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Glyphosate-resistant (GR) giant ragweed (Ambrosia trifida L.) has been confirmed in several states across the mid-western and mid-southern U.S. Greenhouse and lab studies were conducted to investigate possible mechanism of glyphosate resistance in a suspect population from Monroe County, Mississippi. Translocation of 14C-glyphosate in the susceptible biotype was 77%, compared to 12% in the resistant biotype at 120 hours after treatment, suggesting that the glyphosate resistance mechanism for this giant ragweed biotype is reduced translocation. Dose response studies were conducted to confirm and characterize glyphosate resistance in suspect biotypes from Mississippi (MS-R) and Tennessee (TN-R). The ED50 for MS-R and TN-R were 3.9- and 6.3-fold higher than a susceptible biotype. Results from a fallow field study conducted in 2016 in Monroe County revealed PRE and POST treatments containing dicamba and mesotrione alone and in various combinations provided effective control of GR giant ragweed. Studies were conducted to measure fitness, phenotypic, and genetic variation among GR biotypes from MS-R, TN-R, and Ohio (OH-R). Non-destructive measurements of plants over an eight-week period revealed rapid early growth of two GR accessions from MS in the absence of glyphosate. However, no differences in vegetative biomass were recorded after eight weeks with the exception of OH-R biotype which exhibited lower biomass due to photoperiod sensitivity. Vegetative biomass and fecundity were similar. Multivariate and PCA analysis of traits grouped biotypes based on state of origin. Groupings by state of origin can be significant as managers could design similar methods of control to address giant ragweed in these areas. Simple sequence repeat (SSR) markers were used to record genetic diversity among and within biotypes. Genetic diversity values were high at 0.514, 0.502, and 0.525 within biotypes from MS, TN, and OH, respectively. However, genetic diversity did not differ due to glyphosate response or level of glyphosate resistance. High levels of genetic variation can be an indicator of the ability of giant ragweed biotypes to adapt to changing environments and conditions.

No description available.

Resumo: O fator de transcrição mitocondrial A (gene Tfam), uma proteína fundamental para a ocorrência do processo de transcrição mitocondrial e um participante na replicação do genoma dessa organela, atua indiretamente na biogênese e oxidação de lipídeos. O objetivo do presente estudo foi de caracterizar e verificar a relação entre polimorfismos na região promotora do gene Tfam com características de carcaça em novilhas Nelore. Foram analisadas 272 novilhas da raça Nelore pertencentes a três rebanhos experimentais da Estação Experimental de Zootecnia de Sertãozinho - (EEZS), unidade de pesquisa do Instituto de Zootecnia, dois deles selecionados (NeS e NeT) para peso ao ano (machos) e ao sobreano (fêmeas) e um rebanho controle (NeC), em que os animais são selecionados para a média desses pesos. O DNA genômico foi extraído a partir do sangue dos animais, seguindo-se a amplificação do fragmento analisado e posterior digestão com enzimas de restrição. As medidas de gordura de cobertura foram obtidas por ultra-sonografia entre 555 e 800 dias de idade. Pela análise de PCR-RFLP-Hae III foi observada a presença de três genótipos (AA, CA e CC) e, pela análise de PCR-RFLP-Mbo I foram observados apenas dois genótipos (CC e TC). Não houve associação significativa entre os polimorfismos observados no gene Tfam, com as enzimas Hae III e Mbo l, e a característica espessura de gordura de cobertura. Entretanto, os resultados sugerem uma associação entre o polimorfismo detectado pela enzima Hae III e a área do músculo Longissimus dorsi. / Abstract: The mitochondrial transcription factor A (Tfam) is an essential protein for the transcription and genome replication processes of this organelle which also has indirect effects on lipids biosynthesis and oxidation. This study was conducted on intent to verify the existence of polymorphisms on the promoter region of Tfam gene and verify their possible relationships with carcass traits from Nelore heifers (Bos indicus). Genomic DNA were extracted from blood samples of 272 animals followed by PCR isolation/amplification of Tfam gene and application of the RFLP with thr restriction enzymes Hae III and Mbo I. The carcass traits considered were rib eye and fat thickness and were obtained by ultrasound between the ages of 555 and 800 days. The analysis with PCR-RFLP Hae III and Mbo I showed the migration patterns AA, CA, CC and CC,TC, respectively. There was no significant association among the patterns obtained and the fat thickness. However, the results showed an associated effect among the patterns obtained with PCR-RFLP Hae III and rib eye, which indicates the possible use of Tfam as a candidate gene. / Orientadora: Lúcia Galvão de Albuquerque / Coorientadora: Maria Eugênia Zerlotti Mercadante / Banca: Humberto Tonhati / Banca: Antonio Roberto Otaviano / Mestre

Aquesta tesis doctoral està basada en estudiar la resposta immunològica dels peixos en models d'estrès i d'activació del sistema immune des la genòmica funcional. L'aplicació de tecnologies moleculars com el Differential Display van permetre identificar y clonar por primera vegada en orades (Sparus aurata) y en altres especies de peix, el gen enolasa. Aquest enzim glucolític s'ha plantejat per primera vegada com un bon marcador molecular per estudiar el benestar dels peixos. Per mitjà de l'ús d'una plataforma de microarrays dissenyada específicament per a salmònids, i altres metodologies biomoleculars, es va comprovar que els nivells d'enolasa eren regulats en diferents teixits y en diferents especies de peix, com també en adverses situacions per l'animal. D'altra banda, s'han estudiat diferents gens immunològics candidats a ser possibles gens per l'estudi del sistema immunològic dels peixos. Aquests gens s'han estudiat a nivell d'expressió en teixits de truites (Oncorhynchus mykiss) mitjançant PCR convencional i PCR quantitativa, i l'ús de metodologies biomoleculars i bioinformàtiques. Entre ells, destaca el factor de transcripció PU.1, un gen indispensable per el desenvolupament de l'hematopoesi. Aquest gen, s'ha clonat i caracteritzat per primera vegada en salmònids. L'expressió de PU.1 s'ha estudiat mitjançant l'ús d'hibridacions in situ en ronyó anterior y en cervell de truita. A més, l'ús de microarray en aquest dos teixits han permès fer un estudi exhaustiu i pioner a nivell de transcriptòmica en peixos. Les anàlisis del xip de microarray, ha revelat que grups de gens s'activen o s'inhibeixen com a conseqüència d'un estrès immunològic.En resum, aquesta tesis doctoral ha aplicat el desenvolupament de noves tecnologies moleculars pioneres en peixos, com el microarray, la clonació de noves seqüències gèniques i la bioinformàtica, per estudiar la genòmica funcional dels peixos en situacions d'activació dels mecanismes d'estrès i del sistema immune. / The main results of the present thesis can be integrated to a better understanding the stress and the immune responses in fish at a transcriptional level. The application of functional genomic tools, which encloses from using simple PCR analysis to more modern, sophisticate and fashionable microarray technique, allowed us to identified transcriptional regulations of certain set of genes which are enhanced or repressed under stress conditions. Our findings contribute to increase knowledge of molecular mechanism involved in coping the stress and immune responses in fish and provides a better understanding of fish physiology when fish health is threatened. Furthermore, thesis results may be interesting for aquaculture which looks for good biomolecular markers that may improve fish production and fish quality. The isolation, characterization and gene expression study with further microarray analysis of the enolase gene, allowed us to describe enolase as a possible biomolecular marker to determine fish welfare. The in situ hybridization study of the hematopoietic transcription factor PU.1, contributed to amplify the knowledge of the development of the fish immune system. Throughout this thesis, DNA sequences and mRNA expression levels of several genes studied, have contributed to enlarged genomic fish database. In summary, this thesis described from a transcriptional level, gene expression and molecular mechanisms activated or repressed when fish welfare is threatened and contributes to a better understanding of transcriptiomic mechanisms required to cope with the stress.

Made available in DSpace on 2014-06-11T19:26:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-07-29Bitstream added on 2014-06-13T19:33:21Z : No. of bitstreams: 1 ayres_dr_me_jabo.pdf: 301436 bytes, checksum: 880c0c14e46f06bc104b475866c16dcf (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O fator de transcrição mitocondrial A (gene Tfam), uma proteína fundamental para a ocorrência do processo de transcrição mitocondrial e um participante na replicação do genoma dessa organela, atua indiretamente na biogênese e oxidação de lipídeos. O objetivo do presente estudo foi de caracterizar e verificar a relação entre polimorfismos na região promotora do gene Tfam com características de carcaça em novilhas Nelore. Foram analisadas 272 novilhas da raça Nelore pertencentes a três rebanhos experimentais da Estação Experimental de Zootecnia de Sertãozinho – (EEZS), unidade de pesquisa do Instituto de Zootecnia, dois deles selecionados (NeS e NeT) para peso ao ano (machos) e ao sobreano (fêmeas) e um rebanho controle (NeC), em que os animais são selecionados para a média desses pesos. O DNA genômico foi extraído a partir do sangue dos animais, seguindo-se a amplificação do fragmento analisado e posterior digestão com enzimas de restrição. As medidas de gordura de cobertura foram obtidas por ultra-sonografia entre 555 e 800 dias de idade. Pela análise de PCR-RFLP-Hae III foi observada a presença de três genótipos (AA, CA e CC) e, pela análise de PCR-RFLP-Mbo I foram observados apenas dois genótipos (CC e TC). Não houve associação significativa entre os polimorfismos observados no gene Tfam, com as enzimas Hae III e Mbo l, e a característica espessura de gordura de cobertura. Entretanto, os resultados sugerem uma associação entre o polimorfismo detectado pela enzima Hae III e a área do músculo Longissimus dorsi. / The mitochondrial transcription factor A (Tfam) is an essential protein for the transcription and genome replication processes of this organelle which also has indirect effects on lipids biosynthesis and oxidation. This study was conducted on intent to verify the existence of polymorphisms on the promoter region of Tfam gene and verify their possible relationships with carcass traits from Nelore heifers (Bos indicus). Genomic DNA were extracted from blood samples of 272 animals followed by PCR isolation/amplification of Tfam gene and application of the RFLP with thr restriction enzymes Hae III and Mbo I. The carcass traits considered were rib eye and fat thickness and were obtained by ultrasound between the ages of 555 and 800 days. The analysis with PCR-RFLP Hae III and Mbo I showed the migration patterns AA, CA, CC and CC,TC, respectively. There was no significant association among the patterns obtained and the fat thickness. However, the results showed an associated effect among the patterns obtained with PCR-RFLP Hae III and rib eye, which indicates the possible use of Tfam as a candidate gene.

The increasing demand for recombinant therapeutic proteins coupled with advances in technologies allow research to develop approaches to improve the efficiency, yield, and quality of biopharmaceutical products from CHO cells. CHO DG44 cells used in this study were engineered to express erythropoeitin (EPO) as the model recombinant protein in a DHFR-based selection system. From a series of CHO-DG44 cell lines derived from a polyclonal population, one cell line expressed a notable change in growth phenotype during prolonged culture (10 weeks). This cell line (IJ4) exhibited prolonged growth, reached a greater density, and delayed cell death. The change in growth was reflected in an increased total yield of EPO, whilst the specific productivity of cell line IJ4 remained similar. The increased total yield of EPO presents a desirable goal for production and hence detailed ‘omics studies were performed to identify factors associated with better cell growth and survivability. Two different ‘omics analyses were performed (microarray transcriptomic and GC-MS metabolic profiling) to identify potential target genes and key metabolites associated with changes in growth profile. The -omics analyses identified a subset of genes (MMP20, PLA1A, POSTN, SLC46A3, and TOP2A), and a metabolic marker (farnesal) strongly associated with changes in cell growth and nutrient uptake. The use of complementary ‘omics approaches to identify molecular markers has allowed an integrated model to be built, which explains how CHO cell phenotype can adapt to long-term culture, and this defines molecular approaches for cell line screening and engineering.

Maize (Zea mays L.) is one of the world’s largest food crops and thus any pathogens of maize are of great importance. Aspergillus flavus is one of these pathogens and it produces a carcinogenic metabolite called aflatoxin. Efforts to reduce infection by A. flavus and subsequent aflatoxin accumulation include the development of maize lines resistant to aflatoxin accumulation. However, resistant lines that have been developed contain agronomically unfavorable traits. Gene-based markers would allow for easier transfer of resistance from resistant inbred lines into maize lines with good agronomic traits. The focus of this research was the development of gene-based markers for resistance to aflatoxin accumulation. To this end, two genes were characterized for their association with reduced aflatoxin accumulation in maize. A gene coding for a photosytem II3 protein shown to be differentially regulated between maize lines Mp313E (resistant) and Va35 (susceptible) was used to develop the marker MpM1. This marker was shown to be associated with resistance to aflatoxin accumulation in three F2:3 mapping populations derived from Mp313E x B73, Mp313E x Va35, and Mp715 x T173 and identified a new quantitative trait locus (QTL) on chromosome 4. The second gene chosen was the chitinase A gene (chiA), which has been shown to inhibit fungal growth and is differentially regulated between resistant and susceptible lines of maize. ChiA also had an association with reduced aflatoxin accumulation in the three F2:3 mapping populations and identified a new QTL in the Mp313E x Va35 population. Together, MpM1 and chiA were associated with 27% of the phenotypic variation in one environment of the Mp313E x B73 population. These markers represent the first two gene-based markers developed for resistance to aflatoxin accumulation, and the methodology developed in this study can be used to screen other candidate genes for potential use as gene-based makers.

There is a pressing need to develop strategies to reduce the emergence of macrocyclic lactone anthelmintic resistance in sheep flocks. Management practices aimed at maintaining anthelmintic susceptible nematodes in refugia while achieving a satisfactory level of production may prove to be useful. However, sensitive molecular tests are required to monitor the subtle effects of these practices on the frequency of resistance alleles within nematode populations. To-date, conventional studies of candidate genes coding for the known methods of action of macrocyclic lactone anthelmintics have produced a complex picture, highlighting the relevance of different approaches to the identification of resistance markers. This thesis describes the development of a single nematode parent genetic crossing method and discusses its application to identify molecular markers for anthelmintic resistance. Parasitological and molecular verification of successful inbreeding of the MHco3 strain of H. contortus derived from the progeny of a genetic cross between single nematode parents is described. The single parent genetic crossing method has enabled the production of diverse inbred lines of the MHco3 H. contortus and may prove useful for genome assembly, or for the development of a genetic map. The study has afforded insights to the biology of H. contortus and effects of host immunity on nematode parasites. New information is presented concerning the period during which adult female nematodes continue to shed fertilised eggs after removal of males, the development of unfertilised H. contortus eggs, and the population genetics of mixed infections of two different strains of H. contortus. Novel backcrossing experiments initially between a macrocyclic lactone resistant (MHco4 or MHco10) and a susceptible (MHco3) strain of H. contortus and then between ivermectin treated backcross generations and the parental susceptible strain are described. The resources provided by these experiments should enable comparative genomic analysis and conventional molecular biology to identify resistance genes derived from the parental resistant strains in fourth backcross generations that are the same as a parent ivermectin susceptible population, apart from the presence of alleles linked to anthelmintic resistance, derived from parent resistant strains.