Differential effects of low-temperature inhibition on the propylene induced autocatalysis of ethylene production, respiration and ripening of 'Hayward' kiwifruit

Previous studies (Stavroulakis and Sfakiotakis, 1993) have shown an inhibition of propylene-induced ethylene production in kiwifruit below a critical temperature range of 11-14.8 degrees C. The aim of this research was to identify the biochemical basis of this inhibition in kiwifruit below 11-14.8 degrees C. 'Hayward' kiwifruit were treated with increasing propylene concentrations at 10 and 20 degrees C. Ethylene biosynthesis pathways and fruit ripening were investigated. Kiwifruit at 20 degrees C in air started autocatalysis of ethylene production and ripened after 19 d with a concomitant increase in respiration. Ethylene production and the respiration rise appeared earlier with increased propylene concentrations. Ripening proceeded immediately after propylene treatment, while ethylene autocatalysis needed a lag period of 24-72 h. The latter event was attributed to the delay found in the induction of 1-aminocyclopropane-1-carboxylate synthase (ACC synthase) activity and consequently to the delayed increase of l-aminocyclopropane l-carboxylic acid (ACC) content. In contrast propylene treatment induced 1-aminocyclopropane-1-carboxylate oxidase (ACC oxidase) activity with no lag period. Moreover, transcription of ACC synthase and ACC oxidase genes was active only in ethylene-producing kiwifruit at 20 degrees C. In contrast, treatment at 10 degrees C with propylene strongly inhibited ethylene production, which was attributed to the low activities of both ACC synthase and ACC oxidase as well as the low initial ACC level. Interestingly, fruit treated with propylene at 10 degrees C appeared to be able to transcribe the ACC oxidase but not the ACC synthase gene. However, propylene induced ripening of that fruit almost as rapidly as in the propylene-treated fruit at 20 degrees C. Respiration rate was increased together with propylene concentration. It is concluded that kiwifruit stored at 20 degrees C behaves as a typical climacteric fruit, while at 10 degrees C behaves like a non-climacteric fruit. We propose that the main reasons for the inhibition of the propylene induced (autocatalytic) ethylene production in kiwifruit at low temperature (less than or equal to 10 degrees C), are primarily the suppression of the propylene-induced ACC synthase gene expression and the possible post-transcriptional modification of ACC oxidase.

Differential effects of low-temperature inhibition on the propylene induced autocatalysis of ethylene production, respiration and ripening of 'Hayward' kiwifruit

Previous studies (Stavroulakis and Sfakiotakis, 1993) have shown an inhibition of propylene-induced ethylene production in kiwifruit below a critical temperature range of 11-14.8 degrees C. The aim of this research was to identify the biochemical basis of this inhibition in kiwifruit below 11-14.8 degrees C. 'Hayward' kiwifruit were treated with increasing propylene concentrations at 10 and 20 degrees C. Ethylene biosynthesis pathways and fruit ripening were investigated. Kiwifruit at 20 degrees C in air started autocatalysis of ethylene production and ripened after 19 d with a concomitant increase in respiration. Ethylene production and the respiration rise appeared earlier with increased propylene concentrations. Ripening proceeded immediately after propylene treatment, while ethylene autocatalysis needed a lag period of 24-72 h. The latter event was attributed to the delay found in the induction of 1-aminocyclopropane-1-carboxylate synthase (ACC synthase) activity and consequently to the delayed increase of l-aminocyclopropane l-carboxylic acid (ACC) content. In contrast propylene treatment induced 1-aminocyclopropane-1-carboxylate oxidase (ACC oxidase) activity with no lag period. Moreover, transcription of ACC synthase and ACC oxidase genes was active only in ethylene-producing kiwifruit at 20 degrees C. In contrast, treatment at 10 degrees C with propylene strongly inhibited ethylene production, which was attributed to the low activities of both ACC synthase and ACC oxidase as well as the low initial ACC level. Interestingly, fruit treated with propylene at 10 degrees C appeared to be able to transcribe the ACC oxidase but not the ACC synthase gene. However, propylene induced ripening of that fruit almost as rapidly as in the propylene-treated fruit at 20 degrees C. Respiration rate was increased together with propylene concentration. It is concluded that kiwifruit stored at 20 degrees C behaves as a typical climacteric fruit, while at 10 degrees C behaves like a non-climacteric fruit. We propose that the main reasons for the inhibition of the propylene induced (autocatalytic) ethylene production in kiwifruit at low temperature (less than or equal to 10 degrees C), are primarily the suppression of the propylene-induced ACC synthase gene expression and the possible post-transcriptional modification of ACC oxidase.

Effet de Streptococcus Suis sur la capacité de présentation antigénique de cellules dendritiques

Streptococcus suis est un important pathogène porcin et humain, causant méningites et septicémies. Des études suggèrent que S. suis dispose de facteurs de virulence, notamment sa capsule polysaccharidique (CPS), qui lui permettent de moduler les fonctions des cellules dendritiques (DCs), situées à l’interface entre l’immunité innée et adaptative. Les difficultés à développer un vaccin efficace suggèrent aussi une altération de la voie T dépendante. L’objectif général du projet était d’évaluer l’effet de S. suis sur l’activation des cellules T CD4+ ainsi que sur la capacité de présentation antigénique des DCs. Nous avons étudié dans un modèle murin in vivo la réponse T CD4+ mémoire lors d’infections primaire et secondaire. Une faible réponse mémoire centrale a été obtenue, suggérant que la réponse adaptative générée contre S. suis est limitée. Étant donné l’importance du complexe majeur d’histocompatibilité (MHC) de classe II dans la présentation antigénique, nous avons évalué in vitro et in vivo l’expression de ces molécules chez les DCs. Une modulation de l’expression du MHC-II par S. suis a été observée. L’analyse de la transcription de gènes impliqués dans la régulation transcriptionnelle et post-transcriptionnelle du MHC-II nous permet de suggérer que S. suis régule à la baisse la synthèse de nouvelles molécules et favorise leur dégradation lysosomale. Cette stratégie, dans laquelle la CPS ne jouerait qu’un rôle partiel, permettrait à S. suis d’échapper à la réponse adaptative T dépendante. Les résultats de cette étude fourniront de nouvelles perspectives dans la compréhension de la réponse adaptative lors de l’infection par S. suis.

Effet de Streptococcus Suis sur la capacité de présentation antigénique de cellules dendritiques

Streptococcus suis est un important pathogène porcin et humain, causant méningites et septicémies. Des études suggèrent que S. suis dispose de facteurs de virulence, notamment sa capsule polysaccharidique (CPS), qui lui permettent de moduler les fonctions des cellules dendritiques (DCs), situées à l’interface entre l’immunité innée et adaptative. Les difficultés à développer un vaccin efficace suggèrent aussi une altération de la voie T dépendante. L’objectif général du projet était d’évaluer l’effet de S. suis sur l’activation des cellules T CD4+ ainsi que sur la capacité de présentation antigénique des DCs. Nous avons étudié dans un modèle murin in vivo la réponse T CD4+ mémoire lors d’infections primaire et secondaire. Une faible réponse mémoire centrale a été obtenue, suggérant que la réponse adaptative générée contre S. suis est limitée. Étant donné l’importance du complexe majeur d’histocompatibilité (MHC) de classe II dans la présentation antigénique, nous avons évalué in vitro et in vivo l’expression de ces molécules chez les DCs. Une modulation de l’expression du MHC-II par S. suis a été observée. L’analyse de la transcription de gènes impliqués dans la régulation transcriptionnelle et post-transcriptionnelle du MHC-II nous permet de suggérer que S. suis régule à la baisse la synthèse de nouvelles molécules et favorise leur dégradation lysosomale. Cette stratégie, dans laquelle la CPS ne jouerait qu’un rôle partiel, permettrait à S. suis d’échapper à la réponse adaptative T dépendante. Les résultats de cette étude fourniront de nouvelles perspectives dans la compréhension de la réponse adaptative lors de l’infection par S. suis.

Synonymous co-variation across the E1/E2 gene junction of hepatitis C virus defines virion fitness

Hepatitis C virus is a positive-sense single-stranded RNA virus. The gene junction partitioning the viral glycoproteins E1 and E2 displays concurrent sequence evolution with the 3′-end of E1 highly conserved and the 5′-end of E2 highly heterogeneous. This gene junction is also believed to contain structured RNA elements, with a growing body of evidence suggesting that such structures can act as an additional level of viral replication and transcriptional control. We have previously used ultradeep pyrosequencing to analyze an amplicon library spanning the E1/E2 gene junction from a treatment naïve patient where samples were collected over 10 years of chronic HCV infection. During this timeframe maintenance of an in-frame insertion, recombination and humoral immune targeting of discrete virus sub-populations was reported. In the current study, we present evidence of epistatic evolution across the E1/E2 gene junction and observe the development of co-varying networks of codons set against a background of a complex virome with periodic shifts in population dominance. Overtime, the number of codons actively mutating decreases for all virus groupings. We identify strong synonymous co-variation between codon sites in a group of sequences harbouring a 3 bp in-frame insertion and propose that synonymous mutation acts to stabilize the RNA structural backbone.

Insight into the molecular and functional determinants of HP1043, the essential master regulator of Helicobacter pylori.

Helicobacter pylori is one of the most widespread and successful human pathogens, colonizing half of the population stomach mucosa and causing gastric malignancies in 1% of carriers. Due to the increasing number of antimicrobial-resistant strains, in 2017 the WHO included H. pylori among pathogens that pose a major threat for humankind. In this study, we propose as a molecular target for novel antimicrobial strategies HP1043, an orphan response regulator essential for the viability of H. pylori as it orchestrates all the most important cellular processes. Amino acids most relevant for HP1043 dimerization and target DNA recognition were identified and used to guide an in-silico protein-DNA docking and generate a high-resolution structural model of the interacting HP1043 dimer and its target DNA. The model was experimentally validated and exploited to carry out a virtual screening of small molecule libraries, identifying 8 compounds potentially able to interfere with HP1043 function and likely block H. pylori infection. A second line of research aimed at the characterization of the regulatory function of HP1043 and the tight mechanisms of regulation of hp1043 gene expression. In particular, we proved a direct interaction between HP1043 and the housekeeping sigma80 factor of the RNA polymerase. A conditional mutant H. pylori strain overexpressing a synthetic copy of the hp1043 gene altered in nucleotide sequence yet encoding the wild-type protein was generated, achieving increased intracellular levels of HP1043. However, overexpression of HP1043 did not result in an upregulation of target genes transcription nor modulation of hp1043 transcript levels, pinpointing the existence of multiple overlayed mechanisms of regulation that affect both protein levels and functionality as well as maintain steady the amount of hp1043 transcript. Finally, we proposed that a mechanism of post-transcriptional regulation could depend on an antisense transcript to the hp1043 gene which was validated in two different strains.

Genes involved in germline regulative pathways in metazoa: an integrative approach to investigate a key feature of animal biology

In Metazoa, the germline represents the cell lineage devoted to transmission of genetic heredity across generations. Its functions intuitively evoke the crucial roles that it plays in the development of a new organism and in the evolution of the species. Germline establishment is tightly tied to animal multicellularity itself, in which the complex differentiation of cell lineages is favoured by the confinement of totipotency in specific cell populations. In the present thesis, I addressed the subject of germline characterization in animals through different approaches, in an attempt to cover different sides and scales. First, I investigated the extent and nature of shared differentially transcribed molecular factors in 10 different species germline-related lineages. I observed that newly evolved genes are less likely to be involved in germline-related mechanisms and that the mostly shared transcriptional signal across the species considered was the upregulation of genes associated to proper DNA replication, instead of the expected transcriptional and post-transcriptional regulation, that apparently have a higher level of lineage-specificity. I then focused on the evolutionary history of Tudor domain containing proteins, a gene family that underwent germline-associated expansions in animals. Using data from 24 holozoan phyla, I could confirm the previously proposed evolution of the Tudor domain secondary structure. Also, I associated lineage-specific family reductions and expansions to peculiar genomic dynamics and to the evolution of germline-associated piRNA pathway of retrotransposon silencing. Lastly, I characterized and investigated the expression of the Tudor protein TDRD7 in the clam Ruditapes philippinarum. Through immunolocalization, I could compare its expression profiles in gametogenic specimens to the previously characterized germline marker vasa. Combining results with literature, I proposed that, in this species, TDRD7 is involved in the assembly of germ granules, i.e. cytoplasmic structures associated to germline differentiation in virtually all animals, but whose assemblers can be taxon specific.

Characterisation of RNase Y in H. pylori: a non-essential enzyme involved in the processing of cag-PAI non coding RNA 1 (CncR1) sRNA

The importance of Helicobacter pylori as a human pathogen is underlined by the plethora of diseases it is responsible for. The capacity of H. pylori to adapt to the restricted host-associated environment andto evade the host immune response largely depends on a streamlined signalling network. The peculiar H. pylori small genome size combined with its paucity of transcriptional regulators highlights the relevance of post-transcriptional regulatory mechanisms as small non-coding RNAs (sRNAs). However, among the 8 RNases represented in H. pylori genome, a regulator guiding sRNAs metabolism is still not well studied. We investigated for the first time the physiological role in H. pylori G27 strain of the RNase Y enzyme. In the first line of research we provide a comprehensive characterization of the RNase Y activity by analysing its genomic organization and the factors that orchestrate its expression. Then, based on bioinformatic prediction models, we depict the most relevant determinants of RNase Y function, demonstrating a correlation of both structure and domain organization with orthologues represented in Gram-positive bacteria. To unveil the post-transcriptional regulatory effect exerted by the RNase Y, we compared the transcriptome of an RNase Y knock-out mutant to the parental wild type strain by RNA-seq approach. In the second line of research we characterized the activity of this single strand specific endoribonuclease on cag-PAI non coding RNA 1 (CncR1) sRNA. We found that deletion or inactivation of RNase Y led to the accumulation of a 3’-extended CncR1 (CncR1-L) transcript over time. Moreover, beneath its increased half-life, CncR1-L resembled a CncR1 inactive phenotype. Finally, we focused on the characterization of the in vivo interactome of CncR1. We set up a preliminary MS2-affinity purification coupled with RNA-sequencing (MAPS) approach and we evaluated the enrichment of specific targets, demonstrating the suitability of the technique in the H. pylori G27 strain.

Characterization of the molecular mechanisms mediated by the insulin-like growth factor-2 mRNA-binding proteins and Ephrin receptor A2 (EPHA2) signaling in the malignancy of CIC-DUX4 sarcomas

Ewing sarcoma (EWS) and CIC-DUX4 sarcoma (CDS) are pediatric fusion gene-driven tumors of mesenchymal origin characterized by an extremely stable genome and limited clinical solutions. Post-transcriptional regulatory mechanisms are crucial for understanding the development of this class of tumors. RNA binding proteins (RBPs) play a crucial role in the aggressiveness of these tumors. Numerous RBP families are dysregulated in cancer, including IGF2BPs. Among these, IGF2BP3 is a negative prognostic factor in EWS because it promotes cell growth, chemoresistence, and induces the metastatic process. Based on preliminary RNA sequencing data from clinical samples of EWS vs CDS patients, three major axes that are more expressed in CDS have been identified, two of which are dissected in this PhD work. The first involves the transcription factor HMGA2, IGF2BP2-3, and IGF2; the other involves the ephrin receptor system, particularly EphA2. EphA2 is involved in numerous cellular functions during embryonic stages, and its increased expression in adult tissues is often associated with pathological conditions. In tumors, its role is controversial because it can be associated with both pro- and anti-tumoral mechanisms. In EWS, it has been shown to play a role in promoting cell migration and neoangiogenesis. Our study has confirmed that the HMGA2/IGF2BPs/IGF2 axis contributes to CDS malignancy, and Akt hyperactivation has a strong impact on migration. Using loss/gain of function models for EphA2, we confirmed that it is a substrate of Akt, and Akt hyperactivation in CDS triggers ligand-independent activation of EphA2 through phosphorylation of S897. Moreover, the combination of Trabectedin and NVP/BEZ235 partially inhibits Akt/mTOR activation, resulting in reduced tumor growth in vivo. Inhibition of EphA2 through ALWII 41_27 significantly reduces migration in vitro. The project aim is the identification of target molecules in CDS that can distinguish it from EWS and thus develop new targeted therapeutic strategies.

Perspectives in the control of infectious diseases by transgenic mosquitoes in the post-genomic era: a review

Arthropod-borne diseases caused by a variety of microorganisms such as dengue virus and malaria parasites afflict billions of people worldwide imposing major economic and social burdens. Despite many efforts, vaccines against diseases transmitted by mosquitoes, with the exception of yellow fever, are not available. Control of such infectious pathogens is mainly performed by vector management and treatment of affected individuals with drugs. However, the numbers of insecticide-resistant insects and drug-resistant parasites are increasing. Therefore, inspired in recent years by a lot of new data produced by genomics and post-genomics research, several scientific groups have been working on different strategies to control infectious arthropod-borne diseases. This review focuses on recent advances and perspectives towards construction of transgenic mosquitoes refractory to malaria parasites and dengue virus transmission.

Critical role of the transcriptional repressor neuron-restrictive silencer factor in the specific control of connexin36 in insulin-producing cell lines.

Connexin36 (Cx36) is specifically expressed in neurons and in pancreatic beta-cells. Cx36 functions as a critical regulator of insulin secretion and content in beta-cells. In order to identify the molecular mechanisms that control the beta-cell expression of Cx36, we initiated the characterization of the human 5' regulatory region of the CX36 gene. A 2043-bp fragment of the human CX36 promoter was identified from a human BAC library and fused to a luciferase reporter gene. This promoter region was sufficient to confer specific expression to the reporter gene in insulin-secreting cell lines. Within this 5' regulatory region, a putative neuron-restrictive silencer element conserved between rodent and human species was recognized and binds the neuron-restrictive silencing factor (NRSF/REST). This factor is not expressed in insulin-secreting cells and neurons; it functions as a potent repressor through the recruitment of histone deacetylase to the promoter of neuronal genes. The NRSF-mediated repression of Cx36 in HeLa cells was abolished by trichostatin A, confirming the functional importance of histone deacetylase activity. Ectopic expression, by viral gene transfer, of NRSF/REST in different insulin-secreting beta-cell lines induced a marked reduction in Cx36 mRNA and protein content. Moreover, mutations in the Cx36 neuron-restrictive silencer element relieved the low transcriptional activity of the human CX36 promoter observed in HeLa cells and in INS-1 cells expressing NRSF/REST. The data showed that cx36 gene expression in insulin-producing beta-cell lines is strictly controlled by the transcriptional repressor NRSF/REST indicating that Cx36 participates to the neuronal phenotype of the pancreatic beta-cells.

Control dolor post quirúrgic pacients intervinguts hèrnia inguinal per CMA

El dolor post-operatori és un dels problemes més importants que segons la OMS afecta als pacients intervinguts quirúrgicament i el que més els preocupa. Any rere any hi ha un increment del nombre de pacients intervinguts per Cirurgia Major ambulatòria al nostre país de manera que ha de ser el mateix usuari i la família els que facilitin les cures als pacients al propi domicili. Aquest estudi planteja si introduir intervencions educatives al servei disminueix el dolor post-operatori dels usuaris Objectiu general: Dissenyar un programa d’informació que realitzarà infermeria dirigit als cuidadors i pacients del servei de CMA i que han de ser intervinguts de hèrnia engonal. Avaluar l’eficiència del mateix programa educatiu. Metodologia: l’àmbit d’estudi d’aquest treball serà les unitats de Cirurgia Major ambulatòria de qualsevol centre de Catalunya. Es realitzarà un estudi comparatiu quantitatiu entre dos grups independents integrants per 30 participants en cadascun d’ells (total de 60 participants). Grupo A: protocol habitual de la unitat Grup B: intervenció educativa dissenyada Els participants seran pacients majors d’edat, que compleixen els requisits per ser intervinguts per Cirurgia Major Ambulatòria de Hèrnia inguinal. El participant ha de fer-ho amb un familiar responsable de les seves cures. Es realitzaran 3 enquestes al pacient i 3 al familiar responsable de les cures, la primera a les 24 hores després de la intervenció, la segona a la setmana i l’última a les 2 setmanes. Limitacions de l’estudi: en cas de que el pacient que ha de formar part de l’estudi requereixi ingrés hospitalari per alguna complicació durant la cirurgia el pacient deixa de participar en l’estudi automàticament. En cas que el pacient o familiar no contestin al telèfon el dia i l’hora pactada prèviament també deixarà de formar part de la investigació. Per aquest motiu es tindran 10 pacients de reserva per si calgués reemplaçar el subjecte d’estudi.

Effects of competing vegetation and post-planting weed control on the mortality, growth and vole damages to Betula pendula planted on former agricultural land

Abstract

Perspectivas del empleo del control biológico en post-cosecha de fruta

Actualmente se estudian diversos métodos alternativos a los procedimientos habituales de control de plagas y enfermedades de la fruta en post-cosecha, algunos de los cuales se utilizan ya a nivel comercial, como el empleo de microorganismos antagónicos, método en el que se centra el presente artículo. La fruta almacenada en cámaras frigoríficas supone una buena oportunidad para emplear sistemas de lucha biológica en el control de las enfermedades de post-cosecha, puesto que la fruta se encuentra bajo condiciones ambientales controladas, tanto de humedad relativa y temperatura como por la concentración de gases.