Infection of mungbean seed by Macrophomina phaseolina is more likely to result from localized pod infection than from systemic plant infection

The ubiquitous fungal pathogen Macrophomina phaseolina is best known as causing charcoal rot and premature death when host plants are subject to post-flowering stress. Overseas reports of M.phaseolina causing a rapid rot during the sprouting of Australian mungbean seed resulted in an investigation of the possible modes of infection of seed. Isolations from serial portions of 10 mungbean plants naturally infected with the pathogen revealed that on most plants there were discrete portions of infected tissue separated by apparently healthy tissue. The results from these studies, together with molecular analysis of isolates collected from infected tissue on two of the plants, suggested that aerial infection of aboveground parts by different isolates is common. Inoculations of roots and aboveground parts of mungbean plants at nine temperaturexsoil moisture incubation combinations and of detached green pods strongly supported the concept that seed infection results from infection of pods by microsclerotia, rather than from hyphae growing systemically through the plant after root or stem infection. This proposal is reinforced by anecdotal evidence that high levels of seed infection are common when rainfall occurs during pod fill, and by the isolation of M.phaseolina from soil peds collected on pods of mungbean plants in the field. However, other experiments showed that when inoculum was placed within 130mm of a green developing pod and a herbicide containing paraquat and diquat was sprayed on the inoculated plants, M.phaseolina was capable of some systemic growth from vegetative tissue into the pods and seeds.

Reducing seed viability of flaxleaf fleabane, feathertop Rhodes grass and awnless barnyard grass

In the sub-tropical grain region of Australia, cotton and grains systems are now dominated by flaxleaf fleabane (Conyza bonariensis (L.) Cronquist), feathertop Rhodes grass (Chloris virgata Sw.) and awnless barnyard grass (Echinochloa colona (L.) Link). While control of these weed species is best achieved when they are young, previous studies have shown a potential for reducing seed viability and minimising seed bank replenishment by applying herbicides when plants are reproductive. Pot trials were established over two growing seasons to examine the effects of 2,4-D, 2,4-D + picloram, glyphosate and glufosinate which had been successful on other species, along with paraquat and haloxyfop (grasses only). Herbicides were applied at ¾ field rates in an attempt not to kill the plants. Flaxleaf fleabane plants were sprayed at two growth stages (budding and flowering) and the grasses were sprayed at two stages (late tillering/booting and flowering). Spraying flaxleaf fleabane at flowering reduced seed viability to 0% (of untreated) in all treatments except glyphosate (51%) and 2,4-D + picloram (8%). Seed viability was not reduced with the first and second regrowths with the exception of 2,4-D + picloram where viability was reduced to 20%. When sprayed at budding only 2,4-D + picloram reduced seed viability in both trials. Spraying the grasses at late tillering/booting did not reduce viability except for glufosinate on awnless barnyard grass (50%). Applying herbicides at flowering resulted in 0% seed viability in awnless barnyard grass from glufosinate, paraquat and glyphosate and 0% viability in feathertop Rhodes grass for glufosinate. These herbicides were less effective on heads that emerged and flowered after spraying, only slightly reducing seed viability. These trials have shown that attempts to reduce seed viability have potential, however flaxleaf fleabane and feathertop Rhodes grass are able to regrow and will need on-going monitoring and control measures.

Ozone-induced signaling in Arabidopsis thaliana

Tropospheric ozone (O3) is one of the most common air pollutants in industrialized countries, and an increasing problem in rapidly industrialising and developing countries in Asia, Africa and South America. Elevated concentrations of tropospheric O3 can lead to decrease in photosynthesis rate and therefore affect the normal metabolism, growth and seed production. Acute and high O3 episodes can lead to extensive damage leading to dead tissue in plants. Thus, O3 derived growth defects can lead to reduction in crop yield thereby leading to economical losses. Despite the extensive research on this area, many questions remain open on how these processes are controlled. In this study, the stress-induced signaling routes and the components involved were elucidated in more detail starting from visual damage to changes in gene expression, signaling routes and plant hormone interactions that are involved in O3-induced cell death. In order to elucidate O3-induced responses in Arabidopsis, mitogen-activated protein kinase (MAPK) signaling was studied using different hormonal signaling mutants. MAPKs were activated at the beginning of the O3 exposure. The activity of MAPKs, which were identified as AtMPK3 and AtMPK6, reached the maximum at 1 and 2 hours after the start of the exposure, respectively. The activity decreased back to clean air levels at 8 hours after the start of the exposure. Both AtMPK3 and AtMPK6 were translocated to nucleus at the beginning of the O3 exposure where they most likely affect gene expression. Differences were seen between different hormonal signaling mutants. Functional SA signaling was shown to be needed for the full protein levels and activation of AtMPK3. In addition, AtMPK3 and AtMPK6 activation was not dependent on ethylene signaling. Finally, jasmonic acid was also shown to have an impact on AtMPK3 protein levels and AtMPK3 activity. To further study O3-induced cell death, an earlier isolated O3 sensitive Arabidopsis mutant rcd1 was mapped, cloned and further characterized. RCD1 was shown to encode a gene with WWE and ADP-ribosylation domains known to be involved in protein-protein interactions and cell signaling. rcd1 was shown to be involved in many processes including hormonal signaling and regulation of stress-responsive genes. rcd1 is sensitive against O3 and apoplastic superoxide, but tolerant against paraquat that produces superoxide in chloroplast. rcd1 is also partially insensitive to glucose and has alterations in hormone responses. These alterations are seen as ABA insensitivity, reduced jasmonic acid sensitivity and reduced ethylene sensitivity. All these features suggest that RCD1 acts as an integrative node in hormonal signaling and it is involved in the hormonal regulation of several specific stress-responsive genes. Further studies with the rcd1 mutant showed that it exhibits the classical features of programmed cell death, PCD, in response to O3. These include nuclear shrinkage, chromatin condensation, nuclear DNA degradation, cytosol vesiculation and accumulation of phenolic compounds and eventually patches of HR-like lesions. rcd1 was found to produce extensive amount of salicylic acid and jasmonic acid in response to O3. Double mutant studies showed that SA independent and dependent processes were involved in the O3-induced PCD in rcd1 and that increased sensitivity against JA led to increased sensitivity against O3. Furthermore, rcd1 had alterations in MAPK signature that resembled changes that were previously seen in mutants defective in SA and JA signaling. Nitric oxide accumulation and its impact on O3-induced cell death were also studied. Transient accumulation of NO was seen at the beginning of the O3 exposure, and during late time points, NO accumulation coincided with the HR-like lesions. NO was shown to modify defense gene expression, such as, SA and ethylene biosynthetic genes. Furthermore, rcd1 was shown to produce more NO in control conditions. In conclusion, NO was shown to be involved in O3-induced signaling leading to attenuation of SA biosynthesis and other defense related genes.

Stress and developmental responses in Arabidopsis thaliana: regulation through the transcription factor-interacting protein RCD1

Plants constantly face adverse environmental conditions, such as drought or extreme temperatures that threaten their survival. They demonstrate astonishing metabolic flexibility in overcoming these challenges and one of the key responses to stresses is changes in gene expression leading to alterations in cellular functions. This is brought about by an intricate network of transcription factors and associated regulatory proteins. Protein-protein interactions and post-translational modifications are important steps in this control system along with carefully regulated degradation of signaling proteins. This work concentrates on the RADICAL-INDUCED CELL DEATH1 (RCD1) protein which is an important regulator of abiotic stress-related and developmental responses in Arabidopsis thaliana. Plants lacking this protein function display pleiotropic phenotypes including sensitivity to apoplastic reactive oxygen species (ROS) and salt, ultraviolet B (UV-B) and paraquat tolerance, early flowering and senescence. Additionally, the mutant plants overproduce nitric oxide, have alterations in their responses to several plant hormones and perturbations in gene expression profiles. The RCD1 gene is transcriptionally unresponsive to environmental signals and the regulation of the protein function is likely to happen post-translationally. RCD1 belongs to a small protein family and, together with its closest homolog SRO1, contains three distinguishable domains: In the N-terminus, there is a WWE domain followed by a poly(ADP-ribose) polymerase-like domain which, despite sequence conservation, does not seem to be functional. The C-terminus of RCD1 contains a novel domain called RST. It is present in RCD1-like proteins throughout the plant kingdom and is able to mediate physical interactions with multiple transcription factors. In conclusion, RCD1 is a key point of signal integration that links ROS-mediated cues to transcriptional regulation by yet unidentified means, which are likely to include post-translational mechanisms. The identification of RCD1-interacting transcription factors, most of whose functions are still unknown, opens new avenues for studies on plant stress as well as developmental responses.

Binding of carboxylatopillar5]arene with alkyl and aryl ammonium salts in aqueous medium

The complex formation of alkyl ammonium salts by water-soluble carboxylatopillar5] arene (CP5A) in aqueous medium is reported. p-Xylene diammonium salt and a series of secondary alkyl ammonium salts with various alkyl groups have been prepared and investigated for complex formation. All the ammonium salts exhibit strong host-guest complexation with CP5A under neutral aqueous conditions. H-1 NMR, H-1 DOSY and 2D NOESY NMR experiments have been performed to characterize these inclusion complexes. In this study, the hydrophobic and electrostatic interactions govern the complex formation leading to the formation of pseudorotaxane species. Five pseudo2] rotaxanes and one pseudo3] rotaxane were obtained whose association constant values and stoichiometry were evaluated by an NMR titration method. The results indicate the use of ammonium salts as new complimentary synthons for CP5A in aqueous medium, adding to the repertoire of existing recognition motifs such as paraquat and 1,4-bis(pyridinium) derivatives.

Efecto de labranza y métodos de control de malezas sobre la dinámica de las maleza y el crecimiento y rendimiento del frijol común (Phaseolus vulgaris L) postrera, 1994

Con el propósito de evaluar la influencia de los dos sistemas de labranza y tres métodos de control de malezas sobre el comportamiento de las malezas y en el crecimiento y rendimiento del cultivo del frijol común, se llevó a efecto el presente experimento en la finca experimental La Compañía, San Marcos, Carazo. Los factores en estudio fueron A. manejo de suelo, el cual incluyo labranza mínima (chapia más raya de siembra) y labranza cero (chapia más siembra el espeque) y 5: controles de malezas, donde se incluyeron: Pre-emergente más post-emergente (paraquat, 1 día después de le siembra más fluazifob-butil más fomesalen, 21 días después de le siembra), pre-emergente más cobertura (paraquat, 1 día después de la siembra, más cobertura muerta de maíz, 8 días después de la siembra) y pre-emergente más chapia (paraquat, 1 días después de la siembre, más chapia con azadón a los 21 días después de la siembra). Los resultados obtenidos indican que el sistema de labranza cero permitió la mayor abundancia y cobertura de maleza, sin embargo ésta presentó la menor biomasa de malezas. El método de control de maleza que presentó el mejor efecto sobre las malezas fue el pre-emergente más post-emergente, al permitir los menores valores en abundancia, cobertura, biomasa (peso seco) y diversidad. El sistema de labranza mínima presentó el mayor número de vainas por planta, y el mejor rendimiento, aunque no significativamente diferente de labranza cero. Los métodos de control de malezas obtuvieron valores similares en relación a las variables de rendimiento, como número de vainas por plantas, número de granos por vainas y peso de 100 granos, destacándose con un mayor rendimiento el control pre-emergente más cobertura. El método de control pre-emergente más cobertura y el sistema de labranza mínima obtuvieron los mejores rendimientos y también el mejor beneficio neto y la mejor rentabilidad.

Susceptibilidad de especies nobles a diferentes herbicidas, en diferentes dosis en café (Coffea arabica L.)

Con el propósito de evaluar la susceptibilidad de especies nobles a diferentes dosis de herbicidas en cafetales, se condujo el presente trabajo que se llevó a cabo en el Centro Experimental de Café del Pacifico "Mauricio López Munguía", ubicado en Municipio de Masatepe, departamento de Masaya y en la Finca "El doble" Municipio del Crucero, departamento de Managua, a partir de Mayo de1991a Enero de 1992. Las especies nobles estudiadas fueron Commelina díffusa, Panicun trichoides y Desmodium ovalifolium sembradas en las calles del cafeto. Commelina diffusa y Oplismenus burmannii, establecidas de forma natural en las calles del cafeto. Se probaron tres herbicidas: Paraquat (Gramoxone), Glifosato (Round up) y 2.4-D evaluándose en cada uno tres dosis que se escogieron partiendo de las dosis comerciales utilizadas por los caficultores. Se usó diseño de bloques completos al azar con arreglo en parcelas subdivididas con 4 repeticiones, 108 tratamientos para el experimento de coberturas sembradas y arreglo en parcelas divididas con 3 repeticiones, 27 tratamientos para Oplismenus burmannii y 3 repeticiones, 18 tratamientos para Commelina diffusa.Se evaluó porcentaje de susceptibilidad a los 8,15,30,45 y 60 días después de aplicación y porcentaje de recuperación a los 45 y 60 días después de aplicación para el experimento de coberturas sembradas. En los otros dos experimentos se tomó porcentaje de susceptibilidad a los 15,30 y 45 días después de aplicación y porcentaje de recuperación a los 45 días después de aplicación. Las diferentes dosis ensayadas no mostraron diferencias significativas. Oplismenus burmannii presentó el menor grado de susceptibilidad y el mayor porcentaje de recuperación a los herbicidas. Commelina diffusa presentó el mayor grado de susceptibilidad a los herbicidas ensayados. 2,4-D no ejerció ningún efecto sobre Oplismenus burmatmii y Panicum trichoides. De forma general en donde se aplicó Paraquat se observó el mayor porcentaje de recuperación de las especies nobles.

Producción de maiz (Zea mays L.) bajo dos sistemas de labranza y tres métodos de control de malezas, efecto sobre la dinámica de las malezas y el crecimiento y rendimiento del cultivo

Durante la época de primera (junio a septiembre de 1995) se llevó a cabo el presente experimento en la finca experimental La Compañía, localizada en el municipio de San Mareos, Carazo. Los objetivos perseguidos fueron evaluar la influencia de dos sistemas de labranza y tres métodos de control de malezas sobre el comportamiento de la malezas y el crecimiento y rendimiento del cultivo de maíz. (Zea mays L.), así como analizar desde el punto de vista económico la factibilidad de los tratamientos evaluados. Los factores incluidos en el experimento fueron establecidos en diseño de parcelas divididas con distribución en bloques completos al azar, con cuatro repeticiones, constituyéndose en la parcela principal , la labranza del suelo (cero y mínima) y los controles de malezas en las sub-parcelas, estos fueron: paraquat más chapia(pre-emergente y control mecánico a los 28 dias después de la siembra), paraquat (pro-emergente) y paraquat más paraquat (pre-emergente y post-emergente). Los resultados obtenidos muestran que el sistema de labranza cero presentó la mayor abundancia, cobertura y biomasa de malezas. El método de control de malezas que reflejó el mejor efecto sobre las malezas fue el control a base de pre más post-emergente, el cual obtuvo los menores valores en abundancia, diversidad, cobertura y biomasa de malezas. Los mejores resultados en cuanto a rendimiento se obtuvieron con labranza cero y control a base de pre-emergente más chapia. En lo que respecta a variables de crecimiento del cultivo, los sistemas de labranzas y controles de malezas no manifestaron diferencias significativas sobre las variables en estudio. El sistema de labranza que ofrece mayores beneficios netos y mejores rendimientos fue labranza cero. En cuanto a los controles de malezas, el mejor rendimiento y mejor rentabilidad se obtuvo con el control a base de pre-emergente más chapia.

Efecto de tres sistemas de labranza y métodos de control de maleza sobre cenosis, plagas y enfermedades en el cultivo de maiz (Zea mays L.)

En el centro experimental La Compañía en el municipio de San Marcos, departamento de Carazo, Nicaragua, se realizó un experimento para evaluar el efecto de tres sistemas de labranzas y tres métodos de control de malezas, sobre el comportamiento de las malezas, plagas, enfermedades y el crecimiento y rendimiento del maíz (Zea mays L). El diseño experimental utilizado fue un bloque completo al azar con tratamientos arreglados en parcelas divididas en los cuales la parcela grande corresponde a las labranzas y la parcela pequeña a los controles. Se evaluaron los sistemas de labranzas cero, mínima y convencional y los métodos de control pre-emergente (Atrazina), post-emergente (Paraquat) y control mecánico Las variables de malezas fueron abundancia, dominancia, biomasa y diversidad. Las variables evaluadas del cultivo fueron altura de planta, diámetro del tallo, número de plantas cosechadas, número de mazorcas cosechadas, diámetro de mazorcas, longitud de mazorcas, número de hileras por mazorcas, número de granos por hileras, peso de cien granos, peso de campo y rendimiento de grano. Los datos se analizaron por medio del análisis de varianza, excepto las variables de diversidad y porcentaje de cobertura de malezas. Se realizó comparaciones de medias utilizando la prueba de rangos múltiples de Duncan al 5 %. El análisis económico se hizo a través de la metodología del presupuesto parcial y análisis marginal de beneficios netos. Las variables de malezas que presentaron diferencias para el factor labranza fueron abundancia (solo el primer muestreo) y biomasa. Mientras las variables del cultivo que presentaron diferencias fueron altura de planta, diámetro del tallo, longitud de mazorca, incidencia de enfermedades, peso de campo, biomasa de rastrojos y rendimiento de granos. El factor control de malezas presentó diferencias significativas en las variables de malezas, abundancia y biomasa. Ninguna de las variables evaluadas del cultivo presentaron diferencias significativas. La labranza con mejor tasa de retomo marginal fue labranza cero, mientras en el factor control la obtuvo el control pre- emergente (Atrazina). Los mejores resultados económicos los presentó la interacción de los factores labranza mínima más control pre emergente de malezas. Este tratamiento presentó la mejor tasa de retomo marginal.

Efecto de dos sistemas de labranza (mínima y cero) y tres métodos de control de malezas sobre la dinámica de las malezas, crecimiento y rendimiento del cultivo del maíz (Zea mays L.) primera 1994

Durante la época de primera de 1994, se estableció un experimento de campo en la estación experimental La Compañia, localizada en el municipio de San Marcos, Carazo, con el propósito de evaluar la influencia de dos sistemas de labranza y tres métodos de control de malezas sobre la dinámica de las malezas, el crecimiento y rendimiento del cultivo de maíz (Zea mays L.). Se utilizó un diseño de parcelas divididas, arreglados en bloques completos al azar la parcela grande correspondió a las labranzas y las pequeñas a los controles, con cuatro repeticiones. Los factores en estudio fueron A:sistema de labranza, con los siguientes niveles al: labranza cero y a2: mínima y B: Control de malezas con los siguientes niveles: b1: control de malezas pre emergente (utilizando paraquat), b2: pre emergente más chapia a los 28 dias después de la siembra, y b3: aplicación pre emergente más post emergente, con la utilización de paraquat a la siembra y a los 28 días después de la siembra. Los resultados indican que la labranza cero, mostró efectos negativos sobre la dinámica de las malezas, sobre todo en cuanto a dominancia y diversidad, no asi en abundancia donde ambos sistemas mostraron similitud. El mejor método de control de malezas fue pre emergente más post emergente, seguido de pre emergente má chapia y el menos efectivo fue el control pre emergente. En las variables de crecimiento, tanto los sistemas de labranzas como de los metodos de control de malezas, no mostraron efectos significativos. La evaluación de los componentes del rendimiento en los sistemas de labranzas no mostraron diferencias significativas, no obstante labranza cero mostró mejores resultados en longitud de mazorca, con un valor de 9.47 cm, número de granos por hilera 20, peso de cien granos 18.5gr. y rendimiento de grano 1681.3kg/ha. Los métodos de control de malezas mostraron pocas diferencias significativas en los componentes del rendimiento. El control pre emergente más pos emergente mostró los mejores resultados en diámetro de mazorca con un valor de 4.02cm., número de hileras por mazorca 13.25, número de granos por hilera 21.25, rendimiento 1881.2kg/ha y peso de paja 1972.4kg/. Existieron diferencias significativas en el rendimiento de grano en los controles de malezas, el mejor comportamiento lo presentó el control pre emergente más post emergente. La mejor rentabilidad la obtuvieron el sistema de labranza cero y el control de malezas pre emergente más post emergente, debido a que estos ofrecen mayores beneficios netos, además obtuvieron loa mejores rendimientos.

Estudio de Cyperus rotundus sobre efecto de tres herbicidas en su control integral y efecto alelopatico en el crecimiento de diferentes cultivos

Durante 1987 sea realizo estudios con el objetivo de evaluar la influencia de diferentes herbicidas en el control de Cyperus rotundus en la producción de tomate industrial (UC-82) y el efecto alelopático sobre el crecimiento de cebolla, tomate, sorgo y maíz. A través de la vados de sustratos sembrados con y sin C rotundus de 30, 60 y 90 días de crecimiento. Los resultados indican que la abundancia de C rotundus se disminuyó en un 37% por el efecto de Methaloclor (12 lts/ha) y en un 32% por glifosato (4.3 lts/ha) al comprarlo con paraquat (1.5 lts/ha.) Además se encontró que la biomasa del Cyperus rotundus, aérea y subterránea fue menor con el uso de glifosato. En cuanto a las sustancias inhibidoras del C rotundus mostraron una tendencia biológica a disminuir el porcentaje de germinación altura y longitud de la radícula de los cultivo evaluado cuando se trataron con sustancias drenadas de los suelos infestados y no infestados con coyolillo y a incrementarse principalmente en arena con coyolillo. Las sustancia alelopáticas extraídas de suelos no infestada sembrado con coyolillo de 30 días de crecimiento disminuyeron el crecimiento significativamente del tomate. En tanto en el sorgo se incrementó con las sustancias lavadas de suelos no infestado y arena sembrados con c.rotundus. También se determinó que las sustancias inhibidoras exudadas por el coyolillo tienen sitio de acción sobre el órgano dependiendo del cultivo y que esta no persistan en suelo. En general se encontró que el c.rotundus puede ser tolerado dentro del cultivo del tomate bajo un manejo integrado y que las sustancias inhibidoras producidas por el c.rotundus tiene su mayor efecto sobre los cultivo cuando se trataron con sustancia extraídas de plantas de coyolillo con crecimiento de 30 y 60 días.

Resposta adaptativa e efeitos das substâncias oxidantes - K2TeO3, H2O2 e paraquate - na produção de biofilme de amostras de Corynebacterium diphtheriae isolados de origens diversas

A difteria é uma doença imunoprevinível que permanece endêmica em diversas regiões do mundo. Além dos casos de difteria clássica, doenças invasivas como endocardite, osteomielite, pneumonia e infecções relacionadas à cateter, tem acometido indivíduos adultos parcialmente imunizados. A natureza multifatorial dos fatores de virulência que favorecem a formação de biofilme e sobrevivência em macrófagos tem sido evidenciada para o Corynebacterium diphtheriae. Entretanto, escassos são os trabalhos que investigaram a influência de condições ambientais na virulência do patógeno. Uma vez que agentes oxidantes são capazes de gerar radicais livres e levar ao estresse oxidativo que, consequentemente, podem resultar em resposta adaptativa e influenciar na formação do biofilme e na virulência bacteriana, o presente trabalho teve como objetivo geral avaliar os efeitos do peróxido de hidrogênio (H2O2), paraquate e telurito de potássio (K2TeO3) em propriedades biológicas de cepas de C. diphtheriae de origens diversas. Os resultados demonstraram que as amostras de C. diphtheriae apresentaram níveis de resistência ao K2TeO3, H2O2 e paraquate, porém em intensidades variadas e independentes da capacidade de produção da toxina diftérica. Algumas amostras, toxinogênicas ou não, isoladas do trato respiratório superior demonstraram resposta adaptativa para H2O2 passando, portanto, a expressar resistência aumentada após uma prévia exposição ao referido agente oxidante. C. diphtheriae não exibiu respostas adaptativas para o K2TeO3 e paraquate. C. diphtheriae foi capaz de produzir biofilme na superfície de substratos abióticos de natureza hidrofílica (vidro) e hidrofóbica (poliestireno) na presença de agentes oxidantes K2TeO3, H2O2 e paraquate. A presença dos agentes oxidantes influenciou na formação de biofilme de algumas amostras. O paraquate inibiu a produção de biofilme de todas amostras. A amostra TR241 teve a produçao de biofilme inibida na presença dos três agentes oxidantes analisados. Por outro lado, a presença de H2O2 e do K2TeO3 estimulou a produção de biofilme de algumas amostras. Para todas as amostras de C. diphtheriae testadas, independentes das origens, biotipos e da capacidade de produção de toxina diftérica, os ensaios moleculares indicaram a presença de sequências gênicas cromossômicas homólogas, codificadores da proteína DIP 0906 (TeR) e da proteína DIP 1421 (OxyR), envolvidos na resistência ao telurito e na proteção contra o estresse oxidativo, respectivamente. Não foi observada correlação da suscetibilidade e a resposta adaptativa aos agentes oxidantes com as diferentes características bacterianas: origem, sítio de isolamento, produção de toxina diftérica, metabolização de sacarose e produção de biofilme. Em conclusão, o estresse oxidativo foi capaz de influenciar fatores de virulência do C. diphtheriae, como a capacidade de produçao de biofilme, que podem estar contribuindo para a patogenia da difteria clássica assim como de doenças invasivas causadas por cepas atoxinogênicas.

Proteomic approach to oxidative stress in Daphnia magna

The keystone aquatic organism Daphnia magna is extensively used to assess the toxicity of chemicals. This has recently lead to an increase in the omics literature focusing on daphnids, an increase fuelled by the sequencing of the Daphnia pulex genome. Yet, no omics study has looked directly at oxidative stress (OS) in daphnids, even though OS is of primary importance in the response of aquatic organisms to their changing environment and is often induced by anthropogenic xenobiotics. This thesis thus focuses on the application of redox-proteomics, the study of the oxidative modification of proteins, to D. magna Specifically, daphnids were exposed to copper or paraquat, two well studied prooxidants, and protein carbonyls were labelled with fluorescein-5-thiosemicarbazide prior to twodimensional electrophoresis (2DE). This showed clearly that both compounds affect a different portion of the proteome. The identified proteins indicated that energy metabolism was affected by paraquat, while copper induced a reduction of the heat shock response (heat shock proteins, proteases and chaperones) a counterintuitive result which may be adaptative to metal toxicity in arthropods. The same approach was then applied to the study of the toxicity mechanism of silver nanoparticles (AgNP), an increasingly utilised form of silver with expected environmental toxicity, and its comparison to silver nitrate. The results demonstrate that, although less toxic than silver ions, AgNP toxicity functions through a different mechanism. AgNP toxicity is thus not a product of silver dissolution and increased protein carbonylation indicates that AgNP cause OS. Interestingly three of the four tested compounds altered vitellogenin levels and oxidation. Vitellogenins could thus represent an interesting subproteome for the detection of stress in daphnids. Finally, an experiment with oxidised BSA demonstrates the applicability of solid phase hydrazide in the enrichment of undigested carbonylated proteins.

Burkholderia cenocepacia C5424 produces a pigment with antioxidant properties using a homogentisate intermediate

Burkholderia cenocepacia is a gram-negative opportunistic pathogen that belongs to the Burkholderia cepacia complex. B. cenocepacia can survive intracellularly within phagocytic cells, and some epidemic strains produce a brown melanin-like pigment that can scavenge free radicals, resulting in the attenuation of the host cell oxidative burst. In this work, we demonstrate that the brown pigment produced by B. cenocepacia C5424 is synthesized from a homogentisate (HGA) precursor. The disruption of BCAL0207 (hppD) by insertional inactivation resulted in loss of pigmentation. Steady-state kinetic analysis of the BCAL0207 gene product demonstrated that it has 4-hydroxyphenylpyruvic acid dioxygenase (HppD) activity. Pigmentation could be restored by complementation providing hppD in trans. The hppD mutant was resistant to paraquat challenge but sensitive to H2O2 and to extracellularly generated superoxide anions. Infection experiments in RAW 264.7 murine macrophages showed that the nonpigmented bacteria colocalized in a dextran-positive vacuole, suggesting that they are being trafficked to the lysosome. In contrast, the wild-type strain did not localize with dextran. Colocalization of the nonpigmented strain with dextran was reduced in the presence of the NADPH oxidase inhibitor diphenyleneiodonium, and also the inducible nitric oxide inhibitor aminoguanidine. Together, these observations suggest that the brown pigment produced by B. cenocepacia C5424 is a pyomelanin synthesized from an HGA intermediate that is capable of protecting the organism from in vitro and in vivo sources of oxidative stress.

Characterization of SodC, a periplasmic superoxide dismutase from Burkholderia cenocepacia

Burkholderia cenocepacia is a gram-negative, non-spore-forming bacillus and a member of the Burkholderia cepacia complex. B. cenocepacia can survive intracellularly in phagocytic cells and can produce at least one superoxide dismutase (SOD). The inability of O2- to cross the cytoplasmic membrane, coupled with the periplasmic location of Cu,ZnSODs, suggests that periplasmic SODs protect bacteria from superoxide that has an exogenous origin (for example, when cells are faced with reactive oxygen intermediates generated by host cells in response to infection). In this study, we identified the sodC gene encoding a Cu,ZnSOD in B. cenocepacia and demonstrated that a sodC null mutant was not sensitive to a H2O2, 3-morpholinosydnonimine, or paraquat challenge but was killed by exogenous superoxide generated by the xanthine/xanthine oxidase method. The sodC mutant also exhibited a growth defect in liquid medium compared to the parental strain, which could be complemented in trans. The mutant was killed more rapidly than the parental strain was killed in murine macrophage-like cell line RAW 264.7, but killing was eliminated when macrophages were treated with an NADPH oxidase inhibitor. We also confirmed that SodC is periplasmic and identified the metal cofactor. B. cenocepacia SodC was resistant to inhibition by H2O2 and was unusually resistant to KCN for a Cu,ZnSOD. Together, these observations establish that B. cenocepacia produces a periplasmic Cu,ZnSOD that protects this bacterium from exogenously generated O2- and contributes to intracellular survival of this bacterium in macrophages.