Rozprawy doktorskie na temat „AGC REGULATORS”

Il lavoro della mia tesi è focalizzato sullo studio di due differenti proteine e del loro coinvolgimento nel controllo dell’omeostasi cellulare: il fattore di trascrizione EB (TFEB) e l’isoforma 1 dello scambiatore mitocondriale per aspartato-glutammato (AGC1). Nella prima parte della tesi presentata (Capitolo 1) ho indagato il ruolo di TFEB e dei lisosomi nell’omeostasi del Ca2+ intracellulare. Come ben noto da letteratura, i lisosomi sono degli organelli rivestiti da membrane, coinvolti principalmente nei processi catabolici e che possono espellere il loro contenuto nell’ambiente extracellulare attraverso esocitosi. Alcuni dei processi che li vedono coinvolti, come la fusione vescicolare e il traffico degli organelli, richiedono calcio per il loro funzionamento. Recentemente è stato dimostrato che le principali funzioni lisosomiali sono regolate dal fattore di trascrizione TFEB attreverso l’attivazione dell’espressione di geni coinvolti nella biogenesi e controllo dell’esocitosi lisosomiali. Così abbiamo studiato gli effetti della modulazione transiente dell’espressione di TFEB in cellule HeLa misurando i livelli di Ca2+ citosolico e le dinamiche del Ca2+ nel reticolo endoplasmico (ER) e direttamente nei lisosomi in risposta all’attivazione dell’influsso capacitativo di Ca2+. I nostri risultati mostrano come la sovra espressione transeinte di TFEB riduca significativamente il livello di Ca2+ citosolico e la velocità di riempimento di Ca2+ del reticolo durante l’attivazione dell’influsso capacitativo, aumentando la capacità di assorbimento del catione stesso da parte dei lisosomi. Inoltre, la ditruzione o il danneggiamento dei lisosomi elimina questi effetti dipendenti da TFEB nel citosol e nel reticolo. Questi risultati suggeriscono un possibile ruolo di tampone Ca2+ dei lisosomi aprendo nuove vie sulle funzionalità di questi organelli nel controllo dell’omeostasi del Ca2+. Nella seconda parte del mio elaborato (capitolo 2) ho focalizzato la mia attenzione su AGC1, un carrier mitocondriale che catalizza l’esportazione Ca2+-inducibile di aspartato nel citosol in cambio di glutammato e rappresenta un componente chiave dello shuttle malato-aspartato che trasferisce equivalenti ridotti del NADH dal citosol al mitocondrio. Sostenendo l’ossidazione del glucosio, AGC1 è ritenuto fondamentale per il rifornimento energetico delle cellule, in particolar modo nel sistema nervoso centrale e nei muscoli dove questa proteina è maggiormente espressa. Mutazioni del gene codificante di AGC1 causano la perdita della proteina stessa, encefalopatia infantile con ritardo nella mielinizzazione e ridotti livelli encefalici di N-acetilaspartato (NAA), precursore della sintesi della mielina nel sistema nervoso centrale. Qui mostriamo come cellule N2A non differenziate con alterati livelli di AGC1, abbiano un significativo deficit di proliferazione, sebbene sia aumentato lo stress ossidativo e persista un’insufficiente sintesi del NAA. I nostri dati suggeriscono che il deficit energetico della cellula dovuto alla mancanza di AGC1 sia associato a livelli di aspartato inappropriati a supportare la proliferazione nueronale quando la glutammina non viene usata come substrato metabolico. Perciò abbiamo proposto che il ritardo di mielinizzazione nei pazienti affetti da mancanza di AGC1 potrebbe essere attribuibile alla perdita neuronale combinata alla mancanza di NAA durante lo svilupo del sistema nervoso.
This thesis focused on two different proteins and their involvement in cellular homeostasis: the transcription factor EB (TFEB) and the mitochondrial aspartate-glutamate carrier isoform 1 (AGC1). The first work (chapter 1) presented is aimed to investigate the roles of TFEB and lysosomes in intracellular Ca2+ homeostasis. As well documented, lysosomes are membrane-bound organelles mainly involved in catabolic processes. In addition, lysosomes can expel their contents outside of the cell via lysosomal exocytosis. Some of the key steps involved in these important cellular processes, such as vesicular fusion and trafficking, require calcium (Ca2+) signaling. Recent data show that lysosomal functions are transcriptionally regulated by transcription factor EB (TFEB) through the induction of genes involved in lysosomal biogenesis and exocytosis. We studied the effect of transient modulation of TFEB expression in HeLa cells by measuring the cytosolic Ca2+ response after capacitative Ca2+ entry activation and Ca2+ dynamics in the endoplasmic reticulum (ER) and directly in lysosomes. Our observations show that transient TFEB overexpression significantly reduces cytosolic Ca2+ levels under a capacitative influx model and ER re-uptake of calcium, increasing the lysosomal Ca2+ buffering capacity. Moreover, lysosomal destruction or damage abolishes these TFEB-dependent effects in both the cytosol and ER. These results suggest a possible Ca2+ buffering role for lysosomes and shed new light on lysosomal functions during intracellular Ca2+ homeostasis. The second work (chapter 2) is focused on AGC1, the isoform 1 of the mitochondrial aspartate-glutamate carrier which catalyzes a Ca2+-stimulated export of aspartate to the cytosol in exchange for glutamate, and is a key component of the malate-aspartate shuttle which transfers NADH reducing equivalents from the cytosol to mitochondria. By sustaining the complete glucose oxidation, AGC1 is thought to be important in providing energy for cells, in particular in the CNS and muscle where this protein is mainly expressed. Defects in the AGC1 gene cause AGC1 deficiency, an infantile encephalopathy with delayed myelination and reduced brain N-acetylaspartate (NAA) levels, the precursor of myelin synthesis in the CNS. Here, we show that undifferentiated Neuro2A cells with down-regulated AGC1 display a significant proliferation deficit associated with reduced mitochondrial respiration, and are unable to synthesize NAA properly. In the presence of high glutamine oxidation, cells with reduced AGC1 restore cell proliferation, although oxidative stress increases and NAA synthesis deficit persists. Our data suggest that the cellular energetic deficit due to AGC1 impairment is associated with inappropriate aspartate levels to support neuronal proliferation when glutamine is not used as metabolic substrate, and we propose that delayed myelination in AGC1 deficiency patients could be attributable, at least in part, to neuronal loss combined with lack of NAA synthesis occurring during the nervous system development.

Jasmonates (JAs) and spermidine (Sd) influence fruit (and seed) development and ripening. In order to unravel their effects in peach fruit, at molecular level, field applications of methyl jasmonate (MJ) and propyl dihydrojasmonate (PDJ), and Sd were performed at an early developmental stage (late S1). At commercial harvest, JA-treated fruit were less ripe than controls. Realtime RT-PCR analyses confirmed a down-regulation of ethylene biosynthetic, perception and signaling genes, and flesh softening-related genes. The expression of cell wall-related genes, of a sugar-transporter and hormone-related transcript levels was also affected by JAs. Seeds from JA-treated fruit showed a shift in the expression of developmental marker genes suggesting that the developmental program was probably slowed down, in agreement with the contention that JAs divert resources from growth to defense. JAs also affected phenolic content and biosynthetic gene expression in the mesocarp. Levels of hydroxycinnamic acids, as well as those of flavan-3-ols, were enhanced, mainly by MJ, in S2. Transcript levels of phenylpropanoid pathway genes were up-regulated by MJ, in agreement with phenolic content. Sd-treated fruits at harvest showed reduced ethylene production and flesh softening. Sd induced a short-term and long-term response patterns in endogenous polyamines. At ripening the up-regulation of the ethylene biosynthetic genes was dramatically counteracted by Sd, leading to a down-regulation of softening-related genes. Hormone-related gene expression was also altered both in the short- and long-term. Gene expression analyses suggest that Sd interfered with fruit development/ripening by interacting with multiple hormonal pathways and that fruit developmental marker gene expression was shifted ahead in accord with a developmental slowing down. 24-Epibrassinolide was applied to Flaminia peaches under field conditions early (S1) or later (S3) during development. Preliminary results showed that, at harvest, treated fruit tended to be larger and less mature though quality parameters did not change relative to controls.

Jasmonates (JAs) and spermidine (Sd) influence fruit (and seed) development and ripening. In order to unravel their effects in peach fruit, at molecular level, field applications of methyl jasmonate (MJ) and propyl dihydrojasmonate (PDJ), and Sd were performed at an early developmental stage (late S1). At commercial harvest, JA-treated fruit were less ripe than controls. Realtime RT-PCR analyses confirmed a down-regulation of ethylene biosynthetic, perception and signaling genes, and flesh softening-related genes. The expression of cell wall-related genes, of a sugar-transporter and hormone-related transcript levels was also affected by JAs. Seeds from JA-treated fruit showed a shift in the expression of developmental marker genes suggesting that the developmental program was probably slowed down, in agreement with the contention that JAs divert resources from growth to defense. JAs also affected phenolic content and biosynthetic gene expression in the mesocarp. Levels of hydroxycinnamic acids, as well as those of flavan-3-ols, were enhanced, mainly by MJ, in S2. Transcript levels of phenylpropanoid pathway genes were up-regulated by MJ, in agreement with phenolic content. Sd-treated fruits at harvest showed reduced ethylene production and flesh softening. Sd induced a short-term and long-term response patterns in endogenous polyamines. At ripening the up-regulation of the ethylene biosynthetic genes was dramatically counteracted by Sd, leading to a down-regulation of softening-related genes. Hormone-related gene expression was also altered both in the short- and long-term. Gene expression analyses suggest that Sd interfered with fruit development/ripening by interacting with multiple hormonal pathways and that fruit developmental marker gene expression was shifted ahead in accord with a developmental slowing down. 24-Epibrassinolide was applied to Flaminia peaches under field conditions early (S1) or later (S3) during development. Preliminary results showed that, at harvest, treated fruit tended to be larger and less mature though quality parameters did not change relative to controls.

Neutrophil degranulation, angiogenesis and wound healing are three important host responses during inflammation. In this thesis we studied how the immunocalin α1-acid glycoprotein (AGP), in its role of immunomodulatory acute phase protein, affected this processes. We studied bovine neutrophil degranulation within the frame of acute inflammation and we studied angiogenesis and wound healing on endothelial human primary cells. We found that AGP exhorts an important role downregulating both spontaneous and ZAS activated degranulation of secondary bovine neutrophil granules but not on primary granules, and it does it in a dose dependent way. Carbohydrate moiety was found to be critical since desialylated AGP did not have any effects on secondary granules exocytosis. When tested on endothelial cells AGP clearly inhibited Matrigel induced angiogenesis, and modulated endothelial cell adhesion in a biphasic way, inhibiting it at high concentrations. Migration into a wound was also inhibited by high concentrations of AGP in a reversible way. On the modified Boyden chambers AGP acted both as a chemokinetic and as a chemoattractant with results similar to those obtained when FBS was used as a chemoattractant. In our last set of experiments we tested human PMN adhesion to an endothelial monolayer, and we saw that AGP clearly inhibits fMLP/CytoB activated PMN’s to the monolayer whether this is LPS activated or not. Taken together this results support the hypothesis that AGP is heavily involved in the fine tuning of neutrophil activity in the inflammatory environment and endothelial cell behavior.

Gambling constitutes an inherent part of British cultural landscape but due to its potential to cause significant detriments it remains controversial. The Gambling Act 2005 liberalised the UK gambling industry and created an environment where commercial gambling, although regulated, can be offered within a relatively free market setting and its consumption can be stimulated by advertising. The task of the law is to provide a framework where the need for customer choice, a flourishing market, and the respect for private liberties can be adequately balanced with the duty to protect vulnerable individuals such as minors. The Gambling Act has been positioned as containing sufficient protective measures to prevent minors from being harmed by gambling but there is still a relative paucity of research that focuses specifically on how this regime affects this age group. This thesis fills some of the gaps by analysing whether the existing legal and regulatory framework reconciled the conflicting priorities adequately. It uniquely combines legal doctrinal analysis with empirical evidence collected from a sample of British pupils to expose that the liberalisation of gambling has brought severe limitations on protecting minors that are not sufficiently counterbalanced by existing measures. This thesis demonstrates that the legal definition of prohibited gambling does not incorporate all activities that may lead to gambling-related harm. While the age verification measures adopted by online gambling providers appear to be successful, young people continue to have easy access to gambling in land-based venues and are exposed to significant volumes of gambling advertising that appeals to them but these factors are not sufficiently compensated by any holistic regulatory strategy. However, the thesis indicates that the correlation between fun and real gambling games should not be attributed to overlaps in minor's motivations for engaging in either form or to minors' lack of accurate differentiation between them.

Cell signaling is central to integration of internal and external cues that regulate development and homeostasis. Most development is thought of as pre-adult, but limited developmental processes occur in adults. Gametogenesis incorporates elements of both these facets, with a distinct developmental plan for gamete synthesis which is regulated by integration of homeostatic inputs such as nutrient status, and environmental cues. Signaling pathways integrate and transduce information from these cues to evoke a response. A decline in homeostasis and subsequent cues occurs over time, in the case of reproductive tissues leading to a progressive loss of fertility. The Janus Kinase and Signal Transducer and Activator of Transcription or Jak/Stat signaling pathway is conserved between vertebrates and invertebrates and is necessary for numerous functions needed to maintain organism and reproductive homeostasis, as well as contributing to various developmental events. The pathway in the fruit fly Drosophila melanogaster, is composed of a single receptor, Domeless, one Janus kinase, Hopscotch, one known effector, Stat92E, and the Unpaired family of ligands consisting of Upd, Upd2, and Upd3. Jak/Stat signaling is highly pleiotropic in both sexes with involvement in homeostasis and reproduction, making it an ideal model for studying the role of signaling in reproductive aging. Reduction of pathway activity in females results in a higher proportion of unfertilized eggs, which increases with age, and in males leads to a premature onset of infertility. Central to both is integration through cell signaling to evoke an appropriate response. This dissertation explores two of the requirements for Jak/Stat signaling: the pleiotropic requirement for Jak/Stat activity during oogenesis and male reproductive maintenance. Jak/Stat functions from the beginning of oogenesis, in the stem cell niche. From there it participates in multiple functions including specification of a subset of somatic cells called the border cells through the polar cells, a pair of cells at either pole of the egg. Pathway stimulation in the border cells drives their migration with the polar cells to the oocyte boundary, where the polar cells each form an extension in a coordinated manner into the micropyle, the means for sperm entrance during fertilization. Loss of Jak/Stat activity in the border cells prevents border cell migration. While border cell migration has been well studied, polar cell involvement after completion of border cell migration is less well known. To investigate the requirements for polar cell activity and Jak/Stat activity after the completion of border cell migration, we reduced Jak/Stat signaling in the polar cells which, while having no effect on border cell migration, results in blocked micropyles due to loss of coordination of extensions during their outgrowth. Reduced function in the polar cells did not significantly affect expression of adhesion molecules. But, the loss of Stat92E is phenocopied by loss of DE-cadherin. Hence, these results indicate a previously unknown autocrine requirement for Jak/Stat activity in the polar cells. The testes also have a continuous requirement for Jak/Stat activity for stem cell maintenance and differentiation of the germline into mature sperm. Reproductive maintenance not only requires sustained production of gametes, but reproductive tissues are also subject to deterioration of homeostatic functions that contribute to organismal aging. Males from thirty-nine lines of the Drosophila Genetic Reference Panel (DGRP), a panel of inbred, fully sequenced lines, were screened for age at infertility. Data were used to perform a genome-wide association study (GWAS) to identify the genetic architecture of reproductive aging. Candidate variants associated with cell signaling regulators, genes with functions in maintaining cell homeostasis, and organism behavior were uncovered. Notably, several SNPs fell in and near Ptp61F, a negative regulator of Jak/Stat activity. While variants in the primary components of the Jak/Stat pathway were not identified, the general classes of candidate loci functions reflect the requirements for homeostasis, metabolism, and development that have been shown by other studies examining the genetics of aging and fecundity. Thus, we show that Jak/Stat has an amazing amount of pleiotropy that encompasses both the real-time functions of fertility and the time related process of aging.

Over the last three decades, international agricultural trade has grown significantly. Technological advances in transportation logistics and storage have created opportunities to ship anything almost anywhere. Bilateral and multilateral trade agreements have also opened new pathways to an increasingly global market place. Yet, international agricultural trade is often constrained by differences in regulatory regimes. The impact of “regulatory asymmetry” is particularly acute for small and medium sized enterprises (SMEs) that lack resources and expertise to successfully operate in markets that have substantially different regulatory structures. As governments seek to encourage the development of SMEs, policy makers often confront the critical question of what ultimately motivates SME export behavior. Specifically, there is considerable interest in understanding how SMEs confront the challenges of regulatory asymmetry. Neoclassical models of the firm generally emphasize expected profit maximization under uncertainty, however these approaches do not adequately explain the entrepreneurial decision under regulatory asymmetry. Behavioral theories of the firm offer a far richer understanding of decision making by taking into account aspirations and adaptive performance in risky environments. This paper develops an analytical framework for decision making of a single agent. Considering risk, uncertainty and opportunity cost, the analysis focuses on the export behavior response of an SME in a situation of regulatory asymmetry. Drawing on the experience of fruit processor in Muzaffarpur, India, who must consider different regulatory environments when shipping fruit treated with sulfur dioxide, the study dissects the firm-level decision using @Risk, a Monte Carlo computational tool.

Over the last three decades, international agricultural trade has grown significantly. Technological advances in transportation logistics and storage have created opportunities to ship anything almost anywhere. Bilateral and multilateral trade agreements have also opened new pathways to an increasingly global market place. Yet, international agricultural trade is often constrained by differences in regulatory regimes. The impact of “regulatory asymmetry” is particularly acute for small and medium sized enterprises (SMEs) that lack resources and expertise to successfully operate in markets that have substantially different regulatory structures. As governments seek to encourage the development of SMEs, policy makers often confront the critical question of what ultimately motivates SME export behavior. Specifically, there is considerable interest in understanding how SMEs confront the challenges of regulatory asymmetry. Neoclassical models of the firm generally emphasize expected profit maximization under uncertainty, however these approaches do not adequately explain the entrepreneurial decision under regulatory asymmetry. Behavioral theories of the firm offer a far richer understanding of decision making by taking into account aspirations and adaptive performance in risky environments. This paper develops an analytical framework for decision making of a single agent. Considering risk, uncertainty and opportunity cost, the analysis focuses on the export behavior response of an SME in a situation of regulatory asymmetry. Drawing on the experience of fruit processor in Muzaffarpur, India, who must consider different regulatory environments when shipping fruit treated with sulfur dioxide, the study dissects the firm-level decision using @Risk, a Monte Carlo computational tool.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2014.
Vita. Cataloged from PDF version of thesis.
Includes bibliographical references.
regulators of gene expression in a wide range of organisms and biological processes. Each miRNA guides Argonaute (Ago) protein complexes to target and repress hundreds of genes in a sequence-dependent manner. To identify all targets of miRNA regulation, we performed UV crosslinking and immunoprecipitation (CLIP) of Ago complexes in mouse embryonic (ESC) and mesenchymal (MSC) stem cell lines. We also captured the genome-wide miRNA-independent binding footprint of Ago by performing CLIP in cells that lack Dicer, an enzyme required for mature miRNA biogenesis. We surprisingly found that Ago bound a similar set of genes in the absence of Dicer, and this overlap in target genes was due partially to residual, unprocessed miRNAs in the Dicer KO cells. Other potential sites of miRNA-independent Ago interactions, such as histone transcripts and poly-A cleavage and polyadenylation sites, were also identified. One Ago CLIP dataset revealed the enrichment for a G-rich sequence motif at Ago target sites. We later demonstrated that the G-motif is not directly bound to Ago but rather is enriched near miRNA-guided Ago binding sites. Its presence near miRNA target sites is associated with stronger repression of Ago-miRNA targets. Fortuitously, the original Ago CLIP dataset that identified the G-motif was later shown to likely contain target sites of another co-immunoprecipitating RNA binding protein (RBP). Using mass spectroscopy of Ago antibody immunoprecipitations from Ago KO cells, we identified a list of interacting RBPs that could potentially augment Ago-miRNA activity through the G-motif. To investigate target competition in miRNA networks, we related our CLIP analysis of genome-wide, quantitative Ago binding to measurements of absolute miRNA and target RNA concentrations. We found that all miRNAs other than the miR-290 family in ESCs and let-7 family in MSCs were expressed at concentrations below their total target pool. However, 8-12 miRNA families were expressed at near or greater than equimolar ratios with their pool of high affinity targets, and this affinity-partitioned stoichiometry led to significant Ago accumulation and repression of high affinity target sites despite little consequential binding at low affinity sites. Single-cell reporter assays demonstrated that high expressed miRNAs are not susceptible to physiological inductions of competing target transcripts but targets of lower expressed miRNAs are derepressed in a weakly threshold-like manner upon increased target pool levels. In summary, we identify a network of confidently bound targets of miRNA regulation in ESCs and MSCs, reveal the extent of miRNA-independent binding in these two cell types, provide a list of potential miRNA enhancer RBPs, and create a quantitative context for evaluating target competition in miRNA networks.
by Andrew D. Bosson.
Ph. D.

The accessory gene regulator (agr) quorum-sensing system is one of the major regulators of virulence factor production in the pathogen Staphylococcus aureus. Activation of the system depends on the production and sensing of a cyclic peptide signal called the autoinducing peptide (AIP). The biosynthesis of AIP depends on the coordinated action of the AgrB integral membrane endopeptidase and SpsB signal peptidase to process the peptide precursor AgrD into the final signal structure. The primary goal of this dissertation was to gain further insight on the role of AgrD and AgrB in the AIP biosynthesis mechanism. Studies in Chapter II were undertaken to better understand the role of AgrD domains in AgrB-mediated processing. A series of truncation and site-directed mutagenesis studies identified key residues in the AgrD C-terminus that were essential for AgrB processing and AIP production. In parallel, genetic manipulation of the N-terminal leader and AIP-encoding sequence revealed a role for these segments in AIP processing. For the first time, a complex of AgrD covalently linked to AgrB was identified, supporting proposals that this intermediate is an important precursor to AIP production. In Chapter III structure-function studies were performed on AgrB to gain further insight into the AIP biosynthetic mechanism. Initially, the agrBD genes were subjected to random mutagenesis and screened for deficiencies in AIP production. Single-site mutations at 20 different residues within AgrB and another 14 in AgrD were isolated. Interestingly, new mutations in the AgrD N-terminal leader were identified that affect AIP biosynthesis at different steps. In AgrB, most of the mutations blocked peptidase activity, but charge alterations to the K129-K131 region were defective in a later pathway step, separating the peptidase function from AIP ring formation and transport. To localize the AgrB mutations, we reevaluated the membrane topology using the substituted cysteine accessibility method. Our new model predicts four transmembrane helices and a reentrant loop, with both termini located outside of the cell. Finally, co-immunoprecipitation studies indicate that AgrB forms oligomeric structures within the membrane. Taken together, these findings provide a better understanding of the functional role of specific AgrD and AgrB regions in AIP biosynthesis.

Efficient skeletal muscle regeneration requires the accumulation of Foxp3+CD4+ regulatory T (Treg) cells. Muscle Tregs have a transcriptome and T cell receptor repertoire distinct from Tregs found in other tissues. The gene, Areg, was enriched in muscle Tregs and this molecule enhanced regeneration at the level of the satellite cell, a myogenic precursor. Il1rl1, which encodes for the interleukin(IL)-33 receptor, ST2, was also enriched in muscle Tregs. Our findings showed that muscle Tregs required expression of ST2 to both accumulate in injured muscle and potentiate repair. When we examined the producers of IL-33, we uncovered IL-33 was primarily produced by fibro/adipocyte progenitor cells, often in close association with neural structures, e.g. nerve tracts, bundles and muscle spindles, a muscle structure specialized for proprioception. Aging severely impairs skeletal muscle regeneration. Interestingly, although the Treg fraction in lymphoid organs increased with age, we found Treg accumulation in the injured muscle from aged mice was severely limited. Diminished Treg accumulation was due to impaired immigration from circulation, defective proliferation, and less efficient retention within muscle. Additionally, aging was accompanied by a decrease in IL-33-producing cells. When we administered exogenous IL-33, the aged muscle Treg population was restored to levels found in young mice, and regeneration was enhanced. In summary, these studies expand our understanding of this numerically small, but potent immune cell population. We uncover how aging impairs the Treg regenerative response, and in turn, highlight how IL-33 may potentially address age-related sacropenia and other muscle regeneration defects.
Medical Sciences

The evolutionary conserved insulin and nutrient signaling network regulates growth andmetabolism. Nutrients are directly utilized for growth or stored, mostly as triglycerides. InDrosophila, activation of insulin/nutrient signaling in the fat body (the fly equivalent of liverand adipose tissue), causes an increase in fat stores composed of several small-size lipiddroplets (LDs). Conversely, fasting produces an increase in LD size and a decrease in fatcontents. The TOR kinase and its substrate S6 kinase (S6K) play a central role in this response,and particularly in Drosophila, they have been shown to orchestrate cell-autonomous andhormone-controlled growth. However, despite extensive research studies on different modelorganisms (mouse, fly, worm) to decipher the molecular and physiological functions of S6K,nothing is known about how its degradation is regulated.Taking advantage of the inducible RNA interfering (RNAi) library from NIG (Japan), we haveperformed three genetic screens to identify novel regulators of steroidogenesis, lipidmetabolism and dS6K-dependent growth. First, RNAi lines were screened in the ring gland; anorgan that controls the progression of the developmental steps by producing the steroidhormone ecdysone. Out of 7,000 genes screened, 620 positive candidates were identified toproduce developmental arrest and/or overgrowth phenotypes. Then, we challenged 4,000 genesby RNAi screening able to recapitulate the larger sized LD phenotype as obtained uponstarvation, leading to the identification of 24 potential candidates. Finally, the RNAi lines werescreened for their ability to enhance a growth phenotype dependent of the Drosophila S6K(dS6K). Out of 7,000 genes screened, 45 genes were identified as potential negative regulatorsof dS6K. These genes were further used to design a novel protein-protein interaction networkcentered on dS6K through the available data from yeast-2-hybrid (Y2H) assay. The most potentinteractors were then analyzed by treatment of cultured S2 cells with the corresponding doublestrand RNA (dRNA). Western blotting thus, allowed us to discriminate between the geneproducts that regulate dS6K levels versus those that regulate its phosphorylation, as a hallmarkfor its kinase activity. Interestingly, archipelago (ago), which encodes a component of an SCFubiquitinligase known to regulate the degradation of dMyc, Cyclin E and Notch, was identifiedas a negative regulator of dS6K-dependent growth. Based on the Y2H available data showingthat Ago and dS6K interact each other and the presence of a putative Ago-interaction motif indS6K, we hypothesized that Ago causes an ubiquitin-mediated degradation of dS6K. Ourmolecular data showed that loss of ago caused an elevated level of dS6K, which confirms arole of Ago in controlling dS6K degradation. Altogether our findings emphasize the importanceof the saturating screening strategies in Drosophila to identify novel regulators of metabolicand signaling pathways.

The innate mammalian response to injury involves the initiation of activation of two major blood-borne proteolytic systems; coagulation and complement. Recent studies have revealed that there is considerable crosstalk and interplay between these two systems. Polyphosphate (polyP) is a naturally occurring inorganic linear polymer that co-regulates these two systems, acting as a promoter of coagulation and an inhibitor of complement. This thesis aims to further characterize the mechanisms by which polyP regulates the complement system, and to test its physiological relevance in a model of human disease, age-related macular degeneration (AMD), the pathogenesis of which involves excess complement activation and oxidative stress. Based on data from our lab and studies in bacteria that polyP dampens complement activation and interferes with oxidative stress, I hypothesized that polyP would protect against AMD. To test this hypothesis, I used hemolytic assays to measure the complement activity in response to polyP, in vitro studies with AMD-associated cell lines to examine protective properties of polyP, and an in vivo model of AMD to evaluate the therapeutic efficacy of polyP. I showed that polyP dampens complement activation by interfering with the terminal pathway of complement, and that it also interferes with oxidative stress-induced cellular damage. The mechanisms by which it exerts this effect have not yet been determined. However, in vivo, in rodent models of AMD, polyP protects against laser-induced choroidal neovascularization (CNV), a feature of AMD, with reduced deposition of complement. An agent such as polyP, that simultaneously suppresses complement activation and protects against oxidative stress, holds potential therapeutic value. The findings in this thesis raise awareness of the potential importance of a ubiquitous, naturally occurring inorganic compound that has largely been overlooked. Most important, the findings reveal a promising use for polyP as a treatment for AMD, a common and devastating disease that affects millions of people worldwide.
Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
Graduate

Mutations in the eukaryotic integral membrane protein Cystic Fibrosis Transmembrane conductance Regulator (CFTR) cause the hereditary disease cystic fibrosis (CF). CFTR functions as an ion channel at the surface of epithelial cells and regulates the movement of chloride ions and water across the plasma membrane. CFTR is difficult to express and purify in heterologous systems due to its propensity to form insoluble aggregates and its susceptibility to degradation. Obtaining good yields of highly purified CFTR has proven problematic and contributes to our limited understanding of the structure and function of the protein. The most prevalent disease causing mutation, F508del, results in misfolded CFTR which is particularly unstable and is quickly targeted for degradation by the host system and is prevented from being trafficked to the plasma membrane. There are limited treatment options for patients with the F508del mutation and it is therefore of significant interest within CF research. New methods and assays are required to identify potential compounds which could correct the F508del mutation. This thesis investigates the use of Saccharomyces cerevisiae to express and purify codon optimised recombinant CFTR. The use of a green fluorescent protein (GFP) tag enabled quick and simple detection of CFTR in whole cells and after extraction from the plasma membrane. By optimising the culture conditions for CFTR expression and detergent solubilisation conditions, relatively high yields of full-length protein were obtained. When used as a chemical chaperone at the time of inducing CFTR expression, glycerol increased yields of full-length protein. Degradation of CFTR could be limited by inducing expression at an optimal cell density and by harvesting cells within a specific time window. CFTR was extracted by solubilisation in the mild detergent dodecyl-β-D-maltopyranoside (DDM) in the presence of up to 1 M NaCl with up to ~87% efficiency in some cases. Using a gene optimisation strategy in which additional purification tags and a yeast Kozak-like sequence were added, the human CFTR (hCFTR) protein was expressed and purified. Fluorescence microscopy revealed CFTR localisation at the periphery of yeast cells. Immunoaffinity chromatography facilitated by the GFP tag at the C terminus of CFTR produced protein of up to 95% purity. An assessment of the thermal stability of this highly purified CFTR using a fluorescent probe binding assay revealed a denaturation midpoint (Tm) of ~43 degC. The ability of this assay to determine the stability of CFTR is encouraging and there is the potential to further develop it in a high-throughput manner to identify compounds which stabilise the F508del protein and which may hold the key to developing new treatments for CF.

O tratamento de infecções por Staphylococcus aureus tem sido problemático devido ao surgimento de cepas resistentes a múltiplios antibióticos. O antibiótico de escolha para o tratamento de infecções por S. aureus resistente a oxacilina é o glicopeptídeo vancomicina. Desde o primeiro isolamento de cepas com sensibilidade reduzida a vancomicina (VISA) em 1997, tem havido crescente preocupação com a disseminação da resistência a este antibiótico. Os mecanismos moleculares que levam à resistência de baixo nível a vancomicina ainda não foram elucidados. A detecção deste fenótipo na rotina de laboratório clínico é laboriosa, pois as técnicas disponíveis são de difícil execução e interpretação. Até agora, não há relato de transmissão horizontal de infecção por VISA, e todas as cepas com este fenótipo foram isoladas de pacientes que faziam o uso prolongado de vancomicina. Uma deficiência no locus regulador de genes acessórios (agr) foi postulado como fator de risco para a aquisição do fenótipo VISA por uma cepa sensível a este antibiótico. Para este estudo, foram selecionadas 47 cepas de S. aureus, com sensibilidades variadas a vancomicina, inclusive 5 cepas VISA isoladas no Brasil. Determinou-se nas cepas as concentrações inibitórias mínimas de vancomicina e oxacilina, a atividade hemolítica em ágar sangue de carneiro e de coelho, a capacidade de aderir ao poliestireno e o polimorfismo do locus agr. Determinou-se a integridade do locus agr por PCR-RFLP e sequenciamento de bases em 13 cepas representativas das 47 estudadas. A integridade do locus regulador acessório sarA também foi avaliada por sequenciamento de bases nestas 13 cepas. Foram escolhidas 18 cepas sensíveis a vancomicina com variadas características fenotípicas e estas foram submetidas à indução de resistência a vancomicina através da passagem seriada em concentrações crescentes deste antibiótico. A taxa de mutação que leva à capacidade de crescimento em 6 µg/mL de vancomicina foi avaliada em 8 cepas através de ensaios de flutuação. Não observou-se correlação entre a aquisição de resistência a vancomicina com as atividades hemolíticas ou capacidade de adesão das cepas. A maioria das cepas (82,9%) apresentou-se como pertencente ao grupo polimórfico agr I, inclusive as cepas VISA. Duas cepas não conseguiram ser induzidas à resistência a vancomicina. O tempo levado para a aquisição de resistência não se correlacionou com nenhuma característica fenotípica ou genotípica de um grupo de cepas. A taxa de mutação que leva à capacidade de crescimento em 6µg/mL de vancomicina apresentou-se maior para uma cepa pertencente ao clone endêmico brasileiro (CEB) cujo locus agr pertence ao grupo I e não apresentou variação de acordo com funcionalidade ou tipo do locus agr. Apenas uma das cepas VISA apresentou uma mutação no locus agr que o torna disfuncional. Os loci agr das outras cepas estudadas apresentaram-se íntegros. O locus sarA das cepas estudadas apresentou-se íntegro e com polimorfismos funcionais agrupados de acordo com a linhagem clonal das cepas. Pôde-se concluir que a integridade funcional do locus agr não é uma condição sine qua non para a aquisição de resistência de baixo nível a vancomicina por parte de uma cepa sensível a este antibiótico. O grupo polimórfico agr II não tem maior predisposição à aquisição de resistência de baixo nível a vancomicina, como havia sido sugerido por alguns trabalhos disponíveis na literatura.
The treatment of staphylococcal infections has lately been a strenuous undertaking due to the resistance of Staphylococcus aureus to multiple antibiotics. The antimicrobial drug of choice for the treatment of methicillin resistant S. aureus (MRSA) is the glycopeptide vancomycin. Since the first isolation of S. aureus with reduced susceptibility to vancomycin (VISA) in 1997, there has been growing concern as to the dissemination of this resistance phenotype among isolates of this species. The molecular mechanisms that result in low level resistance to vancomycin have not yet been completely elucidated. The correct detection of this phenotype in the clinical laboratory is tricky, for the techniques available for this purpose are hard to execute and interpret. Until now, lateral transmission (dissemination) of VISA has not been reported and all strains bearing this phenotype have been isolated from patients who had been making prolonged use of vancomycin. A deficiency in the accessory gene regulator (agr) has been proposed as a risk factor for the acquisition of a VISA phenotype by a susceptible strain. For this study, 47 nosocomial VISA strains, that had been isolated in another study, were used. These strains were isolated from multiple geographical regions of Brazil, and included 5 VISA strains. The minimal inhibitory concentrations (MIC) of vancomycin and oxacillin, as well as haemolysis in sheep and rabbit agar, adhesion to polystyrene and agr polymorphism were determined in all of these strains. The integrity of the agr locus was determined by PCR-RFLP and by nucleotide sequencing in a sample of 13 strains chosen to be representative of the 47 strains studied. The integrity of the Staphylococcal accessory regulator sarA was also determined by nucleotide sequencing in these 13 strains. Another representative sample of 18 strains that were susceptible to vancomycin were submitted to induction of resistance to vancomycin by serial passage in increasing concentrations of this drug. The mutation rate of a mutation that leads to the ability of growing in a concentration of 6 µg/mL of vancomycin was determined for 8 strains by fluctuation assays. There was no correlation between the acquisition of resistance to vancomycin with either haemolysis or adhesion to polystyrene. Most strains (82.9%) bore a group I agr polymorphism, including all of the VISA strains. Two strains could not be induced to resistance. The time taken for each strain to acquire resistance to vancomycin did not correlate with any phenotypic or genotypic characteristic pertaining to a group of strains. The rate of mutation that leads to the ability of growing in 6µg/mL of vancomycin proved to be higher for a strain belonging to the Brazilian Endemic Clone (BEC) bearing an agr group I polymorphism, and did not vary according to presence or type of agr locus. Only one of the VISA strains presented a mutation in the agr locus that renders it disfunctional. The agr loci of the other strains studied presented themselves to be intact. The sarA loci of the strains evaluated were intact however presented functional polymorphisms that were groups according to the clonal lineage of the strains. It can thus be concluded that the functional integrity of the agr locus is not a sine qua non condition for the acquisition of low level resistance to vancomycin by a susceptible strain. Bearing of an agr group II polymorphism does not predispose a strain to acquire resistance to vancomycin, as has been previously suggested in literature.

Four experiments were conducted with the objectives of (i) comparing the suitability of various H. bulbosum clones for haploid production, (ii) determining the parental effects of H. bulbosum and barley genotypes on percentage of pollinated florets yielding caryopses with rescuable embryos and on embryo viability, (iii) comparing different stages for embryo culture and caryopsis culture, and (iv) attempting to produce barley haploids directly from cultured immature caryopses. The results demonstrated: that reproductive characteristics of H. bulbosum clones varied with environmental conditions; that the hybrid H. bulbosum clones MBC-3 and MBC-4 were superior to their parents Cb2920 and Cb2929 as pollen donors; that both parental genotypes and date of harvesting after pollination had large effects on percentage of pollinated florets yielding caryopses with rescuable embryos and on embryo viability; that haploid plantlets can be generated from haploid caryopsis culture without embryo rescue, but only at a low frequency and with a slow rate of germination.

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he accessory gene regulator (agr) is a S. aureus global regulator of virulence factors, as the staphylococcal enterotoxins (SE), responsible for the staphylococcal food poisoning. There are four different agr groups due to the polymorphism in the amino acids sequence of the agrC and agrD. In the literature is described a relationship among agr groups and virulence factors, diseases, preferential host and antibiotic resistance. In this context, it was aimed to characterize the agr groups through biplex PCR, and relationship among agr groups, enterotoxigenic and antimicrobian profiles of S. aureus isolated from foods and bovine mastitis milk. A total of 115 strains were used to characterize the agr groups , being 30 isolated from f bovine mastitis milk and 85 isolated from several sources of foods. To assess the relationship between agr groups and enterotoxin production were used 14/85 strains previously characterized for the presence of some enterotoxins (sea, seb, sec, sed e cluster egc). To determine the profile of antibiotics resistance were used 71/115 strains. We observed a prevalence of agr group II with19.1% (22/115 strains), followed by the agr I with 8.6% (10/115 strains), agr III with 7.8% (9 / 115 strains), and agr IV with6.0% (7 / 115 strains). Among the strains isolated from bovine mastitis milk agr group I was prevailed with 20% (6/30 strains), whereas in the strains isolated from several food sources was observed prevalence of agr group II with 32.7% (18/85 strains), especially among those from chicken meat. Among the 14 strains (14/85) that contained enterotoxin genes, the majority of them contained the cluster egc (70%) belonged to agr II, whereas no relationship was found with those who had the genes for the classical SE (sea, seb, sec, sed). Considering the antibiotic resistance 100% of bovine mastitis milk strains and from various sources of food were resistant to penicillin, ampicillin, cefoxitin, and vancomycin. Relationship was observed between food strains, which were resistant to vancomycin and agr II, however, no relationship was found between antibiotic profile and agr groups among the strains isolated from bovine mastitis milk. These results demonstrated the prevalence of agr II among food strains and agr I among bovine mastitis milk strains. Moreover, the strains that carried the cluster egc were predominant agr II, which could indicate the occurrence of a clonal group among those. Another important result obtained in this study was the high rate of S. aureus multiresistant strains isolated from food, which emphasizes the importance of dissemination of these strains among foods.
O accessory gene regulator (agr) é um regulador global de fatores de virulência em S. aureus, como as enterotoxinas estafilocócicas (EE), responsáveis pela intoxicação alimentar estafilocócica. São conhecidos quatro distintos grupos agr devido ao polimorfismo na seqüência dos aminoácidos de agrC e agrD. Na literatura descreve-se relação entre fatores de virulência, patogenias, hospedeiro preferencial e perfil de resistência a antibióticos com grupos agr. Neste contexto, objetivou-se caracterizar grupos agr através de biplex PCR, e relacioná-los com os perfis enterotoxigênico e antimicrobiano de cepas de S. aureus isoladas em alimentos e leite de vacas mastíticas. Para caracterização dos grupos agr foram utilizadas 115 cepas, sendo 30 isoladas em leite de vacas mastíticas e 85 em diversas fontes de alimentos. Para a relação entre grupos agr e produção de enterotoxina foram utilizadas 14/85 cepas previamente caracterizadas quanto à presença de alguma enterotoxina (eea, eeb, eec, eed e cluster egc), já para determinar o perfil de resistência a antibióticos utilizaram-se 71/115 cepas. Observou-se uma prevalência do grupo agr II, com 19,1% (22/115 cepas), seguido do agr I, com 8,6% (10/115 cepas), agr III 7,8% (9/115 cepas), e agr IV, 6,0% (7/115 cepas). Entre as cepas isoladas em leite de vacas com mastite houve predomínio do grupo agr I, com 20% (6/30 cepas); já nas cepas isoladas de diversas fontes de alimentos observou-se prevalência do grupo agr II, com 32,7% (18/85 cepas), especialmente entre as provenientes de carne de frango. Entre as 14/85 cepas que carreavam genes de enterotoxinas, a maioria que albergava o cluster egc (70%), pertencia ao grupo agr II, enquanto nenhuma relação foi observada com aquelas que possuíam os genes para as EE clássicas (eea, eeb, eec, eed). Com relação ao perfil de resistência antimicrobiana, 100% das cepas isoladas de leite de vacas com mastite e das diversas fontes de alimentos apresentaram resistência à penicilina, ampicilina, cefoxitina e vancomicina. Observou-se relação entre cepas isoladas de alimentos, que eram resistentes à vancomicina e grupo agr II, entretanto, nenhuma relação foi observada entre perfil antimicrobiano e grupos agr entre as cepas isoladas em leite de vacas com mastite. Através destes resultados demonstra-se a prevalência do grupo agr II entre as cepas isoladas de alimentos e do grupo agr I em cepas isoladas de leite de vacas mastíticas. Além disso, nas cepas que carreiam o cluster egc houve predominância do grupo agr II, podendo indicar um grupo clonal entre essas. Outro resultado relevante obtido neste estudo foi à elevada taxa de cepas de S. aureus multiresistentes isoladas em alimentos, o que ressalta a importância da disseminação de cepas multiresistentes entre os alimentos.

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly in developed countries. Aggressive research is underway to elucidate putative molecular targets for therapy for both the atrophic and neovascular forms of this disease. Current pharmacotherapy is effective in some patients but not sufficient to halt disease progression or repair damage that has already occurred. Drug intervention and retinal cell replacement represent the two most promising potential treatment avenues. The purpose of this thesis was to investigate the role of a recently identified regulator of neural development, SIRT1, in retinogenesis and to further investigate whether pharmacological inhibition of this protein represents a possible treatment option in neovascular AMD. Via immunohistochemistry and immunocytochemistry we evaluated the expression and subcellular localization of SIRT1 and its innate inhibitor, DBC1, in mouse and human fetal and adult retinas. We further studied SIRT1 in mouse- and human-derived retinal progenitor cells, the former being using in small interfering RNA studies. We found SIRT1 to be widely expressed in developing and adult retinas and to be a regulator of key retinal development genes, namely PAX6, Nestin and CRX. Moreover, we found that photoreceptor precursor cells were among the smallest cells in the heterogeneous population of mouse retinal progenitors. Collectively, these results provide the foundation for manipulating SIRT1 expression in small retinal progenitors as a means of increasing the yield of photoreceptors for transplantation in models of retinal degeneration.We further found SIRT1 to be highly expressed in human-derived choroidal neovascular membranes and sought to pharmacologically inhibit its activity via the drug Nicotinamide. We found Nicotinamide to be a potent regulator of angiogenic and hypoxic signaling in a human retinal pigment epithelial cell line at both the protein level using angiogenesis arrays and at the RNA level using whole genome microarrays. These results point to the SIRT1 inhibitor, Nicotinamide, as a possible agent for treatment of neovascular AMD. Further studies of Nicotinamide are warranted in animal models of AMD. To the best of our knowledge, this is the first time that a detailed analysis of SIRT1 as a regulator of both retinal development and choroidal neovascularization has been reported.
La dégénérescence maculaire liée à l'âge (DMLA) est la principale cause de cécité chez les personnes âgées dans les pays développés. Une recherche dynamique est en cours pour élucider des cibles moléculaires potentiels pour le traitement de la dégénérescence à la fois pour la forme atrophique et la forme néovasculaire de cette maladie. L'actuelle pharmacothérapie est efficace chez certains patients mais pas suffisante pour arrêter la progression de la maladie ou la réparation des dommages qui ont déjà eu lieu. La decouverte de nouveaux medicaments et le remplacement de cellules rétiniennes représentent les deux avenues les plus prometteuses de traitements possibles. Le but de cette thèse est d'étudier le rôle d'un régulateur récemment identifié du développement neuronal, SIRT1, dans le developpement de la retine et de rechercher si l'inhibition pharmacologique de cette protéine représente une option de traitement possible dans la DMLA. Via l'immunohistochimie et l'immunocytochimie, nous avons évalué l'expression et la localisation subcellulaire de SIRT1 et de son inhibiteur inné, DBC1, chez la souris et les humains dans les retines fœtales et adultes. Nous avons également étudié SIRT1 dans les cellules souches de la rétine chez la souris et l'homme. Nous avons trouvé SIRT1 largement exprimé dans la rétine en développement et des adultes et à un régulateur de gènes clés du développement de la rétine, à savoir PAX6, Nestin et CRX. En outre, nous avons constaté que les cellules précurseurs des photorécepteurs ont été parmi les plus petites cellules dans la population hétérogène de cellules progénitrices. Collectivement, ces résultats fournissent la base pour la manipulation de l'expression SIRT1 dans les petits progéniteurs rétiniens comme un moyen d'augmenter le rendement des photorécepteurs à la transplantation dans les modèles de dégénérescence rétinienne. Nous avons en outre constaté que SIRT1 est fortement exprimé dans les membranes néovasculaires humaines et avons cherché à inhiber son activité pharmacologique par le nicotinamide. Nous avons trouvé que Nicotinamide est un puissant régulateur de l'hypoxie et de l'angiogenèse au niveau de la protéine et de l'ARN. Ces résultats indiquent que l'inhibiteur de SIRT1, Nicotinamide est un agent possible pour le traitement de la DMLA néovasculaire. D'autres études de la nicotinamide devraient être poursuivit dans des modèles animaux de la DMLA. Au meilleur de notre connaissance, c'est la première fois qu'une analyse détaillée de SIRT1 l'identifie comme un régulateur du développement tant de la rétine et de la néovascularisation choroïdienne.

Peach (Prunus persica) is one of the most important fleshy fruit crops worldwide and model species for drupe plant species. Peach fruit development is characterized by a tight relationship between seed and pericarp during the early stages, followed in later stages by an uncoupling in the pattern of development due to the lignification of the endocarp. Diverse peach cultivars may have fruit developmental periods of very different length, while having a similar development for the seed. Understanding the relationship between seed and pericarp sheds light onto the mechanism regulating fruit development. Transcriptomic approach is a powerful tool to investigate this relationship, as it gives broad information on the transcription of a large amount of genes in a single experiment. Chapter II is a published article regarding the use of the µPEACH1.0 array for the understanding of the relationships between seed and mesocarp and between early and late stages of development in the cultivar Fantasia. Peach mRNA samples were taken from early and late developmental stages of the two organs and then hybridized on the 4 806 probes of the µPEACH1.0 array. The transcriptomic data obtained from these samples were then cross-compared. Marker genes for the four peach developmental stages (S1 stage: fruit cells division and enlargement, S2: lignification of the endocarp, S3: mesocarp cell expansion, S4: ripening) were found for both mesocarp and seed and their expression confirmed by qRT-PCR. Stage- specific markers found for the mesocarp were a RD22-like protein, a serin- carboxypeptidase, a senescence-related protein and an Aux/IAA, for S1, S2, S3 and S4 stages, respectively, while seed markers were a lipid transfer protein(LTP1), a pathogenesis-related (PR) protein, a prunin and Late Embryogenesis Abundant (LEA) protein, for S1, S2, S3 and S4 stages, respectively. By qRT-PCR it was confirmed that these genes act as markers also in an early cultivar (SpringCrest) and a slow ripening genotype (slr). Then, the data were analyzed with the HORMONOMETER tool in order to indirectly measure the relative amounts of hormones in the different organs and developmental stages. It was found that auxins, cytokinins, and gibberellins may be involved in signaling during the early development, when there is cross-talk between the two organs. Chapter III is an unpublished article in which it is described how a new microarray platform, µPEACH3.0, was employed to study peach mesocarp and seed development. The recent publication of the peach genome allowed the development of a whole-genome microarray which overcame the problem of having an array assessing gene expression of only one part of the genome. In respect of the study described in Chapter II, also the number of samples were increased: three biological replicates for each of six time-points for each of the two organs were used, giving a larger overview on the development of these two tissues. The whole genome microarray, µPEACH3.0, performed well, with a correlation with qRT-PCR data of 0.77, a number similar to that found for other arrays. The transcriptomic data easily distinguished the two tissues and the six time-points, as shown by principal component analysis. 69% of the probes gave a significant signal from at least one of the samples. Anyway, considering that the number of functioning probes diminishes if only the samples of one tissue are taken into account, it is probable that testing the microarray with mRNA coming from other tissues (such as leaves or roots) will increase the number of significant signals coming from the array. Global analysis of gene activity was focused into the early stages of development. Data allowed us to identify several genes involved in cell cycle processes that occur at the onset of both mesocarp and seed development. In particular genes of the TITAN family were found to be active in the endosperm containing seed. The analysis of the cell cycle genes in the mesocarp showed the existence of two different patterns of expression: while mitosis related genes were expressed only in stage S1, DNA replication genes showed a double peak of expression, in S1 and then in S3/S4, suggesting that events of endoreduplication may occur in these late stages. By qRT-PCR the expression levels of these genes were tested also in other cultivars, the data obtained suggest that the lack of endoreduplication may be involved in the slow rate of growth in S3 stage of the slr genotype. The patterns of expression of transcription factors (TFs) families were then assessed, as transcription factors are thought to be the proteins with the most important regulatory roles during development. It was found that TFs of the SQUAMOSA promoter Binding Protein (SBP) family have an high expression level at the beginning of the development of both the organs considered, which then quickly decreases. The transcription of Growth Regulating Factors (GRFs) has been discovered to be induced in the mature seed. The data were confirmed by qRT-PCR also in an ‘SpringCrest’ and the slow ripening genotype slr. Given that the mRNA abundance of genes belonging to these TFs families is regulated by specific microRNAs (miRNAs) in other plant species, the expression of the peach homologues of these miRNAs was measured. In three different cultivars a negative correlation in the RNA abundance was found for the following miRNA/target TF couples: miR156/SBP, miR396/GRF and mir167/ARF8, suggesting not only that these miRNAs have the same activity also in peach, but also that miRNAs are deeply involved in the regulatory network underlying the peach fruit development. Appendix is a published study in which µPEACH3.0 is used to study the effects of wounding in two peach cultivars with different tolerance to this stress. RNA samples from wounded and unwounded mesocarps of melting cultivar Glohaven (GH) and slow melting cultivar BigTop (BT) were used. Transcriptomic data, confirmed by qRT-PCR analysis, showed the involvement of WRKY, AP2/ERF and HSP20 transcription factors in the GH response to wounding. Along with them, also genes involved in response to stresses, cell wall metabolism, phenilpropanoid and triterpenoid biosynthesis were found to be up regulated in the wounded GH mesocarp.
Il pesco (Prunus persica) è uno dei più importanti alberi da frutto al mondo e la specie modello per le drupacee. Lo sviluppo del frutto di pesco è caratterizzato da un stretto rapporto tra il seme e il pericarpo durante i primi stadi, seguito negli stadi successivi da un disaccoppiamento nello schema di sviluppo dovuto alla lignificazione dell’endocarpo. Le varie cultivar di pesco possono avere dei periodi di sviluppo del frutto dalla lunghezza estremamente variabile, pur avendo un seme che si sviluppa in maniera simile. Per questo, comprendere la relazione tra seme e pericarpo può chiarire il meccanismo che regola lo sviluppo del frutto nel suo complesso. L’approccio transcrittomico è uno strumento potente per analizzare questa relazione, dato che produce un gran numero di informazioni sulla trascrizione di un gran quantitativo di geni in un singolo esperimento. Il Capitolo II consiste in un articolo pubblicato che descrive l’uso dell’array µPEACH1.0 nello studiare la relazione tra seme e mesocarpo e tra stadi iniziali e finali di sviluppo nella cultivar Fantasia. Campioni di mRNA di pesco sono stati raccolti dagli stadi iniziali e finali dei due organi e ibridizzati sulle 4 806 sonde dell’array µPEACH1.0. I dati trascrittomici ottenuti da questi campioni sono stati quindi confrontati. Sono stati trovati dei geni marcatori per i quattro stadi di sviluppo del pesco (Stadio S1: divisione ed espansione cellulare nel frutto, S2: lignificazione dell’endocarpo, S3: espansione cellulare nel mesocarpo, S4: maturazione) sia per il mesocarpo che per il seme e la loro espressione confermata con la qRT-PCR: I marcatori stadio-specifici per il mesocarpo sono rispettivamente per S1, S2, S3 e S4: una proteina RD22-like, una serin-carbossipeptidasi, una proteina correlata alla senescenza e una Aux/IAA; mentre per il seme sono, rispettivamente: una proteina trasportatrice di lipidi (LTP1), una proteina correlata alla patogenesi (PR), una prunina e una proteina LATE EMBRYOGENESIS ABUNDANT (LEA). La qRT-PCR ha confermato che questi geni sono marcatori anche in una cultivar precoce (SpringCrest) e in un genotipo a maturazione lenta (slr). Quindi i dati sono stati analizzati con lo strumento HORMONOMETER al fine di misurare indirettamente il quantitativo relativo di ormoni nei vari organi e stadi di sviluppo. E’ emerso che l’auxina, le citochinine e le gibberelline possono essere coinvolte nella segnalazione durante l’inizio dello sviluppo, quando vi è comunicazione tra i due organi. Il Capitolo III è un articolo non pubblicato nel quale viene descritto come venga utilizzata una nuova piattaforma microarray (µPEACH3.0) nello studiare lo sviluppo del seme e del mesocarpo di pesco. La recente pubblicazione del genoma di pesco ha permesso lo sviluppo di un microarray che copre l’intero genoma, superando così il problema di avere un array che misura l’espressione genica solo di una parte del genoma. Rispetto allo studio descritto nel Capitolo I, anche il numero di campioni è stato incrementato: sono state usate tre repliche biologiche per sei diversi momenti per ciascuno dei due organi, dando così una visuale più vasta sullo sviluppo di questi due tessuti. L’array µPEACH3.0 ha funzionato bene, dando una correlazione con i dati di qRT-PCR pari a 0.77, un numero simile a quello trovato per altri array. I dati trascrittomici hanno facilmente distinto i due tessuti e i sei campionamenti, come mostrato dall’analisi delle componenti principali. Il 69% delle sonde ha prodotto un segnale significativo in almeno uno dei campioni, ciò nonostante, considerando che il numero di sonde funzionanti decresce se si prende in considerazione un solo tessuto, è probabile che testando il microarray con mRNA proveniente da altri tessuti (come le foglie o le radici) aumenti il numero di segnali significativi provenienti dall’array. L’analisi globale dell’attività genica è stata indirizzata ai primi stadi di sviluppo. I dati hanno permesso di indentificare parecchi geni coinvolti nei processi del ciclo cellulare che si verificano all’inizio dello sviluppo sia del mesocarpo che del seme. In particolare, è stato trovate che geni della famiglia TITAN sono attivi nel seme contenente endosperma. L’analisi dei geni del ciclo cellulare nel mesocarpo ha mostrato l’esistenza di due diversi profili di espressione: mentre i geni relativi alla mitosi erano espressi solo nello stadio S1, i geni della replicazione del DNA hanno mostrato un doppio picco di espressione, in S1 e poi in S3/S4, suggerendo che in questi stadi possono verificarsi eventi di endoreduplicazione. Con l’utilizzo di qRT-PCR, i livelli d’esrpessione di questi geni sono stati testati anche in altre cultivar; i dati ottenuti suggeriscono che nel genotipo slr la mancanza di endoreduplicazione possa essere coinvolta nel basso tasso di crescita durante lo stadio S3 di questo genotipo. Sono stati quindi valutati i profili d’espressione di famiglie di fattori di trascrizione (FT), dato che si ritiene che i fattori di trascrizione siano le proteine con i ruoli più importanti nella regolazione durante lo sviluppo. E’ stato trovato che FT delle famiglia SQUAMOSA promoter Binding Protein (SBP) hanno un alto livello di espressione all’inizio dello sviluppo di entrambi gli organi considerati, il quale successivamente diminuisce velocemente. E’ stato scoperto che nel seme maturo è indotta la trascrizione di FT di tipo Growth-Regulating Factor (GRF). Questi dati sono stati confermati con l’utilizzo di qRT-PCR in ‘SpringCrest’ precoce e nel genotipo a lenta maturazione slr. Dato che in altre specie vegetali l’abbondanza dell’mRNA di geni appartenenti a queste famiglie di FT è regolata da microRNA (miRNA) specifici, è stata misurata l’espressione degli omologhi di pesco di questi miRNA. In tre diverse cultivar è stata trovata una correlazione negativa nel contenuto di RNA per le seguenti coppie microRNA/FT: miR156/SBP, miR396/GRF e mir167/ARF8, suggerendo non solo che questi miRNA posseggono la stessa attività un pesco, ma anche che i miRNA sono profondamente coinvolti nella rete regolativa sottostante lo sviluppo del frutto di pesco. In appendice vi è uno studio pubblicato nel quale viene descritto l’uso di µPEACH3.0 nello studiare gli effetti delle ferite su due cultivar con diversa tolleranza a questo stress. Sono stati utilizzati campioni di RNA estratti da mesocarpi feriti o intatti della cultivar “melting” Glohaven (GH) e della cultivar “slow melting” BigTop (BT). I dati trascrittomici, confermati dall’analisi tramite qRT-PCR, hanno mostrato il coinvolgimento di fattori di trascrizione di tipo WRKY, AP2/ERF, e HSP20 nella risposta di GH alla ferite. Insieme a questi, è stato trovato che nel mesocarpo ferito di GH viene indotta l’espressione anche di geni coinvolti nella risposta agli stress, nel metabolismo della parete cellulare, nella biosintesi dei fenilpropanoidi e triterpenoidi.

Abstract S. aureus has 16 predicted two-component systems (TCS) that respond to a range of environmental stimuli, and allow for adaptation to stresses. Of these 16, three have no known function, and are not homologous to any other TCS found in closely related organisms. NsaRS is one such element, and belongs to the intramembrane-sensing histidine kinase (IM-HK) family, which is conserved within the Firmicutes. The regulators are defined by a small sensing domain within their histidine kinase, suggesting that they do not sense external signals, but stress in or at the membrane. Our characterization of NsaRS in this work reveals that, as with other IM-HK TCS, it responds to cell-envelope damaging antibiotics, including phosphomycin, ampicillin, nisin, gramicidin, CCCP and penicillin G. Additionally; we reveal that NsaRS regulates a downstream transporter, NsaAB, during nisin-induced stress. Phenotypically, nsaS mutants display a 200-fold decreased ability to develop resistance to another cell-wall targeting antibiotic, bacitracin. Microarray analysis reveals the transcription of 245 genes is altered in a nsaS mutant, with the vast majority down-regulated. Included within this list are genes involved in transport, drug-resistance, cell-envelope synthesis, transcriptional regulation, amino acid metabolism and virulence. Using ICP-MS, a decrease in intracellular divalent metal ions was observed in an nsaS mutant, when grown under low abundance conditions. Characterization of cells using electron microscopy reveals that nsaS mutants also have alterations in cell-envelope structure. Finally, a variety of virulence related phenotypes are impaired in nsaS mutants, including biofilm formation, resistance to killing by human macrophages and survival in whole human blood. Thus NsaRS is important in sensing cell wall damage in S. aureus, and functions to reprogram gene expression to modify cell-envelope architecture, facilitating adaptation and survival. Interestingly, in our microarray analysis, we observed a more than 30-fold decrease in transcription of an ABC transporter, SACOL2525/2526, in the nsaS mutant. This transporter bears strong homology to nsaAB, and is currently uncharacterized. Exploration of the role of SACOL2525/2526 revealed that, along with NsaRS, it too responds to cell-envelope damaging antibiotics. Specifically, its expression was induced by phosphomycin, daptomycin, penicillin G, ampicillin, oxacillin, D-cycloserine and CCCP. Mutation of this transporter results in increased sensitivity to the antibacterial agent daptomycin, and decreased sensitivity to free fatty acids. These findings are perhaps explained by altered membrane fluidity in the mutant strain, as the transporter null-strain is more readily killed in the presence of organic solvents, such as toluene. In addition, SACOL2525/2526 mutants have a decreased ability to form spontaneous mutants in response to several other peptidoglycan synthesis targeting antibiotics, suggesting a role for SACOL2525/2526 in antibiotic resistance. Inactivation of this transporter alters the cell envelope, and produces similar effects to those observed with the nsaS mutant, with increased capsule production, that may provide resistance to lysostaphin. Interestingly, the nsaS microarray revealed that this TCS negatively regulates only 34 genes, including 6 out of the 10 major secreted proteases. Despite a number of reports in the literature describing these enzymes as virulence factors, the data is often conflicting. Therefore, the contribution of proteases to CA-MRSA pathogenesis was investigated, by constructing a strain lacking all 10 extracellular protease genes. Analysis of this strain using murine models of infection reveals secreted proteases significantly impact virulence in both localized and systemic infections. Additionally, inactivation of these enzymes strongly influences survival in whole human blood, and increases sensitivity to antimicrobial peptides. Using a proteomics approach, we demonstrate that the contribution of secreted proteases to pathogenicity is related to differential processing of a large number of surface-associated virulence factors and secreted toxins. Collectively these findings provide a unique insight into the role of secreted proteases in CA-MRSA infections.

N-acetylaspartate (NAA) is one of the most abundant molecules in the mammalian central nervous system (CNS). The current paradigm suggests that NAA is synthesized in neurons by the enzyme N-acetyltransferase 8-like (NAT8L) and hydrolyzed into aspartate and acetate by the enzyme aspartoacylase (ASPA) in oligodendrocytes. Although the function of NAA is not well understood, several hypotheses have been proposed since its discovery several decades ago. Among the most cited theory is the concept of acetate delivery to oligodendrocytes via NAA for the synthesis of fatty acids for myelin lipids and myelination. Another concept suggests that NAA functions as a molecular water pump to remove molecular water from oxidative phosphorylation. In contrast, disruption of NAA metabolism has been associated with oxidative stress contributing to neurodegeneration, as seen in Canavan disease, a monogenic disorder associated with loss-of-function mutations in ASPA. Accumulation of NAA in the CNS and peripheral organs is pathognomonic for Canavan disease (CD) and is used clinically to diagnose this rare disease. Symptoms typically occur within months after birth and primarily manifest in the CNS with spongy degeneration of the white matter. Initially, affected patients present with poor feeding, lack of head control, hydrocephalus; later, they miss developmental milestones and develop seizures. Only supportive treatment is available possibly helping patients to survive past the first couple of years. Gene therapy has been considered early on for the treatment of CD. The first trial in humans demonstrated safety but did not result in symptomatic improvement. In addition to gene therapy for the treatment of CD, NAA has gained increasing interest in neurodegenerative and psychiatric disorders, but also in adipose tissue. Here, we are investigating the function of NAA in the context of ASPA deficiency, aka Canavan disease. We found that impaired NAA metabolism caused by ASPA mutations is characterized by a neurometabolic profile that suggests cellular shift from glucose towards fatty acid metabolism for energy production. Although, we found a similar metabolic signature in asymptomatic mice within days after birth, longitudinal comparison suggest that disease progression leads to fatty acid depletion, which is not present in asymptomatic mice, potentially challenging the concept that NAA-derived acetate is essential for lipid synthesis in the myelinating brain. Using rAAV to determine the reversibility of this metabolic phenotype, we found that early treatment prevents loss of myelin, normalizes the neurometabolic phenotype and keeps Canavan mice asymptomatic; in contrast, later treatment only allows for partial normalization of the neurometabolome, despite adequate ASPA gene delivery by rAAV, independent of ubiquitous or astrocyte-restricted hASPA expression. Furthermore, we found that non-enzymatically active hASPA might play a ubiquitous role in glucose uptake regulation in vivo. Importantly, we identified brain regions with metabolic changes that also correspond to the areas with significant histopathologic alterations. Finally, we confirmed the glycolytic changes in a Canavan disease patient cell line using Seahorse metabolic analyzer, demonstrating the decreased rate of glycolysis for energy production. Overall, our findings reveal a novel metabolic phenomenon in Canavan disease and NAA metabolism that allows to assign a novel function of N-acetylaspartate.

Despite the implementation of legal prohibitions and regulatory policies to limit the commercial availability of lottery products to minors, published research continues to document a high prevalence of participation in and ease of access to lottery playing amongst adolescents. This study systematically investigated the influence of individual-level factors in vendor compliance with youth access statutes and policies for lottery and alcohol products. Six underage youths each attempted to purchase a lottery ticket, a beer, or both products together in the same 313 convenience stores, for a total of 1,219 purchase attempts. The results revealed that only a moderate proportion of vendors surveyed in this study were compliant with existing statutes and policies, and that gender and vendor age variables playa significant role in youth purchasing of lottery tickets and alcohol. These findings were interpreted in terms of their implications for strengthening regulatory policies and future research.

The plasma protein complement factor H (fH, 155 kDa) regulates the activity of the alternative pathway of complement activation. Factor H is monomeric, and its 20 CCP modules are arranged in a predominantly elongated conformation, joined by linking sequences that vary in length, with the longest linkers occurring in the central portion of the molecule. CCP modules 1 through 4 of fH host its capacity to act as a cofactor for fI-mediated proteolytic degradation of C3b and its ability to accelerate the decay of the C3 convertase, C3bBb, thereby regulating the so-called tick-over activation of the alternative pathway. Mutations in this part of fH might compromise its function and lead to underregulation of the alternative pathway. It is hypothesized that this can cause predisposition to diseases such as atypical haemolytic uraemic syndrome (aHUS) and age-related macular degeneration (AMD). In the current work, the known disease-associated mutations R53H and R78G were compared to wild-type in terms of fluid-phase cofactor assays, C3b-binding affinity and the ability to accelerate the decay of the convertase. In addition, the protective variant, I62, was also inspected because its protective role might be explained by an increased regulatory activity. The second, linked, aim of this project was to employ Forster resonance energy transfer (FRET) to study the link between conformation and function in fH. FRET is valuable for obtaining long-distance restraints up to a maximum of 100 °A and is therefore particularly useful for inferring domain orientations within multidomain proteins. This approach to measure long-range inter- and intramolecular distances is a convenient way to complement NMR-based structural investigations, which rely on short-range restraints. It is also a valuable complement to X-ray crystallography since it is a solution technique that can be conducted under physiological conditions. By using site-directed mutagenesis in the current work, free cysteines were introduced into CCP modules 1-4 at strategic points, which were then used for attachment of fluorescent tags. C3 possesses an internal thioester which can be labelled with a fluorophore upon activation to C3b. Intermolecular FRET measurements were thus undertaken to gain information about the interaction between the two proteins that is crucial for understanding functional activity. The CCP modules in the centre of fH may be responsible for introducing a bend into fH that brings the N-teminus close to the C-terminus (the latter is important for host versus non-host discrimination) joined by the longest linkers occurring in the whole molecule. This coincidence of two relatively small CCP modules, 12 and 13, with the highest number of eight amino acids between them, is hypothesised to reflect some unique architectural features. To explore the structural details of this portion of fH by FRET, single-labelled cysteine mutants were further modifed to provide a recognition site for transglutaminase (TGase), which can be enzymatically labeled with a second fluorophore. This stoichiometrically-labelled protein was used for intramolecular FRET studies.

C1QTNF5 is a 25kDa short chain collagen of unknown function which is mutated in late-onset retinal macular degeneration (L-ORMD). L-ORMD is an autosomal dominant disease characterised by sub-retinal pigment epithelial deposits leading to photoreceptor death and visual loss and shows several similarities to age-related macular degeneration (AMD). A Tyr402His polymorphism in complement factor H (CFH), a regulatory protein in the innate immune system, has been associated with increased risk of AMD. C1QTNF5 and CFH are both expressed and secreted by the retinal pigment epithelium (RPE) which supports photoreceptors and is responsible for phagocytosis of shed rod photoreceptor outer segments (ROS). The properties of the normal C1QTNF5 and disease-associated Ser163Arg mutation were examined in detail, including protein characterisation, cellular processing and function. Recombinant wild type and mutant C1QTNF5 were produced and their multimerisation and solubility functions compared. Both proteins were found to be soluble and to form similar multimeric species which were resistant to reducing conditions, as seen in other short chain collagens. Due to the similarities between LORMD and AMD, a proposed interaction between C1QTNF5 and CFH was investigated. CFH is composed of 20 short consensus repeats (SCR) and interactions were confirmed between C1QTNF5 and both CFH and SCR modules 7-8 and 19-20. CFH showed a greater affinity for mutant C1QTNF5 compared with wild type on the basis of surface plasmon resonance assays. Stably transfected RPE-derived cell lines were created which expressed either wild type or mutant C1QTNF5. Both proteins were found to be secreted and showed similar cellular processing with no evidence of aggregation or retention of the mutant protein within the endoplasmic reticulum. In order to investigate C1QTNF5 function, phagocytosis of ROS by the stably transfected cell lines was carried out. Cells expressing wild type C1QTNF5 showed greater ROS phagocytosis compared with mutant C1QTNF5-expressing or untransfected cells. Addition of anti-C1QTNF5 antibody increased ROS phagocytosis further. In summary, it is proposed that wild type and mutant C1QTNF5 are secreted by the RPE where they interact with CFH. C1QTNF5 is also shown to have a role in ROS phagocytosis, with mutation in C1QTNF5 affecting phagocytosis efficiency, which may contribute to sub-RPE deposit formation. The results suggest that CFH may also be involved in this process, suggesting a common pathogenic pathway between L-ORMD and AMD.

This thesis addresses the question of how data protection law should respond to the challenges arising from the ever-increasing prevalence of big data. The investigation is conducted with the case study of online behavioural advertising (OBA) and within the EU data protection legal framework, especially the General Data Protection Regulation (GDPR). It is argued that data protection law should respond to the big data challenges by leveraging the regulatory options that are either already in place in the current legal regime or potentially available to policymakers. With the highly complex, powerful and opaque OBA network, in both technical and economic terms, the use of big data may pose fundamental threats to certain individualistic, collective or societal values. Despite a limited number of economic benefits such as free access to online services and the growth of the digital market, the latent risks of OBA call for an effective regulatory regime on big data. While the EU's GDPR represents the latest and most comprehensive legal framework regulating the use of personal data, it has still fallen short on certain important aspects. The regulatory model characterised by individualised consent and the necessity test remains insufficient in fully protecting data subjects as autonomous persons, consumers and citizens in the context of OBA. There is thus a pressing need for policymakers to review their regulatory toolbox in the light of the potential threats. On the one hand, it is necessary to reconsider the possibilities to blacklist or whitelist certain data uses with mechanisms that are either in place in the legal framework or can be introduced additionally. On the other hand, it is also necessary to realise the full range of policy options that can be adopted to assist individuals in making informed decisions in the age of big data.

The biological membrane surrounding the living cell provides a sealed barrier that tightly regulates the interactions with the outside environment. A large number of integral membrane proteins mediate these interactions and are involved in a wide variety of biological processes. An increasing number of studies have led to the conclusion that lipids provide more than a hydrophobic solvent for membrane proteins, and that interactions between lipids and proteins are required to allow protein function. ABC transporters are one of the most important family of membrane proteins. However, the importance of their lipidic environment is largely unknown. Only a few studies showed that their activity was dependent on the lipidic composition of the surrounding bilayer. The bacterial ABC transporter HorA was used as a model to probe the influence of the lipidic environment on that class of membrane proteins.

HorA is a multidrug transporter expressed in Lactobacillus brevis, a Gram-positive beer spoilage bacterium. It turned out that phosphatidylethanolamine (PE) was indispensable to maintain both the activity and the structural integrity of HorA.

Surprisingly, replacement of PE by the chemically related PC (phosphatidylcholine) did not led to the suppression of HorA activity, but to an unexpected phenotype. Whereas the cytoplasmic domains of HorA were still able to hydrolyze ATP, the membrane parts of the transporter were unable to use that energy to mediate substrate transport. Using several biophysical methods particularly adapted to the study of reconstituted systems, we showed that the structure of HorA is strongly altered by this lipid replacement. In particular, the structural organization of the transmembrane domains of the protein is strongly affected.

Doctorat en Sciences agronomiques et ingénierie biologique
info:eu-repo/semantics/nonPublished

La région fos de la souche d’Escherichia coli pathogène aviaire BEN2908 permet le métabolisme des fructanes, des prébiotiques largement utilisés en alimentation humaine et animale. Ce métabolisme contribue à l’implantation de BEN2908 dans son réservoir, l’intestin. La région fos, située au sein de l’îlot génomique AGI-3, est composée de 6 gènes codant pour un transporteur de sucre et des enzymes impliquées dans le métabolisme des fructanes, et d’un gène transcrit de façon divergente codant pour FosR, un régulateur transcriptionnel de la famille LacI/GalR. Nous avons montré que l’expression des gènes fos est réprimée par FosR, contrôlée par la répression catabolique et induite en présence de fructanes. FosR se lie à 2 séquences opératrices de la région promotrice de l’opéron fos et cette liaison est inhibée en présence de fructanes, surtout par le 1-kestose. La région fos confère un avantage de croissance en présence de contenu cæcal et contribue à la colonisation des cæca in vivo. De plus, AGI-3 est mobile et transférable, ce qui suggère une possible diffusion du métabolisme des fructanes au sein de l’espèce E. coli
The fos region of the avian pathogenic Escherichia coli strain BEN2908 is involved in fructan metabolism, prebiotics widely used commercially in food products for both humans and animals. This metabolism contributes to the establishment of BEN2908 in its reservoir, the intestine. The fos region, located within the genomic island AGI-3, is composed of six genes encoding a sugar transporter and enzymes involved in fructan metabolism, and of a divergently transcribed gene encoding a transcriptional regulator, FosR, belonging to the LacI/GalR family. We demonstrated that the expression of fos genes is repressed by FosR, controlled by catabolite repression and induced in the presence of fructans. FosR binds to two operator sequences of the fos operon promoter region. This binding to DNA is inhibited in the presence of fructans, especially by 1-kestose. The fos region strongly benefits growth on cecal content and colonization of the ceca in vivo. Moreover, AGI-3 is mobile and transferable, suggesting a possible dissemination of fructan metabolism within the species E. coli

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O trigo (Triticum aestivum L.) é uma cultura de destaque econômico e um dos cereais de inverno mais cultivados no sul do Brasil. Entretanto, suas sementes apresentam dormência, sendo um fator limitante em programas de melhoramento genético, quando se deseja rápida germinação pós-colheita, necessitando dessa forma, viabilizar tecnologias para potencializar a germinação. A pesquisa teve como objetivo avaliar a eficácia de doses de ácido giberélico na superação de dormência de sementes de trigo. As sementes de trigo da cultivar TBIO Toruk foram tratadas com inseticida Cropstar imidacloprid+thiodicarb 600 SC), fungicida Spectro (difenoconazol 150 SC) seguido de adição na calda do TS de GA3 nas respectivas dosagens. Os tratamentos empregados consistiram em doses de ácido giberélico, sendo: Pro-Gibb® (50 mg Kg-1 de GA3); Pro-Gibb® (100 mg Kg-1 de GA3); Pro-Gibb® (150 mg Kg-1 de GA3); Pro-Gibb® (200 mg Kg-1 de GA3); Pro-Gibb® (250 mg Kg-1 de GA3); Pro-Gibb® (300 mg Kg-1 de GA3) e testemunha (semente sem tratamento). Para avaliar a eficácia do GA3 as variáveis analisadas foram germinação, primeira contagem da germinação, velocidade de germinação, índice e coeficiente de velocidade de germinação. Os resultados obtidos permitem concluir que o ácido giberélico influenciou de forma positiva na superação da dormência de sementes de trigo, onde foi constatado maior porcentagem de germinação e vigor. A dose de ácido giberélico de 50 mg Kg-1, propiciou maior porcentagem de germinação (91 %), com incremento de 35 % na germinação de sementes de trigo quando comparado com sementes não tratadas. Por outro lado, constatou-se que doses de ácido giberélico, acima de 250 mg Kg-1, não são recomendadas para superação de dormência de sementes de trigo.
Wheat (Triticum aestivum L.) is a crop that has highlight of economic and one of the most important winter cereals grown in southern Brazil. However, the seeds present dormancy, limiting factor in breeding programs, considering that is necessary germination immediately after the harvest, requiring viable technologies to enhance germination. The research aimed to evaluate the effectiveness of gibberellic acid doses in overcoming wheat seed dormancy, It was used wheat seeds cultivar TBIO Toruk, Were treated with Cropstar insecticide (imidacloprid + thiodicarb 600 SC), Spectro fungicide (difenoconazole 150 SC) followed by the addition of the TS with GA3 in the dosis evaluated. The treatments consisted in the use of gibberellic acid doses, as follows: Pro-Gibb® (50 mg Kg-1 GA3); Pro-Gibb® (100 mg Kg-1 GA3); Pro- Gibb® (150 mg Kg-1 GA3); Pro-Gibb® (200 mg Kg-1 GA3); Pro-Gibb® (250 mg Kg-1 GA3); Pro-Gibb® (300 mg Kg-1 GA3) and Witness without treatment. To evaluate the effectiveness of GA3 it was analyzed the variables: germination, first germination count, germination rate, index and coefficient of germination speed. The obtained results allow us to conclude that gibberellic acid positively influenced the physiological quality of wheat seeds, where was found greater percentage of germination and vigor. A gibberellic acid dose of 50 mg Kg-1, show greater germination percentage (91 %), an increase of 35 % in wheat seeds germination when compared to untreated seeds. Gibberellic acid doses above 250 mg Kg-1, are not recommended for overcoming wheat seed dormancy.

ABCB4 est exprimé à la membrane canaliculaire des hépatocytes où il sécrète un composant majeur de la bile : la phosphatidylcholine. Plus de 500 mutations d’ABCB4 sont associées à des maladies biliaires. La pathologie la plus sévère est la PFIC3, qui se développe tôt dans l’enfance et progresse rapidement vers l’insuffisance hépatique. La transplantation hépatique reste la seule thérapie efficace. Le développement d’alternatives représente donc un enjeu majeur. Cette thèse s’intéresse à l’effet de cinq mutations décrites chez des patients et situées dans les sites de liaison à l’ATP d’ABCB4. En combinant la modélisation 3D avec des études in vitro, nous avons montré que ces mutations sont responsables d’un défaut d’activité d’ABCB4. Nous avons mis en évidence que le VX-770, un médicament approuvé en clinique pour traiter les patients atteints de mucoviscidose, restaure l’activité des cinq mutants. Ces travaux ouvrent des perspectives de repositionnement du VX-770 pour le traitement ciblé de patients atteints de pathologies biliaires liées à ABCB4. La seconde partie de cette thèse consiste à étudier le rôle de l’interaction du domaine N-terminal d’ABCB4 avec la kinase MRCKalpha. L’inhibition ou l’extinction de cette kinase montrent que MRCKalpha joue un rôle dans la régulation de l’expression membranaire d’ABCB4 en contrôlant son internalisation depuis la membrane plasmique. En inhibant la protéine MLC2, effecteur de MRCKa, nous avons ensuite montré que l’effet de MRCKalpha sur l’expression d’ABCB4 passe par MLC2, qui est un partenaire du domaine linker d’ABCB4. Notre travail montre un rôle commun de ces deux partenaires dans la régulation de l’internalisation d’ABCB4
ABCB4 is exclusively expressed at the canalicular membrane of hepatocytes where its function is to translocate phosphatidylcholine (PC) into bile. Variations in ABCB4 gene sequence are associated with several chronic and progressive liver diseases. The most severe is PFIC3 which develops early in childhood and most often requires liver transplantation. Less severe diseases are the intrahepatic cholestasis of pregnancy and the low phospholipid- associated cholelithiasis syndrome which occur in young adults. Up to now, about 500 disease-causing ABCB4 variants have been reported. A challenge is to find pharmacological treatments for the severe forms of the diseases. We have studied the effect of five disease-causing variations that reside in the highly conserved motifs of ABC transporters, involved in ATP binding. Using three-dimension structural modeling and in vitro studies, we showed that the five mutants were normally processed and targeted to the plasma membrane, whereas their PC secretion activity was dramatically decreased. PC secretion activity of the mutants was rescued by the clinically approved CFTR potentiator ivacaftor (VX-770). These results pave the way for personalized therapy in ABCB4-related diseases.The second part of my project was aimed at investigating the potential role of two ABCB4 partners, the kinase MRCKalpha and its effector the myosin light chain II (MLCII) in the expression and function of ABCB4. We found that downregulation of both partners didn’t affect the canalicular localization of ABCB4 but led to a reduction of its endocytosis. Our results open new insights into the mechanisms underlying the regulation of ABCB4 expression and function

Concern about privacy is an important consideration for users of information and communication technologies (ICT), particularly when using computer-mediated communication (CMC), i.e. Internet usage. Several researchers have studied privacy issues by taking into account the views of users to include individuals, organisations, privacy policy makers, governments and trust organisations. This thesis investigates whether an individual's perspectives about privacy are culturally relevant when using the Internet. This research used a survey in the form of a questionnaire in two countries, namely, Saudi Arabia and Malaysia to compare online privacy perspectives of young and mature (male and female) Saudi and Malaysian students. The research examines the relationship of the effect of the cultural background including the effect of social norms, religious belief, Internet regulation and IT skills of these Internet users upon their attitude towards privacy online and their perspectives about privacy. It also examines the effect of nationality (Malaysian and Saudi), gender and age groups. In this study, online privacy perspectives are a synthesis of three perceptions; what is 'personal' information online, the online privacy concerns and the Internet trust, whereby the cultural effects are the effect of religious beliefs, social norms, Internet regulation and IT skills in the privacy attitudes of keeping personal information safe, caring about their and others' privacy online and when revealing personal information. The demographic factors in this research are nationality, gender and age. To study these relationships, the research uses t-test, ANOVA, and single regression methods as data analysis techniques. The results show that the level of concern and degree of trust exhibited by Malaysian students with regard to submitting personal information via the Internet was affected the most by their gender, and social norms upon their online privacy attitudes. For Saudi students, the level of concern and trust with regard to submitting personal information via the Internet was found to be related to the effect of their age, gender, and religious beliefs on their online privacy attitudes. The other cultural factors, i.e. Internet regulation in force in each country and the IT skills of participants, are likely to have equal effects on both Malaysian and Saudi privacy perspectives. This research adds the cultural background, age and gender effects to the model of the calculus of the privacy concern that is proposed by Dinev and Hart (2006, pp. 63-64). The research also establishes what is 'private' in Malaysia and Saudi Arabia, by identifying 'what counts as personal information with regard to Internet users' and provides a comparison in this concept between the two countries, their gender and age groups. For examples, Malaysian students consider name, e-mail address, date of birth, nationality and religion as 'personal' information and Saudi students consider home address, phone number, photographic image and credit card number as 'personal' information. In addition Saudi females tend to consider, particularly, home address, phone number, and photographic image as 'personal' information more than Saudi males. These findings should help both web designers and Internet policy makers in Saudi Arabia and Malaysia to consider these cultural effects when designing the privacy policies of their websites.

In Anlehnung an das Self-Regulated-Strategy-Development-Modell von Harris und Graham (1996) wurde das Selbstregulatorische Aufsatztraining (SAT) zur Förderung der Schreibkompetenz bei Grundschülern der 4. und 5. Klasse entwickelt. SAT integriert die Vermittlung von Schreibstrategien (hier: „Erzählendes Schreiben“) mit Merkmalen selbstgesteuerten Lernens (Zielsetzung, strategisches Planen, Selbstbewertung und Selbstkorrektur).

Die Wirksamkeit des Trainings wurde in drei Studien untersucht: 1. Eine Pilotstudie diente der Überprüfung der prinzipiellen Eignung von SAT zur Förderung von Schreibleistungen bei Grundschülern der 5. Klassen (N = 42) und der Optimierung seiner Teilkomponenten und Vorgehensweisen. 2. In der Hauptuntersuchung wurden die Effektivität und Nachhaltigkeit des SAT-Programms bei Schülern der 4. Klasse (N = 154) im Vergleich zu zwei Bedingungen getestet: (a) der isolierten Einübung von Schreibstrategien (Aufsatztraining) und (b) konventionellem Aufsatzunterricht (Unterrichtskontrollgruppe). 3. In einer weiteren Studie wurde die Wirksamkeit des Trainings speziell bei Schülern mit ungünstigen Lernvoraussetzungen überprüft; die Studie diente zudem der Illustration des dabei gewählten Vorgehens am Einzelfall (N = 6).

Die ermittelten Befunde sprechen übereinstimmend dafür, dass die Kombination aus strategischem plus selbstregulatorischem Training (SAT) die stärksten und nachhaltigsten Effekte auf die Schreibleistung erzielt. Der Trainingseffekt generalisiert zudem auf die Erinnerungsleistung bei der freien Wiedergabe einer Kurzgeschichte. Schüler mit schwachen Aufsatzleistungen und ungünstigen Lernvoraussetzungen profitieren von dem SAT-Programm in besonderem Maße.

In der Diskussion werden Aufgaben für die zukünftige Forschung erörtert. Forschungsbedarf besteht u.a. hinsichtlich (a) einer stärkeren Verknüpfung von Schreibtrainings mit der kognitionspsychologischen Forschung; (b) der Dekomposition und gezielten Überprüfung der einzelnen Trainingskomponenten; (c) der Ausweitung des SAT-Programms auf andere Textgenre; (d) der Integration verfeinerter Revisionsstrategien in das Förderprogramm; und (e) dessen Implementierung in den Regelunterricht.
Extending on Harris and Graham′s (1996) Self-Regulated-Strategy-Development-Model, I designed an curriculum-integrated intervention program (SAT) to promote the composition skills of elementary school-age students. SAT combines the instruction of task strategies required to write good narratives with the explicit instruction of self-regulation procedures (goal setting, strategic planning, self-evaluation, self-correction).

Three studies examined the effectiveness of the training: 1. A pilot study investigated the viability of the SAT-program among 5th graders (N = 42) and served to refine its components and procedures. 2. In a sample of 4th graders (N = 154), the main study tested the strength and stability of the SAT effects in relation to two comparison groups: (a) Students who were taught the same set of task strategies but received no instruction in self-regulation procedures (strategy-only condition); (b) students who received conventional classroom teaching in composing (control condition). (3.) A third study served to examine the effectiveness of the SAT-program in a group of low achieving 5th graders and to illustrate its instructional steps in a number of single cases (N = 6).

Results obtained from these studies converge in showing that a writing program that conjointly addresses both task strategies and self-regulation procedures (SAT) is most effective in producing strong and lasting effects on elementary school students′ composing skills and generalization performance. Among all students, low achievers were most likely to benefit from the SAT-program.

The discussion highlights a number of issues for future research on writing. Specifically, it is argued that there is a need to (a) further explore the cognitive and meta-cognitive processes underlying good writing, (b) examine the effectiveness of specific training components incorporated in the present version of SAT, (c) crossvalidate the reported SAT effects with respect to various writing genre, (d) incorporate more elaborated revision strategies into the training program, and (e) implement components and procedures specified in SAT into conventional classroom teaching.

Chez Streptococcus thermophilus, le cycle de vie des signaux peptidiques appelés phéromones contribuant aux mécanismes de communication dit quorum sensing (QS) est divisé en quatre étapes. La synthèse des phéromones est suivie par leur maturation par la protéase Eep et leur sécrétion. Enfin, leur re-internalisation par le transporteur d’oligopeptides Ami est indispensable pour leur détection intracellulaire par les régulateurs transcriptionnels dit Rgg-like contrôlant l’expression des gènes cibles. L’objectif de ma thèse était de valider le rôle du transporteur PptAB dans la sécrétion des phéromones avant d’identifier les gènes dont l’expression est dépendante de ce transporteur chez S. thermophilus.En premier, nous avons confirmé le rôle du transporteur PptAB dans l’activation de trois systèmes de QS impliquant les régulateurs Rgg-like. Pour cela nous avons utilisé des fusions transcriptionnelles entre un gène rapporteur codant la luciférase et le promoteur de trois gènes cibles de ces sytèmes. Nous avons ensuite montré que le transporteur PptAB n’exporte probablement que la forme mature des phéromones par LC-MS/MS. Enfin, nous avons découvert que l’expression d’un ensemble des gènes situé en aval des gènes codant les régulateurs Rgg-like est dépendante du transporteur PptAB mais aussi du transporteur Ami et de la protéase Eep par une approche globale transcriptomique. Ainsi, les transporteurs PptAB et Ami et la protéase Eep contrôlent ces cibles par un même mécanisme.Notre travail a mis en évidence des interférences entre ces systèmes qui devront être élucidées
In Streptococcus thermophilus, the life cycle of signaling peptides called pheromones contributing to communication mechanisms named quorum sensing (QS) is divided into four steps. The synthesis of pheromones is followed by their maturation by the protease Eep and their secretion. At last, their re-internalisation by the oligopeptide transporter Ami is essential for their intracellular detection by transcriptional regulators called Rgg-like controlling the expression of target genes. The aim of my thesis was to valid the role of the transporter PptAB in the secretion of pheromones before identifying genes whose expression is dependent of this transporter in S. thermophilus.First, we confirmed the role of the transporter PptAB in the activation of three QS systems involving Rgg-like regulators. For that we used transcriptional fusions between a reporter gene coding the luciferase and the promoter of three target genes of these systems. We then showed that the transporter PptAB exports more likely only the mature form of pheromones by LC-MS/MS. Finally, we discovered that the expression of a set of genes located downstream of genes coding Rgg-like regulators is dependant of the transporter PptAB and also of the transporter Ami and the protease Eep by a global transcriptomic approach. Thereby, the transporters PptAB and Ami and the protease Eep regulate these targets by a same mechanism.Our work brought light on cross-talks between these systems which need to be deciphered

The aim of this thesis is to explore possibilities of rapid control prototyping, describe the concept of creating the software application in MATLAB/Simulink environment with use for development kit Texas instruments LaunchPad and create an application for DC and induction motor control in this environment. This work describes the application for unipolar/bipolar control H-Bridge of power converter for DC motor, measurement of output currents, speed and its displaying in real time using serial control interface. This thesis also desribes scalar and vector control of induction motor. All software applications with measurements are created in MATLAB/Simulink and attached to the thesis.

More than 80% of vineyards around the world use grafted plants: a scion of Vitis vinifera grafted onto a rootstock of single or interspecific hybrids of American Vitis species, resistant or partially resistant to Phylloxera (Daktulosphaira vitifoliae (Fitch, 1856)). The genetic variability of grapevine rootstocks plays a fundamental role in their adaptation to the environment (Serra et al., 2013). In the climate change scenario, predicting an increase of aridity in the near future (Dai, 2013), the more frequent and severe drought events may represent the major constrain for the future of viticulture (IPCC, 2018; Schultz, 2000). Therefore, the selection of new rootstocks able to cope with unfavourable environmental condition is a key asset, as well as a strategy to improve crop yield/vegetative growth balance on scion behaviour (Corso and Bonghi, 2014). So far, the influence of rootstock on scion physiological performance during water stress has always aroused great interest. On the contrary, the scion impact on rootstock response is still less debated. Therefore, the effect of grafting on rootstock behaviour have been investigated. Phenotypical and large-scale whole transcriptome analyses on two genotypes, a drought-susceptible (101-14) and a drought-tolerant (1103 P), own-rooted and grafted with Cabernet Sauvignon, subjected to a gradual water shortage in semi-controlled environmental conditions have been performed. The ungrafted condition affected photosynthesis and transpiration, meaning the decisive role of scion in modulation of gas exchanges and in general in plant adaptation. Molecular evidence highlighted that the scion delays the stimulus perception and rootstock reactivity to drought. Since 1985, the DiSAA research group operating at the University of Milan is carrying on a rootstock crossbreeding program which has led to the release of four genotypes: M1, M2, M3 and M4. They show from moderate to high tolerance to drought (M4 > M1 = M3 > M2). In order to characterize their performance during water stress, their physiological (gas exchanges and stem water potential) and transcriptome response (genes involved in ABA-synthesis and ABA-mediated responses to drought) under well-watered and water stress conditions were examined. The behaviour of M-rootstocks (M1, M2 and M3) was compared with that of other commercial genotypes largely used in viticulture, either tolerant (140 Ru, 41 B, 110 R, 1103 P), less tolerant (SO 4, K 5BB) and susceptible (420 A and Schwarzman). Discriminant analysis (DA) showed that when water availability starts to decrease, rootstocks firstly perceives the stress activating a transcriptome response, consequently physiological changes have been observed. It also demonstrated that the three M-rootstocks were clearly discriminated: M4 was grouped with the most tolerant genotypes while M3 with the less tolerant or susceptible ones from a physiological standpoint, confirming their different attitude to tolerate water stress. M4 has proven to be a promising rootstock due to its ability to adapt to drought conditions. Considering the constant great demand for vine planting materials, the obtainment of genetically homogeneous populations (i.e. clones) from elite individuals through micropropagation represents a rapid alternative to conventional multiplication. For this reason, an efficient high-throughput protocol for M4 in vitro propagation was set up. Its attitude to shooting, root development and callus proliferation was compared to that of other rootstocks largely used in viticulture (K5BB, 1103P, 101-14 and 3309C). Moreover, pro-embryogenic and embryogenic callus from bud explants were also produced, representing a cellular material manipulable with the genetic engineering techniques. In water scarcity condition, among the mechanisms activated by M4, the great ability to scavenge ROS, related to the increased accumulation of stilbenes and flavonoids, may be such as to give it tolerance to the stress. In particular, the higher levels of trans-resveratrol were correlated with the up-regulation of some stilbene synthase genes, mainly VvSTS16, VvSTS18, VvSTS27 and VvSTS29. The over expression of these genes was linked to a structural variation in their promoter region. To confirm that VvSTSs genes may be considered putative factors of M4 better adaptation to water stress, a genome editing protocol based on the CRISPR/Cas9 system, aimed at knock-out the genes, was performed. For testing the gRNAs functionality, a transient assay on in vitro micropropagated plantlets of M4 and 101-14 was performed. The positive results obtained by this experiment will lead to the transformation of somatic embryos and regeneration of whole-edited plants using the vectors developed.

Currently, power system operation and control with AGC are undergoing fundamental changes due to rapidly increasing amount of renewable sources, energy storage system, restructuring and emerging of new types of power generation, consumption and power electronics technologies. Continuous growth in size and complexity, stochastically changing power demands, system modeling errors, alterations in electric power system structures and variations in the system parameters over the time has turned AGC task into a challenging one. Infrastructure of the intelligent power system should effectively support the provision of auxiliary services such as an AGC system from various sources through intelligent schemes. Literature survey shows that performance of AGC of interconnected power system with diverse sources gets improved by changing in controller structure, using intelligent optimization techniques for controller parameters, adding storage system and by considering different participation of diverse sources in multi area power systems. Hence, proposing and implementing new controller approaches using high performance heuristic optimization algorithms to real world problems are always welcomed. Researchers are devising various optimization Algorithms with different behaviors in various systems. Widespread review of the taxonomy of optimization algorithms in power system is explored in this study. Most popular nature inspired algorithm is population based algorithm which has a set of solution which moves towards the goal all together. Meta Heuristic Algorithm is divided into two important groups as trajectory based and population based. Population based algorithm are further divided into six groups depending on bio-inspired, physics, chemistry, social human behavior, plant vi characteristics. Review of various population based algorithm in application to frequency control is studied. Also, critical review of AGC schemes in interconnected multi area power system with diverse sources is done. Performance of many controllers depends on proper selection of certain algorithms and specific control parameters. Hence, the goal of the present study is to propose different types of new supplementary controller to achieve better dynamic performances in multi-area interconnected power system with diverse sources. Based on the extensive literature review on the control designs of AGC of interconnected power system, it has been felt that new control techniques for design of AGC regulators for interconnected power system especially for renewable sources is always welcome and challenging for researchers. The problem of nonlinearity in interconnected power system with diverse sources has also been addressed with suitable control algorithms. The study is conducted by proposing novel human based nature inspired artificial intelligence (AI) technique - Jaya for AGC of two area interconnected thermal-hydro-gas power system with varying participation of sources. Comparative analysis of proposed Jaya based AGC with bio-inspired AI technique like BAT and recently published Bacterial Forging Optimization (BFO) technique justify the stability of controller. The controllers are compared in terms of dynamic response, performance index value and performance. Regular increase in energy demand made the researchers to work towards the realistic hybrid interconnected multi source power system. Undesirable behavior of renewable sources likes hydro and wind is regularly amended by researchers through incorporating different artificial techniques, energy sources, varying participation of sources etc.To make the system realistic, researchers studied nonlinearities and participation factors incorporation in power system. Novel Jaya vii based AI technique is further employed on realistic power system by considering non linearities like Governor Dead band (GDB), Generation Rate Constraint (GRC) and Boiler dynamics. The study is done on Jaya based AGC of two area interconnected thermal-hydro-wind and thermal-hydro-diesel power system with and without nonlinearities by considering step load and random perturbation at different control areas. By proper selection of AI technique based controller for AGC of interconnected multi area multi source power system gives the opportunity to works well with nonlinearities also. Using novel Jaya based PID parameter values, two area interconnected multi source power system without nonlinearities are simulated and set against other bio-inspired based AI techniques like bio inspired IPSO, PSO and BFA and social human behavior based TLBO algorithm. Modern control theory helps in achieving the main objectives of AGC by effective design strategy. Design of optimal AGC regulator is explored for three area interconnected multi source power system with suitable structures of weighting matrices. Linear quadratic regulator (LQR) owns the top spot for Optimal AGC regulator design. The choice of weighting matrices propels the improvement in dynamic performance and stability of system. Designing of optimal AGC regulator for three different three-area interconnected multi source power systems has been planned. In each power system, optimal AGC regulators have been designed by using different structures of cost weighting matrices (Q an R). Three different structures of cost weighting matrices have been inspected for the design of optimal AGC regulators. One of the novel approaches for design of cost weighting matrices is based on the scaling method. To validate the effectiveness of optimal AGC regulator with structures of control and state cost weighting matrices, dynamic response of system for frequency viii and tie line power deviation is procured with 1% slp in each area. Optimal feedback gain matrix, eigen values, dynamic response characteristics and dynamic responses are the assessment to examine the stability of power system. The investigation carried out reveal that optimal AGC regulator based on structures of control and state cost weighting matrices with scaling method offer remarkable improvement in dynamic stability as compared to other designed regulators. Implementation of Superconducting Magnetic Energy Storage System (SMES) in operation and control of AGC of three-area multi source power systems has been studied. Analysis of PSO tuned Integral controller for AGC of three area interconnected multi source power systems with and without SMES by considering step load perturbation at different control areas has bee done. Comparative performance of different bio-inspired artificial technique has been presented on AGC of three area interconnected power system with SMES. With the use of SMES in power system, significant improvement in the dynamic responses of power system has been observed .Degradation of system due to the presence of hydro sources in interconnected multi source power system with SMES is noticeably reduced by considering 1% slp in all area as compared to 1% slp in individually areas. Battery Energy Storage System (BESS) has the feature are to provide and absorb power at the time of load disturbances and have ability to charge and discharge in short span. AGC of three area multi source interconnected power systems by including and excluding Battery Energy Storage System (BESS) at step load perturbation in different control areas has been studied. Application of BESS in power system certainly helps the power system to achieve AGC objectives in astonishingly quick response time even with reneweable sources.

Fruit of Capsicum annuum L. (capsicum or pepper) are one of the major sources of red food colourant and pungency for spice production. In the spice production industry, fruit are mechanically harvested at different ripeness stages and fruit colour needs to be synchronised before being processed. However, even though capsicum ripens normally on the plant it often fails to ripen fully and turn red once harvested at the green stage. Attempts to promote ripening of harvested fruits have had limited success and the reason for this has been unclear. This project, therefore, investigated ripening behaviour on and off the plant of capsicum fruit grown in Australia and examined effects of pre- and postharvest applications on ripening of green harvested fruit. To examine ripening behaviour on and off the plant, capsicum fruit from three different cultivars (a mild paprika type cv. “Papri Queen”, a cayenne chilli cv. “Caysan”, and a sweet type bell pepper cv. “Aries”) were either allowed to ripen naturally on the plant or harvested at three different maturity stages: light green, deep green and breaker. Harvested fruit were stored individually at room temperature and several ripening characteristics including internal ethylene (C2H4) and carbon dioxide (CO2) concentration, extractable colour, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and oxidase activity, and total soluble solid content (TSSC) were studied during storage. There was very limited involvement of C2H4 during ripening of capsicum and the change in ACC synthase and ACC oxidase (two enzymes in C2H4 biosynthesis pathway) activity was not closely related to that of C2H4. However, it appeared that colour development in cv. “Papri Queen” was closely associated with what C2H4 production did occur while a climacteric-like peak of C2H4 could be observed in all fruit from cv. “Caysan”. For all three cultivars, the level of internal CO2 concentration, extractable colour and TSSC were greater in fruit ripened on the plant followed by fruit harvested at the breaker, deep green and light green stage, respectively. Fruit harvested at the light green stage failed to change colour properly and had very low levels of internal CO2 concentration and TSSC while fruit harvested from the breaker stage onwards ripened normally and developed sufficient colour for spice processing. This may suggest a role of external carbon-supply during ripening. To study the effect of the external-carbon supply during ripening, the stem of fruit were cinctured when fruit reached the light green stage and fruit were left to ripen on the plant. Cincturing delayed colour development of fruit by approximately five days but cinctured fruit were still able to turn red and develop extractable colour higher than the acceptable level of 140 ASTA units. Cincturing did not significantly alter other ripening behaviour such as CO2 concentration or TSSC. The lack of external carbon-supply is, therefore, unlikely to play a major role in the failure of green harvested fruit to ripen. To study the effect of application of plant growth regulators (both pre- and postharvest), an effective method of solution application utilising cincturing was firstly developed. Different plant growth regulator solutions including ethephon, naphthalene acetic acid, abscisic acid, jasmonic acid, sucrose, and different combinations of these were applied to fruit at the light green stage to study preharvest effects on ripening parameters during storage. Only treatment with high concentrations of ethephon increased the extractable colour higher than the acceptable level of 140 ASTA units and induced the complete degradation of chlorophyll. To study effects of postharvest application, 10 µL of various plant growth regulators was dropped into the hole created on the stem of harvested fruit for ten consecutive days. Treatment with ethephon significantly increased extractable colour and degraded chlorophyll content of fruit. Pre- and postharvest ethephon treatment strongly up-regulated Capsanthin-capsorubin synthase (Ccs) gene expression in a manner similar to the up-regulation of Ccs observed in fruit ripened on the plant. This explains the effect of C2H4 on colour development and also indicates the possible reason for the failure of green harvested fruit to ripen. However, the Ccs gene expression and chlorophyll degradation induced by ethephon was not visible until 14 days after harvest which indicated it may not be a direct effect and other signal transduction factors may be involved. When fruit are ripened on the plant, colour development may, therefore, be induced by ripening-related factors (other than C2H4) which is possibly inhibited or inactivated when fruit are harvested at the green stage. C2H4 application to fruit at this stage may help to reactivate or recover these factors which in turn induce colour development. Thus, although capsicum fruit show typical non-climacteric behaviour, C2H4 appears to be involved in some aspects of the ripening process.
http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1294648
Thesis(Ph.D.)-- School of Agriculture, Food and Wine, 2007

Fruit of Capsicum annuum L. (capsicum or pepper) are one of the major sources of red food colourant and pungency for spice production. In the spice production industry, fruit are mechanically harvested at different ripeness stages and fruit colour needs to be synchronised before being processed. However, even though capsicum ripens normally on the plant it often fails to ripen fully and turn red once harvested at the green stage. Attempts to promote ripening of harvested fruits have had limited success and the reason for this has been unclear. This project, therefore, investigated ripening behaviour on and off the plant of capsicum fruit grown in Australia and examined effects of pre- and postharvest applications on ripening of green harvested fruit. To examine ripening behaviour on and off the plant, capsicum fruit from three different cultivars (a mild paprika type cv. “Papri Queen”, a cayenne chilli cv. “Caysan”, and a sweet type bell pepper cv. “Aries”) were either allowed to ripen naturally on the plant or harvested at three different maturity stages: light green, deep green and breaker. Harvested fruit were stored individually at room temperature and several ripening characteristics including internal ethylene (C2H4) and carbon dioxide (CO2) concentration, extractable colour, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and oxidase activity, and total soluble solid content (TSSC) were studied during storage. There was very limited involvement of C2H4 during ripening of capsicum and the change in ACC synthase and ACC oxidase (two enzymes in C2H4 biosynthesis pathway) activity was not closely related to that of C2H4. However, it appeared that colour development in cv. “Papri Queen” was closely associated with what C2H4 production did occur while a climacteric-like peak of C2H4 could be observed in all fruit from cv. “Caysan”. For all three cultivars, the level of internal CO2 concentration, extractable colour and TSSC were greater in fruit ripened on the plant followed by fruit harvested at the breaker, deep green and light green stage, respectively. Fruit harvested at the light green stage failed to change colour properly and had very low levels of internal CO2 concentration and TSSC while fruit harvested from the breaker stage onwards ripened normally and developed sufficient colour for spice processing. This may suggest a role of external carbon-supply during ripening. To study the effect of the external-carbon supply during ripening, the stem of fruit were cinctured when fruit reached the light green stage and fruit were left to ripen on the plant. Cincturing delayed colour development of fruit by approximately five days but cinctured fruit were still able to turn red and develop extractable colour higher than the acceptable level of 140 ASTA units. Cincturing did not significantly alter other ripening behaviour such as CO2 concentration or TSSC. The lack of external carbon-supply is, therefore, unlikely to play a major role in the failure of green harvested fruit to ripen. To study the effect of application of plant growth regulators (both pre- and postharvest), an effective method of solution application utilising cincturing was firstly developed. Different plant growth regulator solutions including ethephon, naphthalene acetic acid, abscisic acid, jasmonic acid, sucrose, and different combinations of these were applied to fruit at the light green stage to study preharvest effects on ripening parameters during storage. Only treatment with high concentrations of ethephon increased the extractable colour higher than the acceptable level of 140 ASTA units and induced the complete degradation of chlorophyll. To study effects of postharvest application, 10 µL of various plant growth regulators was dropped into the hole created on the stem of harvested fruit for ten consecutive days. Treatment with ethephon significantly increased extractable colour and degraded chlorophyll content of fruit. Pre- and postharvest ethephon treatment strongly up-regulated Capsanthin-capsorubin synthase (Ccs) gene expression in a manner similar to the up-regulation of Ccs observed in fruit ripened on the plant. This explains the effect of C2H4 on colour development and also indicates the possible reason for the failure of green harvested fruit to ripen. However, the Ccs gene expression and chlorophyll degradation induced by ethephon was not visible until 14 days after harvest which indicated it may not be a direct effect and other signal transduction factors may be involved. When fruit are ripened on the plant, colour development may, therefore, be induced by ripening-related factors (other than C2H4) which is possibly inhibited or inactivated when fruit are harvested at the green stage. C2H4 application to fruit at this stage may help to reactivate or recover these factors which in turn induce colour development. Thus, although capsicum fruit show typical non-climacteric behaviour, C2H4 appears to be involved in some aspects of the ripening process.
Thesis(Ph.D.)-- School of Agriculture, Food and Wine, 2007

L ‘R2R3 MYB è il più grande gruppo di regolatori trascrizionali in pianta. Essi sono coinvolti in differenti processi, come il controllo della produzione di tricomi e peli radicali. Una parte di questi MYB partecipa ad un complesso proteico trascrizionale, che comprende anche una proteina WD40 -repeat (WDR), e una proteina - Helix -Loop - Helix ( bHLH ). I fattori MYB svolgono un ruolo importante in quanto sono i principali responsabili della specificità di azione del complesso. Il complesso MYB - bHLH - WDR può agire sia come un attivatore trascrizionale sia come repressore trascrizionale. La funzione di attivazione comporta la partecipazione del fattore MYB al complesso, che determina l’attivazione della trascrizione di geni target, legandosi direttamente ai loro promotori. Nella repressione, i fattori MYB legandosi al complesso possono reprimere la trascrizione dei geni con diversi meccanismi. In Vitis vinifera il complesso MYB - bHLH - WDR è stato parzialmente studiato in relazione alla via di flavonoidi e alcuni dei fattori MYB hanno un ruolo cruciale nel determinare la regolazione di dell’espressione di enzimi strutturali che appartengono a specifiche ramificazione della via dei flavonoidi. Ad esempio VvMYBA1 e VvMYBA2 regolano la produzione di antociani, che sono i responsabile della colorazione bacca (Walker et al. , 2007), mentre VvMYBPA1 e VvMYBPA2 regolano la produzione di proantocianidine, una via biosintetica che di dirama dalla della via dei flavonoidi. Grazie all’ultima versione del genoma di vite è stato possibile identificare alcuni geni altamente omologhi a regolatori della biosintesi degli antociani giá caratterizzati. Infatti, nell’albero filogenetico composto da R2R3 MYBs e R3 MYBs di Vitis vinifera abbiamo identificato un clade, dove sono presenti VvMYBA1 e VvMYBA2; ad oggi, oltre a queste proteine MYB, nessun altro fattore MYB appartenente a questo clade è stato caratterizzato. Per questo motivo, the gene omologhi e risiedenti sullo stesso cromosoma (cromosoma 2), sono stati nominati VvMYBA5, VvMYBA6, e VvMYBA7 e scelti per le successive caratterizzazioni, grazie anche all’elevata omologia di sequenza con VvMYBA1 e VvMYBA2. Infine, abbiamo ipotizzato che VvMYBA5, VvMYBA6 e VvMYBA7 potrebbero rappresentare dei nuovi regolatori della biosintesi di antociani in organi diversi o in differenti stadi di sviluppo rispetto a VvMYBA1. Abbiamo inoltre identificato un altro clade, chiamato " clade dei repressori " nel quale le proteine sono caratterizzate dalla presenza del motivo repressione. Le informazioni riguardanti i repressori MYB in Vitis vinifera sono scarse e solo un MYB repressore è stato identificato fino ad ora, coinvolto nella repressione della produzione di proantocianidine. Un ulteriore parte del mio progetto di dottorato è la caratterizzazione funzionale di differenti MYB repressori presenti in questo clade, utilizzando approcci diversi come l'espressione eterologa in Petunia hybrida, l’espressione ectopica in radici vite e l’ Yeast two hybrid. I cinque MYB selezionati, denominati VvMYB4a, VvMYB4b , VvMYBC2-L1 , VvMYBC2-L2 e VvMYBC2 - L3, sono stati scelti sulla base della omologia con altri repressori appartenenti a specie diverse e sulla base della presenza di particolari sequenze amminoacidiche caratteristiche dei repressori trascrizionali. Infine, ho iniziato la caratterizzazione di un R3 MYB di vite, chiamato CPC per l’omologia con CPC di Arabidopsis, caratterizzato da
The R2R3 MYB is the largest group of transcriptional regulators in plant. They are involved in different process such as the control of the production of trichomes and root hairs. A part of these MYBs participates to a transcriptional regulatory complex in which includes also a WD40-repeat (WDR) protein and a basic-Helix-Loop-Helix (bHLH) protein. The MYB factor play an important role because it is the major responsible for the specificity of action of the complex. The MYB-bHLH-WDR regulatory complex may act as a transcriptional activator or as a transcriptional repressor: activation function in which MYB activators participate to the complex and activate the transcription of genes by direct binding to their promoters and repression function in which MYB repressors, through different ways, repress the transcription. In Vitis vinifera the MYB-bHLH-WDR complex has been partially studied in relation to the flavonoid pathway, and some MYB factors have been shown to play a role in driving the complex in the activation of specific branches of the pathway. For example, VvMYBA1 and VvMYBA2 are factors responsible for the production of anthocyanin pigments that color the berry skin (Walker et al., 2007), while VvMYBPA1 and VvMYBPA2 are responsible for the production of proanthocyanidins. Thanks to the recently released grape genome we could identify several genes highly homologous to known anthocyanin pathway regulators of other species. In a phylogenetic tree of Vitis vinifera MYB factors there is a clade where VvMYBA1 and VvMYBA2 cluster together with few other MYBs and to date only the MYBA1 and MYBA2 have been characterized. In this clade, three highly homologous genes residing on the same chromosome (Chromosome 2) were named VvMYBA5, VvMYBA6 and VvMYBA7, and chosen for further characterization. Due to the high homology with the characterized VvMYBA1 and VvMYBA2, we hypothesized that they could regulate the anthocyanin synthesis in different organs/stages respect to VvMYBA1. In the same way, we found another clade, called “repressors clade” in witch the proteins are characterized by the presence of the repression motif. The information regarding the MYB repressors in Vitis vinifera is scarce and only one MYB repressor has been identified until now. So another part of my PhD project is the functional characterization of different MYB proteins present in this clade utilizing different approaches as, the heterologous expression in Petunia hybirda, the ectopic overexpression in grapevine hairy roots and the Yeast two hybrid analysis. In details, I choose five different MYB repressors, named VvMYB4a, VvMYB4b, VvMYBC2-L1, VvMYBC2-L2 and VvMYBC2-L3, based on the homology with other repressors belonging to a different species and on particular amino acidic signatures. Moreover, I initiated a study of some MYB proteins belonging to the R2R3 MYB repressor clade, almost completely uncharacterized in grapevine. Finally, I tentatively characterized, a grapevine MYB with a single R3 repeat.

Aging relies on incremental alterations of cell and tissue which contribute to deteriorate organ functions and progressively drives to death. We show herein that midlife brings about a massive transcriptional/metabolic reprogramming, reminiscent of a cell-autonomous activation of an ectopic anti-viral response, which impairs the biological functions on the basis of cell homeostasis and longevity, namely mitochondrial and amino acid biogenesis. Integrated transcriptional analyses indicate IRF7 to be the major driver of these changes reveling an unprecedented, cell-autonomous role of Interferon Regulatory Factor 7 (IRF7) in leading transcriptional, mitochondrial and amino acid alterations with age. We found that the inhibition of IRF7 is sufficient to revert the transcriptional profiling of “old cell”, determining diminished interferon signaling, reverted transcriptional derangement, restored mitochondrial function and partially reestablish the amino acid pool. Our results reveal IRF7 as a major regulator of aging-related transcriptional and metabolic alterations and point it out as an ideal candidate for the development of effective therapies against aging and aging-related diseases.

Dissertação de mestrado em European and Transglobal Business Law
Global consideration on money laundering has its origins in narco-trafficking of 1980s which raised public awareness and took international regulatory body’s attention. Throughout time, due to the socio-economic and political context, legislations on money laundering were transformed in order to introduce an efficient response to new issues. As a need in the aftermath of 9/11, counter-financing of terrorism (CFT) was included in the scope of anti-money laundering (AML) legislations, due to the intertwined nature of these two criminal matters. A new challenge to the AML/CFT legislations was introduced by the technological developments and the emergence of virtual currency. Appearing as an alternative, fast, easy and cheap non-cash payment method, its relation with criminal activities, widespread usage and unregulated operations raised concerns. When traditional approaches to the fight against money laundering and financing of terrorism were circumvented by pseudo-anonymous and decentralized nature of new transaction methods, existing legislations were forced to be transformed once more. European Union, taking its powers for regulating criminal matters from the Treaty of the Functioning of European Union (TFEU), proposed an amendment to the 4th AML, with the purpose of reducing anonymity of virtual currency. Not being accepted yet, its ability to produce an adequate respond to challenges, due to the special nature of virtual currency, is questionable. This thesis analyse European Union’s current Anti-Money Laundering legislation and its responsiveness to the characteristics of virtual currency that are attributable to the risks, with particular attention to crypto-currency, through a critical perspective. It aimed to raise awareness of the subject matter and contribute to the future of AML/CFT reuglations of the EU.
A preocupação internacional com o branqueamento de capitais está ligada ao narcotráfico da década de oitenta. Ao longo do tempo e devido ao contexto sócio económico e político, a legislação relacionada com o branqueamento de capitais foi sendo adaptada, permitindo introduzir uma resposta mais eficiente aos novos desafios. Isto foi particularmente visível na sequência dos ataques de 11 de setembro, momento a partir do qual a prevenção do financiamento do terrorismo passou a estar incluída no domínio do branqueamento de capitais, atendendo à ligação próxima entre estes dois fenómenos. Um novo desafio à legislação sobre branqueamento de capitais surgiu como desenvolvimento tecnológico, nomeadamente com o aparecimento de cripto-moedas. As moedas virtuais surgiram como uma alternativa rápida, fácil e pouco dispendiosa, para realizar pagamentos. Porém, a sua associação a atividades criminosas, uso generalizado e ausência de regulamentação própria conduziram a fortes preocupações por parte das entidades reguladoras. As abordagens tradicionais de combate à lavagem de dinheiro e financiamento do terrorismo tornaram-se obsoletas perante a natureza descentralizada e pseudoanónima destes novos métodos de transações, demandando uma reforma célere da legislação existente. A União Europeia, utilizando o Tratado sobre o Funcionamento da União Europeia como forma de fundamentar os seus poderes, propôs uma alteração à diretiva 4.ª AML, com o objetivo de reduzir o anonimato das cripto-moedas. Não tendo sido ainda aprovada, a capacidade desta alteração produzir a resposta adequada aos desafios apresentados pela natureza especial das moedas virtuais é, no mínimo, questionável. O trabalho aqui apresentado analisa a atual legislação europeia contra o branqueamento de capitais e a sua capacidade de responder às características das moedas virtuais, às quais se atribui um elevado risco. Tem também como objetivo salientar questões relativas a esta temática e despertar maior interesse, assim como contribuir para o futuro da regulamentação AML/CFT da União Europeia.

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Although the regulation of pigmentation is well characterized, it remains unclear whether cell-autonomous controls regulate the cyclic on–off switching of pigmentation in the hair follicle (HF). As human HFs and epidermal melanocytes express clock genes and proteins, and given that core clock genes (PER1, BMAL1) modulate human HF cycling, we investigated whether peripheral clock activity influences human HF pigmentation. We found that silencing BMAL1 or PER1 in human HFs increased HF melanin content. Furthermore, tyrosinase expression and activity, as well as TYRP1 and TYRP2 mRNA levels, gp100 protein expression, melanocyte dendricity, and the number gp100+ HF melanocytes, were all significantly increased in BMAL1 and/or PER1-silenced HFs. BMAL1 or PER1 silencing also increased epidermal melanin content, gp100 protein expression, and tyrosinase activity in human skin. These effects reflect direct modulation of melanocytes, as BMAL1 and/or PER1 silencing in isolated melanocytes increased tyrosinase activity and TYRP1/2 expression. Mechanistically, BMAL1 knockdown reduces PER1 transcription, and PER1 silencing induces phosphorylation of the master regulator of melanogenesis, microphthalmia-associated transcription factor, thus stimulating human melanogenesis and melanocyte activity in situ and in vitro. Therefore, the molecular clock operates as a cell-autonomous modulator of human pigmentation and may be targeted for future therapeutic strategies.