystallization experiments so as to avoid or limit the need for high throughput screening efforts. In summary, this thesis describes the development of microfluidic platforms for protein crystallization with in situ analysis capabilities. Coupling the ability to perform in situ analysis with the small scale, fine control, and the high throughput nature of microfluidic platforms has tremendous potential to enable a new generation of crystallographic studies and facilitate the structure determination of important biological targets. The development of platforms for in meso membrane protein crystallization is particularly significant because they enable the preparation of highly viscous mixtures at a previously unachievable scale. Work in these areas is ongoing and has tremendous potential to improve not only current the methods of protein crystallization and crystallography, but also to enhance our knowledge of the structure and function of proteins which could have a significant scientific and medical impact on society as a whole. The microfluidic technology described in this thesis has the potential to significantly advance our understanding of the structure and function of membrane proteins, thereby aiding the elucidation of human biology, the development of pharmaceuticals with fewer side effects for a wide range of diseases. References (1) Quick, M.; Javitch, J. A. P Natl Acad Sci USA 2007, 104, 3603. (2) Trubetskoy, V. S.; Burke, T. J. Am Lab 2005, 37, 19. (3) Pecina, P.; Houstkova, H.; Hansikova, H.; Zeman, J.; Houstek, J. Physiol Res 2004, 53, S213. (4) Arinaminpathy, Y.; Khurana, E.; Engelman, D. M.; Gerstein, M. B. Drug Discovery Today 2009, 14, 1130. (5) Overington, J. P.; Al-Lazikani, B.; Hopkins, A. L. Nat Rev Drug Discov 2006, 5, 993. (6) Dauter, Z.; Lamzin, V. S.; Wilson, K. S. Current Opinion in Structural Biology 1997, 7, 681. (7) Hansen, C.; Quake, S. R. Current Opinion in Structural Biology 2003, 13, 538. (8) Govada, L.; Carpenter, L.; da Fonseca, P. C. A.; Helliwell, J. R.; Rizkallah, P.; Flashman, E.; Chayen, N. E.; Redwood, C.; Squire, J. M. J Mol Biol 2008, 378, 387. (9) Hansen, C. L.; Skordalakes, E.; Berger, J. M.; Quake, S. R. P Natl Acad Sci USA 2002, 99, 16531. (10) Leng, J.; Salmon, J.-B. Lab Chip 2009, 9, 24. (11) Zheng, B.; Gerdts, C. J.; Ismagilov, R. F. Current Opinion in Structural Biology 2005, 15, 548. (12) Lorber, B.; Delucas, L. J.; Bishop, J. B. J Cryst Growth 1991, 110, 103. (13) Talreja, S.; Perry, S. L.; Guha, S.; Bhamidi, V.; Zukoski, C. F.; Kenis, P. J. A. The Journal of Physical Chemistry B 2010, 114, 4432. (14) Chayen, N. E. Current Opinion in Structural Biology 2004, 14, 577. (15) He, G. W.; Bhamidi, V.; Tan, R. B. H.; Kenis, P. J. A.; Zukoski, C. F. Cryst Growth Des 2006, 6, 1175. (16) Zheng, B.; Tice, J. D.; Roach, L. S.; Ismagilov, R. F. Angew Chem Int Edit 2004, 43, 2508. (17) Li, L.; Mustafi, D.; Fu, Q.; Tereshko, V.; Chen, D. L. L.; Tice, J. D.; Ismagilov, R. F. P Natl Acad Sci USA 2006, 103, 19243. (18) Song, H.; Chen, D. L.; Ismagilov, R. F. Angew Chem Int Edit 2006, 45, 7336. (19) van der Woerd, M.; Ferree, D.; Pusey, M. Journal of Structural Biology 2003, 142, 180. (20) Ng, J. D.; Gavira, J. A.; Garcia-Ruiz, J. M. Journal of Structural Biology 2003, 142, 218. (21) Talreja, S.; Kenis, P. J. A.; Zukoski, C. F. Langmuir 2007, 23, 4516. (22) Hansen, C. L.; Quake, S. R.; Berger, J. M. US, 2007. (23) Newman, J.; Fazio, V. J.; Lawson, B.; Peat, T. S. Cryst Growth Des 2010, 10, 2785. (24) Newman, J.; Xu, J.; Willis, M. C. Acta Crystallographica Section D 2007, 63, 826. (25) Collingsworth, P. D.; Bray, T. L.; Christopher, G. K. J Cryst Growth 2000, 219, 283. (26) Durbin, S. D.; Feher, G. Annu Rev Phys Chem 1996, 47, 171. (27) Talreja, S.; Kim, D. Y.; Mirarefi, A. Y.; Zukoski, C. F.; Kenis, P. J. A. J Appl Crystallogr 2005, 38, 988. (28) Yoshizaki, I.; Nakamura, H.; Sato, T.; Igarashi, N.; Komatsu, H.; Yoda, S. J Cryst Growth 2002, 237, 295. (29) Anderson, M. J.; Hansen, C. L.; Quake, S. R. P Natl Acad Sci USA 2006, 103, 16746. (30) Hansen, C. L.; Sommer, M. O. A.; Quake, S. R. P Natl Acad Sci USA 2004, 101, 14431. (31) Lounaci, M.; Rigolet, P.; Abraham, C.; Le Berre, M.; Chen, Y. Microelectron Eng 2007, 84, 1758. (32) Zheng, B.; Roach, L. S.; Ismagilov, R. F. J Am Chem Soc 2003, 125, 11170. (33) Zhou, X.; Lau, L.; Lam, W. W. L.; Au, S. W. N.; Zheng, B. Anal. Chem. 2007. (34) Cherezov, V.; Caffrey, M. J Appl Crystallogr 2003, 36, 1372. (35) Qutub, Y.; Reviakine, I.; Maxwell, C.; Navarro, J.; Landau, E. M.; Vekilov, P. G. J Mol Biol 2004, 343, 1243. (36) Rummel, G.; Hardmeyer, A.; Widmer, C.; Chiu, M. L.; Nollert, P.; Locher, K. P.; Pedruzzi, I.; Landau, E. M.; Rosenbusch, J. P. Journal of Structural Biology 1998, 121, 82. (37) Gavira, J. A.; Toh, D.; Lopez-Jaramillo, J.; Garcia-Ruiz, J. M.; Ng, J. D. Acta Crystallogr D 2002, 58, 1147. (38) Stevens, R. C. Current Opinion in Structural Biology 2000, 10, 558. (39) Baker, M. Nat Methods 2010, 7, 429. (40) McPherson, A. In Current Topics in Membranes, Volume 63; Volume 63 ed.; DeLucas, L., Ed.; Academic Press: 2009, p 5. (41) Gabrielsen, M.; Gardiner, A. T.; Fromme, P.; Cogdell, R. J. In Current Topics in Membranes, Volume 63; Volume 63 ed.; DeLucas, L., Ed.; Academic Press: 2009, p 127. (42) Page, R. In Methods in Molecular Biology: Structural Proteomics - High Throughput Methods; Kobe, B., Guss, M., Huber, T., Eds.; Humana Press: Totowa, NJ, 2008; Vol. 426, p 345. (43) Caffrey, M. Ann Rev Biophys 2009, 38, 29. (44) Doerr, A. Nat Methods 2006, 3, 244. (45) Brostromer, E.; Nan, J.; Li, L.-F.; Su, X.-D. Biochemical and Biophysical Research Communications 2009, 386, 634. (46) Li, G.; Chen, Q.; Li, J.; Hu, X.; Zhao, J. Anal Chem 2010, 82, 4362. (47) Jia, Y.; Liu, X.-Y. The Journal of Physical Chemistry B 2006, 110, 6949. (48) RCSB Protein Data Bank. http://www.rcsb.org/ (July 11, 2010). (49) Membrane Proteins of Known 3D Structure. http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html (July 11, 2010). (50) Michel, H. Trends Biochem Sci 1983, 8, 56. (51) Rosenbusch, J. P. Journal of Structural Biology 1990, 104, 134. (52) Garavito, R. M.; Picot, D. Methods 1990, 1, 57. (53) Kulkarni, C. V. 2010; Vol. 12, p 237. (54) Landau, E. M.; Rosenbusch, J. P. P Natl Acad Sci USA 1996, 93, 14532. (55) Pebay-Peyroula, E.; Rummel, G.; Rosenbusch, J. P.; Landau, E. M. Science 1997, 277, 1676. (56) Cherezov, V.; Liu, W.; Derrick, J. P.; Luan, B.; Aksimentiev, A.; Katritch, V.; Caffrey, M. Proteins: Structure, Function, and Bioinformatics 2008, 71, 24. (57) Cherezov, V.; Rosenbaum, D. M.; Hanson, M. A.; Rasmussen, S. G. F.; Thian, F. S.; Kobilka, T. S.; Choi, H. J.; Kuhn, P.; Weis, W. I.; Kobilka, B. K.; Stevens, R. C. Science 2007, 318, 1258. (58) Cherezov, V.; Yamashita, E.; Liu, W.; Zhalnina, M.; Cramer, W. A.; Caffrey, M. J Mol Biol 2006, 364, 716. (59) Jaakola, V. P.; Griffith, M. T.; Hanson, M. A.; Cherezov, V.; Chien, E. Y. T.; Lane, J. R.; IJzerman, A. P.; Stevens, R. C. Science 2008, 322, 1211. (60) Rosenbaum, D. M.; Cherezov, V.; Hanson, M. A.; Rasmussen, S. G. F.; Thian, F. S.; Kobilka, T. S.; Choi, H. J.; Yao, X. J.; Weis, W. I.; Stevens, R. C.; Kobilka, B. K. Science 2007, 318, 1266. (61) Wacker, D.; Fenalti, G.; Brown, M. A.; Katritch, V.; Abagyan, R.; Cherezov, V.; Stevens, R. C. J Am Chem Soc 2010, 132, 11443. (62) Höfer, N.; Aragão, D.; Caffrey, M. Biophys J 2010, 99, L23. (63) Li, L.; Ismagilov, R. F. Ann Rev Biophys 2010. (64) Pal, R.; Yang, M.; Lin, R.; Johnson, B. N.; Srivastava, N.; Razzacki, S. Z.; Chomistek, K. J.; Heldsinger, D. C.; Haque, R. M.; Ugaz, V. M.; Thwar, P. K.; Chen, Z.; Alfano, K.; Yim, M. B.; Krishnan, M.; Fuller, A. O.; Larson, R. G.; Burke, D. T.; Burns, M. A. Lab Chip 2005, 5, 1024. (65) Jayashree, R. S.; Gancs, L.; Choban, E. R.; Primak, A.; Natarajan, D.; Markoski, L. J.; Kenis, P. J. A. J Am Chem Soc 2005, 127, 16758. (66) Wootton, R. C. R.; deMello, A. J. Chem Commun 2004, 266. (67) McPherson, A. J Appl Crystallogr 2000, 33, 397.

Aplicación del modelo Discovering Hands en Argentina

Discovering Hands (DH) es un proyecto que nace en Alemania en el 2006, liderado por el doctor Frank Hoffmann. El programa se desarrolla pensando en el importante problema de salud pública en el cual se ha convertido en el cáncer de mama, pues según la Organización Mundial de la Salud es el mayor causal de muerte en mujeres, tanto en países desarrollados como en vía de desarrollo, y en Alemania esta enfermedad acaba con la vida de aproximadamente 18.000 mujeres cada año. (The Global Journal, 2014) DH entrena y capacita mujeres visualmente impedidas para detectar de manera temprana los signos de cáncer de mama, dado que estas poseen un sentido del tacto más desarrollado que el de una persona que no se encuentre limitada visualmente. Esto les permite localizar el cáncer de forma más rápida que un médico general ya que son capaces de identificar los tumores más pequeños, logrando así reducir notablemente los costos totales del tratamiento de esta enfermedad. Adicional a esto, el capacitar y preparar a mujeres con discapacidad visual para la detección temprana de cáncer de mama, incrementa la fuerza laboral del país, pues estas mujeres pasarían a ser parte de la población económicamente activa del mismo (PEA) y lograrían que las personas dejen de percibir esta condición como una discapacidad y por el contrario la vean como una ventaja. Después de unos años de prueba, el programa ha sido mejorado y extendido tanto en Alemania como en otros países (Austria), razón por la cual se realizó el estudio de factibilidad del proyecto en países como Colombia - donde se quiere llevar a cabo un proyecto piloto en la ciudad de Cali - México y Argentina. El presente trabajo se enfoca en Argentina, por medio del cual se busca proponer aportes para disminuir las causas de muertes originadas por esta enfermedad y los altos costos que estas le generan al sector de la salud de este país. Con el estudio se logró identificar la factibilidad de la implementación del modelo de negocio, evidenciando que Argentina cuenta con unas particularidades en su sistema de gobierno que pueden hacer que la puesta en práctica del proyecto sea más compleja que en otros países.

Registro de pacientes con distrofinopatías en Colombia

INTRODUCCIÓN. La distrofia muscular de Duchenne es una enfermedad neuromuscular con una herencia recesiva ligada al X que afecta a 1 de cada 3500 niños nacidos vivos. Se produce por mutaciones en el gen DMD que codifica para la distrofina. Se caracteriza por manifestaciones cl��nicas variables típicas de una distrofia muscular proximal progresiva. OBJETIVO. Realizar el primer registro en Colombia de los pacientes identificados con distrofinopatías, teniendo en cuenta características cl��nicas y paracl��nicas, así como las mutaciones causales de esta patología. METODOLOGÍA Es un estudio descriptivo, transversal, de la revisión de historias cl��nicas de los pacientes con diagnóstico de DMD atendidos en la consulta de Genética de la Universidad del Rosario durante los años 2006 a 2015. RESULTADOS Se identificaron 99 pacientes, de los cuales 56 (56,56%) corresponden al fenotipo Duchenne y 12 (12,12%) al Becker. No fue posible clasificar a 31 pacientes (31,3%) por falta de datos cl��nicos. La edad de inicio de los síntomas fue en promedio de 4,41 años. Las mutaciones más frecuentes fueron las deleciones (69%), seguidas por las mutaciones puntuales(14%), las duplicaciones (11%) y por otras mutaciones (4%). CONCLUSIONES Este registro de distrofinopatías es el primero reportado en Colombia y el punto de partida para conocer la incidencia de la enfermedad, caracterización cl��nica y molecular de los pacientes, garantizando así el acceso oportuno a los nuevos tratamientos de medicina de precisión que permitan mejorar la calidad de vida de los pacientes y sus familias.

Seguridad del monitoreo hemodinámico invasivo versus mínimamente invasivo en pacientes con choque cardiogénico en cuidado intensivo adultos. Revisión sistemática

Introducción: El monitoreo hemodinámico es una herramienta para diagnosticar el choque cardiogénico y monitorear la respuesta al tratamiento; puede ser invasivo, mínimamente invasivo o no invasivo. Se realiza rutinariamente con catéter de arteria pulmonar (CAP) o catéter de Swan Ganz; nuevas técnicas de monitoreo hemodinámico mínimamente invasivo tienen menor tasa de complicaciones. Actualmente se desconoce cuál técnica de monitoreo cuenta con mayor seguridad en el paciente con choque cardiogénico. Objetivo: Evaluar la seguridad del monitoreo hemodinámico invasivo comparado con el mínimamente invasivo en pacientes con choque cardiogénico en cuidado intensivo adultos. Diseño: Revisión sistemática de la literatura. Búsqueda en Pubmed, EMBASE, OVID - Cochrane Library, Lilacs, Scielo, registros de ensayos cl��nicos, actas de conferencias, repositorios, búsqueda de literatura gris en Google Scholar, Teseo y Open Grey hasta agosto de 2016, publicados en ingl��s y español. Resultados: Se identificó un único estudio con 331 pacientes críticamente enfermos que comparó el monitoreo hemodinámico con CAP versus PiCCO que concluyó que después de la corrección de los factores de confusión, la elección del tipo de monitoreo no influyó en los resultados cl��nicos más importantes en términos de complicaciones y mortalidad. Dado que se incluyeron otros diagnósticos, no es posible extrapolar los resultados sólo a choque cardiogénico. Conclusión: En la literatura disponible no hay evidencia de que el monitoreo hemodinámico invasivo comparado con el mínimamente invasivo, en pacientes adultos críticamente enfermos con choque cardiogénico, tenga diferencias en cuanto a complicaciones y mortalidad.

Valor diagnóstico del pepsinógeno i/ii como biomarcador de lesiones pre-malignas y malignas gástricas: revisión sistemática

Antecedentes: El cáncer gástrico se diagnostica tardíamente. Sólo en países como Corea y Japón existen pol��ticas de tamizaje, que se justificarían en cualquier país con alta prevalencia de cáncer gástrico como Colombia o Chile. El análisis del pepsinógeno sérico se ha propuesto para el diagnóstico de lesiones premalignas y malignas gástricas, por lo cual se pretende revisar sistemáticamente en la literatura el valor diagnóstico del cociente pepsinógeno I/II como marcador de lesiones premalignas y malignas gástricas. Metodología: Se revisó la literatura hasta septiembre del 2016 con palabras claves lesiones malignas, premalignas gástricas y pepsinógeno en las bases de datos PubMed, OVID, EMBASE, EBSCO, LILACS, OPENGRAY y Dialnet, artículos de prueba diagnóstica que evaluaran el cociente pepsinógeno I/II en relación con los hallazgos histol��gicos. Resultados: Se incluyeron 21 artículos conun total de 20601 pacientes, que demuestranuna sensibilidad entre13.7% - 91.2%, una especificidad entre 38.5% - 100%, un Valor Predictivo Positivo entre 6.3% - 100% y un Valor Predictivo Negativo entre 33.3% - 98.8%del cociente pepsinógeno I/II en relación con el diagnósticode lesiones premalignas y malignas gástricas. Conclusiones: Los valores del cociente pepsinógeno I/II disminuidos se relacionan con la presencia delesiones premalignas y malignas gástricas.Dado que tiene mejor especificidad que sensibilidad, en cuanto prueba para tamizaje, sería útil para la selección de pacientes que se beneficiaríande la EVDA. Se requieren más estudios de prueba diagnóstica para validar un punto de corte específico que pueda ser utilizado como valor estándar.

Epidemiologia do mal das folhas da seringueira. 1. Ponte Nova-MG.

1989

Consideration of problem formulation and option assessment (PFOA) for environmental risk assessment: Bt cotton in Vietnam.

2008

Anthelmintic activity of Cymbopogon martinii, Cymbopogon schoenanthus and Mentha piperita essential oils evaluated in four different in vitro tests

Anthelmintic resistance is a worldwide concern in small ruminant industry and new plant-derived compounds are being studied for their potential use against gastrointestinal nematodes. Mentha piperita, Cymbopogon martinii and Cymbopogon schoenanthus essential oils were evaluated against developmental stages of trichostrongylids from sheep naturally infected (95% Haemonchus contortus and 5% Trichostrogylus spp.) through the egg hatch assay (EHA), larval development assay (LDA), larval feeding inhibition assay (LFIA), and the larval exsheathment assay (LEA). The major constituent of the essential oils, quantified by gas chromatography for M. piperita oil was menthol (42.5%), while for C. martinii and C. schoenanthus the main component was geraniol (81.4% and 62.5%, respectively). In all in vitro tests C. schoenanthus essential oil had the best activity against ovine trichostrongylids followed by C. martini, while M. piperita presented the least activity. Cymbopogon schoenanthus essential oil had LC(50) value of 0.045 mg/ml in EHA, 0.063 mg/ml in LDA, 0.009 mg/ml in LFIA, and 24.66 mg/ml in LEA. The anthelmintic activity of essential oils followed the same pattern in all in vitro tests, suggesting C. schoenanthus essential oil could be an interesting candidate for nematode control, although in vivo studies are necessary to validate the anthelmintic properties of this oil. (C) 2011 Elsevier B.V. All rights reserved.

Estudos de microscopia óptica e de microscopia eletrônica de varredura em folhas de Mentha spicata e de Mentha spicata x suaveolens (Lamiaceae)

O presente trabalho tem como objetivo realizar um estudo de anatomia foliar por meio de microscopia óptica e de microscopia eletrônica de varredura em Mentha spicata L. e Mentha spicata X suaveolens, caracterizando histologicamente a l��mina foliar. Secções transversais e paradérmicas da região mediana do limbo foliar mostraram a presença de epiderme unisseriada, coberta por uma fina camada de cutícula, apresentando tricomas glandulares do tipo capitado e peltado e não glandulares unisseriados multicelulares, não ramificados. O mesofilo de ambas as espécies é dorsiventral, com parênquima paliçádico uniestratificado, com células alongadas e rico em inclusões citoplasmáticas. O parênquima lacunoso é formado por três a quatro camadas de células irregulares. Os tricomas capitados presentes são classificados como do tipo I, e apresentam-se com uma célula basal, uma célula peduncular e uma grande célula apical, cujo formato varia de circular a piriforme. Os tricomas peltados consistem de uma célula basal, uma célula peduncular curta, larga e unicelular, com paredes externas cutinizadas e uma cabeça grande multicelular com 12 células secretoras, distribuídas radialmente em dois círculos concêntricos, o central com 4 células e o externo com 8 células, as quais acumulam o produto da secreção em uma cavidade entre a cutícula e as células secretoras; o pé do tricoma glandular está inserido em 11 células epidérmicas. Há predominância de tricomas capitados em relação aos tricomas peltados em ambas as espécies de Mentha.

Mentha piperita cultivada com variação de cálcio. Trocas gasosas e óleo essencial

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Produtos naturais no controle do ácaro Varroa destructor em abelhas Apis mellifera L. (africanizadas)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

p75 neurotrophin receptor-mediated neuronal death is promoted by Bcl-2 and prevented by Bcl-x(L)

The p75 neurotrophin receptor (p75NTR) has been shown to mediate neuronal death through an unknown pathway. We microinjected p75NTR expression plasmids into sensory neurons in the presence of growth factors and assessed the effect of the expressed proteins on cell survival. We show that, unlike other members of the TNFR family, p75NTR signals death through a unique caspase-dependent death pathway that does not involve the death domain and is differentially regulated by Bcl-2 family members: the anti-apoptotic molecule Bcl-2 both promoted, and was required for, p75NTR killing, whereas killing was inhibited by its homologue BcI-x(L). These results demonstrate that Bcl-2, through distinct molecular mechanisms, either promotes or inhibits neuronal death depending on the nature of the death stimulus.

Bcl-X-L translocation in renal tubular epithelial cells in vitro protects distal cells from oxidative stress

Background. The molecular pathogenesis of different sensitivities of the renal proximal and distal tubular cell populations to ischemic injury, including ischemia-reperfusion (IR)-induced oxidative stress, is not well-defined. An in vitro model of oxidative stress was used to compare the survival of distal [Madin-Darby canine kidney (MDCK)] and proximal [human kidney-2 (HK-2)] renal tubular epithelial cells, and to analyze for links between induced cell death and expression and localization of selected members of the Bcl-2 gene family (anti-apoptotic Bcl-2 and Bcl-X-L, pro-apoptotic Bax and Bad), Methods. Cells were treated with 1 mmol/L hydrogen peroxide (H2O2) Or were grown in control medium for 24 hours. Cell death (apoptosis) was quantitated using defined morphological criteria. DNA gel electrophoresis was used for biochemical identification. Protein expression levels and cellular localization of the selected Bcl-2 family proteins were analyzed (West ern immunoblots, densitometry, immunoelectron microscopy). Results. Apoptosis was minimal in control cultures and was greatest in treated proximal cell cultures (16.93 +/- 4.18% apoptosis) compared with treated distal cell cultures (2.28 +/- 0.85% apoptosis, P < 0.001). Endogenous expression of Bcl-X-L and Bax, but not Bcl-2 or Bad, was identified in control distal cells, Bcl-X-L and Bax had nonsignificant increases (P > 0.05) in these cells. Bcl-2, Bax, and Bcl-X-L, but not Bad, were endogenously expressed in control proximal cells. Bcl-X-L was significantly decreased in treated proximal cultures (P < 0.05), with Bas and Bcl-2 having nonsignificant increases (P > 0.05). Immunoelectron microscopy localization indicated that control and treated hut surviving proximal cells had similar cytosolic and membrane localization of the Bcl-2 proteins. In comparison, surviving cells in the treated distal cultures showed translocation of Bcl-X-L from cytosol to the mitochondria after treatment with H2O2, a result that was confirmed using cell fractionation and analysis of Bcl-XL expression levels of the membrane and cytosol proteins. Bax remained distributed evenly throughout the surviving distal cells, without particular attachment to any cellular organelle. Conclusion. The results indicate that in this in vitro model, the increased survival of distal compared with proximal tubular cells after oxidative stress is best explained by the decreased expression of anti-apoptotic Bcl-X-L in proximal cells, as well as translocation of Bcl-X-L protein to mitochondria within the surviving distal cells.