Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1990. / Vita. Abstract. Includes bibliographical references (leaves 102-104). Also available via the Internet.

A presença de bactérias patogênicas com capacidade de formar biofilme nos dispositivos de uso médico constitui um dos principais problemas na saúde pública. O biofilme confere proteção contra os mecanismos de defesa do hospedeiro e ação dos antimicrobianos, aumentando a virulência e resistência dos micro-organismos. A erradicação deste tipo de biofilmes é uma das principais estratégias no tratamento de doenças infecciosas associadas a bactérias patogênicas. A erradicação pode ser feita com o uso de biomoléculas produto do metabolismo secundário de diferentes micro-organismos. O objetivo deste estudo foi procurar novas moléculas com o potencial de erradicar biofilmes patogênicos associados a implantes médicos a partir de bactérias associadas ao género Drosera usando como bactérias alvo os patógenos Pseudomonas aeruginosa e Staphylococcus epidermidis. Para isto, foram crescidos 193 isolados bacterianos associados à planta Drosera que cresce na região litoral de Nova Tramandaí (RS) no sul do Brasil. Do total de isolados bacterianos foram usados os 36 que apresentaram atividade proteolítica para a produção de sobrenadantes bioativos. Os sobrenadantes bioativos foram usados para testes de erradicação de biofilme usando placas de 96 poços e a técnica de Cristal Violeta nas duas bactérias alvo. Nove isolados bacterianos associados à Drosera apresentaram atividade de erradicação de biofilme por acima de 40%, três deles para P. aeruginosa e seis para S. epidermidis. Destes isolados foi escolhido o isolado com melhor atividade de erradicação para P. aeruginosa e foram realizados testes para determinação da concentração mínima de erradicação de biofilme, antiformação de biofilme, viabilidade celular e microscopia eletrônica de varredura junto com a identificação da bactéria por médio de genes 16S, bem como o fracionamento bioguiado do sobrenadante. Adicionalmente, o sobrenadante bioativo foi testado in vitro em um modelo de dispositivo médico (cateter). A bactéria Gram-positiva que apresentou maior atividade de erradicação em seu sobrenadante possui 99% de similaridade com o Bacillus pumilus. Esta atividade de erradicação de biofilme de P. aeruginosa, pode ser atribuída à presença de uma molécula o composto de aproximadamente 34 kDa. Esta biomolécula o composto pode constituir uma alternativa complementária ao uso dos antibióticos convencionais. A perspectiva de purificar e caracterizar esta biomolécula ou composto para P. aeruginosa permitirá estudar seu mecanismo de ação e o uso no tratamento deste biofilme patogénico na clínica. / The presence of pathogenic bacteria with the ability to form biofilm on medical devices is one of the major problems in public health. The biofilm protects against the host defense mechanisms and the action of antibiotics, increasing the virulence and resistance of microorganisms. The elimination of this type of biofilms is a main strategy in the treatment of infectious diseases associated with pathogenic bacteria. This elimination can be done by using biomolecules from the secondary metabolism product of different micro-organisms. The objective of this study was to look for new molecules with the potential to eradicate pathogenic biofilms associated with medical implants from bacteria associated with gender Drosera using bacteria as targets Pseudomonas aeruginosa and Staphylococcus epidermidis pathogens. For this, were grown 193 bacterial isolates associated with Drosera plant that grows in the coastal region of New Tramandaí (RS) in southern Brazil. Of total bacterial isolates were used which had 36 proteolytic activity for the production of bioactive supernatants. The bioactive supernatants were used for biofilm eradication tests using 96- well plates and Crystal Violet technique in both target bacteria. Then the supernatant with the best eradication activity to P. aeruginosa was chosen to determine the minimum concentration to eradicate biofilm, biofilm inhibition, cell viability and scanning electron microscopy together with the identification of the bacteria by means of 16S and bioguided the fractionation of the supernatant. Additionally, the bioactive supernatant was tested in an in vitro model using the clinical device. Nine bacterial isolates associated with Drosera showed biofilm eradication activity by over 40%, three for P. aeruginosa and six for S. epidermidis. The Gram-positive bacterium with 99% similarity to the Bacillus pumilus showed the best biofilm eradication activity to P. aeruginosa capacity allocated to the presence of a molecule composed of approximately 34 kDa. These biomolecules can be an alternative to complement the use of conventional antibiotics or the direct use of antibiotic activity is presented as the case of bioactive found for P. aeruginosa supernatant. The prospect of purify and characterize this biomolecule or compound to P. aeruginosa will allow us to study this mechanism of action and the use in the treatment of this pathogenic biofilm in the clinic.

A presença de bactérias patogênicas com capacidade de formar biofilme nos dispositivos de uso médico constitui um dos principais problemas na saúde pública. O biofilme confere proteção contra os mecanismos de defesa do hospedeiro e ação dos antimicrobianos, aumentando a virulência e resistência dos micro-organismos. A erradicação deste tipo de biofilmes é uma das principais estratégias no tratamento de doenças infecciosas associadas a bactérias patogênicas. A erradicação pode ser feita com o uso de biomoléculas produto do metabolismo secundário de diferentes micro-organismos. O objetivo deste estudo foi procurar novas moléculas com o potencial de erradicar biofilmes patogênicos associados a implantes médicos a partir de bactérias associadas ao género Drosera usando como bactérias alvo os patógenos Pseudomonas aeruginosa e Staphylococcus epidermidis. Para isto, foram crescidos 193 isolados bacterianos associados à planta Drosera que cresce na região litoral de Nova Tramandaí (RS) no sul do Brasil. Do total de isolados bacterianos foram usados os 36 que apresentaram atividade proteolítica para a produção de sobrenadantes bioativos. Os sobrenadantes bioativos foram usados para testes de erradicação de biofilme usando placas de 96 poços e a técnica de Cristal Violeta nas duas bactérias alvo. Nove isolados bacterianos associados à Drosera apresentaram atividade de erradicação de biofilme por acima de 40%, três deles para P. aeruginosa e seis para S. epidermidis. Destes isolados foi escolhido o isolado com melhor atividade de erradicação para P. aeruginosa e foram realizados testes para determinação da concentração mínima de erradicação de biofilme, antiformação de biofilme, viabilidade celular e microscopia eletrônica de varredura junto com a identificação da bactéria por médio de genes 16S, bem como o fracionamento bioguiado do sobrenadante. Adicionalmente, o sobrenadante bioativo foi testado in vitro em um modelo de dispositivo médico (cateter). A bactéria Gram-positiva que apresentou maior atividade de erradicação em seu sobrenadante possui 99% de similaridade com o Bacillus pumilus. Esta atividade de erradicação de biofilme de P. aeruginosa, pode ser atribuída à presença de uma molécula o composto de aproximadamente 34 kDa. Esta biomolécula o composto pode constituir uma alternativa complementária ao uso dos antibióticos convencionais. A perspectiva de purificar e caracterizar esta biomolécula ou composto para P. aeruginosa permitirá estudar seu mecanismo de ação e o uso no tratamento deste biofilme patogénico na clínica. / The presence of pathogenic bacteria with the ability to form biofilm on medical devices is one of the major problems in public health. The biofilm protects against the host defense mechanisms and the action of antibiotics, increasing the virulence and resistance of microorganisms. The elimination of this type of biofilms is a main strategy in the treatment of infectious diseases associated with pathogenic bacteria. This elimination can be done by using biomolecules from the secondary metabolism product of different micro-organisms. The objective of this study was to look for new molecules with the potential to eradicate pathogenic biofilms associated with medical implants from bacteria associated with gender Drosera using bacteria as targets Pseudomonas aeruginosa and Staphylococcus epidermidis pathogens. For this, were grown 193 bacterial isolates associated with Drosera plant that grows in the coastal region of New Tramandaí (RS) in southern Brazil. Of total bacterial isolates were used which had 36 proteolytic activity for the production of bioactive supernatants. The bioactive supernatants were used for biofilm eradication tests using 96- well plates and Crystal Violet technique in both target bacteria. Then the supernatant with the best eradication activity to P. aeruginosa was chosen to determine the minimum concentration to eradicate biofilm, biofilm inhibition, cell viability and scanning electron microscopy together with the identification of the bacteria by means of 16S and bioguided the fractionation of the supernatant. Additionally, the bioactive supernatant was tested in an in vitro model using the clinical device. Nine bacterial isolates associated with Drosera showed biofilm eradication activity by over 40%, three for P. aeruginosa and six for S. epidermidis. The Gram-positive bacterium with 99% similarity to the Bacillus pumilus showed the best biofilm eradication activity to P. aeruginosa capacity allocated to the presence of a molecule composed of approximately 34 kDa. These biomolecules can be an alternative to complement the use of conventional antibiotics or the direct use of antibiotic activity is presented as the case of bioactive found for P. aeruginosa supernatant. The prospect of purify and characterize this biomolecule or compound to P. aeruginosa will allow us to study this mechanism of action and the use in the treatment of this pathogenic biofilm in the clinic.

A presença de bactérias patogênicas com capacidade de formar biofilme nos dispositivos de uso médico constitui um dos principais problemas na saúde pública. O biofilme confere proteção contra os mecanismos de defesa do hospedeiro e ação dos antimicrobianos, aumentando a virulência e resistência dos micro-organismos. A erradicação deste tipo de biofilmes é uma das principais estratégias no tratamento de doenças infecciosas associadas a bactérias patogênicas. A erradicação pode ser feita com o uso de biomoléculas produto do metabolismo secundário de diferentes micro-organismos. O objetivo deste estudo foi procurar novas moléculas com o potencial de erradicar biofilmes patogênicos associados a implantes médicos a partir de bactérias associadas ao género Drosera usando como bactérias alvo os patógenos Pseudomonas aeruginosa e Staphylococcus epidermidis. Para isto, foram crescidos 193 isolados bacterianos associados à planta Drosera que cresce na região litoral de Nova Tramandaí (RS) no sul do Brasil. Do total de isolados bacterianos foram usados os 36 que apresentaram atividade proteolítica para a produção de sobrenadantes bioativos. Os sobrenadantes bioativos foram usados para testes de erradicação de biofilme usando placas de 96 poços e a técnica de Cristal Violeta nas duas bactérias alvo. Nove isolados bacterianos associados à Drosera apresentaram atividade de erradicação de biofilme por acima de 40%, três deles para P. aeruginosa e seis para S. epidermidis. Destes isolados foi escolhido o isolado com melhor atividade de erradicação para P. aeruginosa e foram realizados testes para determinação da concentração mínima de erradicação de biofilme, antiformação de biofilme, viabilidade celular e microscopia eletrônica de varredura junto com a identificação da bactéria por médio de genes 16S, bem como o fracionamento bioguiado do sobrenadante. Adicionalmente, o sobrenadante bioativo foi testado in vitro em um modelo de dispositivo médico (cateter). A bactéria Gram-positiva que apresentou maior atividade de erradicação em seu sobrenadante possui 99% de similaridade com o Bacillus pumilus. Esta atividade de erradicação de biofilme de P. aeruginosa, pode ser atribuída à presença de uma molécula o composto de aproximadamente 34 kDa. Esta biomolécula o composto pode constituir uma alternativa complementária ao uso dos antibióticos convencionais. A perspectiva de purificar e caracterizar esta biomolécula ou composto para P. aeruginosa permitirá estudar seu mecanismo de ação e o uso no tratamento deste biofilme patogénico na clínica. / The presence of pathogenic bacteria with the ability to form biofilm on medical devices is one of the major problems in public health. The biofilm protects against the host defense mechanisms and the action of antibiotics, increasing the virulence and resistance of microorganisms. The elimination of this type of biofilms is a main strategy in the treatment of infectious diseases associated with pathogenic bacteria. This elimination can be done by using biomolecules from the secondary metabolism product of different micro-organisms. The objective of this study was to look for new molecules with the potential to eradicate pathogenic biofilms associated with medical implants from bacteria associated with gender Drosera using bacteria as targets Pseudomonas aeruginosa and Staphylococcus epidermidis pathogens. For this, were grown 193 bacterial isolates associated with Drosera plant that grows in the coastal region of New Tramandaí (RS) in southern Brazil. Of total bacterial isolates were used which had 36 proteolytic activity for the production of bioactive supernatants. The bioactive supernatants were used for biofilm eradication tests using 96- well plates and Crystal Violet technique in both target bacteria. Then the supernatant with the best eradication activity to P. aeruginosa was chosen to determine the minimum concentration to eradicate biofilm, biofilm inhibition, cell viability and scanning electron microscopy together with the identification of the bacteria by means of 16S and bioguided the fractionation of the supernatant. Additionally, the bioactive supernatant was tested in an in vitro model using the clinical device. Nine bacterial isolates associated with Drosera showed biofilm eradication activity by over 40%, three for P. aeruginosa and six for S. epidermidis. The Gram-positive bacterium with 99% similarity to the Bacillus pumilus showed the best biofilm eradication activity to P. aeruginosa capacity allocated to the presence of a molecule composed of approximately 34 kDa. These biomolecules can be an alternative to complement the use of conventional antibiotics or the direct use of antibiotic activity is presented as the case of bioactive found for P. aeruginosa supernatant. The prospect of purify and characterize this biomolecule or compound to P. aeruginosa will allow us to study this mechanism of action and the use in the treatment of this pathogenic biofilm in the clinic.

The impact of nutrient addition on growth and nutrient accumulation in insectivorous plants was studied in field populations and greenhouse plantings. Drosera rotundifolia was studied in the field, and D. binata var. multifida and D. capensis were studied in long-established plantings in the greenhouse. In each case, experiments were performed by enclosing insects and/or adding phosphorus and/or nitrogen to the soil. None of the species significantly benefitted from insect capture nutritionally or energetically in nutrient-poor or rich soils. Added nutrients to the soil or by foliar insect feeding decreased phosphorus retention in hibernacula by 50% (D. rotundifolia). Nutrient additions reduced D. rotundifolia vegetative growth in both N and P addition treatments. In addition, reproductive output (inflorescences) decreased flowering by 98% when N was added to the soil. Nutrient addition to soil increased nutrient concentration significantly in D. rotundifolia (N and P), and to a greater extent in D. capensis (N and P) and D. binata (P), and increased growth in D. capensis (N and P). In natural settings, insectivory was not found to be a significant source of nutrients for the species of Drosera studied. Larger subtropical species such as D. capensis and D. binata var. multifida is found in relatively richer (nutrient) soil than cool temperate species (D. rotundifolia) and are better able to utilize available nutrients in a substrate by high absorption rates and luxury consumption. / Master of Science

O gênero Drosera (Droseraceae) compreende cerca de 200 espécies, 30 delas ocorrendo no Brasil. Nesta dissertação, é realizada a revisão taxonômica do clado tetraploide-brasileiro de Drosera, compreendendo 17 espécies e uma variedade. São apresentados e discutidos dados sobre morfologia, ecologia, distribuição geográfica e status de conservação para estas espécies, com mapas de distribuição, ilustrações, fotografias e tabelas comparativas. Os complexos D. graminifolia, D. Montana e D. villosa são discutidos nos capítulos 1, 2 e 3, respectivamente. Além disso, essas três espécies são recircunscritas com base em evidências morfológicas e de sua ecologia. Drosera ascendens, D. spiralis, D. tomentosa e suas duas variedades são restabelecidas, e D. villosa var. latifólia é elevada ao status específico. Drosera chrysolepis é lectotipificada e três novas espécies são descritas. Drosera camporupestris, D. grantsaui, D. graomogolensis, D. quartzicola, D. tentaculata e D. schwackei também pertencem ao clado e são aqui tratadas. Na seção Taxonomia, é apresentada uma chave de identificação e, no anexo II, uma lista com todos os táxons de Drosera ocorrentes no Brasil e aqui aceitos / The genus Drosera (Droseraceae) comprises around 200 species, 30 occurring in Brazil. In this dissertation is carried out the taxonomic revision of the Brazilian-tetraploid clade of Drosera, comprising 17 species and a variety. Here are presented and discussed data on the morphology, ecology, geographic distribution, and conservation status for these species, with distribution maps, drawings, photographs, and comparative tables. The complexes D. graminifolia, D. Montana, and D. villosa are discussed in chapters 1, 2, and 3, respectively. These three species are recircumscribed based on of morphological and ecological evidences. Drosera ascendens, D. spiralis, D. tomentosa and its two varieties are reestablished, and D. villosa var. latifolia is raised to specific rank. Drosera chrysolepis is lectotypified and three new species are described. Drosera camporupestris, D. grantsaui, D. graomogolensis, D. quartzicola, D. tentaculata, and D. schwackei also belong to this clade and are here treated. In section Taxonomy it is presented a dicotomic key and, in appendix II, a list with all Drosera taxa occurring in Brazil here accepted

O gênero Drosera (Droseraceae) compreende cerca de 200 espécies, 30 delas ocorrendo no Brasil. Nesta dissertação, é realizada a revisão taxonômica do clado tetraploide-brasileiro de Drosera, compreendendo 17 espécies e uma variedade. São apresentados e discutidos dados sobre morfologia, ecologia, distribuição geográfica e status de conservação para estas espécies, com mapas de distribuição, ilustrações, fotografias e tabelas comparativas. Os complexos D. graminifolia, D. Montana e D. villosa são discutidos nos capítulos 1, 2 e 3, respectivamente. Além disso, essas três espécies são recircunscritas com base em evidências morfológicas e de sua ecologia. Drosera ascendens, D. spiralis, D. tomentosa e suas duas variedades são restabelecidas, e D. villosa var. latifólia é elevada ao status específico. Drosera chrysolepis é lectotipificada e três novas espécies são descritas. Drosera camporupestris, D. grantsaui, D. graomogolensis, D. quartzicola, D. tentaculata e D. schwackei também pertencem ao clado e são aqui tratadas. Na seção Taxonomia, é apresentada uma chave de identificação e, no anexo II, uma lista com todos os táxons de Drosera ocorrentes no Brasil e aqui aceitos / The genus Drosera (Droseraceae) comprises around 200 species, 30 occurring in Brazil. In this dissertation is carried out the taxonomic revision of the Brazilian-tetraploid clade of Drosera, comprising 17 species and a variety. Here are presented and discussed data on the morphology, ecology, geographic distribution, and conservation status for these species, with distribution maps, drawings, photographs, and comparative tables. The complexes D. graminifolia, D. Montana, and D. villosa are discussed in chapters 1, 2, and 3, respectively. These three species are recircumscribed based on of morphological and ecological evidences. Drosera ascendens, D. spiralis, D. tomentosa and its two varieties are reestablished, and D. villosa var. latifolia is raised to specific rank. Drosera chrysolepis is lectotypified and three new species are described. Drosera camporupestris, D. grantsaui, D. graomogolensis, D. quartzicola, D. tentaculata, and D. schwackei also belong to this clade and are here treated. In section Taxonomy it is presented a dicotomic key and, in appendix II, a list with all Drosera taxa occurring in Brazil here accepted

Le travail présenté porte sur le développement d’une nouvelle technologie innovante, nommée PAT Friday®, visant à produire des protéines recombinantes au sein des sécrétions extracellulaires de plantes carnivores génétiquement modifiées. Deux objectifs ont été fixés : Réaliser la preuve de concept de la technologie sur le modèle expérimental Drosera rotundifolia, en transformant la plante avec des gènes marqueurs et humains afin de mettre en évidence la présence des protéines recombinantes dans la glu ; et développer, après évaluation, la technologie sur un modèle potentiellement industrialisable, Nepenthes alata. Les résultats ont indiqué la présence des deux protéines marqueurs GFP et GUS dans les tissus et dans la glu de Drosera rotundifolia transformées. Les plantes ont également été transformées génétiquement avec les gènes humains de l’interféron gamma et du facteur intrinsèque. Les protéines recombinantes humaines ont été mises en évidence au sein des tissus végétaux. Le potentiel industriel du modèle Nepenthes alata a ensuite été étudié : 10 à 15 kg de protéines totales par hectare et par an peuvent être produits, grâce notamment à des récoltes successives non destructrices, et la possibilité de contrôler l’activité des protéases digestives naturelles. L’élaboration d’un protocole de régénération de la plante a été entreprise par embryogénèse somatique et organogénèse indirecte, en vue de sa transformation génétique. La technologie PAT Friday®, avec des étapes simplifiées d’extraction et de purification des protéines d’intérêt produites dans le liquide digestif, offre de nouvelles perspectives dans le domaine des protéines thérapeutiques produites à partir de plantes / The present work focuses on the development of a new innovating technology, called PAT Friday®, aiming at producing recombinant proteins into the extra-foliar fluid of modified carnivorous plants. Two objectives were assigned to this work : 1- to realize a proof of concept of the technology on the experimental model Drosera rotundifolia, transformed with marker and human genes, to confirm the occurence of the recombinant proteins into glu ; and 2 - to evaluate and develop, the technology on the model Nepenthes alata, more adapted to industrial scaling-up. The results indicate the presence of two marker proteins GUS and GFP inside the tissues and into the glu of modified Drosera rotundifolia plants. The same plant species has also been transformed with human gamma interferon and intrinsic factor genes. The corresponding human recombinant proteins have been detected into the plant tissues. Potential industrial scaling-up has been studied with the species Nepenthes alata. The results show a potential productivity of 10 to 15 kg of total proteins per hectare per year, thanks to non-destructive repeated harvests, and possibility to efficiently control the natural proteinase activity. The elaboration of a regeneration protocol has been undertaken through indirect organogenesis and somatic embryogenesis, with a view to transform genetically this plant. PAT Friday® technology, with simplified extraction and purification methods of the proteins of interest targeted into the liquid secretions, opens new perspectives in the field of therapeutical proteins produced in plants

Nitrogen (N) is an essential element to plants for growth, maintenance and reproduction, however most N does not exist in a form that is biologically available to plants. In order to maximise the acquisition and retention of N, plants have evolved a variety of morphological and physiological adaptations and life history strategies, as well as the ability to respond plastically to changes in resource availability in ecological time. Determining the ecophysiological responses of plants to changes in root N availability is crucial to further understanding of the mechanisms underlying competitive interactions between plants, and between plants and other organisms, that ultimately contribute to community structure and ecosystem functioning. Carnivorous plants are ideal systems for investigating ecophysiological responses to N availability as:- (i) they share a unique adaptation for obtaining supplemental N from captured prey, therefore ecological stoichiometry and energetic cost/benefit models may be explored; (ii) the trait of botanical carnivory is widely considered to have independently co-evolved as a response to N-deficient, sunny and wet environments, therefore resource allocation trade-offs between plant investment in N and carbon (C) acquisition may be observed, and (iii) they are extremely sensitive to changes in root N availability in ecological time. In this research, the carnivorous plant Drosera rotundifolia (round-leaved sundew) was used to address several unanswered ecophysiological and evolutionary questions relating to patterns and processes of prey capture and the N nutrition of carnivorous plants. Furthermore, the potential for reducing uncertainty in the calculation of plant reliance on carnivory using a δ15N natural abundance multi-level linear mixing model was explored. A combined approach of in-situ and ex-situ studies was employed, using co-occurring non-carnivorous plants or carnivorous plant species with differing evolutionary lineages or prey capture mechanisms respectively to provide context. Results show that the adaptations of carnivory, high reproductive investment and a relatively short life span enable Drosera rotundifolia to survive and thrive in an extreme, N deficient environment. Phenotypically plastic responses by the plant to light and root N availability provide evidence of resource allocation trade-offs between investment in carnivory for N acquisition and in photosynthesis for C acquisition. Plants invested less heavily in prey capture (measured as the stickiness of leaf mucilage) as N availability increased or light availability decreased. These results show that the energetic costs associated with carnivory are avoided by the plant when less costly sources of N are available for uptake and that the production of carbon-rich mucilage is only made under nutrient-limited and well-lit conditions. Results obtained from the comparison of captured insect prey with background invertebrates of potential prey indicate that Drosera rotundifolia is a dietary generalist, where the quantity of prey captured per plant is positively correlated with leaf stickiness and total leaf area. Plant reliance on prey-derived N decreased with increasing root N availability, providing evidence that carnivory is only of net benefit to the plant in N-deficient and well-lit environments, as the photosynthetic costs of investment in the trait are not exceeded by the energetic gain from prey N uptake in shady or dry habitats. A more accurate and precise method for calculating plant reliance on botanical carnivory is presented which incorporates the insect diet of the plant. This method has wider significance for reducing uncertainty in the calculation of relative source contributions to a mixture for most natural abundance applications using a multi-level linear mixing model. To conclude, results from this research further understanding of the ecophysiological mechanisms underlying plant responses to changes in resource availability and the selective pressures driving the evolution of plant adaptations. These results therefore assist with predicting how plants and plant communities may respond to sustained N deposition inputs and future environmental scenarios.