A citricultura brasileira se destaca no mercado global de citros, apresentando altos valores de produção e produtividade. Dentre as diversas doenças que afetam a cultura, a podridão floral dos citros (PFC) vem ganhando destaque com o deslocamento de áreas produtoras para regiões mais favoráveis à ocorrência desta doença. A PFC, causada por espécies dos complexos Colletotrichum acutatum e C. gloeosporioides, é especialmente problemática em anos de temperaturas amenas e alta umidade. Os citricultores realizam pulverizações preventivas para o controle da PFC todos os anos, embora condições climáticas favoráveis à doença ocorram apenas ocasionalmente. Além do impacto econômico, as frequentes pulverizações com fungicidas aumentam a pressão de seleção sobre isolados resistentes, o que pode interferir na eficiência do controle químico. A utilização de sistemas de previsão de epidemias pode evitar que pulverizações desnecessárias de fungicidas sejam realizadas caso não haja condições favoráveis à ocorrência da doença. Para o desenvolvimento destes sistemas, é imprescindível determinar um limiar de ação para a aplicação de fungicidas nos pomares. Dessa forma, o presente trabalho teve por objetivos: (i) estabelecer um limiar de ação para a aplicação de fungicidas com base na germinação de conídios de C. acutatum que permita o controle a doença igualmente ou de forma mais eficiente do que o sistema de pulverização adotado pelos citricultores do sudoeste paulista; (ii) caracterizar a sensibilidade de isolados dos complexos C. gloeosporioides e C. acutatum à trifloxistrobina e ao tebuconazol in vitro e molecularmente, para identificar possíveis mudanças de sensibilidade das espécies a estes fungicidas. Para a definição do limiar de ação, tratamentos baseados em índices de risco foram comparados ao tratamento testemunha e ao calendário fixo de aplicações, usualmente adotado pelos produtores. A aplicação de fungicidas quando limiar de 15% de germinação de conídios era atingido, foi eficiente em controlar a doença e reduzir o número de aplicações de fungicidas. Nos ensaios de sensibilidade a fungicidas dois métodos foram utilizados para a determinação da CE50: o da diluição em gradiente espiral para trifloxistrobina e tebuconazol e o da inibição da germinação de conídios para a trifloxistrobina. Foram utilizados isolados coletados entre 1999 e 2016. A CE50 média da coleção de isolados de acordo com o método da diluição em gradiente espiral variou de 0,158 a 0,297 μg/ml e 0,1 a 0,182 μg/ml para trifloxistrobina e tebuconazol, respectivamente. Para a trifloxistrobina, de acordo com o método da inibição da germinação, a CE50 média foi de 0,002 μg/ml. Não foram verificadas características moleculares nem valores de CE50 atrelados à mudança de sensibilidade dos isolados. / Brazilian citrus industry represents a significant share in the global citrus market. Amongst several diseases that affect the crop, postbloom fruit drop (PFD) has been gaining prominence in Sao Paulo with the displacement of citrus areas to regions in which weather conditions are more favorable to the occurrence of this disease. PFD, caused by species of the complexes Colletotrichum acutatum and C. gloeosporioides, is especially problematic in years of mild temperatures and high humidity. Citrus growers spray the orchards preventively for PFD control every year, although favorable climatic conditions do not occur regularly. In addition to the economic impact, this practice increases the selection pressure of fungicide resistant isolates, and may decrease the efficiency of chemical control in a long term. The use of disease forecasting systems is able to prevent unnecessary fungicide sprays. For the development of such systems, it is essential to determine an action threshold for the application of fungicides in the orchards. The objectives of this work were: (i) to establish an action threshold for fungicide sprays based on the germination of C. acutatum conidia; (ii) to characterize the sensitivity of C. gloeosporioides and C. acutatum isolates to trifloxystrobin and tebuconazole in vitro and molecularly. Regarding the definition of the action threshold, treatments based on risk indices were compared to the control treatments and calendar based sprays, usually adopted by growers. The 15% conidia germination threshold was efficient in controlling the disease and reducing the number of fungicide applications. In the fungicide sensitivity tests, two methods were used to determine the EC50, the spiral gradient dilution for trifloxystrobin and tebuconazole, and the method of conidial germination inhibition for trifloxystrobin. Isolates collected between 1999 and 2016 were used. The mean EC50 of the isolate collection determined by the spiral gradient dilution method ranged from 0.158 to 0.297 μg/ml and from 0.1 to 0.182 μg/ml for trifloxystrobin and tebuconazole, respectively. Mean EC50 of trifloxystrobin estimated by the conidial germination inhibition method was 0.002 μg/ml. No mutations or EC50 values indicated shifts of fungicide sensitivity on the isolates.

Rhizoctonia blight (sensu lato) is a common and serious disease of many turfgrass species. The most widespread causal agent R. solani consists of several genetically different anastomosis groups (AGs) and subgroups. Though anastomosis or hyphal fusion reactions have been used to group Rhizoctonia species, they are time consuming and sometimes difficult to interpret. Anastomosis reactions are incapable of identifying isolates belonging to different AG subgroups within an AG. This study evaluated molecular techniques in comparison with traditional anastomosis grouping (AG) to identify and group isolates of Rhizoctonia. More than 400 Rhizoctonia isolates were collected from diseased turfgrass leaves from eight geographic areas in Virginia and Maryland. A random sample of 86 isolates was selected and initially characterized by colony morphology, nuclei staining and anastomosis grouping. Molecular identification was performed by analysis of rDNA-ITS region and DNA fingerprinting techniques universally primed PCR (UP-PCR) and amplified fragment length polymorphism (AFLP). The cladistic analysis of ITS sequences and UP-PCR fragments supported seven clusters. Isolates of R. solani AG 1-IB (n=18), AG 2-2IIIB (n=30) and AG 5 (n=1) clustered separately. Waitea circinata var. zeae (n=11), and var. circinata (n=4) grouped separately. A cluster of six isolates (UWC) did not fall into any known Waitea group. Most of the binucleate Rhizoctonia-like fungi (BNR) (n=16) grouped separately. AFLP grouping also largely agreed with the above results. However, UWC isolates clustered into two groups. Molecular analyses corresponded well with traditional anastomosis grouping by clustering isolates within an AG or AG subgroup together. UP-PCR cross-hybridization could distinguish closely related Rhizoctonia isolates to their infraspecies level. Genetically related isolates belonging to the same AG subgroups cross-hybridized strongly, while isolates of different AGs did not cross-hybridize or did so weakly. Sequence-characterized amplified region (SCAR) markers were generated from UP-PCR products to identify isolates of major pathogenic groups AG 1-IB and AG 2-2IIIB. Specific primer pairs successfully distinguished isolates of AG 1-IB and AG 2-2IIIB from isolates of other AGs. Sensitivity of Rhizoctonia species and AGs was tested in vitro to commercial formulations of iprodione, triticonazole and pyraclostrobin. W. circinata isolates were moderately sensitive to iprodione while isolates of R. solani and BNR were extremely sensitive. Isolates of AG 2-2IIIB showed less sensitivity to triticonazole than other Rhizoctonia isolates. W. circinata var. zeae isolates were moderately sensitive to pyraclostrobin while most of the other isolates were extremely sensitive. / Ph. D.

The amphibian decline phenomenon now involves in excess of a third of the roughly 6000 species of amphibians on the planet. The problems that drive the declines are diverse with no end in sight. The Amphibian Conservation Action Plan (ACAP) aims to stem amphibian decline through four recommended actions by researchers and conservation biologists: (1) Expand scientific understanding of amphibian declines and extinctions; (2) continue to document amphibian diversity and ecology and how they are changing; (3) develop and implement long-term conservation programs; (4) prepare emergency response actions for eminent crises. This Dissertation focused on two of those recommendations: expanding scientific understanding of amphibian declines and extinctions and continuing to document amphibian diversity and ecology and how they are changing. The first chapter is a review of the amphibian decline phenomenon. The second, third, and fourth chapters focus on expanding scientific understanding of amphibian diversity and ecology with the description of a formerly unknown species (chapter 2), and ecological papers on two poorly known species (chapters 3 and 4). Chapter five focuses on the first ACAP recommendation in improving scientific understanding of the causes behind amphibian decline. The chapter is an experimental examination of two related species and their developmental reactions to common heavy metal contaminants. The goal of this Dissertation is to contribute toward the general amphibian knowledge base relative to the recommendations of ACAP.

Mefenoxam has been a premier compound for Phytophthora disease control in the nursery industry for 30 years. The primary objectives of this research were to examine whether Phytophthora species have developed resistance to this compound and to investigate fungicide resistance management strategies. Phytophthora nicotianae, a destructive pathogen of numerous herbaceous and some woody ornamental plants, was used as a model system. P. cinnamomi, a major pathogen of a wide range of tree species and shrub plants, was also included for comparison. Twenty-six isolates of P. nicotianae were highly resistant to mefenoxam with a mean EC50 value of 326.5 µg/ml while the remaining 70 were sensitive with an EC50 of <0.01 µg/ml (Label rate: 0.08µg/ml). All resistant isolates were recovered from herbaceous annuals and irrigation water in 3 Virginia nurseries. Resistant isolates were compared with sensitive ones using seedlings of Lupinus "Russell Hybrids" in the absence of mefenoxam for relative competitive ability. Resistant isolates out-competed sensitive ones within 3 to 6 sporulation cycles. Resistant isolates exhibited greater infection rate and higher sporulation ability than sensitive ones. No mefenoxam resistant isolates were identified in P. cinnamomi. All 65 isolates of P. cinnamomi were sensitive to mefenoxam with an EC50 of < 0.04 ï ­g/ml. Attempts to generate mutants with high resistance to mefenoxam through UV mutagenesis and mycelial adaptation were not successful. However, there were significant reductions in sensitivity to mefenoxam; those slightly resistant mutants carried fitness penalties, which may explain why P. cinnamomi remains sensitive to mefenoxam. The effect of propamocarb hydrochloride on different growth stages of Phytophthora nicotianae was evaluated in search for an alternative fungicide. Propamocarb greatly inhibited sporangium production, zoospore motility, germination and infection. However, it has little inhibition of mycelial growth and infections. Propamocarb can be used as an alternative fungicide to mefenoxam where mefenoxam resistance has become problematic. However, it must be used preventively; i.e. before infections occur. The genetic inheritance of mefenoxam resistance in P. nicotianae was studied using F1 progenies of a cross between resistant and sensitive isolates. The F1 progenies segregated for mefenoxam resistance in ratio of 1R:1S, indicating the mefenoxam resistance is controlled by a single dominant gene. One RAPD marker putatively linked to resistant locus in repulsion phase was obtained by bulked segregant analysis and was converted to the SCAR marker. This marker is capable of differentiating mefenoxam resistant populations from sensitive populations included in this study. / Ph. D.

7 ABSTRACT Crystallization of the zebrafish P2X4 receptor in both open and closed states revealed conformational differences in the ectodomain structures, including the dorsal fin and left flipper domains. The role of these domains in forming of ATP-binding pocket and receptor function was investigated by using alanine scanning mutagenesis of the R203- L214 (dorsal fin) and the D280-N293 (left flipper) sequences of the rat P2X4 receptor and by examination of the responsiveness to ATP and orthosteric analog agonists 2- (methylthio)adenosine 5'-triphosphate, adenosine 5'-(γ-thio)triphosphate, 2'(3'-O-(4- benzoylbenzoyl)adenosine 5'-triphosphate, and α,β-methyleneadenosine 5'- triphosphate. ATP potency/efficacy was reduced in 15 out of 26 alanine mutants. The R203A, N204A, and N293A mutants were essentially non-functional, but receptor function was restored by ivermectin, an allosteric modulator. The I205A, T210A, L214A, P290A, G291A, and Y292A mutants exhibited significant changes in the responsiveness to orthosteric analog agonists. In contrast, the responsiveness of L206A, N208A, D280A, T281A, R282A, and H286A mutants to analog agonists was comparable to that of the wild type receptor. These experiments, together with homology modeling, indicate that residues of the first group located in the upper part of...

Study of biosensors has become an essential part of research in biotechnology. Biosensors as fast, portable, highly sensitive, and low-cost bioanalytical detection devices have been utilized in many fields of human activity. The first part of the presented work focuses on electrochemical biosensors for rapid environmental screening of herbicides as water pollutants. A sol-gel immobilization method for a photosystem II (PSII) complex is studied in order to enhance the sensitivity and the signal strength and stability of a PSII-based biosensor. Computer simulations of a PSII biosensor are employed with the aim to find out how the immobilization membrane properties influence the biosensor parameters. Newly developed immobilization by a thin-layer membrane based on the results of computer simulations and revised measurement protocols are presented. The second part of the work is devoted to synthesis and electrochemical detection of newly developed metal labels for electrochemical immunosensors. The synthesis of dendrimer-encapsulated silver nanoparticles and biorecognition properties of biotin-nanocomposite conjugates are discussed. For detection of synthesized labels, a microfluidic detector was manufactured and tested and different approaches to packing of a microfluidic chip employing polydimethylsiloxane (PDMS) were investigated. Newly designed microstructures for a microfluidic separator of magnetic beads (MBs) were studied by computer simulations. The separator was made and trapping of MBs for the further employment in MBs-based immunoassays are presented

Magister Scientiae (Medical Bioscience) - MSc(MBS) / This study investigated the effects of TKIs on the growth and proliferation of MCF-7 breast carcinoma cells in culture. MCF-7 cells were exposed to different concentrations of TKIs alone and in combination with each other. Inhibition of cell growth by TKIs used individually occurred in a dose- and time-dependent manner. When EGFR Inhibitor I, EGFR Inhibitor II/BIBX1382 and the multi-specific EGFR/ErbB-2/ErB-4 Inhibitor were used in combination with each other at equimolar log dose concentrations, the combined effects on cell growth was significantly different to inhibitors used individually as reflected in a decreased EC50 (IC50) during combination treatments. Generally, for the combinations with DOX, CPL and the TKIs, synergistic as well as antagonistic effects were observed at isoeffective concentrations with resultant decreases in dose reduction indices (DRIs) implying greater efficacies with the respective combinations. In this study, conventional PCR was used to detect and illustrate the presence of the EGFR gene in the samples, while RT-qPCR was used to determine the mRNA expression levels of this gene in MCF-7 breast carcinoma cells