NmlR of Neisseria gonorrhoeae: a novel redox responsive transcription factor from the MerR family

A MerR-like regulator (NmlR -Neisseria merR-like Regulator) identified in the Neisseria gonorrhoeae genome lacks the conserved cysteines known to bind metal ions in characterized proteins of this family. Phylogenetic analysis indicates that NmlR defines a subfamily of MerR-like transcription factors with a distinctive pattern of conserved cysteines within their primary structure. NmlR regulates itself and three other genes in N. gonorrhoeae encoding a glutathione-dependent dehydrogenase (AdhC), a CPx-type ATPase (CopA) and a thioredoxin reductase (TrxB). An nmlR mutant lacked the ability to survive oxidative stress induced by diamide and cumene hydroperoxide. It also had > 50-fold lower NADH-S-nitrosoglutathione oxidoreductase activity consistent with a role for AdhC in protection against nitric oxide stress. The upstream sequences of the NmlR regulated genes contained typical MerR-like operator/promoter arrangements consisting of a dyad symmetry located between the -35 and -10 elements of the target genes. The NmlR target operator/promoters were cloned into a beta-galactosidase reporter system and promoter activity was repressed by the introduction of NmlR in trans. Promoter activity was activated by NmlR in the presence of diamide. Under metal depleted conditions NmlR did not repress P-AdhC (or P-CopA) promoter activity, but this was reversed in the presence of Zn(II), indicating repression was Zn(II)-dependent. Analysis of mutated promoters lacking the dyad symmetry revealed constitutive promoter activity which was independent of NmlR. Gel shift assays further confirmed that NmlR bound to the target promoters possessing the dyad symmetry. Site-directed mutagenesis of the four NmlR cysteine residues revealed that they were essential for activation of gene expression by NmlR.

The human stress-activated protein kinase-interacting 1 gene encodes JNK-binding proteins

The orthologous proteins of the stress-activated protein kinase-interacting 1 (Sin1) family have been implicated in several different signal transduction pathways. In this study, we have investigated the function of the full-length human Sin1 protein and a C-terminally truncated isoform, Sin 1 alpha, which is produced by alternative splicing. Immunoblot analysis using an anti-Sin 1 polyclonal antibody showed that full-length Sin I and several smaller isoforms are widely expressed. Sin 1 was demonstrated to bind to c-Jun N-terminal kinase (JNK) in vitro and in vivo, while no interaction with p38- or ERK1/2-family MAPKs was observed. The Sin1 alpha isoform could also form a complex with JNK in vivo. Despite localizing in distinct compartments within the cell, both Sin1 and Sin1 alpha co-localized with JNK, suggesting that the Sin1 proteins could recruit JNK. Over-expression of full-length Sin1 inhibited the activation of JNK by UV-C in DG75 cells, as well as basal JNK-activity in HEK293 cells. These data suggest that the human Sin1 proteins may act as scaffold molecules in the regulation of signaling by JNK. (c) 2004 Elsevier Inc. All rights reserved.

Stress in veterinary science students: A study at the University of Queensland

This paper reports on the results of a survey of selected University of Queensland (UQ) veterinary students aimed at elucidating factors causing stress during the five undergraduate years of the program. Students from each of the five years were asked to form six- or seven-member focus groups. Each focus group was then interviewed and their opinions sought on causes of ongoing stress and the ranking of those causes into predetermined categories. They were also asked to give their opinions on counseling services available within the university and what, if any, services they would like to see in place to help students with stress-related problems. Students in the first, third, and fourth years of the program rated academic issues as the most likely causes of ongoing stress, while students in the second and fifth years of the program ranked lifestyle and financial issues as more likely to cause ongoing stress. in most cases, students coped well with these causes of stress and tended not to use counseling services available to all UQ students. When faced with stressful issues, students looked to their classmates or family members for help and not to university counseling services. Students were also happy to approach staff members in the Veterinary School when faced with a problem. The authors nevertheless conclude that mechanisms set in place at the undergraduate level to help veterinary students cope with stress should particularly benefit those students when they become new graduates and are faced with the stresses of veterinary practice.

Adjustment in mothers of children with Asperger syndrome: An application of the double ABCX model of family adjustment

The present study examined the applicability of the double ABCX model of family adjustment in explaining maternal adjustment to caring for a child diagnosed with Asperger syndrome. Forty-seven mothers completed questionnaires at a university clinic while their children were participating in an anxiety intervention. The children were aged between 10 and 12 years. Results of correlations showed that each of the model components was related to one or more domains of maternal adjustment in the direction predicted, with the exception of problem-focused coping. Hierarchical regression analyses demonstrated that, after controlling for the effects of relevant demographics, stressor severity, pile-up of demands and coping were related to adjustment. Findings indicate the utility of the double ABCX model in guiding research into parental adjustment when caring for a child with Asperger syndrome. Limitations of the study and clinical implications are discussed.

Tissue transglutaminase and the stress response

The expression of the protein crosslinking enzyme tissue transglutaminase (TG2, tTG), the ubiquitous member of transglutaminase family, can be regulated by multiple factors. Although it has been suggested that TG2 can be involved in apoptotic cell death, high levels of enzyme have also been associated with cell survival in response to different stimuli. Furthermore, evidence indicates that increases in TG2 production cause enzyme translocation to cell membrane. Cell stress can also lead to TG2 accumulation on the cell surface and in the extracellular matrix resulting in changes in cell-matrix interactions. Here, we discuss the underlying mechanisms of TG2 up-regulation induced by various stimuli including glutamate exposure, calcium influx, oxidative stress, UV, and inflammatory cytokines. These findings agree with a postulated role for transglutaminases in molecular mechanisms involved in several diseases suggesting that cross-linking reactions could be a relevant part of the biochemical changes observed in pathological conditions. © 2007 Springer-Verlag.

Stressors, mediators, and health care outcomes of home enteral nutrition in family caregivers and their older adult care recipients

Family caregivers manage home enteral nutrition (HEN) for over 77% of an estimated 1 of every 400 Medicare recipients. Increasing usage of HEN in older adults combined with reliance on family caregivers raises concerns for the quality, outcomes, and costs of care. These concerns are relevant in light of Medicare limitations on nursing assistance and non-reimbursement for nutrition services, despite annual costs of over $600 million. This study applied stress process theories to assess stressor, mediator, and outcome variables salient to HEN and caregiving. In-home structured interviews occurred with a multi-ethnic sample of 30 caregiving dyads at 1–3 months after discharge on HEN. Care recipients were aged ≥60 (M = 68.4 years) and did not have dementia. Caregivers were aged ≥21, unpaid, and lived within 45 minutes of care recipients. Caregivers performed an average of 19.7 tasks daily for 61.9 hours weekly. Training needs were identified for 33 functional, care management, technical, and nutritional tasks. Preparedness scores were low (M = 1.73/4.0), and positively correlated with competence, self-rated quality of care and positive feelings, and negatively with overload, role captivity, and negative feelings (Ps < .05). Caregivers had multiple changes in lifestyle and dietary behaviors. Lifestyle changes positively correlated with overload, and negatively with preparedness and positive feelings. Dietary changes positively correlated with number of tasks, overload, role captivity and negative feelings, and negatively with preparedness (Ps < .01). Fifty-seven percent of caregivers aged >50 were at nutrition risk. Care recipients fared worse. Average weight change was −4.35 pounds (P < .001). Physical complications interrupted daily enteral infusions. Water intake was half of fluid need and associated with signs of dehydration (P < .001). Physical and social function was poor, with older subjects more impaired ( P < .04). Those with better prepared or less overloaded caregivers had higher functionality and QOL (P < .002). Complications, type of feeding tube, and caregiver preparedness correlated with frequency of health care utilization (Ps < .05). Efficacy of HEN in older adults requires specialized caregiver training, attention to caregivers' needs, and frequent monitoring from a highly skilled multidisciplinary team including dietitians. ^

Temporal changes in immigration stress, adaptation, and the factors contributing to the immigration -adaptation process in newly immigrant parents

The adaptation process to a new land can be an arduous transition for families who migrate from their countries in an attempt to evade negative life conditions. Family-based immigration has been the cornerstone of immigration policy for the U.S. However, there has been a relative lack of attention given in immigration studies to the impact of immigration particularly on parents. Furthermore, little is known about their adjustment to their post-migration circumstances, particularly the initial phase of migration, where the psychological impact of immigration tends to be concentrated. It is even rarer that investigators have addressed longitudinally the dynamic process of parents' adaptation to a new ecology, which can shed a great deal of light on its mechanisms. In this dissertation, changes over time in levels of stress, adjustment (affect balance and life satisfaction), and the factors (social support, economic hardship, and discrimination) contributing to stress and adjustment were examined in newly immigrant parents from Argentina, Colombia, Cuba, Haiti, and the West Indies. Moderating effects of gender and country-of-origin were examined as well. This study also aimed to investigate to what extent the contributing factors impacted stress and adjustment, not only concurrently, but also over the first three years of post-migration. Analysis of variance results showed that both affect balance and social support increased whereas life satisfaction decreased over time. There was no significant change in stress, however. Both gender and group effects were also observed. Mothers experienced higher stress whereas fathers experienced higher discrimination. Among groups, Haitians appeared at the greatest risk in terms of stress, discrimination, and economic hardship. A structural equation modeling analysis showed that the relative importance of contributing factors changed over time in the process of immigrants' adaptation. Yet, social support emerged as a powerful protective factor in that its effects carried over time, and discrimination was a primary mediator through which other predictors were related to stress and adjustment. These findings shed light on the "hows and whys" of the immigration-adaptation process, by demonstrating the significance of specific conditions of life change to psychological outcomes as newly immigrant parents adapt to their post-migration ecology.

The Influence of Psychological Predictors and Cognitive Behavioral Stress Management Intervention on Antiretroviral Therapy (ART) Adherence Among HIV-Positive Female Haitian Alcohol Users

Purpose: Over half the HIV-infected persons in the Caribbean, the second most HIV-impacted region in the world, live in Haiti. Using secondary data from a parent study, this research assessed the effects of psychological and social factors on antiretroviral therapy (ART) adherence among Haitian, HIV-positive, female alcohol users. Theoretical Foundation and Research Questions: Using the Theory of Planned Behavior/Reasoned Action and the Information, Motivation, Behavior skills model as guiding theoretical frameworks, the study examined the effectiveness of an adapted cognitive behavioral stress management (CBSM-A) intervention in improving ART adherence. The effect of psychological factors (depression, anxiety, beliefs about medicine, and social support), social factors (stigma, relationship status, and educational attainment), and alcohol on adherence to ART was assessed. Methods: The sample consisted of 116 female ART patients who were randomly assigned to the CBSM-A intervention or the wait-list control group. Participants completed intervention sessions as well as pre- and post-test assessments. Analyses of variance, t-tests, and point biserial correlations were used to test hypotheses. Results: Surprisingly, ART adherence rates significantly decreased for both groups combined [F (1, 108) = 8.79, p = .004]; there was no significant difference between the intervention and control groups with regard to the magnitude of change between baseline and post assessment. On average, depression decreased significantly among participants in the CBSM-A group only [(t (62) = 5.54, p < .001)]. For both groups combined, alcohol use significantly decreased between baseline and post-assessment [(F (1, 78) = 34.70, p < .001)]; there was no significant difference between the intervention and control groups with regard to the magnitude of change between baseline and post-assessment. None of the variables were significantly correlated with ART adherence. Discussion: Adherence to ART did not improve in this sample, nor were any of the variables significantly associated with adherence. The findings suggest that additional supportive and psychological services may be needed in order to promote higher adherence to ART among HIV-positive females. More research may be needed on this sample; a focus on mental health issues, partner conflict, family and sexual history may allow for better targeting and more successful interventions.

Parent's emotional expressiveness and child, parent, and family functioning

This study investigated associations between parents' expressed emotion during a series of play tasks with their child, and psychological assessments of parent, child, and family functioning. Parent and child dyads included 41 families with a child between ages 4 and 12 who sought a developmental assessment at the Youth and Family Development Program laboratory at Florida International University. Videotaped dyadic play tasks were rated, using an Emotional Expressiveness Rating Scale (EERS) developed for this study, for parents' communication of criticism, hostility, emotional over-involvement, indifference, and warmth toward their child. Associations between parent expressed emotion and parent, child, and family functioning were assessed. Negative expressed emotion in parents, particularly criticism, was correlated with externalizing child problems, high parental stress, and family conflict; parent warmth was correlated with parents' feeling rewarded by their child, and with family cohesion. The implications for mental health research and practice with parents and children are discussed.

Equilíbrios Difíceis: stress laboral, equilíbrio trabalho/família e diferenças de género entre os colaboradores de uma empresa de tecnologias da informação

O stress é não apenas inerente para o ser humano, como indispensável para a sua sobrevivência. À medida que as sociedades humanas evoluíram, assim também se alteraram as principais fontes de stress. Actualmente o stress organizacional é uma das principais áreas de investigação, bem como as suas relações com a família e a vida pessoal. Em foco estão ainda as variáveis individuais que servem como moderadoras da experiência de stress, como o coping e o suporte social. Neste trabalho procurou-se conhecer os níveis de stress experienciados pelos colaboradores da Cisco Systems Lda, identificando diferenças de género e entre os tipos de trabalho. Pretendeu-se ainda relacionar o stress no trabalho com o equilíbrio trabalho/família, equlíbrio vida pessoal/trabalho, suporte social e coping. Através de um estudo não experimental transversal, descritivo/descritivo correlacional, com 42 sujeitos, utilizando o PMI – Pressure Management Indicator, foi possível identificar quatro factores, com α a partir de 0,531 para as variáveis moderadoras ate 0,904 para as variáveis stressoras. Os trabalhadores da amostra trabalham em média 49,7 horas por semana, apontando como principal razão “para que o trabalho seja feito”; 31,7% consideram estar a sofrer de pressão negativa iniciada há mais de 3 meses. Estão muito satisfeitos com a organização, mas sentem pressão devido ao volume de trabalho, ao relacionamento interpessoal e às dificuldades em desligar do trabalho quando em casa, fazendo uso de estratégias focadas no problema e de suporte social. Existem diferenças estatisticamente significativas entre homens e mulheres nas subescalas “Estado de espírito” e “Nível de confiança”, indicando níveis mais altos de segurança e satisfação com o seu estado mental para o grupo dos homens. No que respeita aos tipos de trabalho, foi possível identificar diferenças entre as categorias Sénior gerência/profissional e Manual/hábil nas subescalas “Nível de energia”, “Volume de trabalho” e “Equilíbrio vida/trabalho”. Existe correlação positiva entre as variáveis stressoras e a subescala “Equilíbrio trabalho/família” (r=0,890; p<0,000) e entre as variáveis stressoras e a escala de Coping (r=0,748; p<0,000), sugerindo que níveis de stress elevados interferem com a vida familiar e conduzem a maior utilização das estratégias de coping. Apesar das políticas de recursos humanos da empresa no sentido de promover a conciliação entre trabalho e vida pessoal e familiar, há ainda margem para intervir e melhorar. / Stress is not only inherent to the human being, it’s essential to survival. As the humans societies evolved, so did the major sources of stress. These days, organizational stress is one of the main areas of research, as it is it’s relation with family and personal life. Under the spotlight are also individual variables that moderate the stress experience, such as coping and social support. With this paper, we intended to know the levels of stress experienced by the workers of Cisco Systems Lda, identifying gender and work type differences. We were also intending to relate work stress with home/work balance, life/work balance, social support and coping. Using a cross sectional correlational study, with n=42, using the PMI, we were able to identify four factors, with α starting on 0,531 for moderator variables to 0,904 for stressor variables. The workers on this sample work an average of 49,7 hours per week, naming “to get the job done” as the main reason for it. 31,7% consider to be suffering from negative pressure that started more than 3 months ago. They are very satisfied with the organization, but feel pressure due to workload, interpersonal relationship and the difficulties in switching off from work when at home. They use mostly Problem focus strategies and social support.There are statically significative differences between men and women in the subscales “State of mind” and “Confidence level”, which indicate higher levels of security and satisfaction with their state of mind for men. Regarding work types, we were able to identify differences between senior Management and Manual categories on subscales “Energy level”, “Workload” and “Life/work balance”. There is a positive correlation between stressor variables and the subscales “Home/work balance” (r=0,890; p<0,000) and between stressor variables and Coping scale (r=0,748; p<0,000), suggesting that high levels of stress interfere with family life and lead to higher use of coping mechanisms. Despite the company’s human resources policies to promote the balance between work and personal and family life, there is still place to intervene and improve.

Suporte, Stress e Solidão: um estudo sobre o apoio offline e online percecionado pelas mães portuguesas

Uma “rede social” diz respeito a uma estrutura constituída por pessoas ou organizações que partilham interesses, motivações e valores comuns. Um ponto partilhado pelos diversos tipos de rede social é a troca de informações, conhecimentos, interesses e esforços na tentativa de atingir objectivos comuns, muitas vezes potencializando forças e recursos em situações de crise. Recentemente tem-se ouvido falar bastante deste conceito aplicado à internet. A maternidade constitui, por si só, um momento de crise na vida de cada família e de cada mulher, requerendo adaptações e mudanças. O estudo realizado teve o intuito de avaliar o suporte online e offline (denominando-se essas ligações de redes sociais), no caso específico de assuntos ligados à maternidade. Pretendeu-se verificar também se existia uma correlação entre estes tipos de suporte e os níveis de stress e solidão sentidos pelas mães. Por último, aferiu-se ainda a influência que a idade dos filhos teria nesta adesão a grupos de suporte à maternidade, no Facebook. Este estudo teve uma amostra constituída por 170 mulheres (mães), estando as suas idades compreendidas entre os 25 e os 62 anos. Os resultados revelaram que apesar de não haver diferenças estatisticamente significativas entre o apoio online e offline, as mulheres que pertencem a grupos do Facebook tendem a sentir mais suporte por parte das plataformas de suporte disponíveis. Constatou-se ainda que não existem diferenças significativas entre os níveis de solidão e stress, entre mães que pertencem a grupos e mães que não pertencem. Foi interessante verificar que as mulheres com filhos mais novos (faixa etária dos 0-4 anos) têm uma presença mais significativa em grupos de apoio à maternidade, no Facebook. Pensamos que isso se deve às dúvidas e ao stresse gerado por um primeiro filho ou pela introdução de mais um filho no núcleo familiar, nos primeiros anos em que tal ocorre, procurando por isso mais activamente grupos que ajudem como rede de suporte, para partilhas, apoio emocional e esclarecimento de dúvidas. / A “social network” refers to a structure formed by individuals or organisations that share interests, motivations and common values. A feature shared by several types of social network is the exchange of information, knowledge, interests and efforts, in an attempt to achieve common goals, often potentiating strengths and resources in crisis situations. Recently, we have heard of this concept applied to the internet. Motherhood is, by itself, a moment of crisis in the life of every family and every woman, requiring adaptations and changes. This study aimed to assess online and offline supports (being these connections themselves denominated social networks), in the specific case of motherhood-related issues. It was also intended to confirm whether there was a correlation between these types of support and stress and loneliness levels sensed by mothers. Finally, it was evaluated as well the influence that the children’s age would have on this adherence to motherhood support groups on Facebook. This study had a sample of 170 women (mothers), with ages between 25 and 62 years. The results showed that, although there are no statistically significant differences between the online and offline support, women who belong to Facebook groups tend to feel more support from the available support platforms. It was shown as well that there are no significant differences between loneliness and stress levels among mothers who belong to groups and mothers who do not belong to those groups. It was interesting to find that women with young children (aged 0-4 years old) have a more significant presence in motherhood support groups on Facebook. We think that this is due to the doubts and the stress generated by a first child or the introduction of another child in the household, therefore making sense that they seek, in the early years of the child’s life, groups that help as a support network, for shares, emotional support and answering questions.

Vivre avec un syndrome de stress post-traumatique : l'expérience et l'impact du diagnostic pour des militaires français

Ce mémoire de maitrise porte sur deux axes principaux : la mise en diagnostic et les enjeux que cela représente pour l’individu ainsi que la question du syndrome de stress post-traumatique (SSPT) dans une population particulière, celle des militaires français. À travers une étude de différents concepts anthropologiques tels que la sous-culture militaire, l’anthropologie du corps, de la mémoire et du discours, le travail de terrain a permis de mettre de l’avant l’expérience et l’impact du diagnostic du syndrome de stress post-traumatique. Les différentes étapes de la mise en diagnostic sont présentées afin de découvrir comment est vécue l’officialisation de ce trouble. Il est alors expliqué le paradoxe propre à ce diagnostic qui est posé, peu importe le type de choc traumatique à l’origine du SSPT. Cela permet de comprendre pourquoi le diagnostic du syndrome de stress post-traumatique reste un véritable enjeu lorsqu’il est question de la population militaire. Bien que de plus en plus de recherches existent sur le sujet au Canada ou aux États-Unis, la question en France reste encore en développement. C’est pourquoi cette recherche permet de découvrir la perception du trouble pour l’individu atteint et ses proches, la prévention et la prise en charge du stress post-traumatique à travers le diagnostic proposé actuellement.

STRESS AND INTERNATIONAL ENTREPRENEURHSIP: The dark side of international entrepreneurship

Entrepreneurship attracts people with high job satisfaction and financial independence. Unfortunately, being deceived by this image, people do not pay attention to side effects of entrepreneurship. This ignorance usually turns into devastating results for entrepreneur’s health and venture performance. Therefore, it is required to seek ways to avoid these situations. The interest of the study lies in understanding of stress influence on international entrepreneurs by considering stress as a negative side effect of international entrepreneurship. To cover the concept of entrepreneurial stress completely, the study was divided into three section presented by following research questions: 1. What are the antecedents of entrepreneurial stress? 2. What are the consequences of entrepreneurial stress? 3. What coping strategies are applied to address entrepreneurial stress? Systematic literature review has been chosen as scientific approach to answer above questions due to the reason that it enables to minimise inconsistencies of both concepts of international entrepreneurship and stress. This method has afforded an opportunity to distinguish such stress causes as role conflict, overload, and ambiguity. Additionally, the study has covered the notion of stress moderators. The author argues that entrepreneurial traits, venture environment, and social support can have influence on degree of stress perception. Further, it has been proven that unaddressed stress could lead to reduction of entrepreneur’s psychological and physiological health. It should be taken into consideration that degree of both job satisfaction and performance would depend on the level of perceived stress. The last part of study emphasises the coping strategies. The author argues that it is important for an international entrepreneur to comprehend his or her and others’ emotions in order to overcome negative consequences of stress. In addition, the author suggests that an international entrepreneur needs to practise job sharing to reduce the amount of work to be completed. Moreover, it is believed that job sharing can help to overcome work-family conflict that prevails among entrepreneurs. The author anticipates that results of study can be beneficial for entrepreneurs who aim to achieve great results.

Transcription of alternative oxidase (AOX) and plastid terminal oxidase (PTOX) during stress-regulated root tissue growth in Daucus carota L. - An approach to identify functional marker candidates for breeding on carrot yield stability

A presente tese explora a hipótese de utilização dos genes da oxidase alternativa (AOX) e da oxidase terminal da plastoquinona (PTOX) como genes-alvo para o desenvolvimento de marcadores funcionais (MF) para avaliar a performance do crescimento em cenoura, fator determinante da produtividade. Para avaliar se os referidos genes estão associados com o crescimento da cenoura procedeu—se ao seu isolamento e posterior análise dos seus perfis de transcrição em diversos sistemas biológicos. O sistema in vitro selecionado, denominado sistema de culturas primárias, permitiu avaliar alterações na quantidade de transcritos desses genes durante os processos de reprogramação celular e crescimento. Ao nível da planta foi também estudado o efeito do frio na expressão precoce dos genes AOX. Ambos os genes DcAOX1 e DcAOX2a revelaram uma resposta rápida e um padrão semelhante apos stresse (inoculação in vitro e resposta ao frio). Foi igualmente verificado um incremento na expressão do gene DcPTOX durante a fase inicial do processo de reprogramação celular. Estudos de expressão dos genes AOX durante o desenvolvimento da raiz da cenoura revelaram que o gene DcAOX2a será potencialmente o gene mais envolvido neste processo. De modo a avaliar a hipótese de envolvimento do gene DcPTOX no crescimento da raíz procederam—se a estudos de expressão ao nível do tecido meristemático. Todavia, para um mais completo entendimento da ligação entre DcPTOX e o crescimento secundário e/ou acumulação de carotenos, a expressão do gene DcPTOX foi também avaliada em raízes de cenoura durante o desenvolvimento, utilizando cultivares caracterizadas por distintos conteúdos de carotenos. Os resultados obtidos demonstraram a associação do gene DcPTOX a ambos os processos. O envolvimento da PTOX no crescimento adaptativo da raiz foi analisado com um ensaio que permitiu identificar, no tecido meristemático, uma resposta precoce do gene DcPTOX face a uma diminuição da temperatura. Adicionalmente, foi efetuada a seleção de genes de referência para uma analise precisa da expressão génica por RT-qPCR em diversos sistemas biológicos de cenoura, e a importância do seu estudo ao nível do sistema biológico foi realçada. Os resultados desta tese são encorajadores para prosseguir os estudos de utilização dos genes AOX e PTOX como MF no melhoramento da performance do crescimento adaptativo em cenoura, fator determinante para a produtividade; ABSTRACT: This thesis explores the hypothesis of using the alternative oxidase (AOX) and theplastid terminal oxidase (PTOX) as target genes for functional marker (FM) development for yield-determining growth performance in carrot. To understand if these genes are associated to growth, different AOX gene family members and the single PTOX gene were isolated, and their expression patterns evaluated in diverse carrot plant systems. An in-vitro primary culture system was selected to study AOX and PTOX transcript changes during cell reprogramming and growth performance. At plant level, a putative early response of AOX to chilling was also evaluated. In fact, both DcAOXl and DcAOXZa were early responsive and showed similar patterns under stress conditions (in vitro inoculation and chilling). A role for DcPTOX during earliest events of cell reprogramming was also suggested. Next, the expression profiles of AOX gene family members during carrot tap root development were investigated. DcAOXZa was identified as the most responsive gene to root development. In order to evaluate if DcPTOX is associated with carrot tap root growth performance, DcPTOX transcript levels were measured in the central root meristem. To further understand whether DcPTOX is associated with secondary growth and/or carotenoids accumulation, DcPTOX expression was also studied in deveIOping carrot tap roots in cultivars with different carotenoids contents. The results indicated that DcPTOX associates to both carotenoid biosynthesis and secondary growth during storage root development. To obtain further insights into the involvement of PTOX on adaptive growth, the early effects of temperature decrease were explored in the root meristem, where a short—term early response in DcPTOX was found, probably associated with adaptive growth. Furthermore, a selection of the most suitable reference genes for accurate RT—qPCR analysis in several carrot experimental systems was performed and discussed. The present research provides the necessary toolbox for continuing studies in carrot AOX and PTOX genes as promising resources for FM candidates in order to assist breeding on yield—determining adaptive growth performance.

Studying the role of the strawberry Fra protein family in the flavonoid metabolism during fruit ripening

Strawberry fruits are highly appreciated worldwide due to their pleasant flavor and aroma and to the health benefits associated to their consumption. An important part of these properties is due to their content in secondary metabolites, especially phenolic compounds, of which flavonoids are the most abundant in the strawberry fruit. Although the flavonoid biosynthesis pathway is uncovered, little is known about its regulation. The strawberry Fra a (Fra) genes constitute a large family of homologs of the major birch pollen allergen Bet v 1 and for which no equivalents exist in Arabidopsis. Our group has shown that Fra proteins are involved in the formation of colored compounds in strawberries (Muñoz et al., 2010), which mainly depends on the production of certain flavonoids; that they are structurally homologs to the PYR/PYL/RCAR Arabidopsis ABA receptor, and that they are able to bind flavonoids (Casañal et al., 2013). With these previous results, our working hypothesis is that the Fra proteins are involved in the regulation of the flavonoids pathway. They would mechanistically act as the ABA receptor, binding a protein interactor and a ligand to regulate a signaling cascade and/or act as molecular carriers. The main objective of this research is to characterize the Fra family in strawberry and gain insight into their role in the flavonoid metabolism. By RNAseq expression analysis in ripening fruits we have identified transcripts for 10 members of the Fra family. Although expressed in all tissues analyzed, each family member presents a unique pattern of expression, which suggests functional specialization for each Fra protein. Then, our next approach was to identify the proteins that interact with Fras and their ligands to gain knowledge on the role that these proteins play in the flavonoids pathway. To identify the interacting partners of Fras we have performed a yeast two hybrid (Y2H) screening against cDNA libraries of strawberry fruits at the green and red stages. A protein that shares a 95% homology to the Heat stress transcription factor A-4-C like of Fragaria vesca (HSA4C) interacts specifically with Fra1 and not with other family members, which suggests functional diversification of Fra proteins in specific signaling pathways. The Y2H screening is not yet saturated, so characterization of other interacting proteins with other members of the Fra family will shed light on the functional diversity within this gene family. This research will contribute to gain knowledge on how the flavonoid pathway, and hence, the fruit ripening, is regulated in strawberry; an economically important crop but for which basic research is still very limited. References: Muñoz, C, et al. (2010). The Strawberry Fruit Fra a Allergen Functions in Flavonoid Biosynthesis. Molecular Plant, 3(1): 113–124. Casañal, A, et al (2013). The Strawberry Pathogenesis-related 10 (PR-10) Fra a Proteins Control Flavonoid Biosynthesis by Binding Metabolic Intermediates. Journal of Biological Chemistry, 288(49): 35322–35332.