O Origannum vulgare L. (orégano) é uma erva com potencial terapêutico, havendo, entre seus extratos, maior atividade detectada no óleo essencial (OEO). Dentre os constituintes estudados neste extrato estão monoterpenos responsáveis por atividades farmacológicas, incluindo ação antibiótica sobre diversos organismos, entre os quais, Aspergillus spp, os mais importantes fungos em medicina veterinária. A aspergilose é a micose de maior morbimortalidade em aves, e a principal doença respiratória nesses animais. A espécie mais envolvida em casos clínicos é o A. fumigatus, devido principalmente à sua ampla capacidade de disseminação ambiental, ao reduzido tamanho de seus propágulos e à produção de metabólitos secundários. O tratamento da micose é considerado pouco eficiente, devido, entre outros fatores, ao número limitado de antifúngicos, à alta toxicidade destes fármacos, e à resistência por parte do Aspergillus spp. Em vista desta realidade o presente estudo objetivou avaliar o potencial terapêutico do OEO no tratamento e prevenção da aspergilose crônica experimental por A. fumigatus em frangos (Gallus gallus). Para tanto realizou-se, primeiramente testes de sensibilidade in vitro de Aspergillus spp. frente ao OEO; de toxicidade do óleo e desenvolvimento experimental de aspergilose em frangos. Os testes de sensibilidade in vitro foram ensaiados com A. fumigatus, A. flavus e A. niger, iniciando com OEO na concentração 900mg/mL (6%) passando por dez diluições seriadas com base log2. Foram realizados testes de toxicidade aguda sistêmica (24h de exposição) e toxicidade subcrônica sistêmica (30 dias de exposição) com OEO nas concentrações 225mg/mL (1,5%), 450mg/mL (3,0%) e 675mg/mL (4,5%). A infecção experimental foi realizada em três etapas, utilizando para tanto diferentes concentrações, volumes e números de inoculações. E tanto para tratamento quanto para profilaxia da aspergilose utilizou-se grupo tratado com OEO (O), com o antifúngico padrão itraconazol (I), com ambos os produtos associados (I+O), e grupo sem tratamento (C). A partir da análise química do óleo foram observados 15 diferentes compostos ativos, sendo 4-terpineol (18.4%), hidrato de sabinene (15,6%) e timol (13,6%), os componentes majoritários. No teste de sensibilidade in vitro o óleo essencial apresentou tanto atividade fungicida quanto fungistática frente a Aspergillus spp., com CIM variando entre 28,125mg/mL (0,1875%) e 450mg/mL (3,0%) e CFM entre 112,5mg/mL (0,75%) e 450mg/mL (3,0%). E CIM 50 de 112,5 mg/mL (0,75%) e CIM 90 de 450mg/mL (3,0%). Observouse que em todas as concentrações testadas, o óleo essencial de O. vulgare não causou nenhuma alteração clínica nem anatomopatológica. Para reproduzir aspergilose experimental crônica A. fumigatus em frangos de 15 dias definiu-se que deve-se utilizar concentração de 12x108 UFC/mL em três inoculações seriadas, com intervalos de 24h. O primeiro dia de infecção foi considerado o dia três. Na avaliação do uso d OEO para tratamento e profilaxia da aspergilose, I+O apresentou melhores resultados, com menos sinais clínicos, anatomopatológicos, microscópicos e menor contagem de Unidades Formadoras de Colônia/g/órgão. I e O apresentaram resultados, de forma geral, inferiores à I+O, porém superiores à C, não sendo possível avaliar qual grupo obteve maior eficácia no tratamento e/ou profilaxia da micose. / Origannum vulgare L. (oregano) is a herb with therapeutic properties. In its extract, higher activity is detected on the essential oil (OEO). Among the extract components are monoterpenes with pharmacological properties, e.g. antibiotic action over several microorganisms, including Aspergillus spp., which are fungi of great importance in veterinary medicine. Avian aspergillosis is a mycosis causing the highest morbidmorbidity in captive birds. It is also the main respiratory disease observed on this group. A. fumigatus is the pathogen more commonly detected in clinical cases, mainly due to its high capacity of being environmentally disseminated and producing secondary metabolites, as well as due to its reduced propagule size. The mycosis treatment is inefficient mainly due to the limited availability of antifungal compounds, the high toxicity found on these compounds and the resistance of Aspergillus species. Thus, the aim of this work was to evaluate the therapeutic potential of OEO in both the treatment and prevention of experimental chronic aspergillosis, caused by A. fumigatus in poultry (Gallus gallus). Initial experiments were carried out to test the in vitro OEO-sensitivity of Aspergillus spp., oil toxicity, and the experimental progression of disease in poultry. In vitro sensitivity testes were carried out with A. fumigatus, A. flavus and A. niger, starting with a concentration of 900mg/mL (6%) OEO, going through ten serial dilutions (log2 scale). Systemic acute toxicity (24h of exposure) and systemic subchronic toxicity (30h of exposure) tests were carried out, with OEO concentrations of 225mg/mL (1.5%), 450mg/mL (3.0%) and 675mg/mL (4.5%). The experimental infection was accomplished in three stages, with diverse inoculation concentrations, volumes and frequencies being tested. Both in aspergillosis treatment and prevention, groups tested were either treated with OEO (O), with the standard anti-fungal itraconazol (I), with both products combined (I+O), or with none of them (C). Fifteen different active compounds were observed in the chemical analysis of the oil, being 4- terpineol (18.4%), sabinene hydrate (15.6%) and thymol (13.6%) the major components found. At the in vitro sensitivity test, the essential oil displayed both fungicide and fungistatic effect towards Aspergillus spp., with MIC varying between 28.125mg/mL (0.1875%) and 450mg/mL (3%) and MFC varying between 112.5mg/mL (0.75%) and 450mg/mL (3%). In all concentrations tested, the essential oil from O. vulgare did not cause any clinical or anatomopathologic alteration. Experimental reproduction of chronic A. fumigatus aspergillosis in 15 day-old poultry was achieved by using an inoculum concentration of 12x108 Colonies Forming Units/mL (CFU) in three serial inoculations at 24h intervals, being day three considered the first day of infection. The use of OEO in aspergillosis treatment and prevention was best observed in I+O, resulting in less clinical, anatomopathologic and microscopic signs, as well as a smaller count of CFU/g/organ. Overall, I and O were less effective than I+O, but more effective than C. Nevertheless, it was not possible to evaluate which group increased the mycosis treatment and/or prevention efficacy.

O Origannum vulgare L. (orégano) é uma erva com potencial terapêutico, havendo, entre seus extratos, maior atividade detectada no óleo essencial (OEO). Dentre os constituintes estudados neste extrato estão monoterpenos responsáveis por atividades farmacológicas, incluindo ação antibiótica sobre diversos organismos, entre os quais, Aspergillus spp, os mais importantes fungos em medicina veterinária. A aspergilose é a micose de maior morbimortalidade em aves, e a principal doença respiratória nesses animais. A espécie mais envolvida em casos clínicos é o A. fumigatus, devido principalmente à sua ampla capacidade de disseminação ambiental, ao reduzido tamanho de seus propágulos e à produção de metabólitos secundários. O tratamento da micose é considerado pouco eficiente, devido, entre outros fatores, ao número limitado de antifúngicos, à alta toxicidade destes fármacos, e à resistência por parte do Aspergillus spp. Em vista desta realidade o presente estudo objetivou avaliar o potencial terapêutico do OEO no tratamento e prevenção da aspergilose crônica experimental por A. fumigatus em frangos (Gallus gallus). Para tanto realizou-se, primeiramente testes de sensibilidade in vitro de Aspergillus spp. frente ao OEO; de toxicidade do óleo e desenvolvimento experimental de aspergilose em frangos. Os testes de sensibilidade in vitro foram ensaiados com A. fumigatus, A. flavus e A. niger, iniciando com OEO na concentração 900mg/mL (6%) passando por dez diluições seriadas com base log2. Foram realizados testes de toxicidade aguda sistêmica (24h de exposição) e toxicidade subcrônica sistêmica (30 dias de exposição) com OEO nas concentrações 225mg/mL (1,5%), 450mg/mL (3,0%) e 675mg/mL (4,5%). A infecção experimental foi realizada em três etapas, utilizando para tanto diferentes concentrações, volumes e números de inoculações. E tanto para tratamento quanto para profilaxia da aspergilose utilizou-se grupo tratado com OEO (O), com o antifúngico padrão itraconazol (I), com ambos os produtos associados (I+O), e grupo sem tratamento (C). A partir da análise química do óleo foram observados 15 diferentes compostos ativos, sendo 4-terpineol (18.4%), hidrato de sabinene (15,6%) e timol (13,6%), os componentes majoritários. No teste de sensibilidade in vitro o óleo essencial apresentou tanto atividade fungicida quanto fungistática frente a Aspergillus spp., com CIM variando entre 28,125mg/mL (0,1875%) e 450mg/mL (3,0%) e CFM entre 112,5mg/mL (0,75%) e 450mg/mL (3,0%). E CIM 50 de 112,5 mg/mL (0,75%) e CIM 90 de 450mg/mL (3,0%). Observouse que em todas as concentrações testadas, o óleo essencial de O. vulgare não causou nenhuma alteração clínica nem anatomopatológica. Para reproduzir aspergilose experimental crônica A. fumigatus em frangos de 15 dias definiu-se que deve-se utilizar concentração de 12x108 UFC/mL em três inoculações seriadas, com intervalos de 24h. O primeiro dia de infecção foi considerado o dia três. Na avaliação do uso d OEO para tratamento e profilaxia da aspergilose, I+O apresentou melhores resultados, com menos sinais clínicos, anatomopatológicos, microscópicos e menor contagem de Unidades Formadoras de Colônia/g/órgão. I e O apresentaram resultados, de forma geral, inferiores à I+O, porém superiores à C, não sendo possível avaliar qual grupo obteve maior eficácia no tratamento e/ou profilaxia da micose. / Origannum vulgare L. (oregano) is a herb with therapeutic properties. In its extract, higher activity is detected on the essential oil (OEO). Among the extract components are monoterpenes with pharmacological properties, e.g. antibiotic action over several microorganisms, including Aspergillus spp., which are fungi of great importance in veterinary medicine. Avian aspergillosis is a mycosis causing the highest morbidmorbidity in captive birds. It is also the main respiratory disease observed on this group. A. fumigatus is the pathogen more commonly detected in clinical cases, mainly due to its high capacity of being environmentally disseminated and producing secondary metabolites, as well as due to its reduced propagule size. The mycosis treatment is inefficient mainly due to the limited availability of antifungal compounds, the high toxicity found on these compounds and the resistance of Aspergillus species. Thus, the aim of this work was to evaluate the therapeutic potential of OEO in both the treatment and prevention of experimental chronic aspergillosis, caused by A. fumigatus in poultry (Gallus gallus). Initial experiments were carried out to test the in vitro OEO-sensitivity of Aspergillus spp., oil toxicity, and the experimental progression of disease in poultry. In vitro sensitivity testes were carried out with A. fumigatus, A. flavus and A. niger, starting with a concentration of 900mg/mL (6%) OEO, going through ten serial dilutions (log2 scale). Systemic acute toxicity (24h of exposure) and systemic subchronic toxicity (30h of exposure) tests were carried out, with OEO concentrations of 225mg/mL (1.5%), 450mg/mL (3.0%) and 675mg/mL (4.5%). The experimental infection was accomplished in three stages, with diverse inoculation concentrations, volumes and frequencies being tested. Both in aspergillosis treatment and prevention, groups tested were either treated with OEO (O), with the standard anti-fungal itraconazol (I), with both products combined (I+O), or with none of them (C). Fifteen different active compounds were observed in the chemical analysis of the oil, being 4- terpineol (18.4%), sabinene hydrate (15.6%) and thymol (13.6%) the major components found. At the in vitro sensitivity test, the essential oil displayed both fungicide and fungistatic effect towards Aspergillus spp., with MIC varying between 28.125mg/mL (0.1875%) and 450mg/mL (3%) and MFC varying between 112.5mg/mL (0.75%) and 450mg/mL (3%). In all concentrations tested, the essential oil from O. vulgare did not cause any clinical or anatomopathologic alteration. Experimental reproduction of chronic A. fumigatus aspergillosis in 15 day-old poultry was achieved by using an inoculum concentration of 12x108 Colonies Forming Units/mL (CFU) in three serial inoculations at 24h intervals, being day three considered the first day of infection. The use of OEO in aspergillosis treatment and prevention was best observed in I+O, resulting in less clinical, anatomopathologic and microscopic signs, as well as a smaller count of CFU/g/organ. Overall, I and O were less effective than I+O, but more effective than C. Nevertheless, it was not possible to evaluate which group increased the mycosis treatment and/or prevention efficacy.

Made available in DSpace on 2014-06-12T23:02:35Z (GMT). No. of bitstreams: 2 arquivo8568_1.pdf: 1675980 bytes, checksum: 5e811a808b181cc918a70ed7ba334651 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2006 / Origanum vulgare L. (orégano) têm sido reconhecido como uma espécie vegetal possuidora de várias propriedades terapêuticas, de modo que atualmente seu potencial antimicrobiano vem recebendo um grande interesse científico. Este estudo objetivou analisar a efetividade do óleo essencial de O. vulgare em inibir o crescimento de cepas de microrganismos de interesse em alimentos, bem como avaliar a interferência de diferentes tratamentos térmicos sobre a sua efetividade antimicrobiana e composição química. O screening do potencial antimicrobiano do óleo essencial foi realizado através da técnica de difusão em meio sólido, a Concentração Inibitória Mínima-CIM foi determinada através da técnica de difusão em meio sólido e microdiluição, enquanto a avaliação da interferência da CIM sobre a viabilidade microbiana foi realizada através da contagem de células viáveis em placa. A identificação e quantificação dos componentes do óleo essencial foram realizadas através de cromatografia gasosa espectrometria de massa. O óleo essencial de O. vulgare mostrou proeminente poder de inibição do crescimento de todas as cepas microbianas ensaiadas. Os valores de CIM oscilaram entre 80 e 5 &#956;L/mL quando determinada através da técnica de difusão em meio sólido, enquanto o método da microdiluição mostrou valores de CIM entre 5 e 0.62 &#956;L/mL. A CIM determinada pela técnica de difusão em meio sólido foi capaz de causar significante (p<0,05) efeito inibitório da viabilidade de todas as cepas de bactéria e de leveduras ensaiadas. De outra forma, a CIM determinada pela técnica de microdiluição não mostrou eficiência na inibição da viabilidade da maioria das cepas ensaiadas. O óleo essencial em diferentes concentrações (80, 40, 20 &#956;L/mL) mostrou capacidade de inibição da microbiota autóctone de carne moída armazenada sob refrigeração. A exposição do óleo essencial a diferentes tratamentos térmicos (temperatura ambiente, 60, 80, 100, 120°C/1hora) não apresentou interferência (p<0,05) sobre seu potencial antimicrobiano e composição química. A análise da composição do óleo essencial mostrou uma predominância de terpenos e compostos terpênicos, apresentando carvacrol e p-cimeno como componentes majoritários. Conclui-se, que o óleo essencial de O. vulgare apresenta-se como uma potencial fonte de agentes antimicrobianos para uso na conservação de alimentos

Seed germination and early plant development is a crucial phase of plants' life. Multiple internal and external stimuli influence germination progress and have a serious impact on a plant's survival and vitality. Biotic and abiotic stimuli trigger a whole range of changes, both on molecular and developmental levels, but the complex molecular mechanisms regulating these responses are far from being resolved. This thesis reviews the seed germination process and outlines the role of external stimuli in its progress. The experimental part describes the development of a method for seed germination monitoring, provides new insight into the role of hydrogen peroxide in germination, and analyzes effects of cadmium ions, temperature, salt and drought on proteome and metabolome of germinating seeds of Hordeum vulgare. In total, 2000 proteins and 800 metabolites were identified. The analyses revealed over 95 putative abiotic stress markers, including 63 and 36 proteins and metabolites, respectively

Salinity is a critical challenge facing productivity of crops around the world, causing major reduction in growth, yield, and quality. It is necessary to produce varieties with the ability to tolerate salinity. However, the lack of genetic variation among H. vulgare genotypes prevents progress in developing salt tolerant varieties. H. bulbosum is a source of tolerance to stress conditions. Consequently, five accessions of domestic barley and six of wild barley were used in this study. Accessions were screened for salinity tolerance. Genotypes 7, 9, and 10 germinated at 2% NaCl. Lines of H. vulgare showed reduction of root and shoot length greater than H. bulbosum. Crosses were made between diploid and tetraploid H. vulgare ♀ and tetraploid H. bulbosum ♂. Immature embryos were rescued. Murashige and Skoog medium was found to be generally better for most crosses. Number of successful crosses varied among families. Female 5, for diploid crosses, and female 2, for tetraploid crosses, have high GCA and compatibility with bulbosum males, and 17.9% and 17.6% of their progeny exceeding the mean grain yield, respectively. Parent 9 had also high GCA and compatibility with vulgare parents (2x), and its progeny seem to exceed the mean in many cases with most families. During germination screening, progeny of diploid females 1 and 2 were found to be highly desirable for saline tolerance. Among the tetraploid crosses, Family 1 had greatest percentage of superior progeny (18.8%), while Families 2 and 3 had greatest number of superior individuals (8 and 7, respectively). For seedling growth, diploid Families 2 and 3 crosses had the greatest shoot dry weight and tolerant saline index (SSI<1). Families 2 and 4 of tetraploid crosses had 12 of 39 and eight of 26 crosses show greatest shoot dry weight and tolerance as measured by SSI, respectively. Final germination percentage (FG%) showed positive association with plant height, while associating negatively with tiller number, fertility, cSW, and grain yield. In diploid crosses, FG% associated positively with tiller number. Shoot dry weight showed negative association with plant height, while it associated positively with tiller number, fertility, cSW, and grain yield.

Stomata sind kleine Poren in der Blattoberfläche, die Pflanzen eine Anpassung ihres Wasserhaushalts an sich ändernde Umweltbedingungen ermöglichen. Die Öffnungsweite der Stomata wird durch den Turgordruck der Schließzellen bestimmt, der wiederum durch Ionenflüsse über die Membranen der Zelle reguliert wird. Ein Netzwerk von Signaltransduktionswegen sorgt dafür, dass Pflanzen die Stomabewegungen an die Umgebungsbedingungen anpassen können. Viele molekulare Komponenten dieser Signaltransduktionketten in Schließzellen von Angiospermen sind inzwischen bekannt und Calcium spielt darin als Signalmolekül eine wichtige Rolle. Weitgehend unbekannt sind dagegen die Mechanismen, die zur Erzeugung von transienten Erhöhungen der Calciumkonzentration führen. Auch die molekularen Grundlagen der Regulierung der Stomaweite in Nicht-Angiospermen-Arten sind bisher nur wenig verstanden. Um zur Aufklärung dieser Fragestellungen beizutragen, wurden in dieser Arbeit Mechanismen zur Erhöhungen der cytosolischen Calciumkonzentration sowie elektrophysiologische Eigenschaften von Schließzellen untersucht. Der Fokus lag hierbei insbesondere auf der Visualisierung cytosolischer Calciumsignale in Schließzellen. Im ersten Teil der Arbeit wurde durch die Applikation hyperpolarisierender Spannungspulse mittels TEVC (Two Electrode Voltage Clamp) gezielt eine Erhöhung der cytosolischen Calciumkonzentration in einzelnen Schließzellen von Nicotiana tabacum ausgelöst. Um die Dynamik der cytosolischen Calciumkonzentration dabei zeitlich und räumlich hoch aufgelöst zu visualisieren, wurde simultan zu den elektrophysiologischen Messungen ein Spinning-Disc-System für konfokale Aufnahmen eingesetzt. Während der Applikation hyperpolarisierender Spannungspulse wurde eine transiente Vergrößerung des cytosolischen Volumens beobachtet. Diese lässt sich durch einen osmotisch getriebenen Wasserfluss erklären, der durch die Veränderung der Ionenkonzentration im Cytosol verursacht wird. Diese wiederum wird durch die spannungsabhängige Aktivierung einwärtsgleichrichtender Kaliumkanäle in der Plasmamembran der Schließzellen und durch den Kompensationsstrom der eingestochenen Mikroelektrode hervorgerufen. Mit Hilfe des calciumsensitiven Farbstoffs Fura-2 konnte gezeigt werden, dass die Erhöhung der freien cytosolischen Calciumkonzentration während der Applikation hyperpolarisierender Spannungspulse durch zwei Mechanismen verursacht wird. Der erste Mechanismus ist die Aktivierung hyperpolarisationsaktivierter, calciumpermeabler Kanäle (HACCs) in der Plasmamembran, die schon 1998 von Grabov & Blatt beschrieben wurde. Zusätzlich zu diesem Mechanismus der Calciumfreisetzung, konnte ein zweiter bislang unbekannter Mechanismus aufgedeckt werden, bei dem Calcium aus intrazellulären Speichern in das Cytosol freigesetzt wird. Dieser Mechanismus hängt mit der oben beschriebenen Vergrößerung des cytosolischen Volumens zusammen und ist wahrscheinlich durch die Änderungen der mechanischen Spannung der Membran bzw. der Osmolarität innerhalb der Zelle bedingt. Diese könnten zu einer Aktivierung mechanosensitiver, calciumpermeabler Kanäle führen. Der zweite Teil der Arbeit beschäftigt sich mit den molekularen Grundlagen der Regulierung von Stomata in Nicht-Angiospermen. In Schließzellen von Polypodium vulgare konnten durch die Anwendung der TEVC-Technik ähnliche spannungsabhängige Ströme über die Plasmamembran gemessen werden wie in Angiospermen. Ebenso wurden durch die Applikation hyperpolarisierender Spannungspulse an Schließzellen von Polypodium und Asplenium Erhöhungen der cytosolischen Calciumkonzentration ausgelöst, die auf die Existenz spannungsabhängiger, calciumpermeabler Kanäle in der Plasmamembran hinweisen. Die Diffusion von Fluoreszenzfarbstoffen in die Nachbarschließzellen nach der iontophoretischen Beladung in Polypodium, Asplenium, Ceratopteris und Selaginella zeigte, dass in diesen Arten eine symplastische Verbindung zwischen benachbarten Schließzellen besteht, die an Schließzellen von Angiospermen bisher nicht beobachtet werden konnte. Anhand elektronenmikroskopischer Aufnahmen von Polypodium glycyrrhiza Schließzellen konnte gezeigt werden, dass diese Verbindung wahrscheinlich durch Plasmodesmata zwischen benachbarten Schließzellen gebildet wird. Durch die Analyse der Calciumdynamik in benachbarten Schließzellen nach hyperpolarisierenden Spannungspulsen stellte sich heraus, dass die Calciumhomöostase trotz symplastischer Verbindung in beiden Schließzellen unabhängig voneinander reguliert zu werden scheint. Im Rahmen der Untersuchungen an Farnschließzellen wurde desweiteren eine Methode zur Applikation von ABA etabliert, die es erlaubt mithilfe von Mikroelektroden das Phytohormon iontophoretisch in den Apoplasten zu laden. Im Gegensatz zu den Schließzellen von Nicotiana tabacum, die auf eine so durchgeführte ABA-Applikation mit dem Stomaschluss reagierten, wurde in Polypodium vulgare auf diese Weise kein Stomaschluss ausgelöst. Da die ABA-Antwort der Farnstomata aber auch von anderen Faktoren wie Wachstumsbedingungen abhängig ist (Hõrak et al., 2017), kann eine ABA-Responsivität in dieser Farnart trotzdem nicht vollkommen ausgeschlossen werden. Die Freisetzung von Calcium aus intrazellulären Speichern, wie sie in dieser Arbeit gezeigt wurde, könnte eine wichtige Rolle bei der Regulierung der Stomaweite spielen. Zur Aufklärung dieser Fragestellung wäre die Identifizierung der Kanäle, die an der osmotisch/mechanisch induzierten Calciumfreisetzung aus internen Speichern beteiligt sind, von großem Interesse. Weiterführende Studien an Schließzellen von Farnen könnten die physiologische Bedeutung der aus Angiospermen bekannten Ionenkanäle für die Stomabewegungen in evolutionär älteren Landpflanzen aufklären und so maßgeblich zum Verständnis der Evolution der Regulierunsgmechanismen von Stomata beitragen. Außerdem stellt sich die Frage, welche Rolle die hier gezeigte symplastische Verbindung der Nachbarschließzellen durch Plasmodesmata für die Funktion der Stomata spielt. / Stomata are small pores in the leaf surface that allow plants to adapt their water balance to changing environmental conditions. The turgor pressure of the guard cells determines the width of the stomatal aperture and is regulated by ion fluxes in or out of the guard cell. A network of different signal transduction pathways is necessary for the adaption of stomatal movements to ambient conditions. Many of these transduction pathways have been described in detail and many of their components have been identified. It is a well known fact that calcium acts as a second messenger in pathways regulating stomatal movements. However, the mechanisms that lead to transient elevations of the cytosolic calcium concentration are largely unknown. The molecular basis of the regulation of stomatal aperture in non-angiosperm species is also poorly understood. In order to gain new insights into these topics, mechanisms of calcium elevation and electrophysiological properties of guard cells were studied, focussing especially on the visualization of the cytosolic calcium concentration in guard cells. In the first part of this study, the application of hyperpolarizing voltage pulses by means of TEVC (Two Electrode Voltage Clamp) was used to specifically trigger an increase in the cytosolic calcium concentration in individual guard cells in the angiosperm model plant Nicotiana tabacum. To visualize the dynamics of the cytosolic calcium concentration with high temporal and spatial resolution, a spinning disc system for confocal imaging was used simultaneously with the electrophysiological recordings. During the application of hyperpolarizing voltage pulses a transient increase in cytosolic volume was observed. This increase can be explained by an osmotically driven water flux caused by changes of the cytosolic ion concentration. These in turn are caused by the voltage-dependent activation of inward rectifying potassium channels in the guard cell plasma membrane and by the compensating current from the impaled microelectrode. Using the calcium-sensitive dye Fura-2, it could be shown that two mechanisms lead to the elevation of the cytosolic calcium concentration during the application of hyperpolarizing voltage pulses. The first mechanism is the activation of hyperpolarization-activated calcium permeable channels (HACCs) in the plasma membrane, which has already been described in 1998 by Grabov & Blatt. In addition to this mechanism of calcium release, a second previously unknown mechanism was discovered in which calcium is released into the cytosol from intracellular stores. This mechanism is related to the increase in cytosolic volume we described above and is probably caused by changes in membrane tension or osmolarity within the cell. These changes could lead to an activation of mechanosensitive calciumpermeable channels. The second part of this thesis deals with the molecular basis of the regulation of stomata in non-angiosperms. In guard cells of Polypodium vulgare voltage-dependent currents across the plasma membrane similar to those described in angiosperm model plants could be measured using TEVC. Furthermore, the application of hyperpolarizing voltage pulses induced increases in cytosolic calcium concentration in guard cells of Polypodium and Asplenium indicating the existence of voltage-dependent calcium permeable channels in the plasma membrane. The diffusion of iontophoretically injected fluorescent dyes into the neighboring guard cells in Polypodium, Asplenium, Ceratopteris and Selaginella showed that in these species a symplastic connection between neighboring guard cells exists, which could not be observed in guard cells of angiosperms. Electron microscopic images of Polypodium glycyrrhiza guard cells showed that this connection is probably formed by plasmodesmata between adjacent guard cells. Analysis of the calcium dynamics in neighboring guard cells after hyperpolarizing voltage pulses revealed that calcium homeostasis seems to be regulated independently in both guard cells despite their symplastic connection. As part of the investigations on guard cells of ferns, a new method for the application of ABA was established, which allows the phytohormone to be charged iontophoretically into the apoplast with the aid of microelectrodes. In contrast to the guard cells of Nicotiana tabacum, which reacted with loss of turgor and subsequential stomatal closure to this method of ABA-application, no closure of the stomata could be induced in Polypodium vulgare in this way. However, since the ABA response of fern stomata is also dependent on other factors such as growth conditions (Hõrak et al., 2017), an ABA-responsiveness in this fern species can still not be completely excluded. The release of calcium from intracellular stores, as shown in this work, could play an important role for the regulation of stomatal aperture. To clarify this question, the identification of the channels involved in osmotically/mechanically induced calcium release from internal stores would be of great interest. Further studies on fern guard cells could clarify the physiological significance of ion channels known from angiosperms for the stomatal movements in early land plants, and thus contribute significantly to the understanding of the evolution of stomatal regulation. In addition, the question arises as to what role the symplastic connection of the neighboring guard cells through plasmodesmata plays for the function of stomata.

To prove the post-natal depression model, the antidepressant sertraline, was assessed in rat mothers (n=14) divided into Prenatally Stressed (PS) and Non-Stressed (NS) groups. The data failed to support the hypothesis that ‘the progeny of 10mg of sertraline-treated PS mothers displayed less anxiety than the progeny of vehicle-treated PS mothers’. The forced swim test (FST) was used to examine depressive-like behaviour in mice. Barley successfully increased mobility in mice exposed to the FST. Barley was antidepressant at low doses (0.8g/kg and upwards) if used subchronic; and at high doses(6.4g/kg and 12.8g/kg) if used acutely;(n=113,56acute,57 subchronic- treated). Barley (6.4g/kg) was also able to alleviate the depressive-behaviour in mice induced by the Reserpine Test (n=114, 58 reserpinised, 56 non-reserpinised) and Social ‘Defeat’ Test (n=24, 8 vehicle undefeated, 8 barley defeated, 8 vehicle defeated mice). To confirm that the anti-depressant effects of barley(6.4g/kg) were not simply due to increased locomotor activity in the FST, an Open Field Test(OFT) was undertaken (n=14,7 vehicle, 7 barley). Barley had no effect on locomotor activity and also caused no significant changes in weight (n=16, 8vehicle, 8 barley). In mice,Barley(6.4g/kg) significantly delayed the tremorogenic effects of Physostigmine (n=18, 6 control,6 Physostigmine, 6 Physostigmine with barley); reduced bradykinesia induced by reserpine (n=18,6 control, 6 vehicle, 6 barley treated);and was analgesic in nociception tests (n =20, 5 control, 5 barley, 5 pain, 5 pain with barley). Overall, barley was seen to have many useful properties, though its effect in PND remains to be assessed. / Saudi Cultural Bureau in London; Medical Services Department of the Ministry of Interior in Riyadh, Saudi Arabia. / The full text of this thesis is embargoed indefinitely.

Net blotch is one of the most devastating diseases of barley (<i>Hordeum vulgare</i> L.) and occurs in two distinct forms, net-type net blotch (NTNB) and spot-type net blotch (STNB), caused by the fungal pathogens <i>Pyrenophora teres</i> f. sp. <i>teres</i> Smedeg. and <i>P</i>. <i>teres</i> f. sp. <i>maculata</i> Smedeg., respectively. Several sources of resistance have been previously reported, however, few barley cultivars with high levels of resistance have been developed from these sources. Efficient utilization of available resistance sources is dependent upon successful characterization of genes governing resistance in each resistant parent. Five net blotch resistant parents and one susceptible parent were crossed to identify novel resistance genes, postulate gene number and mode of inheritance, and conduct linkage mapping of novel genes for net blotch resistance. Results indicate that the highly resistant spring barley lines CIho 2291 and CIho 5098, and the winter barley cultivar Nomini each have single dominant genes for NTNB resistance. Resistance to NTNB in CIho 5098 is controlled by the same dominant gene conferring resistance in Nomini. Resistance to NTNB in CIho 2291 is controlled by one dominant gene which putatively is the same gene conferring resistance in ND B112, but differs from the resistance genes carried by the other parents in this study. An F2 population of 238 individuals derived from a cross between Nomini and the susceptible parent "Hector", and an F2 population of 193 individuals derived from a cross between CIho 2291 and Hector were used to map the genes governing NTNB resistance in Nomini and CIho 2291. The dominant gene governing resistance in Nomini, temporarily designated <i>Rpt-Nomini</i>, was mapped to a 9.2 cM region near the centromere of barley chromosome 6H between the flanking microsatellite markers Bmag0344a (r2=0.70) and Bmag0103a (r2=0.90), which were 6.8 cM and 2.4 cM away from <i>Rpt-Nomini</i>, respectively. The dominant gene governing resistance in CIho 2291, temporarily designated <i>Rpt-CIho2291</i>, was mapped to the distal region of barley chromosome 6H between the flanking microsatellite markers Bmag0173 (r2=0.65) and Bmag0500 (r2=0.26), which were 9.9 cM and 24.4 cM from <i>Rpt-CIho2291</i>, respectively. Previous studies have reported genes governing net blotch resistance in this region; however, allelism tests have not been conducted to determine the relationship between these genes. Identification of the chromosomal location of <i>Rpt-Nomini</i> and <i>Rpt-CIho2291</i> will facilitate future efforts in pyramiding multiple independent genes for net blotch resistance. / Ph. D.

Made available in DSpace on 2017-07-25T19:29:59Z (GMT). No. of bitstreams: 1 Marina Senger.pdf: 672810 bytes, checksum: 4349ab10f1f5d2436d6c99566564a482 (MD5) Previous issue date: 2013-07-22 / The arrangement of plants spaced paired is one of the management practices being used in some cultures and has resulted in increased yield. Aiming to evaluate the arrangements and plant density on agronomic characteristics, yield components and yield of two cultivars of wheat and barley, there were four experiments, differing in culture and farming in the city of PontaGrossa, PR, 2012. The experimental design used in was randomized blocks in a 2x4 factorial design with four replications. The treatments consisted of two plant arrangements (single spaced and paired) and four plant densities (45, 60, 75 and90plantsm-1). Treatments were applied in two wheat cultivars (BRS-Tangará and Marfim) and two barley (BRSCauêandMN-743). Increasing plant density reduced the number of tillers, stem diameter and number of spikelets per spikein wheat and barley, promoted height increaseonly inBRSCauêand lowerthousand grain weightin cultivarsBRS-Tangará e Marfim. In bothwheat cultivarsspacingpairedpromotedfewerspikeper square meter, higherthousand grain weightandlower yield. In the twobarley cultivarsin the spacing paired occurred fewer spikes persquare meter.On BRS-Cauê pacingpairedincreasedyieldandMN-743 pairedspacinghigher yieldsin the density of90plantsper meter. / O arranjo de plantas em espaçamento pareado é uma das práticas de manejo que está sendo utilizada em algumas culturas e tem resultado em aumento de produtividade. Com o objetivo de avaliar arranjos e densidades de plantas em características agronômicas, componentes de produção e produtividade de duas cultivares de trigo e de cevada, realizaram-se quatro experimentos, diferindo pela cultura e cultivar, no município de Ponta Grossa, PR, no ano de 2012. O delineamento experimental foi de blocos ao acaso, em esquema fatorial 2x4 com quatro repetições. Os tratamentos constaram de dois arranjos de plantas (espaçamento simples e pareado) e quatro densidades de plantas (45, 60, 75 e 90 plantas.m-1). Os tratamentos foram aplicados em duas cultivares de trigo (BRS-Tangará e Marfim) e em duas de cevada (BRSCâue e MN-743). O aumento da densidade de plantas reduziu o número de perfilhos, o diâmetro do caule e o número de espiguetas por espiga no trigo e na cevada,promoveu aumento da altura apenas na cultivar BRS-Câue e menor massa de mil grãos nas cultivares Marfim e BRS-Tangará. Nas duas cultivares de trigo o espaçamento pareado promoveu menor número de espiga por metro quadrado, maior massa de mil grãos e menor produtividade. Nas duas cultivares de cevada no espaçamento pareado ocorreu menor número de espigas por metro quadrado. Na cultivar BRS-Câue o espaçamento pareado aumentou a produtividade e para MN-743 o espaçamento pareado promoveu maior produtividade na densidade de 90 plantas por metro.

Made available in DSpace on 2017-07-25T19:30:47Z (GMT). No. of bitstreams: 1 Ariadne Waureck.pdf: 1552798 bytes, checksum: d207d66f92bd28ac1d16d99ac88a1093 (MD5) Previous issue date: 2015-02-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The seeds classification and the appropriate storage conditions are critical to maintaining quality, otherwise, the previous efforts to develop material and cultural techniques for seed production will not be valid. Accordingly, the objective of this study was to evaluate the interference of classification and storage on quality of barley seeds. Seed samples of four lots of BRS Cauê were used. The design was completely randomized with the treatments: group I - control - seeds unclassified; treatment II - seeds retained on the sieve 2.2 mm width x length 22.0mm; treatment III - seeds retained on the sieve 2.2 mm width x length 22.0mm + table of gravity; IV treatment - seeds retained on the sieve 2.5 mm width x length 22.0mm; treatment V - seeds retained on the sieve 2.5 mm width x length 22.0mm + table gravity. The evaluations were performed at the beginning of storage and 45, 90 , 150, 210 and 270 days after storage. The seeds remained stored in natural environment. To determine the quality of seeds were evaluated: the water content , the volumetric weight, thousand seed weight , the amount of infected seeds, germination, vigor, health and protein content. The infestation , test weight , moisture content and protein content , were not subjected to statistical analysis , the first three repetitions performed with two replications . The results of percentage of sanity test were transformed into arcsin √ ( x / 100 ) , and the other variables were subjected to analysis of variance and means were compared by Tukey test at 5 % significance level. In general, the results of seeds classified in sieve 2.5 x 22.0 mm associated with gravity table presented thousand seed weight and higher test weight in relation to the results of other treatments. However, these results were not observed for physiological and health parameters and protein content . Thus, it is concluded that , right for the barley seeds is the classification in the sieve 2.5 mm + table gravity. Besides, there was a reduction in the quality of barley seeds and the incidence of field fungi from 90 days of storage in a natural environment . / A classificação das sementes e as condições adequadas de armazenamento são fundamentais para manutenção da qualidade, caso contrário, os esforços anteriores para o desenvolvimento do material genético e as técnicas culturais para a produção das sementes não serão válidos. Nesse sentido, o objetivo desse trabalho foi avaliar a interferência da classificação e do armazenamento na qualidade das sementes de cevada. Foram utilizadas sementes de quatro lotes da cultivar BRS Cauê. O delineamento utilizado foi o inteiramente casualizado, sendo os tratamentos: I - controle – sementes não classificadas; II – sementes retidas na peneira 2,2 mm de largura x 22,0mm de comprimento; III – sementes retidas na peneira 2,2 mm de largura x 22,0mm de comprimento + mesa de gravidade; IV – sementes retidas na peneira 2,5 mm de largura x 22,0mm de comprimento; V – sementes retidas na peneira 2,5 mm de largura x 22,0mm de comprimento + mesa de gravidade. As avaliações foram realizadas no início do armazenamento e aos 45, 90, 150, 210 e 270 dias após armazenamento. As sementes permaneceram armazenadas em ambiente natural. Para a determinar a qualidade das sementes foram avaliados: o teor de água, o peso volumétrico, o peso de mil sementes, a quantidade de sementes infestadas, a germinação, o vigor, a sanidade e o teor de proteína. A infestação, peso hectolítrico, teor de água e teor de proteína, não foram submetidos a análise estatística, sendo os três primeiros realizados com duas repetições repetições. Os resultados de porcentagem do teste de sanidade foram transformados em arcsen √(x /100), e as demais variáveis foram submetidas a análise de variância e as médias comparadas pelo teste de Tukey a 5% de significância. Em geral, os resultados das sementes classificadas na peneira 2,5 X 22,0 mm associadas à mesa de gravidade apresentaram peso de mil sementes e peso hectolítrico superiores em relação aos resultados dos demais tratamentos. Porém, esses resultados não foram observados para os parâmetros fisiológico, sanitário e teor de proteína. Dessa forma, conclui-se que, o indicado para as sementes de cevada é a classificação na peneira 2,5mm + mesa de gravidade. Além disso, houve redução da qualidade das sementes de cevada e da incidência de fungos de campo a partir de 90 dias de armazenamento em ambiente natural. A classificação das sementes e as condições adequadas de armazenamento são fundamentais para manutenção da qualidade, caso contrário, os esforços anteriores para o desenvolvimento do material genético e as técnicas culturais para a produção das sementes não serão válidos. Nesse sentido, o objetivo desse trabalho foi avaliar a interferência da classificação e do armazenamento na qualidade das sementes de cevada. Foram utilizadas sementes de quatro lotes da cultivar BRS Cauê. O delineamento utilizado foi o inteiramente casualizado, sendo os tratamentos: I - controle – sementes não classificadas; II – sementes retidas na peneira 2,2 mm de largura x 22,0mm de comprimento; III – sementes retidas na peneira 2,2 mm de largura x 22,0mm de comprimento + mesa de gravidade; IV – sementes retidas na peneira 2,5 mm de largura x 22,0mm de comprimento; V – sementes retidas na peneira 2,5 mm de largura x 22,0mm de comprimento + mesa de gravidade. As avaliações foram realizadas no início do armazenamento e aos 45, 90, 150, 210 e 270 dias após armazenamento. As sementes permaneceram armazenadas em ambiente natural. Para a determinar a qualidade das sementes foram avaliados: o teor de água, o peso volumétrico, o peso de mil sementes, a quantidade de sementes infestadas, a germinação, o vigor, a sanidade e o teor de proteína. A infestação, peso hectolítrico, teor de água e teor de proteína, não foram submetidos a análise estatística, sendo os três primeiros realizados com duas repetições repetições. Os resultados de porcentagem do teste de sanidade foram transformados em arcsen √(x /100), e as demais variáveis foram submetidas a análise de variância e as médias comparadas pelo teste de Tukey a 5% de significância. Em geral, os resultados das sementes classificadas na peneira 2,5 X 22,0 mm associadas à mesa de gravidade apresentaram peso de mil sementes e peso hectolítrico superiores em relação aos resultados dos demais tratamentos. Porém, esses resultados não foram observados para os parâmetros fisiológico, sanitário e teor de proteína. Dessa forma, conclui-se que, o indicado para as sementes de cevada é a classificação na peneira 2,5mm + mesa de gravidade. Além disso, houve redução da qualidade das sementes de cevada e da incidência de fungos de campo a partir de 90 dias de armazenamento em ambiente natural.