The broad aim of the work described in this thesis was to use the arbuscular mycorrhizal (AM) defective rmc tomato to explore the development and function of different types of fungus-plant interfaces (phenotypes) and to characterize the cellular modifications preceding colonization of rmc by a range of different AM fungi. Three main patterns of colonization with rmc have been described: 1) Pen- phenotype in which the AM fungus is restricted to the root surface with several attempts to penetrate the epidermal cells without success; 2) Coiphenotype where AM fungi penetrate the epidermis but cannot develop cortical colonization; and 3) Myc+ phenotype (with G. intraradices WFVAM23), where the AM fungus penetrates the cortex and forms a “normal” colonization after a delayed penetration of the epidermal cells (Review of literature). Little is known about cellular interactions, nutrient transfer or the ability of the fungi to complete their life cycles in the different phenotypes. These aspects were the main foci of this work. In addition further fungal isolates were screened to asses their ability to colonize rmc. The first experiments involved compartmented pots to follow the fungal life cycle, production of external mycelium and spores in the different rmc phenotypes (Chapter 3). The results showed that in the Pen- and Coiphenotypes, AM fungi are unable to form spores to complete the life cycle. However, in the Coi-phenotype, the fungus remained alive up to week 18, suggesting that some C transfer occurred. The fungus forming the Myc+ phenotype, G. intraradices WFVAM23, was able to produce spores, although they were significantly smaller than those produced with the wild-type tomato. The results suggested that arbuscules are essential for completion of the fungal life cycle. Labeled 32P was used to determine whether arbuscules are also essential for P transfer (Chapter 4). A compartmented pot system was used in which only fungal hyphae but not roots could obtain 32P. 32P was found in the shoots of rmc inoculated with S. calospora (Coi- phenotype), indicating that interfaces other than arbuscules can be involved in transfer of P. A nurse pot system was used to obtain synchronized colonization to determine how long AM fungi stay alive during the interactions with rmc and to elucidate the cellular modifications preceding colonization of rmc by a range of different AM fungi (Chapter 5). The results showed that rmc did attract the AM fungi, that the plant nucleus moved to the middle of the plant cell only after fungal penetration of plant roots and that callose deposition in rmc was not involved in blocking the AM fungi. Fourteen AM fungi with different taxonomic affiliations and fourteen different G. intraradices isolates were screened to try to relate phylogeny of AM fungi with phenotypes in rmc (Chapter 6). There were a large number of interactions, depending on the inoculated AM fungi, and although there were some similarities in the rmc phenotypes within phylogenetic groups, there was no clear relationship between phylogeny and development of interactions with rmc. This study showed the following. 1) Arbuscules/arbusculate coils are necessary for the completion of the AM fungal cycle. However, intraradical hyphae also participate in transfer of both P and C as demonstrated with the Coi- phenotype. 2) rmc clearly attracted AM fungi and the fungi stay alive and induce plant cellular responses such as nuclear movement only after penetrating rmc roots. 3) Plant defense responses such as callose deposition are not involved in blocking AM fungi in rmc; and 4) there was no relationship between the phenotypes described in rmc and phylogeny of the Glomeromycota. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1292816 / Thesis(Ph.D.)-- School of Earth and Environmental Sciences, 2007.

Ce projet illustre cinq études, mettant l'emphase sur le développement d'une nouvelle approche diagnostique cardiovasculaire afin d'évaluer le niveau d’oxygène contenu dans le myocarde ainsi que sa fonction microvasculaire. En combinant une séquence de résonance magnétique cardiovasculaire (RMC) pouvant détecter le niveau d’oxygène (OS), des manœuvres respiratoires ainsi que des analyses de gaz artériels peuvent être utilisés comme procédure non invasive destinée à induire une réponse vasoactive afin d’évaluer la réserve d'oxygénation, une mesure clé de la fonction vasculaire. Le nombre de tests diagnostiques cardiaques prescrits ainsi que les interventions, sont en pleine expansion. L'imagerie et tests non invasifs sont souvent effectués avant l’utilisation de procédures invasives. L'imagerie cardiaque permet d’évaluer la présence ou absence de sténoses coronaires, un important facteur économique dans notre système de soins de santé. Les techniques d'imagerie non invasives fournissent de l’information précise afin d’identifier la présence et l’emplacement du déficit de perfusion chez les patients présentant des symptômes d'ischémie myocardique. Néanmoins, plusieurs techniques actuelles requièrent la nécessité de radiation, d’agents de contraste ou traceurs, sans oublier des protocoles de stress pharmacologiques ou physiques. L’imagerie RMC peut identifier une sténose coronaire significative sans radiation. De nouvelles tendances d’utilisation de RMC visent à développer des techniques diagnostiques qui ne requièrent aucun facteur de stress pharmacologiques ou d’agents de contraste. L'objectif principal de ce projet était de développer et tester une nouvelle technique diagnostique afin d’évaluer la fonction vasculaire coronarienne en utilisant l' OS-RMC, en combinaison avec des manœuvres respiratoires comme stimulus vasoactif. Ensuite, les objectifs, secondaires étaient d’utilisés l’OS-RMC pour évaluer l'oxygénation du myocarde et la réponse coronaire en présence de gaz artériels altérés. Suite aux manœuvres respiratoires la réponse vasculaire a été validée chez un modèle animal pour ensuite être utilisé chez deux volontaires sains et finalement dans une population de patients atteints de maladies cardiovasculaires. Chez le modèle animal, les manœuvres respiratoires ont pu induire un changement significatif, mesuré intrusivement par débit sanguin coronaire. Il a été démontré qu’en présence d'une sténose coronarienne hémodynamiquement significative, l’OS-RMC pouvait détecter un déficit en oxygène du myocarde. Chez l’homme sain, l'application de cette technique en comparaison avec l'adénosine (l’agent standard) pour induire une vasodilatation coronarienne et les manœuvres respiratoires ont pu induire une réponse plus significative en oxygénation dans un myocarde sain. Finalement, nous avons utilisé les manœuvres respiratoires parmi un groupe de patients atteint de maladies coronariennes. Leurs myocardes étant altérées par une sténose coronaire, en conséquence modifiant ainsi leur réponse en oxygénation. Par la suite nous avons évalué les effets des gaz artériels sanguins sur l'oxygénation du myocarde. Ils démontrent que la réponse coronarienne est atténuée au cours de l’hyperoxie, suite à un stimuli d’apnée. Ce phénomène provoque une réduction globale du débit sanguin coronaire et un déficit d'oxygénation dans le modèle animal ayant une sténose lorsqu’un supplément en oxygène est donné. En conclusion, ce travail a permis d'améliorer notre compréhension des nouvelles techniques diagnostiques en imagerie cardiovasculaire. Par ailleurs, nous avons démontré que la combinaison de manœuvres respiratoires et l’imagerie OS-RMC peut fournir une méthode non-invasive et rentable pour évaluer la fonction vasculaire coronarienne régionale et globale. / This project encompasses five studies, which focus on developing a new cardiovascular diagnostic approach for assessing myocardial oxygenation and microvascular function. In combination with oxygenation-sensitive cardiovascular magnetic resonance (OS-CMR) imaging, breathing maneuvers and altered arterial blood gases can be used as a non-invasive method for inducing a vasoactive response to test the oxygenation reserve, a key measurement in vascular function. The number of prescribed cardiac diagnostic tests and interventions is rapidly growing. In particular, imaging and other non-invasive tests are frequently performed prior to invasive procedures. One of the most common uses of cardiac imaging is for the diagnosis of significant coronary artery stenosis, a critical cost factor in today’s health care system. Non-invasive imaging techniques provide the most reliable information for the presence and location of perfusion or oxygenation deficits in patients with symptoms suggestive of myocardial ischemia, yet many current techniques suffer from the need for radiation, contrast agents or tracers, and pharmacological or physical stress protocols. CMR imaging can identify significant coronary artery stenosis without radiation and new trends in CMR research aim to develop diagnostic techniques that do not require any pharmacological stressors or contrast agents. For this project, the primary aim was to develop and test a new diagnostic technique to assess coronary vascular function using OS-CMR in combination with breathing maneuvers as the vasoactive stimulus. Secondary aims then used OS-CMR to assess myocardial oxygenation and the coronary response in the presence of altered arterial blood gases. An animal model was used to validate the vascular response to breathing maneuvers before translating the technique to human subjects into both healthy volunteers, and a patient population with cardiac disease. In the animal models, breathing maneuvers could induce a significant change in invasively measured coronary blood flow and it was demonstrated that in the presence of a haemodynamically significant coronary stenosis, OS-CMR could detect a myocardial oxygen deficit. This technique was then applied in a human model, with healthy participants. In a direct comparison to the infusion of the coronary vasodilator adenosine, which is considered a standard agent for inducing vasodilation in cardiac imaging, breathing maneuvers induced a stronger response in oxygenation of healthy myocardium. The final study then implemented the breathing maneuvers in a patient population with coronary artery disease; in which myocardium compromised by a coronary stenosis had a compromised oxygenation response. Furthermore, the observed effects of arterial blood gases on myocardial oxygenation were assessed. This demonstrated that the coronary response to breath-hold stimuli is attenuated during hyperoxia, and this causes an overall reduction in coronary blood flow, and consequently an oxygenation deficit in a coronary stenosis animal model when supplemental oxygen is provided. In conclusion, this work has improved our understanding of potential new diagnostic techniques for cardiovascular imaging. In particular, it demonstrated that combining breathing maneuvers with oxygenation-sensitive CMR can provide a non-invasive and cost-effective method for assessing global and regional coronary vascular function.

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