Fungi are a crucial component of most ecosystems and are responsible for decomposing organic matter, distributing nutrients through the environment and supporting plants and animal life through symbiotic relationships. Certain species of fungi are common pathogens causing disease and infection in plants and animals. The highly integrated nature of fungi in relation to the environment and all life emphasises the importance of developing a greater understanding of the growth and morphology of such organisms. Mathematical modelling has provided a means through which key processes can be isolated to analyse and simulate a target system to allow observations and form predictions regarding unknown phenomena. Numerous models of fungal colonies have been produced and are generally categorised into two main groups; continuous and discrete. The following study combines the approaches so that the constructed hybrid model comprises a discrete network that represents the fungal mycelia and a continuous component to account for the continuous substrates and other compounds crucial to fungal growth and development. Key processes such as uptake, translocation and anastomosis are included in addition to the implementation of a flexible hyphal orientation scheme that facilitates a variety of tropisms to different influential factors. The hybrid model is used to investigate several scenarios such as the polarisation of growth in response to isolated nutrient resources, competition between multiple colonies and fungal development and persistence in polluted environments. These investigations demonstrate the versatility of the hybrid model and highlight the potential for further applications.

No description available.

The recently identified causative agent of White-Nose Syndrome (WNS), Geomyces destructans, has been responsible for the mortality of an estimated 5.7 million North American bats since its emergence in 2006. A primary focus of the National Response Plan, established by US Fish and Wildlife in 2011, was the identification of biological and chemical control options. In an effort to identify potential biological and chemical control options for WNS, six previously described bacterially produced volatile organic compounds (VOCs) and multiply induced Rhodococcus rhodochrous DAP96253 were screened for anti-Geomyces destructans activity. Geomyces destructans conidia and mycelial plugs were exposed to the VOCs and induced Rhodococcus in a closed air space at 15°C and 4°C and evaluated for inhibition of conidia germination and mycelial extension. Additionally, in situ application methods for induced Rhodococcus, such as fixed cell catalyst and fermentation cell paste in non-growth conditions, were screened with positive results. Rhodococcus was assayed for ex vivo activity via exposure to bat tissue ex-plants inoculated with G. destructans conidia. All VOCs inhibited radial growth of mycelial plugs and growth from conidia at both temperatures, with the greatest effect at low temperature (4°C). Induced Rhodococcus completely inhibited growth from conidia at 15°C and had a strong fungistatic effect at 4°C. Induced Rhodococcus inhibited Geomyces destructans growth from conidia when cultured in a shared air space with bat tissue explants inoculated with Geomyces destructans conidia. During the evaluation diffusible brown pigment was observed in G. destructans cultures exposed to induced Rhodococcus or select VOCs. The pigment was induced by light and oxidative challenge and hypothesized to be melanin. Traditional microbiological methods, as well as copper sulfide-silver staining and ultraviolet-visible spectroscopy, were utilized to confirm this hypothesis. This was a noteworthy result as melanin is a known virulence factor in other pathogenic fungi and may play a significant role in WNS. The identification of bacterially produced VOCs and inducible biological agents with anti-Geomyces destructans activity expands the pool of potential biological and chemical control options for WNS and provides wildlife management personnel with tools to combat this devastating disease.

As part of a project designed to utilize common dairy waste products profitably, reconstituted dry whey permeate and delactosed whey were tested as growth substrates for mycelia of the edible mushroom Lentinus edodes. This mushroom was chosen because it is possible to profitably cultivate it due to its popular culinary appeal and perceived medical benefits. Growth experiments were performed in petri dishes containing either reconstituted dry whey permeate or delactosed whey as a growth substrate, and the measured response was the size of the growing mycelia colony. When reconstituted dry whey permeate was utilized as a growth substrate, the factors of substrate concentration, pH, and growth temperature were controlled in an effort to determine the optimal growth conditions for the mushroom mycelia. These conditions were determined by applying an analytical method known as response surface methodology (RSM). RSM is a collection of mathematical techniques that is able to determine optimal values for many variables run simultaneously in an experiment. Mycelia were also grown on delactosed whey at different substrate concentrations in an effort to determine if this substrate would be suitable for the growth of mushroom mycelia. Results: RSM was successfully utilized to determine the optimal growth conditions for L. edodes when grown on reconstituted dry whey powder. These conditions were 40 g/L substrate concentration, pH 4 .97, and temperature 23.6°C Delactosed whey was successfully utilized as a growth substrate for L. edodes. However, delactosed whey concentrations above 40% v/v were lethal to the mushroom mycelia, suggesting a possible use for delactosed whey as a fungicide.

The cyclic trihydroxamic acid, N, N', N' '-triacetylfusarinine C, produced by Mycelia sterilia EP-76, is shown to be a ferric ionophore for this organism. The association constant for ferric-N, N', N' '-triacetylfusarinine C complex was determined to be log K=32.5. Other iron chelating agents, such as rhodotorulic acid, citric acid, or the monomeric subunit of triacetylfusarinine C, N-acetyl- fusarinine, delivered iron to the cells by an indirect mechanism involving iron exchange into triacetylfusarinine C. In vitro ferric ion exchange was found to be rapid with triacetylfusarinine C. Gallium uptake rates comparable to those of iron were observed with the chelating agents that transport iron into the cell. Ferrichrome, but not ferrichrome A, was also capable of delivering iron and gallium to this organism, but not by an exchange mechanism. Unlike triacetylfusarinine C, the 14C-ligand of ferrichrome was retained by the cell. A mid-point potential of -690 mV versus the saturated silver chloride electrode was obtained for the ferric-N, N', N' '-triacetylfusarinine C complex, indicating that an unfavorable reduction potential was not the reason for utilizing a hydrolytic mechanism of intracellular iron release from the ferric triacetylfusarinine C chelate. The iron transport system recognizes only the  -cis coordination isomer of ferric-N, N’, N' '-triacetylfusarinine C metal ligand complex even though the -cis configuration predominates in solution. Ferrichrome and ferric-N, N', N' '-triacetylfusarinine C are both transported by the same receptor.

<p>Since the beginning of mycology, studies of species concept in fungi have been mainly based on morphology, partially due to the history of mycology as part of botany. Current advances in biochemical and molecular research have provided mycologists with powerful tools that can be used for delineation of fungal taxa. Recently, an integrated approach to fungal taxonomy involving both morphological and molecular traits has found a wide application for identification of species, especially in anamorphic (mitosporic) fungi.</p><p>In this thesis, I have tried to use this approach for identification of species units in two rather unrelated groups of organisms. One of the case studies concerned <i>Colletotrichum acutatum</i> – a worldwide economically important plant pathogenic anamorphic fungus, which is exhibiting a high level of variation in both morphological and molecular features. This fungus has been intensively studied during the past decades, and several attempts have been made to find reliable markers to separate it from other closely related species of <i>Colletotrichum</i>. The second case studied in this thesis was <i>Mycelia Sterilia</i> – an artificial group of fungi, which are deficient in production of spores of any kind, therefore lacking the main morphological feature used for assigning them to any certain fungal taxon below class level. Due to this peculiarity, <i>Mycelia Sterilia</i> have usually been neglected, and currently there is no working species concept applicable to these fungi. </p><p>In this work, I have tried to clarify the relationships among <i>C. acutatum</i> and several other anamorphic (<i>C. gloeosporioides</i> and <i>C. fructigenum</i>) and teleomorphic (<i>Glomerella acutata</i>, <i>G. cingulata</i> and <i>G. miyabeana</i>) taxa that are closely related to each other. For this purpose, examination of morphological traits was employed in combination with comparison of DNA sequencing data from three loci and subsequent phylogenetic analysis. As a result, re-description of <i>C. acutatum</i> and separation of (at least) two new species was proposed.</p><p>For studies of <i>Mycelia Sterilia</i>, a large collection of sterile strains was screened in search for biologically interesting organisms. One novel pathogen has been found, and two plant growth promoting strains with antifungal properties were selected. Attempt for tentative identification of those fungi was made based on their morphological, physiological and molecular features. Sequencing of several genes and spacers of the ribosomal DNA array revealed that the plant pathogenic strain is closely related to the teleomorphic basidiomycete genus <i>Campanella</i>, and plant growth-promoting isolates were identified as belonging to the anamorphic ascomycete genus <i>Phoma</i>. However, assigning the sterile strains to any existing species was not possible.</p><p>The main conclusion of the thesis is that species in anamorphic fungi should be defined based on a combination of morphological and molecular methods, both equally important, involving as many aspects of fungal biology as is possible at our current state of knowledge. </p>

Since the beginning of mycology, studies of species concept in fungi have been mainly based on morphology, partially due to the history of mycology as part of botany. Current advances in biochemical and molecular research have provided mycologists with powerful tools that can be used for delineation of fungal taxa. Recently, an integrated approach to fungal taxonomy involving both morphological and molecular traits has found a wide application for identification of species, especially in anamorphic (mitosporic) fungi. In this thesis, I have tried to use this approach for identification of species units in two rather unrelated groups of organisms. One of the case studies concerned Colletotrichum acutatum – a worldwide economically important plant pathogenic anamorphic fungus, which is exhibiting a high level of variation in both morphological and molecular features. This fungus has been intensively studied during the past decades, and several attempts have been made to find reliable markers to separate it from other closely related species of Colletotrichum. The second case studied in this thesis was Mycelia Sterilia – an artificial group of fungi, which are deficient in production of spores of any kind, therefore lacking the main morphological feature used for assigning them to any certain fungal taxon below class level. Due to this peculiarity, Mycelia Sterilia have usually been neglected, and currently there is no working species concept applicable to these fungi. In this work, I have tried to clarify the relationships among C. acutatum and several other anamorphic (C. gloeosporioides and C. fructigenum) and teleomorphic (Glomerella acutata, G. cingulata and G. miyabeana) taxa that are closely related to each other. For this purpose, examination of morphological traits was employed in combination with comparison of DNA sequencing data from three loci and subsequent phylogenetic analysis. As a result, re-description of C. acutatum and separation of (at least) two new species was proposed. For studies of Mycelia Sterilia, a large collection of sterile strains was screened in search for biologically interesting organisms. One novel pathogen has been found, and two plant growth promoting strains with antifungal properties were selected. Attempt for tentative identification of those fungi was made based on their morphological, physiological and molecular features. Sequencing of several genes and spacers of the ribosomal DNA array revealed that the plant pathogenic strain is closely related to the teleomorphic basidiomycete genus Campanella, and plant growth-promoting isolates were identified as belonging to the anamorphic ascomycete genus Phoma. However, assigning the sterile strains to any existing species was not possible. The main conclusion of the thesis is that species in anamorphic fungi should be defined based on a combination of morphological and molecular methods, both equally important, involving as many aspects of fungal biology as is possible at our current state of knowledge.

In the presented study, dimorphic yeasts were applied as model organisms to study the growth of fungal mycelia. When environmental conditions are chosen appropriately, yeast colonies are built up of well separated individual cells. Thus, in contrast to fungal mycelia the translocation of nutrients and information within the colony can be neglected. The study focuses on the question of how the growth behaviour of a population of single cells is regulated, and which differences can be expected when nutrient translocation actually occurs. To answer this question, at first, an effective method for the highly resolved estimation of biomass distributions inside the colonies was developed. This method facilitates a dynamic non-invasive monitoring of colony development. Furthermore, mathematical models were established which describe the development of the colonies based on the behaviour of discrete individual cells. Growth simulations allow a quantitative prediction, and, thereby, an in silico testing of hypothetic regulatory mechanisms. The growth behaviour of yeast colonies was investigated applying the model organisms Candida boidinii and Yarrowia lipolytica. The yeasts were cultivated on solid agar substrates at various degrees of carbon and nitrogen limitation, respectively. The highest gain of understanding was achieved for the growth of both yeasts on glucose as the limiting carbon source: Investigations showed that mycelial yeast colonies adapt to declining nutrient concentrations by decreasing the cell density in their mycelium while the growth rate of the colony diameter remains constant. Under glucose limitation, the yeast C. boidinii grows diffusion-limited, i.e., the growth of the population is controlled by the amount of nutrient that diffuses towards the colony. The cessation of growth coincides with the depletion of the primary nutrient source glucose from the growth substrate. In contrast to these findings, it was shown that Y. lipolytica colonies continue to extend even after the complete consumption of glucose. In the absence of the primary nutrient source, the yeast assimilates biomass from the inner colony regions to facilitate the growth of the population. The suggested mechanism of coupled extension and decay processes was verified by a number of experiments. However, the mechanism which facilitates the transport of decay products to the growing colony boundary, i.e., the actual nature of the decay process, remains unclear. Mathematical simulations show that a continuous colony extension on the decay products of dying cells cannot be explained by the assumption that colonies are built up of uncoordinatedly growing single cells. Therefore, a hypothesis for the growth of Y. lipolytica colonies was derived which suggests that these populations are built up of tube-like hyphal cells. Accordingly, the measured drop of biomass density in the inner colony areas is the consequence of a cytoplasm transport towards the growing edge of the mycelium where it is assimilated as a secondary nutrient resource in the absence of glucose. It has to be emphasized that this hypothesis also provides a mechanistic explanation for the vacuolisation of hyphae in mycelia of higher fungi. / In der vorgestellten Arbeit wurden dimorphe Hefen als Modellorganismen für die Untersuchung des Wachstums von Pilzmyzelien eingesetzt. Bei geeigneter Wahl der Umgebungsbedingungen sind Hefekolonien aus Einzelzellen aufgebaut, wodurch im Gegensatz zu Myzelien höherer Pilze der Transport von Nährstoffen und Informationen innerhalb der Kolonie vernachlässigt werden kann. Im Mittelpunkt der Untersuchungen stand die Frage, wie das Wachstumsverhalten einer Population individueller Zellen reguliert ist, bzw. welche Unterschiede sich ergeben, wenn ein Nährstofftransport tatsächlich stattfindet. Um diese Fragestellungen bearbeiten zu können, wurde zunächst eine effektive Methode zur hoch ortsaufgelösten Bestimmung der Biomasseverteilung innerhalb der Kolonien entwickelt. Diese Methode ermöglicht ein dynamisches nichtinvasives Monitoring der Entwicklung einer Kolonie. Weiterhin wurden mathematische Modelle entwickelt, die das Wachstumsverhalteeiner Population auf der Grundlage des Verhaltens von diskreten Einzelzellen beschreibt. Die Wachstumssimulationen erlauben quantitative Vorhersagen und damit ein in silico Testen der Auswirkungen von hypothetischen Regulationsmechanismen. Das Wachstumsverhalten von Hefekolonien wurde anhand der Modellorganismen Candida boidinii und Yarrowia lipolytica untersucht. Die Hefen wurden auf festen Agar-Nährböden bei verschieden starker Kohlenstoff- und Stickstofflimitation kultviert. Der größte Erkenntnisgewinn wurde dabei für das Wachstum beider Hefen auf Glukose als limitierender Kohlenstoffquelle erzielt: Die Untersuchungen ergaben, dass myzelartig wachsende Hefekolonien bei sinkenden Nährstoffkonzentrationen eine geringere Zelldichte aber einen konstante Wachstumsgeschwindigkeit des Koloniedurchmessers aufweisen. Die Hefe C. boidinii wächst unter Glukoselimitation diffusionslimitiert, d.h. das Wachstum der Population wird durch die Menge der zur Kolonie diffundierenden Nährstoffe bestimmt. Der Abbruch des Koloniewachstums fällt mit dem Verbrauch der primären Nähstoffquelle Glukose zusammen. Im Gegensatz dazu konnte für das Wachstum von Y. lipolytica gezeigt werden, dass sich die Kolonien auch nach dem vollständigen Verbrauch von Glukose weiter ausdehnen. Im Abwesenheit der primären Nährstoffquelle nutzt die Hefe Zerfallsprodukte eigener Zellmasse aus dem Inneren der Kolonie als Nährstoff, um das weitere Wachstum der Population zu gewährleisten. Während der vorgeschlagene gekoppelte Ausdehnungs- und Zerfallprozess durch eine Reihe von Versuchen experimentell abgesichert wurde, bleibt der Mechanismus des Transports der Zerfallsprodukte zum Kolonierand, bzw. die eigentliche Natur des Zerfallsprozesses unklar. Simulationsrechnungen ergaben, dass eine kontinuierliche Ausdehnung der Kolonie auf Zellzerfallsprodukten sterbender Zellen nicht durch die Annahme erklärt werden kann, dass die Kolonien aus unkoordiniert wachsenden Einzelzellen aufgebaut sind. Aus diesem Grunde wurde für das Wachstum von Y. lipolytica die Hypothese abgeleitet, dass das Myzelium dieser Hefe aus schlauchartigen Hyphenzellen aufgebaut ist. Der gemessene Abfall der Biomassekonzentration im Kolonieinneren ist demnach die Konsequenz des Transports von Zytoplasma hin zum wachsenden Kolonierand, wo es in Abwesenheit von Glukose als sekundäre interne Nährstoffquelle assimiliert wird. Es ist zu beachten, dass diese Hypothese auch eine mechanistische Erklärung für die Ursachen der Vakuolisierung in Myzelien höherer filamentöser Pilze gibt.

Made available in DSpace on 2014-06-11T19:35:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-03-04Bitstream added on 2014-06-13T20:46:30Z : No. of bitstreams: 1 celoto_mib_dr_ilha.pdf: 2236478 bytes, checksum: fe75a04fbb8e850c1386270aaffe5f0e (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Na região de Junqueirópolis, SP, a mancha alvo (Corynespora cassiicola) é a principal doença que vem ocorrendo na cultura da acerola, causando intensa desfolha. Devido a falta de estudos sobre esse patossistema e as dificuldades para a adequação de produtos químicos para uso nesta cultura, os objetivos do presente trabalho foram: 1 – elaborar e validar uma escala diagramática para quantificação da mancha alvo; 2 – avaliar o efeito in vitro da temperatura no crescimento micelial e na germinação de esporos de C. cassiicola e a influência da temperatura e da duração do período de molhamento foliar no desenvolvimento da mancha alvo em mudas de acerola, mantidas em condições de câmara de crescimento; 3 – avaliar o efeito in vitro e in vivo das caldas sulfocálcica, bordalesa e Viçosa sobre C. cassiicola; 4 – avaliar o efeito in vitro de produtos químicos sobre C. cassiicola e no controle da mancha alvo da acerola no campo; 5 – determinar o efeito da poda no controle da mancha alvo e na produção da acerola. A escala diagramática proposta proporcionou bons níveis de acurácia e precisão, mostrando-se adequada para as avaliações da severidade da mancha alvo. As temperaturas ótimas estimadas para o crescimento micelial e a germinação de esporos de C. cassiicola foram de 26,1 e 27,8oC, respectivamente. Para que a infecção ocorra é necessário pelo menos 12h de molhamento foliar. O estabelecimento da mancha alvo ocorre na faixa de 20 a 30oC. As caldas sulfocálcica, bordalesa e Viçosa presentes na superfície das folhas de acerola e in vitro inviabilizaram os esporos de C. cassiicola. Assim, o uso das caldas, na cultura da acerola, pode contribuir na redução de fontes de inóculo do patógeno. Tebuconazole, carbendazim, epoxiconazole + piraclostrobina, DDAC, Nutriphite P + K e Ecolife® apresentaram efeito fungitóxico sobre C. cassiicola in vitro... / In Junqueirópolis, SP, the target spot (Corynespora cassiicola), main leaf disease occurred in barbados cherry, causing intense defoliate. Because few studies about this pathosystem and difficulties for adaptation of chemical products in barbados cherry, the objectives of the work were: 1 – elaborate and validate a diagrammatic scale to quantification of target spot; 2 – effect in vitro of temperature on mycelial growth and spores germination of C. cassiicola and the influence of temperature and the duration of leaf wetness in the development of target spot on seedling of barbados cherry in growthing chamber; 3 – effect in vitro and in vivo of line sulfhur, bordeaux mixture and ‘calda Viçosa’ on C. cassiicola; 4 – effect in vitro of chemical products on C. cassiicola and in the control of target spot of barbados cherry in field condition; 5 – effect of pruning in the control of target spot and production of barbados cherry. The scale provided good levels of accuracy and precision proved to be adequate for severity assessments of target spot of barbados cherry. The estimated maximum temperatures for mycelia growth and for spores germination of C. cassiicola were 26,1oC and 27,8oC, respectively. The presence of free water on the surface of barbados cherry leaves was necessary for the development of target spot, being necessary at least 12h of leaf wetness to infection happened. In the development of lesions of target spot in barbados cherry seedlings, occurs in the range 20 to 30oC. Line sulfhur, Bordeaux mixture and ‘calda Viçosa’ in surface of leaves barbados cherry and in vitro unviability the spores of C. cassiicola. For the reasons, the use of the syrups, in the culture of the acerola, it can contribute in the reduction of sources of inoculum of the pathogen. Tebuconazol, carbendazin, epoxiconazol + piraclostrobin, DDAC, Nutriphite... (Complete abstract click electronic access below)

Orientador: Marli de Fátima Stradioto Papa / Banca: Luiz de Souza Corrêa / Banca: Aparecida Conceição Boliani / Banca: César Júnior Bueno / Banca: Marise Cagnin Martins Parisi / Resumo: Na região de Junqueirópolis, SP, a mancha alvo (Corynespora cassiicola) é a principal doença que vem ocorrendo na cultura da acerola, causando intensa desfolha. Devido a falta de estudos sobre esse patossistema e as dificuldades para a adequação de produtos químicos para uso nesta cultura, os objetivos do presente trabalho foram: 1 - elaborar e validar uma escala diagramática para quantificação da mancha alvo; 2 - avaliar o efeito in vitro da temperatura no crescimento micelial e na germinação de esporos de C. cassiicola e a influência da temperatura e da duração do período de molhamento foliar no desenvolvimento da mancha alvo em mudas de acerola, mantidas em condições de câmara de crescimento; 3 - avaliar o efeito in vitro e in vivo das caldas sulfocálcica, bordalesa e Viçosa sobre C. cassiicola; 4 - avaliar o efeito in vitro de produtos químicos sobre C. cassiicola e no controle da mancha alvo da acerola no campo; 5 - determinar o efeito da poda no controle da mancha alvo e na produção da acerola. A escala diagramática proposta proporcionou bons níveis de acurácia e precisão, mostrando-se adequada para as avaliações da severidade da mancha alvo. As temperaturas ótimas estimadas para o crescimento micelial e a germinação de esporos de C. cassiicola foram de 26,1 e 27,8oC, respectivamente. Para que a infecção ocorra é necessário pelo menos 12h de molhamento foliar. O estabelecimento da mancha alvo ocorre na faixa de 20 a 30oC. As caldas sulfocálcica, bordalesa e Viçosa presentes na superfície das folhas de acerola e in vitro inviabilizaram os esporos de C. cassiicola. Assim, o uso das caldas, na cultura da acerola, pode contribuir na redução de fontes de inóculo do patógeno. Tebuconazole, carbendazim, epoxiconazole + piraclostrobina, DDAC, Nutriphite P + K e Ecolife® apresentaram efeito fungitóxico sobre C. cassiicola in vitro... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In Junqueirópolis, SP, the target spot (Corynespora cassiicola), main leaf disease occurred in barbados cherry, causing intense defoliate. Because few studies about this pathosystem and difficulties for adaptation of chemical products in barbados cherry, the objectives of the work were: 1 - elaborate and validate a diagrammatic scale to quantification of target spot; 2 - effect in vitro of temperature on mycelial growth and spores germination of C. cassiicola and the influence of temperature and the duration of leaf wetness in the development of target spot on seedling of barbados cherry in growthing chamber; 3 - effect in vitro and in vivo of line sulfhur, bordeaux mixture and 'calda Viçosa' on C. cassiicola; 4 - effect in vitro of chemical products on C. cassiicola and in the control of target spot of barbados cherry in field condition; 5 - effect of pruning in the control of target spot and production of barbados cherry. The scale provided good levels of accuracy and precision proved to be adequate for severity assessments of target spot of barbados cherry. The estimated maximum temperatures for mycelia growth and for spores germination of C. cassiicola were 26,1oC and 27,8oC, respectively. The presence of free water on the surface of barbados cherry leaves was necessary for the development of target spot, being necessary at least 12h of leaf wetness to infection happened. In the development of lesions of target spot in barbados cherry seedlings, occurs in the range 20 to 30oC. Line sulfhur, Bordeaux mixture and 'calda Viçosa' in surface of leaves barbados cherry and in vitro unviability the spores of C. cassiicola. For the reasons, the use of the syrups, in the culture of the acerola, it can contribute in the reduction of sources of inoculum of the pathogen. Tebuconazol, carbendazin, epoxiconazol + piraclostrobin, DDAC, Nutriphite... (Complete abstract click electronic access below) / Doutor