Made available in DSpace on 2014-06-11T19:27:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-08-29Bitstream added on 2014-06-13T20:56:14Z : No. of bitstreams: 1 santos_cal_me_arafcf_parcial.pdf: 117464 bytes, checksum: 13283cebdcbc3d9085c0801450c381fd (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / A maioria dos casos de fraturas ósseas é utilizada nas cirurgias, implantes ortopédicos de osteossíntese (placa-parafuso e parafusos) confeccionados em aço inoxidável austeníticos, devido à sua baixa relação custo-benefício. As infecções associadas aos implantes de osteossintese estão relacionadas com o crescimento de microrganismos em biofilmes, resultando em um processo infeccioso de difícil erradicação. O objetivo do estudo foi identificar os microrganismos recuperados do conjunto metálico placaparafuso e parafusos após remoção cirúrgica, avaliar a capacidade de aderência dos microrganismos, verificar a viabilidade celular, caracterizar genotipicamente os isolados Gram-positivos e detectar a resistência aos antimicrobianos. Os conjuntos placa-parafuso e parafusos de aço inoxidável austenítico ASTM F138/F139 e ISO NBR 5832-1/9 foram transportados em bolsa de polietileno para o Laboratório de Microbiologia Clínica. Os implantes foram lavados em solução tampão fosfato-salino, armazenados em bolsa contendo solução Ringer e submetidos ao banho ultrassônico em freqüência de 40±2 kHz por 5 minutos. O fluido sonicado foi transferido para tubos Falcon e centrifugado a 3.000g durante 20 minutos. O sedimento foi resuspendido em nova solução Ringer, homogeneizado por vortex e 10μL semeados em ágar Sangue de carneiro 5%, MacConkey e Sabouraud. Os meios foram incubados em diferentes condições de temperatura por 7 a 14 dias para recuperação de microrganismos. O perfil de resistência dos isolados foi obtido de acordo com CLSI 2011. Para análise da viabilidade celular foi utilizado o kit Live/Dead e microscópio de fluorescência... / The majority of cases of bone fractures is used in surgery, internal fixation of orthopedic implants (screw-plate and screws) manufactured in austenitic stainless steel due to its low cost-benefit. Infections associated with implant fixation are related to the growth of microorganisms in biofilms, resulting in an infection difficult to eradicate. The objective of study was to identify the microorganisms recovered from the set screwplate and screws metallic after surgical removal, evaluate the capacity of adherence of microorganisms, check the cell viability and characterize the isolates genotypically Gram-positive and detect antimicrobial resistance. The sets screw-plate and screws of austenitic stainless steel ASTM F138/F139 and ISO NBR 5832-1/9 were transported in polyethylene bag to the Laboratory of Clinical Microbiology. The implants were washed in phosphate buffered saline solution, stored in bags containing Ringer's solution and submitted to ultrasonic bath at a frequency of 40 kHz ± 2 for 5 minutes. The sonicate fluid was transferred to falcon tubes and centrifuged at 3.000g for 20 minutes The new sediment was re-suspended in Ringer's solution, homogenized by vortex and 10μL seeded agar 5% sheep blood, MacConkey and Sabouraud. The media were incubated at different temperatures for up to 7 days for growth of CFU mL-1. The resistance profile of the isolates was obtained according to CLSI 2011. For analysis of cell viability kit was used for Live/Dead and fluorescence microscope. After microbiological analysis the isolation was observed in some samples of polymicrobial implants. The data obtained showed that bacteria were the most isolated coagulase-negative... (Complete abstract click electronic access below)

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The giant barrel sponge, Xestospongia muta is an iconic and essential species of the coral reefs in South Florida. The sponge has primary roles providing ecosystem services and creating unique habitats for diverse microbial communities. On April 27, 2012 an outbreak of Sponge Orange Band Disease (SOB) was detected off the coast of South Florida. The disease begins with sponge bleaching, followed by mesohyl or “mesohyl” necrosis and often total mesohyl disintegration. Sampling from two diseased populations at Boynton Beach and Fort Lauderdale, FL took place on May 11th and May 29th, 2012. Each of the nine diseased sponges from Boynton Beach and the five diseased sponges from Fort Lauderdale had three separate mesophyl samples collected to examine the effects of disease progression on the microbial community. These included healthy mesohyl from a diseased sponge (HoD), the boundary layer which captured the advancing line of diseased mesohyl (BL) and diseased mesohyl from a diseased sponge (D). Mesohyl from three sponges with no visible signs of SOB disease were also collected from each sampling location to use for healthy controls (HC). Sequencing of the V4 region of the 16S rRNA gene was performed on all of these samples via the “454” pyrosequencing on a Titanium GS FLX platform. The microbial communities associated with the diseased samples revealed a microbiome shift that followed the progression of Sponge Orange Band Disease (SOB) and was dominated by Bacteroidetes, Protebacteria and Chloroflexi. No singular or group of microbes were solely found within the infected mesohyl of Xestospongia muta from both sampling site populations; therefore there is no unequivocal candidate as a definite microbial causative SOB agent. But there were bacteria associated with disease progression that included Armatimonadetes, Caldithrix, Chlorobi, Fibrobacteres, Fusobacteria, GN02, KSB3, OP1, OP2, OP8, Planctomycetes, SR1, TM6, Tenericutes, Verrucomicrobia, WPS-2 and ZB3. Verrucomicrobia and Plantomycetes increased significantly within the D and the BL populations, which was consistent within all the diseased sponges. This study provides a deep sequencing profile of microbial communities within Xestospongia muta affected with SOB Disease and provides a new insight into the sponge healthy microbiome.

Master of Science / Department of Agronomy / Randall S. Currie / Mithila Jugulam / Italian ryegrass (Lolium perenne L. ssp. multiflorum (Lam.) Husnot), one of the problem weeds of the US, evolved resistance to multiple herbicides including glyphosate due to selection in Arkansas (AR). Glyphosate is a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitor and amplification of EPSPS gene, the molecular target of glyphosate confers resistance to this herbicide in several weed species, including Italian ryegrass from AR. The objective of this study was to determine the expression of EPSPS gene and protein as well as distribution of EPSPS copies on the genome of glyphosate-resistant Italian ryegrass (ARR) using a known susceptible Italian ryegrass (ARS) from AR. EPSPS gene copies and expression of ARR and ARS were determined using quantitative PCR with appropriate endogenous controls. EPSPS protein expression was determined using Western blot analysis. Fluorescence in situ hybridization (FISH) was performed on somatic metaphase chromosomes to determine the location of EPSPS copies. Based on qPCR analysis, ARR plants showed a wide range of 12 to 118 EPSPS copies compared to a single copy in ARS. EPSPS gene expression correlated with the gene copy number in both ARR and ARS. Individuals with high EPSPS copies showed high protein expression in Western blot analysis. FISH analysis showed presence of brighter EPSPS signals, distributed randomly throughout the genome of ARR individuals compared to a faint signal in ARS plants. Random distribution of EPSPS copies was previously reported in glyphosate-resistant Palmer amaranth. Overall, the results of this study will help understand the origin and mechanism of EPSPS gene amplification in Italian ryegrass.

A amplificação gênica pode ser compreendida como um mecanismo de regulação de expressão protéica em Leishmania. A exposição a concentrações não letais de diferentes drogas isoladamente, como o metotrexato, a primaquina, cloroquina e antimônio levam à amplificação de regiões específicas como as localizadas no cromossomo 6 e no cromossomo 23. Essas linhagens tornam-se resistentes a estas drogas e eventualmente apresentam resistência cruzada a outras. A terbinafina é uma substância sintética utilizada como antifúngico que age pela inibição da esqualeno epoxidase impedindo a síntese de ergosterol, componente da membrana celular de Leishmania. A exposição do parasita à terbinafina leva à amplificação da região H. A fragmentação da região H permitiu delimitar a resistência a 2,8 kb; fragmento T1. Por mutagênese insercional o gene de resistência à terbinafina foi definido e nomeado HTBF. A proteína codificada pelo HTBF possui uma extremidade N-terminal hidrofílica e C-terminal hidrofóbica com quatro alfa-hélices sendo, supostamente, uma proteína integral de membrana. Possui semelhança com a proteína Yip de Saccharomyces cerevisiae, que participa do transporte vesicular do retículo endoplasmático para o complexo de Golgi. O gene HTBF foi detectado em linhagem sensível de L. major e de outras espécies de Leishmania e seu transcrito em linhagens sensíveis e resistentes de L. major. Levantou-se a hipótese de se tratar de locus de resistência a múltiplas drogas já que as regiões amplificadas, mesmo que induzidas por uma determinada droga, podem levar a resistência a outras. A co-transfecção do gene de resistência ao antimonial MRPA no transfectante portador do HTBF levou à diminuição em até cinco vezes da capacidade de resistência ao antimônio. Apesar dos genes de resistência amplificarem juntos na região H, foi constatado que possuem ação contrária. Sendo que a terbinafina interfere na integridade da membrana celular, levantou-se hipótese na qual a resistência envolveria o mecanismo de reparo de membrana. A perda do fenótipo de resistência pela falta de cálcio sugerindo a inativação desse mecanismo é um fenômeno previsto corroborando a hipótese. / Gene amplification can be recognized as a protein regulation mechanism in Leishmania. Exposure to non-lethal concentrations of different drugs in isolated manner, such as, methotrexate, primaquine, chloroquine and antimony drives the amplification of specific regions. Examples for amplified regions are contained in chromosomes six and 23. The strains become resistant to these drugs and eventu-ally to other drugs as well. Terbinafine is a synthetic substance used as an antifungal agent. Its mechanism of action is by squalene epoxidase inhibition, blocking ergosterol synthesis, a cell membrane component in Leishmania. Parasite exposure to terbinafine elicits H region amplification. H region fragmentation allowed resistance delimitation to 2.8kb, T1 fragment. By insertional mutagenesis terbinafine resistance gene was defined and termed HTBF. HTBF coded protein has a hydrophilic N-terminal and a hydrophobic C-terminal containing four alpha-helixes, putatively, an integral membrane protein. It possesses homology to Saccharomyces cerevisiae Yip protein, which takes part in the vesicular transport from the endoplasmic reticulum to Golgi apparatus. HTBF gene was detected in sensitive L. major strains and other Leishmania species and its transcript was detected in both resistant and sensitive L. major strains. We raised the hypothesis of a multiple drug resistance locus, since amplified regions induced by one drug could elicit resistance to a second drug. Co-transfection of MRPA to the HTBF transfectant led to a five-fold decrease to antimonial resistance level. Even though these resistance genes amplify together in the H region, they have antagonizing mechanisms of action. Since terbinafine action disrupts membrane integrity, we raised a hypothesis in which resistance involves enhancement of membrane repair machinery. Loss of phenotype caused by the lack of calcium suggests the inactivation of this machinery is a predicted phenomenon, corroborating the hypothesis.

The ligand activated transcription factor retinoid X receptor (RXR) forms a DNA binding heterodimer with pregnane X rseceptor (PXR) in response to foreign xenobiotics. In addition to RXR and PXR there are other proteins involved in the RXR/PXR signaling pathway. Many proteins involved in this pathway are still unknown. This study documents the production of RXR and PXR in a bacterial recombinant fusion system. These proteins were expressed in a system that allowed purification with six histidine residues. Once the proteins were expressed and purified from E. coli, they were solublized and tested for function. Different strategies were employed including temperature and inducer studies and denaturing and renaturing techniques to solublize PXR. Following the solubilzation of each protein, all proteins were subjected to a method of functional analysis. RXR function was assessed by electrophoretic mobility shift assay (EMSA) and proved to effectively form a DNA binding heterodimer with PXR. These studies involving RXR and PXR demonstrate that these proteins can be efficiently produced in a functional manner utilizing an inexpensive bacterial system. In addition, this study documents various strategies for combating "inclusion body" formation in the overexpression ofPXR. Also, it describes the production of plasmid pCMV-RXR for transfection into the HepG2 cell line to monitor the levels of cellular RXR in various tissue types.

The resistance of Palmer amaranth (PA) and the tolerance (natural resistance) of pitted morningglory (PM) to glyphosate have made these species among the most common and troublesome weeds in the southeastern U.S. since the adoption of glyphosate-resistant (GR) crops. Populations of GR PA (R1 and R2) were identified in Mississippi. The inheritance of glyphosate resistance was examined in reciprocal crosses (RC) between glyphosate-resistant (R) and -susceptible (S) parents (Female-S × Male-R, S/R, and Female-R × Male-S, R/S), and second reciprocal crosses (2RC) (Female-S/R × Male-S/R, S/R//S/R, and Female-R/S × Male-R/S, R/S//R/S). Dose-response assays resulted in 17- to 4old resistance to glyphosate compared with S. Population S accumulated 325- and 8-times more shikimate at the highest glyphosate dose than in R1 and R2, respectively. cDNA sequence analysis of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene indicated no target site mutation. Genomes of R1, R2, RC, and 2RC contained from 1- to 59old more copies of EPSPS gene than S; EPSPS was highly expressed in R1 and R/S, but was poorly expressed in S, S/R, and R2. EPSPS activity was lower in S and S/R than in R and R/S, glyphosate absent; all were inhibited by glyphosate. Western Blot analysis confirmed an increased EPSPS protein level to EPSPS copy number correlation. Thus, the level of resistance was decidedly influenced by the direction of the cross. R and S female plants were reproductively isolated and seed were still produced, suggesting that PA can produce seed both apomictically and sexually (facultative apomixis). This mode of reproduction determined the low copy number inheritance, as well as guaranteeing the GR trait stability in the R populations. Dose-response assays resulted in 2.6old variability in tolerance to glyphosate between the most tolerant (MT) and the least tolerant (LT) PM populations. The level of tolerance positively correlated with the time of exposure to GR-crop system. Less shikimate was recovered in MT as compared to LT. Levels of aminomethylphosphonic acid (AMPA) were not different between populations and sarcosine was not present in either populations. Consequently, metabolism of glyphosate to AMPA or sarcosine is not a common factor in explaining natural resistance levels.