The work in the dissertation expands the applications of DIEF and describes the development of incorporating DIEF in a microfluidic chip to create a comprehensive proteomics tool. Proof-of-concept DIEF experiments have been done previously, so the focus of this work is to explore the capabilities of DIEF. Dynamic isoelectric focusing (DIEF) is a separation technique invented by Dr. Luke Tolley. It is similar to capillary isoelectric focusing except it uses four high voltage electrodes to form a pH gradient instead of only two. The additional two electrodes are able to manipulate the pH gradient resulting in selection of the region and of the range of pH within a pre-defined sampling or extraction point. One of the first applications described for DIEF was to isolate a single protein from a complex mixture. The protein isolated was a cellulase enzyme capable of degrading multiple cellulose materials over a wide range of environmental conditions. DIEF did isolate the protein in a pH span of 0.005 which is equivalent to 0.075% of the total pH range. Fractions were collected for sequencing analysis, but the fractions were contaminated with keratin both times. DIEF was also successfully performed in an open air channel. Though other electromigration techniques have been successfully done in open air channels, these techniques were severely time and pH limited. In contrast, DIEF in an open air channel is capable of using the entire 3-10 pH range and can perform isolations until the proteins are completely separated. The device developed was also an improvement on increasing sample capacity. The channel was significantly bigger than the traditional glass capillaries used. Since the channel was open, fraction collection was made simpler by collecting using a pipette. This work also demonstrated that DIEF can be made through the use of silicone molding compounds and polyurethane. The amount of milling needed is reduced, the pieces are produced quickly, and a single mold can produce several pieces. Machining pieces with fragile bits is not needed to be done as much since only one acrylic piece is required produce a mold. The mold can produce several polyurethane pieces. This fabrication method has proven useful for making DIEF holders. The next step was to make DIEF a truly comprehensive proteomic tool by incorporating it into a microfluidic chip. Multiple sample fractions are rapidly generated on chip through the use of multiple bubbles simultaneously injected into the separation channel. This stops the separation and, since each droplet is isolated from others by a bubble on each side, the protein peaks are not able to broaden. This novel use of digital microfluidics is still a work in progress, but the fundamentals have been demonstrated. The fabrication protocol for making molds and PDMS casts was developed using materials and procedures that can be done in a common laboratory environment. DIEF is a separation technique still in its infancy, with a wide variety of available applications. DIEF will continue to be tested in other areas and developed into a comprehensive proteomic tool.

Submitted by JAILA FERREIRA MELO null (jailaferreiramelo@yahoo.com.br) on 2016-01-11T23:05:53Z No. of bitstreams: 1 dissertação corrigida 2.pdf: 744284 bytes, checksum: 8607fad88a80aa948935c605abd7eb0a (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-01-12T11:23:01Z (GMT) No. of bitstreams: 1 melo_jf_jabo_int.pdf: 744284 bytes, checksum: 8607fad88a80aa948935c605abd7eb0a (MD5) / Made available in DSpace on 2016-01-12T11:23:01Z (GMT). No. of bitstreams: 1 melo_jf_jabo_int.pdf: 744284 bytes, checksum: 8607fad88a80aa948935c605abd7eb0a (MD5) Previous issue date: 2015-12-18 / A cultura da laranja no Brasil é afetada por várias doenças, como a gomose, causada por Phytophtora nicotianae Breda de Haan (synonymous whit P. parasítica Dast.), cujo controle é comumente realizado com aplicações de fungicidas e medidas de exclusão. Porém, devido aos custos financeiros e problemas ambientais ocasionados pelo uso intenso destas aplicações, faz-se necessário a busca por métodos alternativos de controle. Este trabalho teve por objetivo avaliar 50 isolados de Trichoderma spp. como antagonista de P. nicotianae, sendo que os isolados foram avaliados, através da produção de compostos antimicrobianos por meio destes microrganismos, além da realização de um bioensaio com plântulas de alfafa. No cultivo pareado observou-se que todos os isolados inibiram o crescimento micelial do fitopatógeno. Na produção de compostos antimicrobianos, 41 isolados produziram compostos voláteis utilizando dextrose como fonte de carbono. No entanto, em estudos posteriores, foi verificado que a utilização de sacarose ou maltose como fontes de carbono, aumentou a produção destes compostos. Sete isolados de Trichoderma produziram compostos termoestáveis e 14 produziram compostos antimicrobianos livres de células do antagônico. No bioensaio com plântulas de alfafa foi observado que quatro isolados de Trichoderma inibiram a formação de esporângios e de micélios do fitopatógeno. Diante dos resultados obtidos, concluiu-se que as avaliações in vitro com relação à produção de compostos antimicrobianos pelos isolados de Trichoderma e o bioensaio com plântulas de alfafa mostraram capacidade para selecionar isolados com potencial para o controle de P. nicotianae. Os isolados TB12, TB14, TB28 e TB 30 foram os que proporcionaram maior controle da doença. / Orange crops in the Brazil are affected by various diseases such as gummosis caused by Phytophtora nicotianae Breda de Haan (synonymous whit P. parasítica Dast.), which is controlled by chemical fungicides. However, the high costs and environmental problems caused by the intensive use of these fungicides have led to the search for alternative methods of control. The objective of this study was to evaluate the antagonistic activity of 50 Trichoderma spp. isolates against P. nicotianae by the production of antimicrobial compounds and an alfalfa seedling bioassay. The paired culture showed that all isolates tested inhibited the mycelial growth of the pathogen. In the production of antimicrobial compounds, 41 isolates produced volatile compounds using dextrose as carbon source; however, further assays showed that the use of sucrose or maltose as carbon source increases the production of these compounds. Seven isolates of Trichoderma spp. produced thermostable compounds and 14 isolates produced cell-free culture antimicrobial compounds of the antagonist. In the alfalfa seedling bioassay, four isolates of Trichoderma spp. inhibited the formation of sporangia and mycelia growth. The evaluation in vitro concerning to the antimicrobial compound production by the Trichoderma spp. isolates and the alfalfa seedling bioassay both were able to select biocontrol agents to control of P. nicotianae. The Trichoderma spp. isolates, TB12, TB14, TB28, and TB30 presented the best disease control.

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Made available in DSpace on 2014-10-09T12:34:57Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:04:30Z (GMT). No. of bitstreams: 0 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP / FAPESP:08/00844-6

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Submitted by Claudinei Pracidelli (cpracide@ipen.br) on 2015-04-10T14:16:47Z No. of bitstreams: 0 / Made available in DSpace on 2015-04-10T14:16:47Z (GMT). No. of bitstreams: 0 / Tese (Doutorado em Tecnologia Nuclear) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP