Orientador: Maria Célia Bertolini / Coorientador: Fernanda Zanolli Freitas / Banca: Dulce Helena Ferreira de Souza / Banca: Iran Malavazi / Resumo: Os organismos pertencentes ao reino dos fungos têm exercido um papel fundamental no avanço da compreensão dos mecanismos moleculares de organismos eucariotos devido as suas facilidades de manipulação e o conhecimento das características genéticas e bioquímicas envolvidas em seu ciclo de vida. Neurospora crassa é um fungo cujos mecanismos genéticos e bioquímicos básicos são bem definidos, e por isso tem sido muito usado como um sistema modelo em pesquisas que visam à elucidação de processos celulares complexos. Em trabalhos anteriores desenvolvidos em nosso laboratório foram identificadas algumas proteínas que se ligam à região promotora do gene gsn que codifica a enzima glicogênio sintase, regulatória no processo de síntese de glicogênio. O presente trabalho teve como objetivo estudar algumas dessas proteínas, anotadas como hipotéticas, numa tentativa de entender melhor as suas participações no mecanismo de regulação. Para isto linhagens mutantes nos genes que codificam as referidas proteínas foram adquiridas junto ao Fungal Genetics Stock Center. Análises de determinação do conteúdo de glicogênio nas linhagens mutantes foram realizadas tanto em condições normais de crescimento (30º C) como em situações de choque térmico (45º C). Todas as linhagens mutantes avaliadas apresentaram um perfil de acúmulo de glicogênio semelhante ao da linhagem selvagem, isto é, todas apresentaram uma queda nas quantidades de glicogênio após o choque térmico. Entretanto a linhagem mutante na ORF NCU06679 apresentou o conteúdo de glicogênio mais reduzido, em ambas as situações ambientais (antes e depois do choque térmico). Com o objetivo de verificar o envolvimento das proteínas estudadas na expressão do gene gsn, experimentos de Northern blot foram realizados e revelaram que na situação de estresse térmico, os níveis do transcrito gsn diminuem semelhantemente... (Resumo completo clicar acesso eletrônico abaixo) / Abstract: The organisms belonging to the kingdom of fungi have played a key role in advancing the understanding of the molecular mechanisms of eukaryotic organisms due to their easy handling and the genetic and biochemical mechanisms involved in their life cycle. Neurospora crassa is a fungus whose basic biochemical and genetic mechanisms are well defined, and therefore has been widely used as a model system in studies aimed to elucidate complex cellular processes. In a previous work developed in our laboratory we identified some proteins that bind to the promoter region of the gsn gene that encodes glycogen synthase, the regulatory enzyme in the glycogen synthesis. This work aimed to start studying these proteins, annotated as hypothetical proteins, in an attempt to better understand their roles in the regulation of glycogen metabolism. For this, mutant strains in genes encoding the proteins were purchased from the Fungal Genetics Stock Center. Analysis of glycogen determination were performed in the mutant strains both under normal growth (30º C) and under heat shock condition (45º C). All mutant strains showed a glycogen content profile similar to the wild type strain, that is, all showed a decrease in the amounts of glycogen after heat shock. However the mutant strain in the ORF NCU06679 presented lower glycogen content compared to the wild type strain, in both environmental condition (before and after heat shock). To verify the participation of the proteins in the gsn gene expression, Northern blot experiments were performed and showed that under heat stress the gsn transcript levels decreased similarly to that observed in the wild type strain. However the reduction in the gene expression was higher in the mutant strain containing the ORF NCUO6679 knocked out / Mestre

Made available in DSpace on 2014-06-11T19:23:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-08-10Bitstream added on 2014-06-13T20:29:48Z : No. of bitstreams: 1 santos_ma_me_araiq.pdf: 1654932 bytes, checksum: 6a8607912411fc8812340494300f0501 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Os organismos pertencentes ao reino dos fungos têm exercido um papel fundamental no avanço da compreensão dos mecanismos moleculares de organismos eucariotos devido as suas facilidades de manipulação e o conhecimento das características genéticas e bioquímicas envolvidas em seu ciclo de vida. Neurospora crassa é um fungo cujos mecanismos genéticos e bioquímicos básicos são bem definidos, e por isso tem sido muito usado como um sistema modelo em pesquisas que visam à elucidação de processos celulares complexos. Em trabalhos anteriores desenvolvidos em nosso laboratório foram identificadas algumas proteínas que se ligam à região promotora do gene gsn que codifica a enzima glicogênio sintase, regulatória no processo de síntese de glicogênio. O presente trabalho teve como objetivo estudar algumas dessas proteínas, anotadas como hipotéticas, numa tentativa de entender melhor as suas participações no mecanismo de regulação. Para isto linhagens mutantes nos genes que codificam as referidas proteínas foram adquiridas junto ao Fungal Genetics Stock Center. Análises de determinação do conteúdo de glicogênio nas linhagens mutantes foram realizadas tanto em condições normais de crescimento (30º C) como em situações de choque térmico (45º C). Todas as linhagens mutantes avaliadas apresentaram um perfil de acúmulo de glicogênio semelhante ao da linhagem selvagem, isto é, todas apresentaram uma queda nas quantidades de glicogênio após o choque térmico. Entretanto a linhagem mutante na ORF NCU06679 apresentou o conteúdo de glicogênio mais reduzido, em ambas as situações ambientais (antes e depois do choque térmico). Com o objetivo de verificar o envolvimento das proteínas estudadas na expressão do gene gsn, experimentos de Northern blot foram realizados e revelaram que na situação de estresse térmico, os níveis do transcrito gsn diminuem semelhantemente... / The organisms belonging to the kingdom of fungi have played a key role in advancing the understanding of the molecular mechanisms of eukaryotic organisms due to their easy handling and the genetic and biochemical mechanisms involved in their life cycle. Neurospora crassa is a fungus whose basic biochemical and genetic mechanisms are well defined, and therefore has been widely used as a model system in studies aimed to elucidate complex cellular processes. In a previous work developed in our laboratory we identified some proteins that bind to the promoter region of the gsn gene that encodes glycogen synthase, the regulatory enzyme in the glycogen synthesis. This work aimed to start studying these proteins, annotated as hypothetical proteins, in an attempt to better understand their roles in the regulation of glycogen metabolism. For this, mutant strains in genes encoding the proteins were purchased from the Fungal Genetics Stock Center. Analysis of glycogen determination were performed in the mutant strains both under normal growth (30º C) and under heat shock condition (45º C). All mutant strains showed a glycogen content profile similar to the wild type strain, that is, all showed a decrease in the amounts of glycogen after heat shock. However the mutant strain in the ORF NCU06679 presented lower glycogen content compared to the wild type strain, in both environmental condition (before and after heat shock). To verify the participation of the proteins in the gsn gene expression, Northern blot experiments were performed and showed that under heat stress the gsn transcript levels decreased similarly to that observed in the wild type strain. However the reduction in the gene expression was higher in the mutant strain containing the ORF NCUO6679 knocked out

I collect data on subjects' information acquisition during real and hypothetical risky choices using process-tracing software called Mouselab. I also measure subjects' cognitive ability using the cognitive reflective test (CRT). On average, measured risk preferences are not significantly different across real and hypothetical settings. However, cognitive ability is inversely related to risk aversion when choices are hypothetical, but it is unrelated when the choices are real. This interaction between cognitive ability and hypothetical setting is consistent with the notion that some individuals, specifically higher-ability individuals, treat hypothetical choices as "puzzles" and may help explain why some studies find that subjects indicate that they are more tolerant of risk when they make hypothetical choices than when they make real choices. On average, subjects demonstrate a similar degree of consistency across settings, and there are also no significant differences across settings in the amount of time subjects take to make a choice, the amount of information they acquire, or how they distribute their attention. I also find evidence to suggest that subjects acquire information in a manner consistent with the implicit calculation of expected utility. Specifically, individuals do not merely make choices "as if" they are integrating probabilities and outcomes, it appears that they actually are. Moreover, as they progress through a series of choices in a commonly used risk preference elicitation method, their information acquisition becomes progressively more consistent with integration models. Finally, on average, individuals appear to acquire information in real and hypothetical settings in similar ways.

In a controlled lab experiment, we investigate hypothetical biases in the value of time by comparing stated preference (SP) and revealed preference (RP) values attached to unexpected waiting times. The SP and RP choice sets are identical in terms of design with the only difference being that the RP choices have real consequences in terms of unexpected waiting times and monetary incentives. We find a substantial hypothetical bias with the average SP value of time being only 71% of the corresponding RP value. The bias is mainly driven by participants who have scheduling constraints during the time of the unexpected wait. Scheduling constraints are taken into account to a much lesser extent in the SP setting than in the RP setting, presumably because only in the latter, the consequences of ignoring them are costly. We find evidence that this effect is stronger for persons with relatively low cognitive ability.

Experimental willingness to pay (WTP) studies can be classified as hypothetical or non-hypothetical. In a hypothetical study, such as conjoint analysis, a subject does not need to make a real economic commitment. In contrast, in a non-hypothetical task such as in experimental auctions, a subject may need to actually buy the product. Subjects in hypothetical studies tend to overstate their true WTP. Consequently, researchers need to correct hypothetical values to obtain reliable WTP estimates. Recently, incentive-aligned and cheap talk approaches have been proposed as ways to correct for hypothetical bias. In a hypothetical task, a cheap talk script explicitly reminds the subject about the hypothetical nature of the task and its expected consequences. In an incentive-aligned task (non-hypothetical), subjects are randomly selected to physically buy the product. The objective of our study is to assess and compare the reduction of hypothetical bias in consumers’ willingness to pay for novel products by applying a generic, short, and neutral cheap talk script in a retail setting. To accomplish this objective, we employ non-hypothetical, hypothetical, and hypothetical with cheap talk treatments in our experimental design. We conducted our experimental retail study using conjoint analysis and open-ended elicitation mechanisms, utilizing Becker DeGroot Marshak (BDM) mechanism for the incentive-aligned treatments. Consistently in both elicitation mechanisms, using seemingly unrelated and random-effect Tobit techniques, we find that our cheap talk script is effective in eliminating the hypothetical bias. As expected, the hypothetical WTP values are significantly higher than the non-hypothetical values but the hypothetical values with cheap talk are not significantly different from incentive-aligned or non-hypothetical estimates. In addition, we find that open-ended estimates are significantly higher than conjoint analysis estimates and that emotions and familiarity can have significant impacts on WTP estimates.

I wish to determine when one is justified in paternalistic interferences that override a subject’s autonomy. In order to lay the groundwork for discussing paternalistic interferences with autonomous decisions, I first consider different conceptions of autonomy, welfare, and paternalism, and determine which I mean to use. In particular, I proceed with Dworkin’s characterization of autonomy as a combination of authenticity and self-determination; Nussbaum’s capabilities theory in order to determine welfare; and a definition of paternalism as being an interference with a subject’s liberty or autonomy that is motivated exclusively by consideration for that subject’s own good or welfare. Once I have working definitions for autonomy, welfare, and paternalism, I consider arguments justifying paternalistic interferences. Because I especially wish to determine when paternalistic interferences that conflict with a subject’s autonomous decision are justified, I begin with Scoccia’s arguments for using hypothetical consent — which is based on what would maximize the subject’s welfare — to justify paternalistic acts. Using Scoccia’s argument, I consider a few cases in which concerns for welfare may justify paternalistic acts overriding the subject’s autonomy. However, hypothetical consent does not go very far in justifying paternalistic acts. Therefore, I also consider arguments justifying paternalism in cases where the subject is not necessarily fully autonomous when making or acting upon a decision. For example, Carter argues that paternalistic acts are justified if autonomy has been waived through prior or subsequent consent. Additionally, I look at justifying paternalism when the subject’s autonomy is compromised through involuntariness or incompetence.

Experimental willingness to pay (WTP) studies can be classified as hypothetical or non-hypothetical. In a hypothetical study, such as conjoint analysis, a subject does not need to make a real economic commitment. In contrast, in a non-hypothetical task such as in experimental auctions, a subject may need to actually buy the product. Subjects in hypothetical studies tend to overstate their true WTP. Consequently, researchers need to correct hypothetical values to obtain reliable WTP estimates. Recently, incentive-aligned and cheap talk approaches have been proposed as ways to correct for hypothetical bias. In a hypothetical task, a cheap talk script explicitly reminds the subject about the hypothetical nature of the task and its expected consequences. In an incentive-aligned task (non-hypothetical), subjects are randomly selected to physically buy the product. The objective of our study is to assess and compare the reduction of hypothetical bias in consumers’ willingness to pay for novel products by applying a generic, short, and neutral cheap talk script in a retail setting. To accomplish this objective, we employ non-hypothetical, hypothetical, and hypothetical with cheap talk treatments in our experimental design. We conducted our experimental retail study using conjoint analysis and open-ended elicitation mechanisms, utilizing Becker DeGroot Marshak (BDM) mechanism for the incentive-aligned treatments. Consistently in both elicitation mechanisms, using seemingly unrelated and random-effect Tobit techniques, we find that our cheap talk script is effective in eliminating the hypothetical bias. As expected, the hypothetical WTP values are significantly higher than the non-hypothetical values but the hypothetical values with cheap talk are not significantly different from incentive-aligned or non-hypothetical estimates. In addition, we find that open-ended estimates are significantly higher than conjoint analysis estimates and that emotions and familiarity can have significant impacts on WTP estimates.

Made available in DSpace on 2014-06-11T19:23:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-04-22Bitstream added on 2014-06-13T19:49:29Z : No. of bitstreams: 1 martins_pmm_me_rcla.pdf: 7124946 bytes, checksum: 0e5fc6b0551611dda7e75caf8cd99fad (MD5) / O cancro cítrico é uma doença causada pela bactéria Xanthomonas axonopodis pv. citri (Xac), e que afeta plantas de citros por todo o mundo. O genoma de Xac foi completamente seqüenciado, o que revelou grandes quantidades de ORFs (~30%) codificando para produtos com função desconhecida (proteínas hipotéticas). Baseando-se no princípio de que muitos eventos bioquímicos acontecem em sítios específicos no interior celular, a localização de proteínas em fusão com GFP tem sido amplamente utilizada para a obtenção de informações valiosas a respeito de suas funções. Para iniciarmos estudos de localização de proteínas hipotéticas em Xac, construímos um vetor integrativo capaz de expressá-las em fusão com o polipeptídio GFP, pPM2a. O vetor de expressão para Xac carrega um cassete promotor/repressor de xilose (xylR/pxyl), o gene gfp, um RBS sintético e um fragmento do gene de α-amilase de Xac, para direcionar a integração do sistema de expressão no lócus amy do cromossomo bacteriano. Mostramos aqui a integração estável do vetor no lócus amy de Xac. Além disso, mutantes de Xac expressando o polipeptídio GFP não apresentam nenhuma alteração em seu fenótipo de patogenicidade para o hospedeiro (laranja doce). Mutantes de Xac expressando versões marcadas com GFP para as proteínas ParB e ZapA, ambas codificadas por Xac, foram utilizadas para a padronização dos estudos de localização subcelular. GFP-ZapAXac apresentou um padrão de localização análogo ao de seu ortólogo presente em Bacillus subtilis: uma estrutura semelhante a uma barra, posicionada no meio do bacilo, onde o septo se desenvolve, orientado perpendicularmente com relação ao eixo longitudinal da célula. Este é o primeiro relato de um estudo de localização realizado em Xac. Ao contrário de GFP-ZapAXac, ParBXac-GFP não mostrou nenhum padrão de localização, apesar de a fusão... / Citrus canker is a disease caused by the bacterium Xanthomonas axonopodis pv. citri (Xac), which affects citrus plants worldwide. The genome of Xac was completely sequenced, which unveiled an expressive amount of ORFs (~30%) coding for products of unknown function (hypothetical proteins). Based on the principle that many biochemical events happen at specific sites within the cells, protein localization studies have been extensively used to gather valuable information about function. In order to start subcellular localization studies of hypothetical proteins encoded by Xac using fluorescent microscopy, we constructed an integrative expression vector for GFP-tagging of proteins in this bacterium, pPM2a. The expression vector for Xac carries a xylose repressor/promoter cassette (xylR/pxyl), the gfp gene, a synthetic Ribosome Binding Site (RBS), and a fragment of the α-amylase gene of Xac, to drive the integration of the whole expression system into the amy locus of the bacterial chromosome. We show here stable integration of the expression vector into the amy locus of Xac. Furthermore, Xac mutants expressing the polypeptide GFP do not exhibit any alteration in pathogenicity to the host plant sweet orange. Mutants of Xac expressing GFPtagged versions of ParB and ZapA proteins, both encoded by Xac, were used to standardize the subcellular localization studies. GFP-ZapAXac showed a localization pattern analogous to its ortholog encoded by Bacillus subtilis: a bar-like structure positioned in the middle of the rods, where the septum develops, oriented perpendicularly to the longitudinal axis of the cell. This is the first report of a protein localization study performed in Xac Unlike GFP-ZapAXac, ParBXac-GFP did not display any detectable localization pattern, despite the fact that we were able to detect the production of the fusion ParBXac-GFP in Western blot experiments... (Complete abstract click electronic access below)

Despite recommendations, health services interventions often lack mechanistic evidence. One way of acquiring such evidence is through the use of hypothetical settings. However, there are knowledge gaps about the factors that affect whether hypothetical decisions will correlate to real-world ones. This thesis involves a systematic concept review to identify the factors important to the hypothetical-real-world decision relationship and a case study of the relevance of these factors to the decision aid development context. The 42 identified factors were grouped into 5 categories: Personal Characteristics, Presentation Characteristics, Cognitive Factors, Motivation, and Participant Characteristics. Through cognitive interviews with decision aid developers we found that the interviewees had generally not considered many of the factors thought to affect the external validity of hypothetical data. This thesis contributes a framework that highlights factors for investigators to consider when designing studies in hypothetical settings and that can serve as a foundation for future research.

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