Dissertationen zum Thema „Sols suppressifs“

La réduction de l'utilisation des produits phytosanitaires constitue l'un des axes majeurs de la transition agroécologique. Dans ce contexte, il est urgent de développer des stratégies assurant une gestion efficace et durable de la pression parasitaire, tout en préservant l'environnement. La mobilisation du microbiome du sol, et notamment des communautés de bactéries qui le composent, représente une de ces solutions. S'il a été démontré qu'une partie de ces communautés était capable de limiter l'impact de pathogènes des cultures, les relations entre l'environnement de ce microbiome et sa capacité à réguler les maladies des plantes restent encore largement méconnus. L'ambition de ce projet de thèse était d'évaluer l'impact de facteurs, telles que les conditions pédologiques et les pratiques agricoles, sur la structure et le fonctionnement du microbiome du sol et d'analyser la relation entre ces modifications et la capacité du microbiome à participer à la régulation de la fusariose de l'épi. Ce projet s'articule autour de deux axes de recherche, visant à (1) déterminer quels facteurs expliquent le caractère suppressif des sols vis-à-vis de l'agent pathogène Fusarium graminearum, et (2) évaluer l'influence de l'environnement sur l'assemblage du microbiome du sol et du blé. Pour répondre à ces objectifs, un réseau de 103 parcelles d'agriculteurs de la plaine de Limagne (Puy-de-Dôme, France) a été mobilisé. Les parcelles représentaient une diversité de types de sols et de pratiques agricoles, et se répartissaient soit en agriculture conventionnelle, soit en agriculture biologique, soit en agriculture de conservation. Des échantillons de sol ont été prélevés dans chaque parcelle et caractérisés par (1) les pratiques agricoles (2) des analyses physico-chimiques, (3) leur communauté bactérienne par metabarcoding du gène ADNr 16S et (4) des tests d'inhibition in vitro (fongistase) du champignon pathogène Fusarium graminearum. Parmi ces 103 parcelles, 98 ont servi au prélèvement d'échantillons de rhizosphère et de racines de blé (Triticum aestivum) dont la communauté bactérienne a également été recensée. Enfin, neuf sols parmi les 103 ont été sélectionnés pour la culture et l'infection du blé par F. graminearum en conditions contrôlées. Le test de fongistase a révélé une grande variabilité au sein de l'échantillon ainsi que la capacité de certains sols à inhiber complètement la germination du champignon. Les caractéristiques pérennes du sol et la diversité bactérienne étaient liées à la fongistase des sols. Il existait aussi une corrélation entre l'abondance de Burkholderia spp. et la fongistase. La comparaison des microbiomes bactériens du sol, de la rhizosphère et des racines du blé a révélé des compositions différentes entre les trois compartiments. La composition microbienne dans les sols influençait celles de la rhizosphère et des racines dans une même parcelle. Les caractéristiques physico chimiques et le système de culture influençaient la composition de la communauté bactérienne dans les trois compartiments. L'expérimentation en conditions contrôlées n'a pas révélé de lien entre microbiome (diversité et composition) et symptômes de la fusariose, ni de lien entre fongistase et symptômes in planta sur un même sol. Dans l'ensemble, ces travaux contribuent à évaluer les possibilités d'utilisation des pratiques agricoles comme levier de contrôle agroécologique de la fusariose du blé, à travers la modulation des communautés microbiennes naturelles
Reducing the use of phytosanitary products constitutes one of the major axes of the agroecological transition. In this context, it is urgent to develop strategies ensuring effective and sustainable management of parasitic pressure, while preserving the environment. Mobilization of the soil microbiome, and particularly the bacterial community, represents one of these solutions. Although it has been demonstrated that part of these communities is capable of limiting the impact of crop pathogens, the relationships between the environment of the microbiome and its capacity to regulate plant diseases still remain largely unknown. The ambition of this thesis project was to evaluate the impact of factors, such as soil conditions and agricultural practices, on the structure and functioning of the soil microbiome and to analyze the relationship between these modifications and the capacity of the microbiome to participate in the regulation of Fusarium head blight. This project is structured around two axes of research, aiming to (1) determine which factors explain the suppressive nature of soils with respect to the pathogen Fusarium graminearum, and (2) evaluate the influence of the environment on the assembly of the soil and wheat microbiome. To meet these objectives, a network of 103 plots in the Limagne plain (Puy-de-Dôme, France) was mobilized. The plots represented a diversity of soil types and agricultural practices, and were divided into either intensive agriculture, organic agriculture, or soil conservation agriculture. Soil samples were taken from each plot and characterized by (1) agricultural practices (2) physicochemical analyses, (3) their bacterial community by metabarcoding of the 16S rDNA gene and (4) in vitro inhibition tests (fungistasis) of the pathogenic fungus Fusarium graminearum. Among these 103 plots, 98 were used to collect samples of wheat (Triticum aestivum) rhizosphere and roots, that were also described through their bacterial community. Finally, nine soils among the 103 were selected for the cultivation and infection of wheat by F. graminearum under controlled conditions. The fungistasis test revealed great variability within the sample as well as the ability of certain soils to completely inhibit the germination of the fungus. Perennial soil characteristics and bacterial diversity were related to soil fungistasis. There was also a correlation between the abundance of Burkholderia spp. and fungistasis. Comparison of bacterial microbiomes from soil, wheat rhizosphere and roots revealed different compositions between the three compartments. The microbial composition in the soil influenced those of the rhizosphere and roots of the same plot. The physicochemical characteristics and the management system influenced the composition of the bacterial community in the three compartments. Experimentation under controlled conditions did not reveal a link between microbiome (diversity and composition) and symptoms of Fusarium head blight, nor a link between fungistasis and in planta symptoms on the same soil. Overall, this work contributes to evaluating the possibilities of using agricultural practices as a lever for agroecological control of Fusarium head blight in wheat, through the modulation of natural microbial communities

Les bactéries du sol produisant des antifongiques comme le 2,4-diacétylphloroglucinol(DAPG) protègent les racines des plantes vis-à-vis des champignons phytopathogènes. Néanmoins, les conditions de fonctionnement de ces populations bactériennes dans le sol restent très mal connues. Dans certains sols, dits résistants aux maladies, ces bactéries phytoprotectrices sont présentes à des effectifs importants et leur activité est suffisante pour protéger la plante malgré la présence du pathogène. L'objectif de cette thèse a été de comprendre la relation entre la résistance des sols à la maladie de la pourriture noire des racines de tabac, et la fonction de synthèse du DAPG chez les bactéries du genre Pseudomonas. Dans la situation de référence de Morens (Suisse), les sols résistants diffèrent des sols sensibles par la présence de vermiculite, argile capable de relarguer du fer. On sait que la présence de vermiculite améliore la phytoprotection assurée par les Pseudomonas producteurs de DAPG, mais les mécanismes moléculaires sous-jacents restent inconnus. Dans un premier temps, la quantification de ces bactéries par une nouvelle méthode de PCR quantitative développée ici, a confirmé que leurs effectifs sont élevés dans les sols résistants, mais aussi dans les sols sensibles, suggérant que la résistance puise plutôt dépendre d'une plus forte expression de la fonction de synthèse du DAPG. Dans un second temps, l'étude de l'expression des gènes de synthèse du DAPG en système de sol artificiel, à l'aide de la souche rapportrice P. protegens phlA-gfp, a montré que la présence de vermiculite dans le sol se traduit par une plus forte biodisponibilité du fer pour les Pseudomonas, induisant une plus forte expression des gènes de synthèse du DAPG et la protection du tabac. En conclusion, la résistance des sols de Morens à la maladie de la pourriture noire des racines est conditionnée par plusieurs facteurs abiotiques et biotiques, dont la biodisponibilité du fer qui régule l'expression des gènes de synthèse du DAPG chez Pseudomonas
Soil bacteria producing antimicrobial compounds like 2,4-diacetylphloroglucinol (DAPG) protect plants from soil-borne phytopathogens. Nevertheless, the functioning of these bacterial populations in the soil is largely unknown. In certain soils, termed disease- suppressive soils, these bacteria are present at high numbers and their activity is sufficient to assure effective plant protection in the presence of the pathogen. The aim of this thesis was to understand the relation between soil suppressiveness towards black root rot of tobacco, and the 2,4-diacetylphloroglucinol synthesis ability of certain Pseudomonas. In Morens region (Switzerland), suppressive soils differ from conducive soil by the presence of vermiculite, an iron-releasing clay. It is known that DAPG-producing Pseudomonas provide better plant protection in the presence of vermiculite, but the molecular basis of this interaction is still unknown. First, the quantification of these bacteria, through a new real-time PCR method developed here, confirmed that high numbers of DAPG-producing Pseudomonas occur in suppressive soils, as well as in conducive ones, raising the possibility that suppressiveness depends rather on a higher expression of DAPG synthetic genes. Second, expression studies of DAPG synthetic genes using a P. protegens ph/A- gfp reporter strain and artificial soil systems, confirmed that the presence of vermiculite in the soil can translate into higher iron bioavailability for Pseudomonas, triggering higher expression of DAPG synthetic genes and effective plant protection. In conclusion, black root rot suppressiveness of Morens soils is determined by several abiotic and biotic factors, among which iron bioavailability regulating the expression of DAPG synthetic genes in plant-protecting Pseudomonas

Root-knot nematodes (RKN), Meloidogyne spp., are the most economically important genus of plant parasitic nematodes that cause considerable damage and yield losses of horticultural crops worldwide. RKN management strategies tend to reduce chemical nematicides by encouraging alternative control methods like the use of plants bearing resistance genes (R-genes) and/or by microbe-inducing plant resistance, and the antagonistic potential of soils. In the thesis, two biological control approaches of Meloidogyne spp. were evaluated: 1) the application of selected nematode antagonists, the fungus Pochonia chlamydosporia and the bacteria Bacillus firmus I-1582 (Bf I-1582), to know its ability to induce plant resistance, and 2) the level of soil suppressiveness of vegetable production sites conducted under organic or integrated standards. Regarding the ability of P. chlamydosporia and Bf I-1582 to induce plant resistance, the results of this thesis provide evidence that two out of five P. chlamydosporia isolates (M10.43.21 and M10.55.6) and Bf I-1582 are able to induce systemic resistance against M. incognita in the susceptible tomato (Solanum lycopersicum) cv. Durinta but not in the cucumber (Cucumis sativus) cv. Dasher II in split-root experiments. In addition, the cardinal temperatures for the Bf I-1582 growth and biofilm formation were determined in order to improve its use in field conditions. Moreover, Bf I-1582 was transformed with GFP to study its effect on nematode eggs and on tomato and cucumber root colonization. In tomato, the number of egg masses and the number of eggs per plant were reduced by M10.43.21 and M10.55.6 P. chlamydosporia isolates and by Bf I-1582. P. chlamydosporia isolates colonized both tomato and cucumber roots, being the M10.43.21 and the M10.55.6 isolates the best root colonizers in tomato and cucumber, respectively. In the case of Bf I-1582, the bacteria colonized endophytically roots of both plants, but highest values were recorded in tomato. The dynamic regulation of genes related to jasmonic acid (JA) and salicylic acid (SA) was determined by RT-qPCR at three different times after nematode inoculation (dani). Bf I-1582 primed tomato plants by both SA and JA at all the times in tomato, but only SA at 7 dani in cucumber. Regarding P. chlamydosporia, isolate M10.43.21, induced the expression of the SA pathway in tomato at 0, 7 and 42 dani. The JA pathway was also up regulated at 7 dani. These results show the similar model of dynamic regulation of these plant hormone pathways related to plant defense mechanisms against the nematode. Also, Bf I-1582 grew and formed biofilm between 15 and 45 ºC, being 35 ºC the optimal temperature. Bf I-1582GFP was adhered to the egg shell and inside the eggs. Also, Bf I-1582GFP colonized root hairs and epidermal cells and some bacteria were found inside the tomato root. In cucumber, few bacteria were observed on epidermal cells and the bacteria were no found inside the root. In relation to the level of soil suppressiveness of vegetable production it was carried out a study in four organic and two integrated vegetables production standards sites located in north-eastern Spain. The fluctuation both of Meloidogyne population density and fungal egg parasitism were determined during the rotation sequences in two years (2015-2016). Five out of six of these sites were suppressive soils to Meloidogyne spp. The percentage of fungal egg parasitism ranged from 11.2 to 55 % and P. chlamydosporia was the only fungal species isolated from the eggs. In parallel, two tomato pots experiments were carried out using sterilized and non-sterilized soils from each site and inoculated with second-stage juveniles (J2) to achieve a rate of 1 J2 cm-3 of soil. In both, in five of them the number of nematode eggs per plant was reduced in all nonsterilized soils compared to the sterilizes ones. Also, P. chlamydosporia was the only fungal species isolated from parasitized nematode eggs.
Meloidogyne spp. (RKN) es el género de nematodos fitopatógenos que causan las mayores pérdidas económicas y que más afectan al rendimiento de cultivos hortícolas a nivel mundial. Las estrategias de manejo de RKN tienden a sustituir la utilización de nematicidas químicos por medidas de control alternativas como son el uso de plantas con genes de resistencia (genes R) y/o mediante la utilización de microorganismos inductores de resistencia, y el potencial antagónico del suelo. En esta tesis, se han evaluado dos enfoques de control biológico: 1) la aplicación de microorganismos antagónicos, el hongo Pochonia chlamydosporia (Pc) y la bacteria Bacillus firmus I-1582 (Bf I-1582) para evaluar su capacidad de inducir mecanismos de resistencia, y 2) el nivel de supresividad del suelo de diferentes lugares bajo estándares de producción orgánica e integrada. Con respecto a la capacidad de Pc y Bf I-1582 para inducir resistencia, los resultados de esta tesis muestran que dos (M10.43.21 y M10.55.6) de los cinco aislados de Pc utilizados y la bacteria Bf I-1582 inducen resistencia sistémica frente a M. incognita en el tomate susceptible (Solanum lycopersicum) cv. Durinta pero no en el pepino (Cucumis sativus) cv. Dasher II usando el modelo split-root. En el caso de Bf I-1582, se determinaron las temperaturas cardinales para el crecimiento y la formación de biofilms de Bf I-1582 con el fin de mejorar su utilización en condiciones de campo y se transformó con la GFP para estudiar su efecto sobre los huevos de RKN y sobre la colonización radicular. Los aislados M10.43.21 y M10.55.6 de Pc y Bf I-1582 redujeron el número de masas de huevos y el número de huevos por planta en tomate. Todos los aislados de Pc colonizaron raíces de tomate y pepino, siendo los aislados M10.43.21 y M10.55.6 los mejores colonizadores en tomate y pepino, respectivamente. En el caso de Bf I-1582, la bacteria colonizó endofíticamente las raíces de ambas plantas, pero los valores más altos se registraron en tomate. La expresión de los genes relacionados con el ácido jasmónico (JA) y el ácido salicílico (SA) se determinó a tres tiempos tras la inoculación de nematodos (dani). En plantas de tomate inoculadas con Bf I-1582 la expresión de los genes relacionados con SA y JA aumentaron en los tres puntos, pero en pepino solo se observó un incremento de expresión en el gen relacionado con la ruta de SA a los 7 dani. Con respecto a Pc, el aislado M10.43.21, indujo la expresión de la vía SA en tomate a los 0, 7 y 42 dani. La vía JA también aumentó su expresión a los 7 dani. Además, Bf I-1582 creció y formó biofilms entre 15 y 45 ºC, siendo 35 ºC la temperatura óptima. Bf I-1582GFP se adhirió a la cubierta y al interior de los huevo de M. incognita. Además, Bf I-1582GFP en tomate colonizó los pelos radiculares, así como las células epidérmicas y se encontraron algunas bacterias en el interior radicular. En el pepino, se observó un menor número de bacterias en las células epidérmicas y no se encontraron bacterias en el interior radicular. En relación con el nivel de supresión del suelo, se realizó un estudio en cuatro lugares de producción hortícola orgánica y dos de producción integrada en el noreste de España. Durante la secuencias de rotación en 2015-2016 se determinó la fluctuación tanto de la densidad de población de Meloidogyne en suelo como del parasitismo de huevos de nematodos. Cinco de estos sitios resultaron ser supresivos a Meloidogyne spp. Paralelamente, se llevaron a cabo dos experimentos en macetas con suelo esterilizado y no esterilizado de cada sitio donde las plantas de tomate se inocularon con juveniles (J2) para lograr una tasa de 1 J2 cm-3 de suelo. En cinco de ellos, el número de huevos de nematodos por planta se redujo en todos los suelos no esterilizados en comparación con los esterilizados. Respecto al parasitismo, Pc fue la única especie aislada de los huevos de Meloidogyne spp. p
Els nematodes formadors de gal·les, Meloidogyne spp., és el gènere més important nematodes fitoparàsits que causen danys considerables i generen pèrdues econòmiques en cultius hortícoles arreu del món. Les estratègies actuals de gestió de Meloidogyne solen reduir l’ús dels nematicides químics fomentant mètodes de control alternatius com l’ús de plantes amb gens de resistència (gens R) i/o l’ús de la resistència vegetal induïda per microorganismes, i el potencial antagonista dels sòls. En la present tesis, dos aproximacions al control biològic de Meloidogyne spp. van ser estudiades: 1) l’aplicació d’antagonistes dels nematodes: el fong Pochonia chlamydosporia i el bacteri Bacillus firmus aïllat I-1582 i es a avaluar la seva capacitat per induir resistència vegetal, i 2) el nivell de supressivitat de sòls de producció vegetal orgànica o integrada. Respecte a la capacitat de P. chlamydosporia i B. firmus I-1582 (Bf I-1582) a induir resistència vegetal, els resultats d’aquesta tesis van donar evidències que dos de cinc aïllats de P. chlamydosporia (M10.43.21 i M10.55.6) i Bf I-1582 induien resistència sistèmica enfront M. incognita en tomàquet susceptible (Solanum lycopersicum) cv. Durinta però no en cogombre (Cucumis sativus) cv. Dasher II en experiments “split-root”. A més, les temperatures cardinals de creixement i de formació de biofilm de Bf I-1582 van ser determinades per tal de millorar el seu ús en condicions de camp. A més, el bacteri va ser transformat amb GFP per estudiar el seu efecte sobre els ous del nematode i la seva colonització sobre les arrels de tomàquet i cogombre per microscopia de rastreig làser confocal. En tomàquet, tant el nombre de masses d'ou com el nombre d'ous per planta es va veure reduït quan s’aplicaven tant els aïllats fúngics com el bacteri. Els aïllats de P. chlamydosporia colonitzaven les arrels de tomàquet i cogombre, però diferien en el nivell de colonització. L’aïllat M10.43.21 va ser el millor colonitzador de les arrels de tomàquet mentre que l’aïllat M10.55.6 ho va ser per cogombre. En el cas de Bf I-1582, el bacteri va ser capaç de colonitzar endofíticament les arrels de les dues plantes, però es va trobar un 61% més de densitat d’ADN de bacteri en arrels de tomàquet. La regulació dinàmica dels gens relacionats amb l’àcid jasmònic (JA) i l’àcid salicílic (SA) a tres temps diferents van ser avaluats: 7 dies després de la inoculació de l’antagonista i just després de la inoculació del nematode (0 dani), 7 dies desprès de la inoculació del nematode (7 dani) i 40 dies desprès de la inoculació del nematode (40 dani). Les dues vies SA (gen PR-1) i JA (gen Lox D) van ser sobre-expressades plantes de tomàquet a 0 dani, reduint el nombre de masses d’ou al final de l’experiment “split-root” quan es va inocular amb Bf I-1582. No obstant, no hi va haver diferencies en l’expressió dels gens relacionats SA (PR 1) i JA (Lox D) en cogombre inoculat amb el bacteri com tampoc en el nombre de masses d’ou produïdes en les arrels de cogombre. A 7 dani, el gen relacionat amb el JA (Lox D) estava sobre-expressat en tomàquet i podria afectar el desenvolupament del nematode i la seva reproducció. En cogombre, la via del SA (Pal I) estava sobre-expressada tant en les plantes inoculades amb M. incognita com en les co-inoculades amb el bacteri i el nematode. A 40 dani, quan va començar l’eclosió dels ous i es van produir noves infeccions a l’arrel, les plantes de tomàquet co-inoculades amb els nematode 2 i Bf I-1582 tenia reprimit el gen relacionat amb el JA (Lox D), mentre que el gen relacionat amb la via del SA (PR 1) estava sobre-expressat en plantes co-inoculades i també amb només Bf I-1582, però va ser reprimit en plantes inoculades només amb el nematode. En cogombre, les dues vies, JA i SA, van ser reprimides en plantes inoculades amb M. incognita però només la JA en plantes co-inoculades. Respecte l’aïllat de P. chlamydosporia M10.43.21, va induir l’expressió de la via del SA en arrels de tomàquet a 0, 7 i 42 dani. La via del JA va ser també sobre-expressada a 7 dani. Per tant, alguns aïllats de P. chlamydosporia i l’aïllat Bf I-1582 poden induir resistència sistèmica envers al nematode, encara que depèn de l’espècie vegetal. Aquests resultats han demostrat el model similar de regulació dinàmica d’aquestes vies d’hormones vegetals relacionades amb mecanismes de defensa de les plantes contra el nematode. El bacteri Bf I-1582 va créixer en el rang de temperatures des de 15 ºC a 45 ºC, sent 35 ºC la temperatura òptima de creixement tant en medi sòlid com en líquid, però no a 10 ºC i 50 ºC. Igualment, es va observar la formació de biofilm entre 15 ºC i 45 ºC però tampoc a 10 ºC ni a 50 ºC, sent més gruixut i uniforme a 35 ºC. La degradació de la closca del nematode i la colonització dels ous per Bf I-1582 GFP va mostrar que a 3 dies desprès de la seva inoculació (dai) el bacteri estava envoltant i degradant l'ou del nematode; a 5 dai, colònies de bacteri es van adherir a la closca de l’ou i es van trobar alguns bacteris dins de l’ou; a 10 dai, el bacteri era completament adherit a la closca de l’ou i dins de l’ou. A més, Bf I-1582GFP va colonitzar les pèls radiculars i cèl·lules epidèrmiques a 5 dai; es van observar colònies de bacteris en pèls radiculars de tomàquet i alguns bacteris dins de l’arrel a 10 dai. En cogombre, es van observar pocs bacteris a les cèl·lules epidèrmiques a 5 dai i no es va trobar el bacteri dins de l’arrel a 10 dai. En relació al nivell de supressivitat del sòl, es va realitzar un estudi a sis parcel·les de producció d’hortalisses localitzades al nord-est d’Espanya. Quatre realitzaven producció orgànica (M10. 16, M10.41, M10.55, i M10.56) i dues (M10.43 i M10.45) producció integrada. La fluctuació de la densitat de població de Meloidogyne i el parasitisme d’ous per part de fongs van ser determinats durant la seqüència de rotació de cultius durant dos anys (2015-2016). Cinc dels sols estudiats eren sòls supressius a Meloidogyne spp. El percentatge de parasitisme d’ous va variar de 11.2 a 55 % i P. chlamydosporia va ser l'única espècie fúngica aïllada dels ous. En paral·lel, dos experiments es van dur a terme utilitzant sòl de cada parcel·la. Una part de cada sòl es va esterilitzar i es va barrejar amb sorra estèril, i una altre part no es va esterilitzar i es va barrejar també amb sorra estèril amb una relació 1:1 i es va col·locar en testos de 3-l. El cultivar susceptible de tomàquet Durinta es va trasplantar en cada test i es va inocular amb juvenils de segon estadi (J2) amb un nivell de 1 J2 cm-3 de sòl. En els dos experiments en testos, els nombre d’ous per planta es va reduir (P<0.05) en tots els sòls no esterilitzats comparats amb els estèrils, excepte en el M10.45. També, P. chlamydosporia va la única espècie fúngica aïllada d’ous parasitats de nematodes. P. chlamydosporia és el fong més freqüent i més prevalent amb una alta plasticitat capaç d’adaptar-se a les pràctiques agronòmiques en un sistema de producció vegetal molt pertorbat.

A number of growth factors and cytokines involved in the local regulation of bone remodelling are either synthesised by osteoblasts or have osteoblasts as their target. These include the RANK-L/OPG system, the gp130 cytokine family, including IL-6, and insulin like growth factors. In addition, aberrant cytokine signalling is strongly linked with pathological states characterised by increased bone resorption, including osteoporosis and renal osteodystrophy. The range of action and potency of these osteotropic cytokines requires that their actions are tightly regulated. Amongst such potential control mechanisms are the suppressors of cytokine signalling (SOCS), the presence and role of which in bone has not been studied in detail. The aim of this thesis was (i) to examine the direct effect of uraemia on cytokine release in human osteoblastic cells; (ii) to determine if the regulatory SOCS genes are expressed in these cells and, if so, (iii) to characterise their functional significance. In initial studies, osteoblastic cells were cultured in media containing sera from either healthy volunteers or haemodialysis treated chronic kidney disease patients. Concentrations of OPG and IL-6 were then measured in harvested supernatants. Additionally, individual serum samples collected prior to, and during, a haemodialysis (HD) session were assayed for IL-6, IL-1β and soluble IL-6 receptor (sIL-6R). HD patients had significantly higher concentrations of IL-6 than normal subjects, but there were no significant differences in either IL-1β or sIL-6R. These concentrations did not change significantly during HD. There were no differences in OPG production by osteoblastic cells after exposure to either normal or uraemic serum. Incubation with untreated sera from normal subjects increased IL-6 production by ~6-fold above control, whereas sera from uraemic subjects increased it only ~2-3-fold. HD did not restore the capacity of uraemic serum to augment IL-6 release to the same degree as normal serum. Further work examined a variety of osteotropic stimuli for their ability to induce SOCS1- 3 and CIS expression in human osteoblastic cells. The utility of both conventional RTPCR and fluorescence-based kinetic real time PCR for this purpose are compared. These SOCS were found to be expressed constitutively and could be induced to a variable degree by relevant growth factors. In general, the temporal pattern of SOCS expression was consistent with a negative feedback function. Potential functionality was explored following transfection with SOCS1 and SOCS3 plasmid DNA. Significantly enhanced IL-6 secretion was found in both the basal and stimulated state, whilst OPG production was enhanced only in the latter. Function was also studied in the context of osteoblastic apoptosis, the regulation of which is highly relevant to skeletal disease. Initial experiments developed a framework for subsequent studies: serum starvation for 24h produced reproducible cell death that could be attenuated in a dose dependent manner by IGF-I. SOCS1 and SOCS3 overexpression had limited influence on osteoblast survival, whereas gene knock down experiments using siRNA indicated that IL-1β-induced cell death is mediated differentially, depending on the type of cell death involved. SOCS1 and SOCS3 are involved in the apoptotic cascade, while IL-1β-induced necrosis appears to be independent of SOCS3. Collectively these studies demonstrate that the augmentation of IL-6 production by osteoblastic cells after exposure to normal serum is greater than after uraemic serum. HD does not correct this disparity; perhaps indicating a non-dialysable inhibitor of IL-6 release is involved in the dysregulated bone turnover of uraemic patients. Further work establishes the constitutive presence of the SOCS family in human osteoblastic cells, as well as their transient inducibility by key osteotropic stimuli. Several novel aspects of SOCS function, including influence on IL-6 and OPG production and involvement within apoptotic pathways are demonstrated.

Orientador: Gustavo Luiz Chagas Manhães de Abreu
Resumo: O presente trabalho propõe uma nova estratégia para supressão ativa robusta do fenômeno Ground Resonance (GR) em Helicópteros. O modelo clássico de análise deste fenômeno é desenvolvido para um rotor isotrópico e a análise de estabilidade é feita no domínio de Coleman, para encontrar as fronteiras de instabilidade. Também é proposta uma nova estratégia para lidar com essas fronteiras de instabilidade e suprimir o GR usando controladores com formulação descrita por conjuntos politópicos convexos. Controladores são projetados via desigualdades lineares matriciais (LMIs, Linear Matrix Inequalities), formulados de acordo com a Teoria de Estabilidade de Lyapunov. Adicionalmente, incertezas paramétricas na frequência de lead-lag das pás e a apresentação de uma falha estrutural nos atuadores são consideradas e, assim, novos controladores robustos são projetados a fim de expandir o envelope operacional da aeronave. Ainda, são considerados diferentes tipos de não-linearidades estruturais na rigidez e amortecimento do trem de pouso do helicóptero e a caracterização da estabilidade não-linear do sistema exibe oscilações em ciclo limite (LCO, Limit Cycle Oscillation), que são determinadas a partir da construção de Diagramas de Bifurcação. Utiliza-se a modelagem Fuzzy-TS do sistema para cada caso de estudo e, com base nas fronteiras de estabilidade não-linear do GR, definidas a partir dos Diagramas de Bifurcação, têm-se o projeto de controladores para supressão das LCOs do sistema. Os res... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The present work proposes a new strategy for robust active suppression of Ground Resonance (GR) phenomenon in Helicopters. The classical model to analysis of this phenomenon is developed for an isotropic rotor and stability analysis is done in Coleman domain, to nd the boundaries of instability. It is also proposed a new strategy for dealing with these boundaries of instability and suppressing GR using controllers with polytopic convex hulls formulation. Controllers are designed via Linear Matrix Inequalities (LMIs), formulated according to the Lyapunov Stability Theory. Additionally, parametric uncertainties in the lead-lag frequency of the blades and actuators faults are considered and thus new robust controllers are designed to expand the aircraft operating envelope. Also, di erent types of structural nonlinearities in the landing gear sti ness and damping of the helicopter are considered, and the characterization of the nonlinear stability of the system exhibits Limit Cycle Oscillation (LCO), which are determined from the construction of Bifurcation Diagrams. Fuzzy-TS modeling is used for each case study and, based on the nonlinear stability boundaries of the GR, de ned from the Bifurcation Diagrams, the controllers to suppress the LCO are designed. The results of numerical simulations, discussions and conclusions are presented and show that the control strategy proposed is an attractive solution to suppress the linear and nonlinear GR problem, being able to expand the o... (Complete abstract click electronic access below)
Doutor

La thèse est dédiée au problème de la stéréophotométrie non-Lambertienne sans connaissance a priori sur les conditions d'illumination et son application aux images de boîte de Pétri. Pour obtenir une bonne reconstruction de surfaces non-Lambertiennes, il est proposé de traiter une séquence d'entrée en deux étapes: premièrement il faut supprimer les effets spéculaires et obtenir ainsi des images de surface 'pseudo-Lambertienne'. Ensuite dans une deuxième étape à partir de ces images une reconstruction stéréophotométrique Lambertienne sans aucune information préalable sur les directions d'illumination est effectuée. Dans ce travail nous proposons deux méthodes originales respectivement pour la suppression de spécularités et la reconstruction de surface sans information a priori. Les méthodes proposées sont appliquées pour la caractérisation des colonies microbiennes.La spécularités est un effet optique lié à la nature physique complexe des objets. Il est utile pour la perception humaine des objets 3D mais il gêne le processus de traitement automatique d'images. Pour pouvoir appliquer le modèle Lambertien à la stéréophotométrie, les spécularités doivent être supprimées des images d'entrée. Nous proposons donc une méthode originale pour la correction des zones spéculaires adaptée pour une reconstruction ultérieure. L'algorithme proposé est capable de détecter les spécularités comme des valeurs anormalement élevées d'intensité dans une image de la séquence d'entrée, et de les corriger en utilisant les informations des autres images de la séquence et une fonction de correction continue. Cette méthode permet de faire la suppression des spécularités en préservant toutes les autres particularités de distribution de lumière qui sont importantes pour la reconstruction de surface.Après nous proposons une technique de reconstruction stéréophotométrique de surface Lambertienne sans connaissance a priori sur l'illumination. Le modèle mis en œuvre consiste en quatre composantes, deux composantes (albédo et normales) permettent de d'écrire des propriétés de surface et deux autres (intensités des sources de lumière et leurs directions) décrivent illumination. L'algorithme proposé de reconstruction utilise le principe de l'optimisation alternée. Chaque composante du modèle est trouvée itérativement en fixant toutes les variables sauf une et en appliquant des contraintes de structures, valeurs et qualité pour la fonction d'optimisation. Un schéma original de résolution permet de séparer les différents types d'information inclus dans les images d'entrée. Grâce à cette factorisation de matrices, la reconstruction de surface est faite sans connaissance préalable sur les directions de lumière et les propriétés de l'objet reconstruit. L'applicabilité de l'algorithme est prouvée pour des donnés artificielles et des images de bases publiques pour lesquelles la vérité terrain sur les surfaces des objets est disponible.La dernière partie de la thèse est dédiée à l'application de la chaine complète proposée pour le traitement d'images de boîte de Pétri. Ces images sont obtenues en utilisant les sources de lumières complexes qui sont supposées être inconnues pour le processus de reconstruction. L'évaluation de surfaces de colonies microbiennes s'est révélée être une étape importante pour l'analyse visuelle et automatique des colonies. La chaine proposée est efficace pour ce type de données et permet de compléter les informations d'images par de la surface 3D.

Take-all, caused by the soilborne fungus, Gaeumannomyces graminis var. tritici (Ggt), is an important root disease of wheat that can be reduced by take-all decline (TAD) in successive wheat crops, due to general and/or specific suppression. A study of 112 New Zealand wheat soils in 2003 had shown that Ggt DNA concentrations (analysed using real-time PCR) increased with successive years of wheat crops (1-3 y) and generally reflected take-all severity in subsequent crops. However, some wheat soils with high Ggt DNA concentrations had low take-all, suggesting presence of TAD. This study investigated 26 such soils for presence of TAD and possible suppressive mechanisms, and characterised the microorganisms from wheat roots and rhizosphere using polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). A preliminary pot trial of 29 soils (including three from ryegrass fields) amended with 12.5% w/w Ggt inoculum, screened their suppressiveness against take-all in a growth chamber. Results indicated that the inoculum level was too high to detect the differences between soils and that the environmental conditions used were unsuitable. Comparison between the Ggt DNA concentrations of the same soils collected in 2003 and in 2004 (collected for the pot trial), showed that most soils cropped with 2, 3 and 4 y of successive wheat had reduced Ggt DNA concentrations (by 195-2911 pg g-1 soil), and their disease incidences revealed 11 of the 29 test soils with potential take-all suppressiveness. Further pot trials improved the protocols, such that they were able to differentiate the magnitudes of suppressiveness among the soils. The first of the subsequent trials, using 4% w/w Ggt inoculum level, controlled conditions at 16°C, 80% RH with alternate 12 h light/dark conditions, and watering the plants twice weekly to field capacity (FC), screened 13 soils for their suppressiveness against take-all. The 13 soils consisted of 11 from the preliminary trial, one wheat soil that had been cropped with 9 y of wheat (considered likely to be suppressive), and a conducive ryegrass soil. The results revealed that 10 of these soils were suppressive to take-all. However, in only four of them were the effects related to high levels of microbial/biological involvement in the suppression, which were assessed in an experiment that first sterilised the soils. In a repeat trial using five of the soils H1, H3, M2, P7 (previously cropped with 3, 3, 4 and 9 y successive wheat, respectively) and H15 (previously cropped with 5 y of ryegrass), three of them (H1, H3 and M2) had reduced Ggt DNA concentrations (>1000 pg g-1 soil reductions), and were confirmed to be suppressive to take-all. A pot trial, in which 1% of each soil was transferred into a γ-irradiated base soil amended with 0.1% Ggt inoculum, indicated that soils H1 and H3 (3 y wheat) were specific in their suppressiveness, and M2 (4 y wheat) was general in its suppressiveness. The microbial communities within the rhizosphere and roots of plants grown in the soils, which demonstrated conduciveness, specific or general suppressiveness to take-all, were characterised using PCR-DGGE, and identities of the distinguishing microorganisms (which differentiated the soils) identified by sequence analysis. Results showed similar clusters of microorganisms associated with conducive and suppressive soils, both for specific and general suppression. Further excision, re-amplification, cloning and sequencing of the distinguishing bands showed that some actinomycetes (Streptomyces bingchengensis, Terrabacter sp. and Nocardioides sp.), ascomycetes (Fusarium lateritium and Microdochium bolleyi) and an unidentified fungus, were associated with the suppressive soils (specific and general). Others, such as the proteobacteria (Pseudomonas putida and P. fluorescens), an actinomycete (Nocardioides oleivorans), ascomycete (Gibberella zeae), and basidiomycete (Penicillium allii), were unique in the specific suppressiveness. This indicated commonality of some microorganisms in the take-all suppressive soils, with a selected distinguishing group responsible for specific suppressiveness. General suppressiveness was considered to be due to no specific microorganisms, as seen in soil M2. An attempt to induce TAD by growing successive wheat crops in pots of Ggt-infested soils was unsuccessful with no TAD effects shown, possibly due to variable Ggt DNA concentrations in the soils and addition of nutrients during the experiment. Increasing numbers of Pseudomonas fluorescens CFU in the rhizosphere of plants, during successive wheat crops was independent of the Ggt DNA concentrations and disease incidence, suggesting that increases in P. fluorescens numbers were associated with wheat monoculture. This study has demonstrated that TAD in New Zealand was due to both specific and general suppressiveness, and has identified the distinguishing microorganisms associated with the suppression. Since most of these distinguishing microorganisms are known to show antagonistic activities against Ggt or other soilborne pathogens, they are likely to act as antagonists of Ggt in the field. Future work should focus on validating their effects either individually, or interactively, on Ggt in plate and pot assays and under field conditions.

This study focuses on the soil- and water-borne plant pathogen Phytophthora cinnamomi Rands and the phenomenon of P. cinnamomi suppressive soil. In particular, this thesis reports on the outcome of field surveys and glasshouse assays undertaken to locate P. cinnamomi suppressive soils and to confirm the involvement of biological processes in suppression. The potential role of cellulase and laminarinase in suppression was investigated and a molecular technique known as length heterogeneity PCR (LH-PCR) was used to analyse the structure and diversity of bacterial and fungal communities in avocado orchard soils that were suppressive and conducive to P. cinnamomi. Four avocado orchards with P. cinnamomi suppressive soils were identified and soils were ã-irradiated to destroy their suppressive capacity, thus confirming biological suppression. Suppression was also partially transferred to ã-irradiated and conducive soils by mixing with 10% suppressive avocado soils. Cellulase and laminarinase activities measured in avocado orchard soils inoculated with P. cinnamomi were not associated with disease severity in lupin seedlings during glasshouse assays involving the same soil samples. Minor shifts in bacterial and fungal community structure were observed in response to mixing conducive and irradiated soils with suppressive soils. This was associated with decreased disease severity in avocado seedlings in these treatments. The shift in bacterial community structure was partially determined by the appearance and increased abundance of several bacterial 16S rDNA sequences, which were unique to the suppressive soils, in the mixed soil treatments. It is suggested that the bacteria and fungi from which these sequences originated may be involved in suppression and further work should be undertaken to determine their identity and confirm their potential role in the development and maintenance of P. cinnamomi suppressive soils.
Doctor of Philosophy (PhD)

This study focuses on the soil- and water-borne plant pathogen Phytophthora cinnamomi Rands and the phenomenon of P. cinnamomi suppressive soil. In particular, this thesis reports on the outcome of field surveys and glasshouse assays undertaken to locate P. cinnamomi suppressive soils and to confirm the involvement of biological processes in suppression. The potential role of cellulase and laminarinase in suppression was investigated and a molecular technique known as length heterogeneity PCR (LH-PCR) was used to analyse the structure and diversity of bacterial and fungal communities in avocado orchard soils that were suppressive and conducive to P. cinnamomi. Four avocado orchards with P. cinnamomi suppressive soils were identified and soils were ã-irradiated to destroy their suppressive capacity, thus confirming biological suppression. Suppression was also partially transferred to ã-irradiated and conducive soils by mixing with 10% suppressive avocado soils. Cellulase and laminarinase activities measured in avocado orchard soils inoculated with P. cinnamomi were not associated with disease severity in lupin seedlings during glasshouse assays involving the same soil samples. Minor shifts in bacterial and fungal community structure were observed in response to mixing conducive and irradiated soils with suppressive soils. This was associated with decreased disease severity in avocado seedlings in these treatments. The shift in bacterial community structure was partially determined by the appearance and increased abundance of several bacterial 16S rDNA sequences, which were unique to the suppressive soils, in the mixed soil treatments. It is suggested that the bacteria and fungi from which these sequences originated may be involved in suppression and further work should be undertaken to determine their identity and confirm their potential role in the development and maintenance of P. cinnamomi suppressive soils.

碩士
國立東華大學
電機工程學系
107
Due to the development trend of IC manufacturing technology, the scaling of wafer process is reduced. In addition, the static power caused by the leakage problem is getting more and more serious. Reducing the leakage current in the standby mode is a way to reduce power consumption, and reducing leakage current by manual detection and adjustment is not in line with efficiency. Therefore, this thesis proposed an environmental detector that can automatically detect the PVT change and adjust voltage with maintenance circuit to keep the circuit in the state of minimum power consumption. This proposed environmental detector can meet the trend of the bootstrapped voltage maintenance circuit. It implemented with TSMC 90 nm CMOS Mixed Signal MS General Purpose Standard Process Low K Cu 1P9M 1.0&3.3V (With UTM) process design, operating voltage is 0.5V. The wafer layout area is 449.52 × 350.74f um2. The environment detector is applied to a dynamic standby controller with the bootstrap voltage maintenance circuit. Accordingly, the test chip shows functions of automatic environmental parameter detection and the lowest power adjustment, and then suppress effectively leakage current generated by the transistor.

Rhizoctonia solani AG8 is a significant soil-borne pathogen of cereal roots in semi-arid Mediterranean regions of Australia and the Pacific North West region in the United States of America, causing severe productivity and economic losses to farmers. During the past twenty years the conversion of many farming systems to conservation tillage has meant that the mycelial network of the pathogen is no longer seasonally disturbed by cultivation which has subsequently increased the potential for greater incidence of Rhizoctonia root rot. There has been some success in reducing incidence by using modifications to direct drill seeding equipment enabling some disturbance to Rhizoctonia at sowing. However, a long term sustainable solution with both economic and environmental benefit, as concluded from a review of the literature (Chapter 1) is to harness the potential for biological control of the disease via natural or induced suppression in soils. Biological suppression to specific disease organisms in soil has been reported worldwide from a range of environments. Further, the development of biological soil-borne suppression to Rhizoctonia root rot has been described for one specific agricultural location (Avon) in South Australia following a decade of stubble retention together with higher than average nutrient inputs (Chapter 1). The studies in this thesis investigate soilborne suppression to Rhizoctonia in agricultural soils from a semi-arid region of South Australia called Eyre Peninsula (EP) that produces 40% of the State’s grain. The context is that historically in Eyre Peninsula farming systems crop residue inputs to soil are inherently low, as are fertiliser N and P inputs. However, recent intensification of these systems with the implementation of continuous cereals and minimum or zero tillage has resulted in greater inputs of stubbles and fertilisers. Rhizoctonia root disease is prevalent in the mainly coarse textured alkaline soils of the region, and the reduction in cultural control associated with adoption of reduced till systems has highlighted a need for alternative control measures. In a broad context, the key question addressed in this thesis is whether the soil ecology to suppress Rhizoctonia is present or can develop in these soils from a region considered an extremely harsh environment climatically as well as edaphically. Specific key questions will be addressed in the discussion section of each chapter. The thesis, through a series of controlled environment studies, examines abiotic-biotic interactions between the soil, the Rhizoctonia solani pathogen and wheat seedlings. The work assesses how the soil organisms involved in disease suppression (both the pathogen Rhizoctonia solani AG8 organism and other antagonists or competitors) are influenced by cereal stubbles and fertilizer inputs to the system. Through a series of preliminary experiments (Chapter 3) the important variables of soil moisture and amount of pathogen inoculum (e.g. number of pathogen infected agar plugs) suitable for a bioassay method were standardised, and used throughout the rest of the work described in this thesis. Two controlled environment bioassay experiments (Chapter 4) were undertaken surveying soils from six sites across the region differing in physico-chemical and biological properties to elucidate the influence of abiotic and biotic factors on plant-soil-pathogen interactions and the potential for suppression of Rhizoctonia. A comparison was made with soil from the long term study site in SA (Avon) reported to be suppressive to Rhizoctonia. Studies growing wheat seedlings in sterilised soils demonstrated that the soils assessed were intrinsically different in terms of the growth supported by the abiotic matrix. Greatest shoot and root dry weight was observed in the soil from a region outside the EP (i.e. Avon) and the least was in an EP soil with extremely high calcium carbonate content (e.g. Streaky Bay) – a clear example of plant-soil abiotic interaction. Avon soil was confirmed as suppressive to Rhizoctonia root rot since the Avon soil inoculated with its own biotic component reduced root infection to 50% from more than 70% in the sterilised abiotic control. Whereas, for plants in the two EP soils with low calcium carbonate root infection was similar in the sterilised abiotic matrix to that in the soils inoculated with their biotic component, suggesting they were not biologically suppressive. Further evidence of the suppressive capability of the biotic component of Avon soil was obtained where it was inoculated into the two EP soils with higher carbonate and reduced root infection in plants grown in these two soils, although not in the lower carbonate content abiotic matrix of Minnipa, another EP soil. Surprisingly, considering the hostile edaphic conditions, root infection was reduced in the high calcium carbonate soil inoculated with its own biotic component, suggesting it was suppressive but not to the same extent as Avon. It was hypothesised this was possibly related to the organic C content in that soil being similar to Avon and higher than the other two EP soils. Shifts in soil organism community structure were observed when plants were grown in sterilised soils inoculated with the biotic component from another soil (i.e. rhizosphere soil from plants grown in another non-sterile matrix). Overall this work suggested there was some biotic potential for suppression in EP soils but low organic C was likely to be a constraint. EP soils were not as suppressive as Avon and abiotic constraints were highly likely, for example, the high carbonate reducing availability of P due to chemical fixation. A long term glasshouse study (Chapter 5) was undertaken to measure the effect of carbon addition to two EP soils, as stubble or young root residues, on the potential to suppress Rhizoctonia. Other measurements in this experiment were microbial biomass carbon and quantitative PCR for DNA of pathogen and other specific micro-organisms implicated as contributing to disease suppression. C input to EP soils suppressed Rhizoctonia infection in wheat seedlings (despite abiotic constraints). C input as young roots increased DNA of Rhizoctonia solani and beneficial soil organisms Microbacterium spp. and Pantoea agglomerans. C input as stubble increased the populations of the beneficial soil organism, Trichoderma spp. A further bioassay experiment (Chapter 6) investigated the effect of N and P fertiliser inputs on plant growth and Rhizoctonia suppression in two EP soils. The bioassay further investigated the interaction of these fertiliser nutrients with added available C in these two EP soils, one of which was highly calcareous. There was a positive plant growth response to added ammonium–N in both soils but no effect on Rhizoctonia infection. Addition of fertilizer P to the highly calcareous soil increased shoot and root growth and also Rhizoctonia infection without compromising effects on plant growth. Addition of available C (sucrose) with P fertiliser in the highly calcareous soil markedly suppressed Rhizoctonia infection. Two final experiments focussed on measuring the changes in pathogen and other microbial communities in response to inputs of fertiliser and C in a highly calcareous EP soil, since Rhizoctonia root rot impacts are considered a particularly big issue in this soil type. In the first experiment (Chapter 7) it was hypothesised that fertiliser P may affect suppression of Rhizoctonia root rot not only via increasing plant growth but also by altering microbial community composition. Results showed that virulence of Rhizoctonia solani was unaltered by P addition although pathogen DNA in soil and plant root infection increased. The effect of P fertiliser on plant growth compensated for the effect of P on increased pathogen population and root infection. Whilst fertiliser P increased microbial activity no shifts were detected in communities so the effects of P on soil organisms involved in suppression of Rhizoctonia root rot were not conclusive. However, in the last experiment (Chapter 8) there were measured shifts in populations of organisms resulting from addition of fertiliser P in conjunction with stubble. The known suppressive soil organisms Pantoea agglomerans and Microbacterium spp. increased whereas Rhizoctonia solani (DNA) remained constant and hence Rhizoctonia infection decreased. In summary, some soils from the EP region of South Australia expressed a degree of suppression to Rhizoctonia root rot via their biotic component in pot culture experiments. Furthermore, some of the soils, although not necessarily the same ones, contained soil micro-organisms implicated by other studies in suppression of Rhizoctonia root rot. The biotic component from some of the EP soils, whilst not suppressive in the soil matrix it was extracted from did demonstrate the potential to suppress Rhizoctonia root rot when transferred into another soil matrix, indicating an abiotic constraint to suppression. It is postulated that important abiotic properties in these EP soils were calcium carbonate content, with organic carbon and to a lesser extent mineral N and P also important since these latter properties bridge the abiotic to biotic divide. Important biotic properties are likely to be microbial activity, microbial community structure and the population of the pathogen, Rhizoctonia solani AG8. Results from this thesis work suggest that suppression to Rhizoctonia root rot can occur in EP soils despite abiotic and biotic constraints of limited C and P. Improvement and maintenance of a high suppressive capacity in soils in this semi-arid environment will require integrated agronomy aimed at maintaining a healthy crop using fertilisers, particularly P. Available carbon appears to be the most limiting constraint to microbe based biological disease suppression of Rhizoctonia root rot in these soils. Therefore it is essential that adequate available C is supplied via stubble input to develop and maintain a highly functioning soil biota. Although these results highlight that disease suppression to Rhizoctonia root rot is indeed possible in the constrained soils of the EP, the time required to develop this suppressive capacity in a field situation remains to be investigated.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2013

AIDS-related human cytomegalovirus (HCMV) retinitis remains the leading cause of blindness among untreated HIV/AIDS patients worldwide. Understanding the pathogenesis of this disease is essential for developing new, safe, and effective treatments for its prevention or management, yet much remains unknown about the virologic and immunologic mechanisms contributing to its pathology. To study such mechanisms, we use a well-established, reproducible, and clinically relevant animal model with retrovirus-induced murine acquired immunodeficiency syndrome (MAIDS) that mimics in mice the symptoms and progression of AIDS in humans. Over 8 to 12 weeks, MAIDS mice become susceptible to experimental murine cytomegalovirus (MCMV) retinitis. We have found in this model that MCMV infection significantly stimulates ocular suppressor of cytokine signaling (SOCS)1 and SOCS3, host proteins which dampen immune-related signaling by cytokines, including antiviral interferons. Herein we investigated virologic and/or immunologic mechanisms involved in this stimulation and how virally-modulated SOCS1 and/or SOCS3 proteins may contribute to MCMV infection or experimental MAIDS-related MCMV retinitis. Through pursuit of two specific aims, we tested the central hypothesis that MCMV stimulates and employs SOCS1 and/or SOCS3 to induce the onset and development of MCMV retinal disease. MCMV-related SOCS1 and SOCS3 stimulation in vivo occurred with intraocular infection, was dependent on method and stage of immune suppression and severity of ocular pathology, was associated with stimulation of SOCS-inducing cytokines, and SOCS1 and SOCS3 were differentially sensitive to antiviral treatment. In vitro studies further demonstrated that SOCS1 and SOCS3 stimulation during MCMV infection occurred with expected immediate early kinetics, required viral gene expression in cell-type-dependent and virus origin-dependent patterns of expression, and displayed differential sensitivity to antiviral treatment. These data suggest that SOCS1 and SOCS3 are stimulated by divergent virologic, immunologic, and/or pathologic mechanisms during MCMV infection, and that they contribute to the pathogenesis of retinal disease, revealing new insights into the pathophysiology of AIDS-related HCMV retinitis.

H παρούσα διατριβή εξετάζει το πρόβλημα της διαρροής μικροφώνου, δηλαδή την αλληλεπίδραση και παρεμβολή μεταξύ ταυτόχρονα ενεργών ηχητικών πηγών σε πολυκαναλικές ηχητικές διατάξεις. Παρ' όλο που είναι ένα πολύ συχνό φαινόμενο με το οποίο οι μηχανικοί ήχου έρχονται αντιμέτωποι καθημερινά, δεν έχουν προταθεί μέθοδοι επεξεργασίας σήματος για την επίλυση του προβλήματος. Εδώ, το πρόβλημα διατυπώνεται για πρώτη φορά στο πλαίσιο της επεξεργασίας σήματος. Αρχικά, διατυπώνεται στο πλαίσιο του τυφλού διαχωρισμού πηγών (blind source separation) και αναλύονται οι περιορισμοί αυτής της προσέγγισης. Στην συνέχεια, το πρόβλημα επαναδιατυπώνεται σαν πρόβλημα σήματος υπό θόρυβο στα πλαίσια της καταστολής θορύβου. Ένα πρωτότυπο γενικευμένο πλαίσιο καταστολής διαρροής μικροφώνου εξάγεται βασιζόμενο σε ένα φίλτρο Wiener με πολυκαναλικό όρο θορύβο, καθώς και την ευρέως χρησιμοποιούμενη τεχνική «κοντινού μικροφώνου». Το ακουστικό σύστημα που μοντελοποιεί την διαδικασία μίξης και αλληλεπίδρασης των πηγών αναλύεται και γίνεται διαχωρισμός των σχετικών κρουστικών αποκρίσεων χώρου (room impulse responses) σε απ' ευθείας ακουστικά μονοπάτια και ακουστικά μονοπάτια διαρροής. Οι ιδιότητες του απ' ευθείας ακουστικού μονο- πατιού, δηλαδή της απόκρισης «κοντινού μικροφώνου» αναλύονται για πρώτη φορά από την προσέγγιση της επεξεργασίας σήματος και της ακουστικής κλειστών χώρων για πρώτη φορά. Οι ιδιότητες του ακουστικού μονοπατιού διαρροής αναλύονται επίσης για πρώτη φορά με την χρήση ακουστικών παραμέτρων. Έχοντας καθορίσει τις βασικές ιδιότητες του ακουστικού συστήματος, μια μέθοδος για την καταστολή διαρροής μικροφώνου αναπτύσσεται για μια διάταξη δύο καναλιών, βασισμένη σε ένα φίλτρο Wiener και μια άμεση εκτίμηση των σχετικών πυκνοτήτων φασματικής ενέργεiας (power spectral density). Η απόδοση της μεθόδου για ηχογραφήσεις σε πραγματικούς χώρους είναι πολύ ικανοποιητική και με βάση αυτά τα αποτελέσματα, η μέθοδος επεκτείνεται για περισσότερες από δύο πηγές και μικρόφωνα σε αυθαίρετες διατάξεις. Η ολοκληρωμένη μέθοδος είναι τυφλή και αυτόματη, καθώς δεν απαιτεί την επέμβαση του χρήση. Δεν κάνει χρήση πρότερης γνώσης ούτε απαιτεί εκπαίδευση και είναι υπολογιστικά απλή. Προτείνεται επίσης μια πρωτότυπη μέθοδος ανίχνευσης χρονικών διαστημάτων όπου μόνο μια πηγή είναι ενεργή (χρονικά διαστήματα «σόλο»), η οποία επιτρέπει την εκτίμηση συντελεστών στάθμισης οι οποίοι αντιστοιχούν στην σχετική μείωση της ηχητικής στάθμης που υφίσταται κάθε ηχητική πηγή καθώς το σήμα διαδίδεται προς τα μικρόφωνα. Αυτή η μέθοδος σε συνδυασμό με μια νεά, πρωτότυπη τεχνική εκτίμησης των πυκνοτήτων φασματικής ενέργεαις, η οποία βασίζεται στην αναγνώριση των κυρίαρχων διακριτών συχνοτήτων, επιτρέπει την εκτίμηση όλων των σχετικών ποσοτήτων σε μια πολυκαναλική ηχητική διάταξη. Από αυτές υπολογίζεται ένα πολυκαναλικό φίλτρο Wiener για κάθε σήμα μικροφώνου, το οποίο δίνει την εκτίμηση του αντίστοιχου σήματος πηγής.
This thesis examines the problem of microphone leakage, that is the interference between simultaneously active sound sources in multichannel audio applications. Despite being a common problem with which sound engineers are confronted every day, almost no signal processing methods have been proposed to address this issue. In this work, the problem is formulated for the first time in a signal processing framework. First, it formulated inside the blind source separation (BSS) context and the limitations of related methods are analysed and reported. Since, BSS methods seem to be inappropriate for this specific problem, it is reformulated as a signal in noise problem inside the well-known noise suppression framework. Based on the widely adopted close-microphone technique a novel, generalized framework for leakage suppression is derived based on a multichannel Wiener filter. The acoustic system that models the mixing process is analysed and the related room impulse responses are discerned in direct and leakage acoustic paths. The properties of the direct acoustic path, that is the close-microphone response are investigated for the first time, from a signal processing point of view as well as a room acoustics perspective. The properties of the leakage acoustic path are also analysed for the first time using room acoustic parameters. After key properties of the acoustic paths have been identified, a method for the suppression of microphone leakage in a two channel audio setup is developed based on aWiener filter and a crude approximation of the related power spectral densities (PSDs). The performance of this method for actual recordings in real reverberant environments is more than adequate and based on these results, the method is extended for more than two sources and microphones in arbitrary arrangements. The complete method is blind and automatic, since it does not require any user input. It does not assume any prior knowledge or require training and is computationally efficient. A novel solo detection method has been developed that allows the estimation of weighting coefficients that correspond to the relative attenuation experienced by sound sources as they travel to each microphone. Combined with a new and advanced PSD estimation method based on the identification of dominant frequency bins, the related PSDs in a multichannel audio application can be identified. From these an appropriate multichannel Wiener filter for each microphone signal can be calculated, which will provide the estimated source signal at its output.