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Doctor of Philosophy / Department of Agronomy / Mithila Jugulam / Palmer amaranth (Amaranthus palmeri) is one of the most aggressive, troublesome and damaging broadleaf weeds in many cropping systems including corn, soybean, cotton, and grain sorghum causing huge yield losses across the USA. As a result of extensive and intensive selection of pre- and -post emergence herbicides, Palmer amaranth has evolved resistance to multiple herbicide modes of action, microtubule-, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS)-, acetolactate synthase (ALS)-, photosystem II (PS II)-, hydroxyphenylpyruvate dioxygenase (HPPD)- and more recently to protoporphyrinogen oxidase (PPO)-inhibitors. A Palmer amaranth population from Kansas was found resistant to HPPD-, PS II-, and ALS-inhibitors. The overall objective of this research was to investigate the target-site and/or non-target-site resistance mechanisms in Palmer amaranth from KS (KSR) to mesotrione (HPPD-inhibitor), atrazine (PS II-inhibitor), and chlorsulfuron (ALS-inhibitor) relative to known susceptible Palmer amaranth from Mississippi (MSS) and KS (KSS). Whole plant dose-response assays showed high level of resistance in KSR to mesotrione, atrazine and chlorsulfuron. KSR was 10-18, 178-237 and >275 fold more resistant to mesotrione, atrazine, and chlorsulfuron, respectively, compared to MSS and KSS. Metabolism studies using [¹⁴C] labeled mesotrione and atrazine demonstrated non-target-site resistance to both herbicides, particularly, enhanced metabolism of [¹⁴C] mesotrione likely mediated by cytochrome P450 monooxygenases and rapid degradation of [¹⁴C] atrazine by glutathione S-transferases (GSTs). In addition, molecular and biochemical basis of mesotrione resistance was characterized by quantitative PCR (qPCR) and immunoblotting. These results showed 4-12 fold increased levels of the HPPD transcript and positively correlated with the increased HPPD protein. Sequencing of atrazine and chlorsulfuron target genes, psbA and ALS, respectively, showed interesting results. The most common mutation (serine264glycine) associated with atrazine resistance in weeds was not found in KSR. On the other hand, a well-known mutation (proline197serine) associated with chlorsulfuron resistance was found in 30% of KSR, suggesting ~70% of plants might have a non-target-site, possibly P450 mediated metabolism based resistance. Over all, KSR evolved both non-target-site and target-site based mechanisms to mesotrione and chlorsulfuron with only non-target-site based mechanism of resistance to atrazine leaving fewer options for weed control, especially in no-till crop production systems. Such multiple herbicide resistant Palmer amaranth populations are a serious threat to sustainable weed management because metabolism-based resistance may confer resistance to other herbicides and even those that are yet to be discovered. The findings of this research are novel and valuable to recommend appropriate weed management strategies in the region and should include diversified tactics to prevent evolution and spread of multiple herbicide resistance in Palmer amaranth.

Human enterovirus 71 (HEV71) is a member of the Human Enterovirus A species within the Family Picornaviridae. Since 1997, HEV71 has emerged as a major cause of epidemics of hand, foot and mouth disease (HFMD) associated with severe neurological disease in the Asia-Pacific region. At the present time, little is known about the pathogenesis of acute neurological disease caused by HEV71. The major aim of this study was to generate infectious cDNA clones of HEV71 and use them as tools for investigating the biology of HEV71 and molecular genetics of HEV71 virulence and pathogenesis. Two infectious cDNA clones of HEV71 clinical isolates, 26M (genotype B3) and 6F (genotype C2) were successfully constructed using a low copy number plasmid vector and an appropriate bacterial host. Transfection of cDNA clones or RNA transcripts derived from these clones produced infectious viruses. Phenotypic characterisation of clone-derived viruses (CDV-26M and CDV-6F) was performed, and CDV-26M and CDV-6F were found to have indistinguishable phenotypes compared to their wild type viruses. Strains HEV71-26M and HEV71-6F were found to have distinct cell culture growth phenotypes. To identify the genome regions responsible for the growth phenotypes of the two strains a series of chimeric viruses were constructed by exchanging the 5„S untranslated region (5„S UTR), structural protein (P1), and nonstructural protein (P2 and P3) gene regions using infectious cDNA clones of both virus strains. Analysis of reciprocal virus chimeras revealed that the 5„S UTR of both strains were compatible but not responsible for the observed phenotypes. Both the P1 and P2-P3 genome regions influence the HEV71 growth phenotype in cell culture, phenotype expression is dependent on specific P1/P2-P3 combinations and is not reciprocal. In the previous study, in order to investigate the pathogenesis of HEV71 infection, a mouse HEV71 model was developed using a mouse-adapted variant of HEV71-26M. Mouse-adapted strain MP-26M caused fore- and/or hindlimb paralysis in mice, whereas HEV71-26M-infected mice did not develop clinical signs of infection at any virus dose or route of inoculation tested. In this study, the molecular basis of mouse adaptation by HEV71 was identified. Nucleotide sequence analysis of HEV71-26M and MP-26M revealed three point mutations in the open reading frame, each resulting in an amino acid substitution in the VP1, VP2 and 2C proteins; no mutations were identified in the untranslated regions of the genome. To determine which of the three amino acid mutations were responsible for the adaptation and virulence of HEV71-26M in mice, recombinant cDNA clones containing one, or a combination of two or three mutations, were constructed. Mouse virulence assays of the mutated viruses clearly demonstrated that a non-conservative amino acid substitution (G710„_E) in the capsid protein VP1 alone was sufficient to confer the mouse virulence phenotype on HEV71. In addition, a mouse oral infection model was established in this study. Oral inoculation with the mouse-adapted HEV71 virus, MP-26M, induced fore-or hindlimb paralysis in newborn mice in an age- and dose-dependent manner. As oral transmission is the natural route of HEV71 infection, this murine HEV71 oral infection model will provide a suitable tool for studying HEV71 pathogenesis, for defining neurological determinants, and for testing vaccine efficacy and immunogenicity in the future.

Erwinia amylovora, a quarantine organism of the European Union (EU), is the causal agent of fire blight. This disease causes substantial economic losses in all countries where it is present and its control turns out difficult, due to the absence of effective chemical and biological treatments and the ability of persistence and dissemination of E. amylovora. Cupric treatments constitute the base of the integrated management of fire blight in the European Union countries, because the antibiotics, although have been proved useful against this disease, are forbidden in the EU for plant treatments. This thesis, mostly performed in a P2 security lab, is aimed to dilucidate molecular mechanisms implicated in the response of E. amylovora to copper sulfate as a stress factor, considering that copper is a well known toxic element for bacterial cells over a certain threshold concentration. The global objective was first addressed with the study of a selection of genes that have been related in other bacterial models with copper stress or with stress in general. The quantification of the rpoS gene expression in presence of copper showed that, at least in long-term survival, this gene may be involved in the E. amylovora response to copper stress. Second, a transcriptomic study was performed by microarray after subdue the bacteria to a copper shock treatment. The analysis of the microarray results showed that 44 genes were differentially expressed in presence of this metal. Each one of these genes was studied by gene ontology and, after comparing them with databases published in NCBI, they were classified in functional categories. The gene expression of twenty-five out of fourty-four differentially expressed genes was validated by real-time PCR. In the validation, copA gene was expressed more than 19-fold in presence than in absence of copper and, because of that, it was selected together with other seven genes (soxS, yjcE, ygcF, yhhQ, galF, arcB, EAM_3469), which also showed an increased expression, to generate mutants of E. amylovora. The responses of mutants to copper, and the fact that the wild phenotype was restored in the complemented mutants, has shown the role of copA, soxS, yjcE, ygcF, arcB and yhhQ genes in the E. amylovora in vitro survival against copper stress. Besides, the implication of copA gene has also been proved in planta, in copper treated shoots from pear trees. Finally, all the results obtained along this thesis have allowed to elaborate a putative model of the different genetic mechanisms that seem are involved in the interaction between E. amylovora and copper. The most important mechanism seems to be to face up reactive oxygen species (ROS) by the activation of the soxS and yjcE genes. The activity of these genes is supported by CopA protein, which pumps copper from inside the cell out to the periplasmic space. The activation of arcB gene, which allows the change from aerobic metabolism to anaerobic metabolism, would also help E. amylovora to reduce ROS. Taking together, the results of this thesis have allowed an approximation to the genetic basis of E. amylovora response to copper stress and they constitute a start point to move forward in the knowledge of the molecular mechanisms underlying that response. / Erwinia amylovora, organismo de cuarentena en la Unión Europea (UE), es el agente causal del fuego bacteriano. Esta enfermedad produce grandes pérdidas económicas en todos los países en los que está presente y su control resulta muy difícil, debido a la carencia de tratamientos químicos y biológicos eficaces y a la persistencia y facilidad de diseminación de E. amylovora. Los tratamientos con compuestos cúpricos constituyen la base de la gestión integrada del fuego bacteriano en los países de la UE, puesto que el uso de antibióticos, aunque se ha demostrado útil contra esta enfermedad, está prohibido en la UE para el tratamiento de bacteriosis en plantas. Esta tesis, realizada en su mayoría en un laboratorio de seguridad biológica P2, pretende dilucidar mecanismos moleculares implicados en la respuesta de E. amylovora al sulfato de cobre como factor de estrés, ya que este metal es un elemento tóxico para las células bacterianas por encima de una determinada concentración umbral. El objetivo global se abordó, en primer lugar, con el estudio de una selección de genes que se han relacionado en otros modelos bacterianos con el estrés que produce el cobre o con el estrés en general. La cuantificación de la expresión del gen rpoS en presencia de cobre mostró que este gen puede estar implicado en la supervivencia a largo plazo de E. amylovora para combatir el estrés que produce este metal. En una segunda aproximación, se realizó un estudio transcriptómico mediante microarray tras someter a la bacteria a un breve tratamiento de cobre. El análisis de los resultados del microarray reveló que 44 genes se expresaban de forma diferencial en presencia del metal. Cada uno de ellos se estudió mediante gene ontology y por comparación con las bases de datos publicadas en el NCBI, y así se clasificaron en categorías funcionales. Las categorías de estrés y transporte fueron las más abundantes, tanto respecto a los genes que aumentaron su expresión tras la aplicación de cobre como a los que la disminuyeron. De los 44 genes que se expresaron de forma diferencial, se validó la expresión de 25 de ellos por PCR en tiempo real. En dicha validación, el gen copA se expresó 19 veces más en presencia que en ausencia de cobre, por lo que fue seleccionado, junto con siete genes más (soxS, yjcE, ygcF, yhhQ, galF, arcB, EAM_3469), en los que el incremento en la expresión fue menos pronunciado, para generar mutantes de E. amylovora. La respuesta de los mutantes a la presencia de cobre, y la restauración de fenotipos al complementar las mutaciones generadas, han revelado el papel de los genes copA, soxS, yjcE, ygcF, arcB y yhhQ en la supervivencia in vitro de E. amylovora frente al estrés por cobre. Además, la implicación del gen copA se ha demostrado también in planta en brotes de peral tratados con cobre. Finalmente, todos los resultados obtenidos han permitido elaborar un posible modelo de los diferentes mecanismos genéticos que parecen estar implicados en la interacción de E. amylovora con el cobre. El mecanismo más importante parece ser combatir las especies reactivas del oxígeno (ERO), mediante la activación de la expresión de los genes soxS e yjcE. La actividad de estos genes está apoyada, además, por la proteína CopA, que bombea cobre desde el interior celular al espacio periplásmico. La activación del gen arcB, que permite el cambio de un metabolismo aerobio a uno anaerobio, también ayudaría a la reducción de las ERO. En definitiva, los resultados han permitido una aproximación al sustrato genético de la respuesta de E. amylovora al estrés por cobre, y constituyen un punto de partida para avanzar en el conocimiento de los mecanismos moleculares implicados en dicha respuesta. / E. amylovora, organisme de quarantena a la Unió Europea (UE), és l'agent causal del foc bacterià. Aquesta malaltia produeix grans pèrdues econòmiques a tots els països on està present, i el seu control resulta molt difícil, a causa de l' absència de productes químics i biològics eficaços i també per la capacitat de persistència i disseminació d'E. amylovora. Els tractaments amb composts cúprics constitueixen la base de la gestió integrada del foc bacterià als països europeus, ja que l'ús d'antibiòtics, tot i que s'ha demostrat eficaç per a combatre aquesta malaltía, està prohibit a la UE per al tractament de bacteriosi en plantes. Aquesta tesi, realitzada majoritàriament a un laboratori de seguretat biològica P2, pretén dilucidar mecanismes moleculars implicats en la resposta d'E. amylovora davant del coure com a factor d'estrés, ja que el coure és un element tòxic per la cèl.lula per damunt d'una determinada concentració umbral. L'objectiu global es va abordar, en primer lloc, amb l'estudi d'una selecció de gens relacionats en altres models bacterians amb l'estrés que produeix el coure, o amb l'estrés en general. La quantificació de l'expressió del gen rpoS en presència de coure va mostrar que aquest gen pot estar implicat en la supervivència a llarg termini d'E. amylovora per a combatre l'estrés que produeix aquest metall. En una segona aproximació, es va realitzar un estudi transcriptòmic mitjançant microarrays després de sotmetre els bacteris a un breu tractament de coure. L'anàlisi dels resultats dels microarrays va revelar que 44 gens s'expressen de forma diferencial en presència del metall. Cadascun d'ells es va estudiar mitjançant gene ontology i, per comparació amb les bases de dades publicades al NCBI, es van classificar en categories funcionals. Les categories d'estrés i transport van ser les més enriquides, tant en els gens que augmentaren la seua expressió després de l'aplicació de coure com en aquells que la van reduir. Dels 44 gens que s'expressaren de forma diferencial, es va validar l'expressió de 25 d'ells per PCR a temps real. En la validació, el gen copA es va expressar 19 vegades més en presència que en absència de coure, per aquesta raó va ser seleccionat junt amb set gens més (soxS, yjcE, ygcF, yhhQ, galF, arcB, EAM_3469), en els que l'increment de l'expressió va ser menys pronunciada, per a generar mutants d'E. amylovora. La resposta dels mutants a la presència de coure, i la restauració dels fenotips originals al complementar les mutacions generades, han revelat el paper dels gens copA, soxS, yjcE, ygcF, arcB i yhhQ en la supervivència in vitro d'E. amylovora davant a l'estrés per coure. A més a més, la implicació del gen copA s'ha demostrat també in planta, en brots de perera tractats amb coure. Finalment, tots els resultats obtinguts han permès elaborar un possible model dels diferents mecanismes genètics que semblen estar implicats en la interacció d'E. amylovora amb el coure. El mecanisme més important sembla ser combatre les especies reactives de l'oxigen (ERO), mitjançant l'activació de l'expressió dels gens soxS i yjcE. L'activitat d'aquestos gens és recolzada també per l'acció de la proteïna copA, que bombeja coure des de l'interior cel.lular a l'espai periplàsmic. L'activació del gen arcB, que permet el canvi d'un metabolisme aerobi a un metabolisme anaerobi, també ajudaria a reduir la producción de les ERO. En conclusió, els resultats han suposat una aproximació al substrat genètic de la resposta d'E. amylovora a l'estrés per coure, i constitueixen un punt de partida per avançar en el coneixement dels mecanismes moleculars implicats en aquesta resposta. / Águila Clares, B. (2017). Elucidating the molecular basis of copper stress in Erwinia amylovora [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/81658 / TESIS

Cytoplasmic membrane proteins CvaB and CvaA and the outer membrane protein TolC form the bacteriocin colicin V (ColV) secretion system in Escherichia coli. CvaB functions as an ATP-binding cassette transporter with nucleotide-binding motifs in the C-terminal domain (CTD). To study the role of CvaB-CTD in the ColV secretion, a truncated construct of this domain was made and over-expressed. Different forms of CvaB-CTD were obtained during purification, and were identified as monomer, dimer, and oligomer on gel filtration. Nucleotide binding was shown critical for the CvaB-CTD dimerization: oligomers could be converted into dimers by nucleotide bindings; the removal of nucleotide from dimers resulted in transient monomers followed by CTD oligomerization and aggregation; no dimer form could be cross-linked from the nucleotide-binding deficient mutant D654H. The spatial proximity of the Walker A site and ABC signature motif in CTD dimer was identified through disulfide cross-linking of mixed CvaB-CTD with mutants A530C and L630C, while mutations did not dimerize individually. Those results indicated that the CvaB-CTD formed a nucleotide-dependent head-to-tail dimer. Molecular basis of differential nucleotide bindings was also studied through bioinformatics prediction and biochemical verification. Through sequence alignment and homology modeling with bound ATP or GTP, it was found that the Ser503 and Gln504 on aromatic stacking region (Y501DSQ-loop) of CvaB-CTD provided two additional hydrogen-bonds to GTP, but not to ATP. Site-directed mutations of the S503A and/or Q504L were designed based on the model. While site-directed mutagenesis studies of Walker A&B sites or the ABC signature motif affected little on the GTP-binding preference, the double mutation (S503A/Q504L) on the Y501DSQ-loop increased both ATP-binding and ATPase activity at low temperatures. The double mutant showed slight decrease of GTP-binding and about 10-fold increase of the ATP/GTP-binding ratio. Similar temperature sensitivity in nucleotide-binding and activity assays were identified in the double mutant at the same time. Mutations on the Y501DSQ-loop did not affect the ColV secretion level in vivo. Together, the Y501DSQ-loop is structurally involved in the differential binding of GTP over ATP.

Streptococcus equi is the causative agent of strangles, a prevalent and highly contagious disease of horses. Despite the animal suffering and economic burden associated with strangles, little is known about the molecular basis of S. equi virulence. Here we have investigated the contributions of a specific lipoprotein and the general lipoprotein processing pathway to the abilities of S. equi to colonize equine epithelial tissues in vitro and to cause disease in both a mouse model and the natural host in vivo. Colonization of air interface organ cultures after they were inoculated with a mutant strain deficient in the maturase lipoprotein (prtM138-213, with a deletion of nucleotides 138 to 213) was significantly less than that for cultures infected with wild-type S. equi strain 4047 or a mutant strain that was unable to lipidate preprolipoproteins (lgt190-685). Moreover, mucus production was significantly greater in both wild-type-infected and lgt190-685-infected organ cultures. Both mutants were significantly attenuated compared with the wild-type strain in a mouse model of strangles, although 2 of 30 mice infected with the lgt190-685 mutant did still exhibit signs of disease. In contrast, only the prtM138-213 mutant was significantly attenuated in a pony infection study, with 0 of 5 infected ponies exhibiting pathological signs of strangles compared with 4 of 4 infected with the wild-type and 3 of 5 infected with the lgt190-685 mutant. We believe that this is the first study to evaluate the contribution of lipoproteins to the virulence of a gram-positive pathogen in its natural host. These data suggest that the PrtM lipoprotein is a potential vaccine candidate, and further investigation of its activity and its substrate(s) are warranted.

This thesis addresses key issues of scientific realism in the philosophy of biology and chemistry through investigation of an underexplored research domain: olfaction theory, or the science of smell. It also provides the first systematic overview of the development of olfactory practices and research into the molecular basis of odours across the 19th and 20th century. Historical and contemporary explanations and modelling techniques for understanding the material basis of odours are analysed with a specific focus on the entrenchment of technological process, research tradition and the definitions of materiality for understanding scientific advancement. The thesis seeks to make sense of the explanatory and problem solving strategies, different ways of reasoning and the construction of facts by drawing attention to the role and application of scientific representations in olfactory practices. Scientific representations such as models, classifications, maps, diagrams, lists etc. serve a variety of purposes that range from the stipulation of relevant properties and correlations of the research materials and the systematic formation of research questions, to the design of experiments that explore or test particular hypotheses. By examining a variety of modelling strategies in olfactory research, I elaborate on how I understand the relation between representations and the world and why this relation requires a pluralist perspective on scientific models, methods and practices. Through this work I will show how a plurality of representations does not pose a problem for realism about scientific entities and their theoretical contexts but, on the contrary, that this plurality serves as the most reliable grounding for a realistic interpretation of scientific representations of the world and the entities it contains. The thesis concludes that scientific judgement has to be understood through its disciplinary trajectory, and that scientific pluralism is a direct consequence of the historicity of scientific development.