Tesi sul tema "Regulator"

This thesis presents the design, simulation, and hardware implementation of a proposed method for improving efficiency of voltage regulator. Typically, voltage regulator used for noise-sensitive and low-power applications involves the use of a linear regulator due to its high power-supply rejection ratio properties. However, the efficiency of a linear regulator depends heavily on the difference between its input voltage and output voltage. A larger voltage difference across the linear regulator results in higher losses. Therefore, reducing the voltage difference is the key in increasing regulator’s efficiency. In this thesis, a pre switching regulator stage with positive voltage tracking cascaded to a linear regulator is proposed to provide an input voltage to a linear regulator that is slightly above the output of the linear regulator. The tracking capability is needed to provide the flexibility in having different positive output voltage levels while maintaining high overall regulator’s efficiency. Results from simulation and hardware implementation of the proposed system showed efficiency improvement of up to 23% in cases where an adjustable output voltage is necessary. Load regulation performance of the proposed method was also overall better compared to the case without the output voltage tracking method.

DC-DC converters can be separated into two main groups: switching converters and linear regulators. Linear regulators such as Low Dropout Regulators (LDOs) are straightforward to implement and have a very stable output with low voltage ripple. However, the efficiency of an LDO can fluctuate greatly, as the power dissipation is a function of the device’s input and output. On the other hand, a switching regulator uses a switch to regulate energy levels. These types of regulators are more versatile when a larger change of voltage is needed, as efficiency is relatively stable across larger steps of voltages. However, switching regulators tend to have a larger output voltage ripple, which can be an issue for sensitive systems. An approach to utilize both in cascaded configuration while providing a negative output voltage will be presented in this paper. The proposed two-stage conversion system consists of a switching pre-regulator that can track the negative output voltage of the second stage (LDO) such that the difference between input and output voltages is always kept small under varying output voltage while maintaining the high overall conversion efficiency. Computer simulation and hardware results demonstrate that the proposed system can track the negative output voltage well. Additionally, the results show that the proposed system can provide and maintain good overall efficiency, load regulation, and output voltage ripple across a wide range of outputs.

In order to proliferate, bacteria must coordinate chromosome replication and chromosome segregation with cell wall remodeling and other growth-related events. Selective inhibition of these incompletely understood control processes can enable antibiotic and therapeutic strategies. In my thesis, I examine molecularly similar mechanisms for regulating chromosome replication in alpha-proteobacterium Caulobacter crescentus and in the Actinobacterial human pathogen Mycobacterium tuberculosis. It was presumed that the response regulator CtrA, a global phospho-relay transcription factor, coordinates asymmetric timing of chromosome replication in C. crescentus. CtrA is absent from replicating stalked cells and abundant in non-replicating 'swarmer' cells, and the presence of five high affinity CtrA binding sites in Cori, the origin of chromosome replication, presumably allows repression of replication in swarmer cells. I tested this long-standing hypothesis in vivo using a DNA replacement strategy that uncouples CtrA's function as a global transcription regulator from its specific function(s) at Cori. My results substantially change the established model. Rather than being the principal timer of chromosome replication, CtrA binding sites in Cori rescue cells challenged by simultaneous growth and antibiotic stresses. I propose that CtrA provides an 'emergency brake' that prevents lethal replication during adverse conditions. Presumably, the evolutionary benefits of such regulation are manifested by improved odds of viability when cells resume growth following stressful conditions. CtrA regulates replication, presumably by targeting DnaA, the major bacterial replication protein. In C. crescentus, regulation of both CtrA and DnaA is dominated by proteolysis. DnaA is inherently unstable and further regulated by a proteolysis that specifically removes DnaA protein from nitrogen or carbon-starved cells. I further defined the cues that prompt DnaA proteolysis by using specific auxotrophic strains, implicating tRNA and the ribosomes in this mechanism. Furthermore, as the chaperone that presents DnaA to the ClpP protease is currently unknown, I also designed and characterized translational fusions with DnaA for genetic screens and for biochemical affinity purification methods. This second strategy also implicated contacts between DnaA and a ribosomal protein.Additional insights into response regulator control of chromosome replication were provided by studies of MtrA (a proposed analog of CtrA) from Mycobacterium tuberculosis. By studying MtrA binding to DNA sequences in vitro, I determined that the spacing between conserved MtrA-binding sequences in oriC is sub-optimal yet highly conserved among Mycobacteria oriC's. I also examined MtrA binding at the promoter for immune-dominant antigen 85B, where I argue MtrA binds cooperatively as a tetramer. As the genes regulated by MtrA remain poorly defined, I also proposed the MtrA regulon. Using computational representations of the MtrA binding site and for eleven of the thirteen M. tuberculosis sigma factors, I identified 54 genes likely to be regulated by MrA. These genes are functionally related, arguing that MtrA coordinates replication control with cell wall remodeling and with metabolic adjustments that exploit host nutrients available during the M. tuberculosis phases of infection and dormancy.
Pour proliférer, les bactéries doivent coordonner la réplication des chromosomes et la ségrégation des chromosomes avec le remodelage de la paroi cellulaire et d'autres événements liés à la croissance. L'inhibition sélective de ces processus de contrôle mal comprise peut permettre à des stratégies antibiotiques et thérapeutiques. Dans ma thèse, je examiner les mécanismes moléculaires similaires de régulation de la réplication des chromosomes dans Caulobacter crescentus alpha-protéobactérie et la tuberculose Mycobacterium Actinobacterial agents pathogènes humains.Il a été présumé que le régulateur de réponse Ctra, un facteur de transcription mondiale phospho-relais, coordonne le calendrier asymétrique de la réplication des chromosomes dans C. crescentus. Ctra est absent de la réplication des cellules de harcèlement et abondante dans les cellules non-réplication »Grouillant, et la présence de cinq sites de haute affinité de liaison au Ctra Cori, l'origine de la réplication des chromosomes, permet sans doute la répression de la réplication dans les cellules Grouillant. J'ai testé cette hypothèse de longue date in vivo en utilisant une stratégie de remplacement d'ADN qui découple la fonction Ctra comme un régulateur de transcription globale de sa fonction spécifique (s) à Cori. Mes résultats sensiblement modifier le modèle établi. Plutôt que d'être la minuterie principale de la réplication des chromosomes, Ctra sites de liaison dans les cellules de sauvetage Cori contestée par la croissance simultanée et souligne antibiotiques. Je propose que Ctra offre «frein de secours" qui empêche la réplication létale dans des conditions difficiles. Vraisemblablement, les avantages de l'évolution de cette réglementation se manifestent par l'amélioration de la viabilité de cotes lorsque les cellules renouer avec la croissance après des conditions stressantes.Ctra régule la réplication, probablement en ciblant DnaA, la protéine majeure de réplication bactérienne. Dans C. crescentus, la réglementation des deux Ctra et DnaA est dominé par protéolyse. DnaA est intrinsèquement instable et plus réglementé par une protéolyse qui élimine spécifiquement la protéine DnaA de l'azote ou de carbone cellules-faim. De plus, je définis les indices qui invite la protéolyse DnaA à l'aide de certaines souches auxotrophes, impliquant ARNt et les ribosomes dans ce mécanisme. En outre, comme le chaperon qui présente DnaA à la protéase ClpP est actuellement inconnue, J'ai également conçu et caractérisé fusions traductionnelles avec DnaA pour les écrans génétiques et biochimiques pour les méthodes de purification d'affinité. Cette seconde stratégie a également impliqué des contacts entre DnaA et une protéine ribosomique.autres renseignements sur commande du régulateur de réponse de la réplication des chromosomes ont été fournies par des études de MTRA (un analogue de la proposition de Ctra) de Mycobacterium tuberculosis. En étudiant MTRA lient à des séquences d'ADN in vitro, j'ai déterminé que l'espacement entre les séquences conservées MTRA-obligatoire dans Oric est sous-optimale encore fortement conservées chez les mycobactéries Oric. J'ai aussi examiné MTRA contraignant au niveau du promoteur pour 85B antigène immuno-dominant, où je soutiens MTRA lie collaboration sous forme de tétramère. Comme les gènes régulés par MTRA restent encore mal définies, j'ai également proposé le régulon MTRA. En utilisant des représentations de calcul de la MTRA site de liaison et de onze des treize facteurs sigma M. tuberculosis, j'ai identifié 54 gènes susceptibles d'être réglementés par l'ARM. Ces gènes sont en relation fonctionnelle, en faisant valoir que MTRA coordonnées contrôle de la réplication avec le remodelage de la paroi cellulaire et avec des adaptations métaboliques qui exploitent des éléments nutritifs de l'hôte disponibles pendant les phases de M. tuberculosis de l'infection et de la dormance.

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The dissertation has as its theme the capture of the regulator, in particular the Regulatory Agencies implemented in the legal system from the American model with the administrative reform carried out with more emphasis in the government of Fernando Henrique Cardoso. In this reform, the regulatory agencies have been placed as a modern model and legal certainty generator, considering that their leaders, because they are technical professionals in the regulated area, would be more protected from outside influence, and thus harder to regulatory capture. Notwithstanding the model "modern" presented the facts demonstrated the capture of leaders of regulatory agencies, not fulfilling these ones promised in the administrative reform of the 1990s in Brazil. They will be analyzed in the first chapter, the historical and economic assumptions of regulatory agencies; in the second chapter, it analyzes will be the regulatory agencies, and in the latter the phenomenon of regulatory capture of the agencies. From the combination of the theoretical framework of the Theory of Economic Regulation (economic theory of capture), George J. Stigler, and the notion of State from Leon Duguit, state-fact theory, the conclusion about the capture of the dynamics will be made of specific regulatory entities, called Independent Regulatory Agencies.
A dissertação tem como tema a captura do agente regulador, em especial das Agências Reguladoras implementadas no ordenamento jurídico a partir do modelo norte-americano com a reforma administrativa realizada com mais ênfase no governo de Fernando Henrique Cardoso. Nessa reforma, as Agências Reguladoras foram colocadas como um modelo moderno e gerador de segurança jurídica, haja vista que os seus dirigentes, por serem profissionais técnicos na área regulada, estariam mais protegidos da influência externa, sendo, assim, mais difícil a captura regulatória. Não obstante o modelo “moderno” apresentado, os fatos demonstraram a captura dos dirigentes das Agências Reguladoras, não cumprindo esses entes o prometido na reforma administrativa da década de 1990 no Brasil. Serão analisados, no primeiro capítulo, os pressupostos históricos e econômicos das Agências Reguladoras; no segundo capítulo, analisarse- ão as Agências Reguladoras, e, no último o fenômeno da captura regulatória das agências. A partir da conjugação dos referenciais teóricos da Teoria da Regulação Econômica (teoria econômica da captura), de George J. Stigler, e da noção de Estado de León Duguit, teoria do Estado-fato, será feita a conclusão acerca da dinâmica da captura dos entes regulatórios específicos, denominados de Agências Reguladoras Independentes.

Thesis (Ph.D.)--University of Tennessee Health Science Center, 2007.
Title from title page screen (viewed on July, 18, 2008). Research advisor: Anjaparavanda Naren, Ph.D. Document formatted into pages (xv, 72 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 66-72).

The growing threat of antibiotic-resistant bacteria necessitates the development of new anti-infective therapeutics. Innate defense regulator (IDR) peptides are a novel class of immunomodulatory agents shown to combat bacterial pathogens in murine models of infection via the augmentation of host immune functions, including the stimulation of chemokine production and enhancement of leukocyte recruitment, while suppressing bacterial-induced inflammation. Although IDR-peptides present the potential for future broad-range anti-infective agents, our limited understanding of how they modulate host immunity remains an obstacle in their development as clinical therapeutics. I hypothesized that IDR-peptides impact host immunity by modulating the immune responses of monocytes, a cell population necessary for IDR-mediated protection against infection. In this study, IDR-1002 was found to be a multi-faceted regulator of monocyte migration. IDR-1002 induced the production of monocyte-specific chemokines MCP-1 and MCP-3, as well as neutrophil-specific chemokines, IL-8 and GRO-α in human peripheral blood mononuclear cells (PBMCs), correlating with the activation of the mitogen-activated protein kinases (MAPK), p38 and extracellular-regulated kinase (ERK)-1/2, in monocytes. IDR-1002 was also found to enhance human monocyte migration towards chemokines through the enhancement of β1-integrin-mediated adhesion to fibronectin via regulation of the phosphatidylinositol-3-kinase (PI3K)-Akt signalling pathway. In addition, IDR-1002 increased monocyte responsiveness to the chemokines MIP-1α and RANTES via modulation of CCR5 expression. These results demonstrate an overall promotion of monocyte motility by IDR-1002. In contrast to the immune-strengthening effects of IDR-1002, the production of pro-inflammatory cytokines in human PBMCs stimulated with bacterial lipopolysaccharide (LPS) was suppressed by the peptide, and correlated with a suppression of LPS-induced NFκB and p38 MAPK signalling and activation of PI3K-Akt signalling in monocytes. These results demonstrate that IDR-peptides are potent modulators of human monocyte function via their extensive regulation of monocyte signalling networks, potentially accounting for their multifunctional effects on host immunity in murine models of bacterial infection.

Cutaneous wound healing is an inherent biological process to every injury, surgical procedure, and cases of trauma. Wound healing is loosely categorized into three overlapping phases: inflammation, epithelialization, and maturation. Dysregulation of any of these steps leads to poor wound healing, as do conditions such as diabetes, obesity, and advanced age. We recently identified the clefting syndrome, Van der Woude (VWS), as another condition resulting in poor healing. VWS and the allelic syndrome, popliteal pterygium (PPS) are caused by mutations in Interferon Regulatory Factor 6 (IRF6). In the mouse, removal of Irf6 leads to major developmental defects, including limb, craniofacial and cutaneous anomalies. As embryonic development and wound healing share common biological processes, we hypothesized that Irf6 is critical to the regulation of differentiation, proliferation, and migration of keratinocytes, processes essential to wound healing. Upon in vitro differentiation, wild type murine keratinocytes exhibited increased Irf6 expression. Fittingly, Irf6-/- keratinocytes exhibited a defect in terminal differentiation and increased capacity for long-term proliferation. We also determined that a craniofacial enhancer of Irf6 is functional in differentiating keratinocytes in vitro and in vivo. As cellular migration is a critical process to wound epithelialization, we tested the role of Irf6 in keratinocyte migration. We report a significant delay in closing an in vitro scratch wound in the absence of Irf6. Analysis of time-lapse microscopy revealed that this delay was due to a reduction in keratinocyte velocity. We report a substrate non-specific reduction in adhesion in Irf6-/- keratinocytes compared to wild type cells. In addition,Irf6-/- keratinocytes exhibited longer and more prominent actin stress fibers that were rescued by the addition of the ROCK inhibitor, Y27632. The active form of RhoA, a GTPase regulating stress fiber formation upstream of ROCK was increased in Irf6-/- keratinocytes, suggesting a role for Irf6 in regulating RhoA-dependent stress fibers. We further identified Arhgap29, a GTPase activating protein deactivator of RhoA, as a downstream effector of Irf6 in skin and keratinocytes. Finally, we used a conditional strategy to delete Irf6 in adult murine epidermis to study in vivo wound healing. We report a significantly smaller macroscopically visible wound that was not confirmed by wound stereological analysis. We also utilized an embryonic model to exclusively test epithelialization in the absence of Irf6. Our results indicate a trend toward faster migration in Irf6-/- e10.5 wounds, yet the difference in wound closure after 6 hours of healing was not significant. Thus we conclude that Irf6 regulates the balance between keratinocyte proliferation and differentiation. Furthermore, it regulates stress fiber formation and speed of migrating keratinocytes in vitro, while Irf6 is dispensable for cutaneous wound epithelialization in vivo.

This thesis considers the applicability of 'new governance' techniques within the field of emerging biotechnologies. Through three contrasting case studies I construct an argument in favour of new governance, contending that the qualities of this regulatory trend (flexibility, reflexivity, nuance, open discourse, and participation - 'regulatory desirables' ) have much to offer the regulation of emerging biotechnologies. The first case study examines the existing European and international regulatory frameworks for genetically modified organisms (GMOs). This case study explores the role of (bio)ethics within the regulatory process through each progressive stage: design, operation, and assessment. The regime's failure to provide adequate space for ethical reflection, and the limited role of ethics throughout the regulatory process prompts a proposal for an alternative approach that recognizes the multiple contexts in which regulation operates, and is able to accommodate the socio-ethical nuances of the GMO products being assessed. This case study analyses a traditionally structured regulatory framework. It exemplifies a number of qualities that I consider undesirable in the context of regulating biotechnologies: inflexibility, lack of reflexivity, lack of nuance within the regime, absence of ethical discussion, absence of participation from all interested/affected parties. In the second and third case studies I show how these 'regulatory undesirables' can be addressed through new governance techniques. The second case study focuses on the international regulation of stem cell research; I propose developing a polycentric, principles-based regulation (PBR) regime. The third case study centres on the international governance of the gene synthesis industry; here I recommend adopting a risk-based regulation (RBR) approach. In both these fields, voluntary, interdisciplinary, international organisations have collaborated to produce guidelines, codes, protocols, standards, and statements addressing matters of practice. I argue that these 'soft law' documents form the ideal starting point for the development of more sophisticated regulatory regimes in both fields. Furthermore, I argue that the informal organisations producing these documents are, in certain instances, best placed to step into the role of 'regulator' due to their in-depth, inside knowledge of the field, and network. Thus, I collapse the regulator-regulatee distinction held in traditional, 'command and control' style systems, as these organisations typically include those who would traditionally be seen as the 'regulatee'. Each case study considers the nuances of context vis-à-vis the regulatory approach advocated. I conclude by engaging in a comparative analysis of these three case studies, drawing out the qualities, characteristics and considerations that I regard as essential to the construction of responsible, facilitative governance frameworks across the field of emerging biotechnologies. I conclude that new governance is best suited to achieving these (aforementioned) 'regulatory desirables'.

Thesis (M. S.)--Electrical and Computer Engineering, Georgia Institute of Technology, 2009.
Committee Chair: Rincón-Mora, Gabriel; Committee Member: Ghovanloo, Maysam; Committee Member: Leach, W. Marshall.

Checkpoint kinase 1 (Chk1) is a key player in the DNA damage response signalling pathway and the mode of Chk1 activation whereby it undergoes ATRdependent phosphorylation at Ser317 and Ser345 is well characterised. It has been suggested that phosphorylation at the ATR sites relieves the auto-inhibitory action conferred by the C-terminal negative regulatory domain on the catalytic core of Chk1. In this study, we show that Chk1 activity can also be stimulated by docking to an N-terminal region of the growth regulator p21waf1 and this docking domain is necessary for efficient Chk1-dependent phosphorylation of p21 at Ser146. In addition, Chk1 and p21 are shown to form a transient interaction by immunoprecipitation. Interestingly, although the isolated p21 docking domain can activate Chk1 in trans, a mutant where the C-terminal 70 amino acids are truncated is refractory to stimulation whereas mutation of the ATR phosphoacceptor sites does not affect docking dependent activation. Furthermore, when the amino acid sequence of the p21 docking domain was aligned with the sequence of Chk1, homology to the F region on the kinase domain was identified. Mutation of two conserved tryptophan residues within the homology region appears to release the C-terminus from intramolecular interactions rendering it susceptible to cleavage and refractory to allosteric stimulation. Furthermore, small peptides based on this region of Chk1, like the p21 docking domain, are able to activate Chk1 in trans and disrupt interaction between the N-terminal and Cterminal domains. Interestingly, peptide microarray showed that Chk1 stimulated by activating peptide is able to phosphorylate novel peptide substrates which are not observed with unstimulated Chk1. The data suggest that the last C-terminal 70 amino acids of Chk1 play an important role in auto-inhibition through interaction with the F region of the core catalytic domain. Binding to p21 is able to activate Chk1 by inhibiting the auto-inhibitory interaction independent of phosphorylation at the Ser317 and Ser345 sites. Furthermore, activating peptide is able to modulate Chk1 specificity towards other substrates.

Myogenesis is highly regulated and its activation in the embryo is controlled by a series of complex transcriptional regulatory networks that ultimately result in the expression of myogenic regulatory factors (MRFs). The MRFS, particularly MyoD and Myf5, are responsible, in concert with a vast range of cofactors, for directing the expression of genes responsible for muscle formation and activity. Several candidate proteins have emerged as being responsible for MRF expression, as well as numerous downstream effectors involved in muscle formation in vivo. Several cis-regulatory elements have been identified for MyoD, but only a handful of factors have been identified that bind these elements. In addition, knockout experiments of these regions do not result in a complete loss of MyoD expression, suggesting a certain level of redundancy and the existence of other yet unidentified cis-regulatory modules. In this study, novel potential regulatory regions within the MyoD upstream genomic locus were identified by comparative genomics. These regions, named ReMos 9, 10 and 11, were conserved in mammals, chick and fish. Reporter assays in C2C12 cells using these regions cloned upstream of the MyoD promoter revealed that they positively enhanced the promoter activity. A synergy was uncovered between ReMo 9 and 10, which have a strong positive effect on promoter activity, but none individually; ReMo 11 seemed to disrupt this synergy. In addition, ReMos 9+10 and the CER enhancer were shown, by double fluorescent RNA in situ hybridisations, to be transcribed and possess cryptic promoter activity. This suggested that these elements acted as alternative promoters and encoded RNAs that regulated MyoD gene expression. Furthermore, the use of a newly engineered database generated predictions of DNA-binding factors interacting with the cis-regulatory regions, as well as protein interaction networks involved in MyoD regulation. These predictions were refined and constrained with biological input data derived by microarrays of single-cells transiently expressing relevant constructs. A list of candidate muscle-specific binding factors was then tested in vitro by siRNA knockdown experiments, and showed that MyoD disrupts the positive synergistic effect of ReMos 9 and 10 on the PRR. In conclusion, this study identified a number of regions that seem to be involved in MyoD regulation, and candidate factors binding to the MyoD cis-regulatory regions. Further in vivo validation will identify their function in MyoD spatio-temporal gene expression.

Cystic fibrosis transmembrane conductance regulator (CFTR) is activated by cAMP-dependent phosphorylation, and functions as an ATP-dependent chloride channel involved predominantly in the movement of chloride ions to maintain cellular ionic homeostasis. Mutations in this channel cause cystic fibrosis, the most common lethal autosomal recessive disease in caucasians. The regulation of CFTR forms a large body of research; this thesis investigated the role of three potential components of the regulatory machinery – nucleoside diphosphate kinase B (NDPK-B), tyrosine phosphorylation and G proteins. This thesis aimed to: 1. Describe the functional relationship between NDPK-B and CFTR. 2. Describe the effect of altered tyrosine phosphorylation in 16HBE14o- cells on CFTR function. 3. Identify the tyrosine phosphatases involved in CFTR regulation. 4. Explain how alterations in tyrosine phosphorylation change CFTR function. 5. Describe the effect of G protein stimulation on CFTR channels which have already been activated by an increase in cellular cAMP. The whole cell patch clamp technique was used to examine ion channel function in two cell types - human bronchial epithelial cells (16HBE14o-) and baby hamster kidney cells (BHK-21). Due to alterations in the function of cultured cells, it was not possible to describe the functional relationship between the histidine kinase NDPK-B and CFTR. Further work is required in this area to elucidate the role of this protein in CFTR regulation. The use of general tyrosine phosphatase inhibitors resulted in a significant decrease in the CFTRinh-172-sensitive conductance in 16HBE14o- cells, and specific inhibitors ruled out the involvement of PTP1B and Shp1/2 phosphatases. Further work is required to explain how tyrosine phosphatase inhibition alters CFTR function. Finally, G protein stimulation in 16HBE14o- after increasing intracellular cAMP had no significant effect on channel function. This suggested that the cAMP-dependent activation of the channel is the predominant mechanism for stimulating channel function.

Many analog systems need a stable power supply voltage that does not vary with temperature and time in order to operate properly. In a battery operated system the battery voltage is not stable, e.g. it decreases with decreasing temperature and with ageing. In that case a voltage regulator must be used, that regulates the battery voltage and generates a stable supply voltage to power other circuitry.

In this thesis a voltage regulator to be used in a battery operated system has been designed which meets the given specification of stability and power capabilities. A voltage reference, which is a commonly used devise in analog circuits, was also designed. The role of a reference voltage in an electrical system is the same as for a tuning fork in a musical ensemble; to set a standard to which other voltages are compared.

A functionality to detect when the lifetime of the battery is about to run out was also developed.

[Truncated abstract] In the last ten years regulator T (Tr) cells have re-emerged as an integral part of the immune system. Research in this field has rapidly demonstrated the role of these cells in the maintenance of immune homeostasis and their involvement in disease. Tr cells are generated in the thymus as a normal part of the developing immune system. Furthermore, antigen-specific Tr cells are induced in the periphery by a mechanism which is yet to be completely elucidated, but is likely to involve dendritic cells. Tr cells play an important role in autoimmune disease, transplantation tolerance, cancer. Most recently Tr cell involvement has been demonstrated in a growing number of infectious diseases. Tr cell induction was reported in Friend Virus infection at the commencement of this study, and subsequent to publication of our findings have also been identified in FIV and HIV. Murine AIDS (MAIDS) is a fatal chronic retroviral infection induced in susceptible strains of mice by infection with BM5d, a replication defective virus, in a viral mixture which is designated LP-BM5. The manipulation of Tr cells detailed in this thesis and the related publication represent the first reported therapy utilising targeted removal of Tr cells. Chapter 1 summarises the literature relevant to this study up to November 2004. Chapter 2 details the materials and methodologies used in this work. Chapter 3 investigates whether Tr cells are involved in the development of murine AIDS, particularly in the early stages of infection. The data presented in this chapter provides evidence of a population of CD4+ Tr cells which express CD25 on their cell surface and secrete TGF-β, some IL-10 and low levels of IL-4 are induced following infection with LP-BM5. These cells were found to arise by day 12 post infection (pi) by flow cytometry and immunosuppressive cytokine expression was found to peak at day 16 pi indicating a role in the early stages of disease progression. Chapter 4 investigates the effect of therapeutically targeting these induced Tr cells using the antimitotic agent Vinblastine during their induction period. The efficacy of treatment was found to be time dependent and was shown to abrogate disease progression maximally when given at day 14 pi. Treatment with anti-CD4 monoclonal antibody was also found to be efficacious at day 14 pi and confirmed the identity of the Tr cells as being CD4+ T cells. Adoptive transfer studies demonstrated that the return of these cells to a successfully treated host results in renewed MAIDS progression, confirming their role in disease progression

In this master thesis, an experimental setup for a stand-alone power system has been installed and tested in the Waterpower Laboratory at NTNU. The experimental test rig consisted of a cross-flow turbine, a synchronous generator and an electronic load controller (ELC) manufactured by Remote HydroLight in Afghanistan. The objective of the experiments was to evaluate the performance of the ELC regarding step response in frequency and generator voltage. The controller used phase angle regulation and triacs to divert excess energy to dump loads. The dump loads were heating elements installed and submerged in the lower reservoir in the laboratory. A similar configuration was installed for varying the user load in the energy system. Several tests were performed to evaluate the performance of the ELC. A weak component in the test rig was the transmission system that started to slip. This resulted in an increase in turbine speed during the experiment and reduced the quality of the results. However, the tests indicated that a rapid single peak appears during abrupt disconnection and connection of loads. This may disturb and damage sensitive electronic equipment. Despite this, the ELC performed well during the diffent tests with stable regulation in voltage and frequency.Introducing pulse width modulation would eliminate the unfavorable influence of the triacs in the generator voltage signal. With this modification it is possible to increase the flexibility in the energy system by introducing inductive or conductive loads like a battery bank.A hybrid stand alone energy system connecting existing test rig with a photovoltaic-module has been developed. For a larger and more complex energy system, a more sophisticated system has been designed. Both systems are based on a common DC-grid and charging of a battery bank. This results in a more flexible, reliable and a more stable energy system.

Microorganisms use diverse mechanisms to control the expression of their genes and many of these processes are essential for cell survival. This dissertation concerns a novel type of regulatory protein, termed BipA, which is found in many bacteria including harmless commensals such as Escherichia coli K-12, symbionts such as Sinorhizobium meliloti, and pathogens such as Salmonella and enteropathogenic and enterohaemorrhagic E. coli. BipA regulates a range of cellular processes that influence both the survival and virulence of microorganisms. In contrast to other global regulatory proteins, however, it functions as a GTPase that interacts with 70S ribosomes, suggesting that it may influence the translation of one or more target mRNA transcripts. It has previously been proposed that BipA directly regulates the translation of another global regulatory protein known as Fis. However, a phenotypic comparison bipA and tis null mutants of E. coli revealed that, while flagellamediated cell motility was impaired in both mutants, the tis mutant successfully formed colonies at temperatures below 30°C whereas the bipA null mutant did not. These observations indicate the involvement of BipA in processes that are not mediated through Fis. They also suggest that BipA is positioned higher up in the regulatory hierarchy than Fis. In addition to comparing bipA and tis phenotypes, the effects of ectopic expression of Fis were studied. Aberrant Fis expression was shown to be deleterious to cell growth, blocking cell division and hence causing filamentation in E. coli. During the course of this study results were obtained that were inconsistent with the findings presented for the control of Fis expression by BipA. Re-examination of some previously reported data indicates it is unsafe to conclude that BipA directly controls the expression of Fis. In view of these circumstances, a search for additional protein targets that are directly or indirectly regulated by BipA was initiated. Proteomic analysis of lag phase cells from a bipA null mutant of Salmonella enterica serovar Typhimurium and its parent strain uncovered candidate proteins whose expression appears to be influenced by BipA, including SipC, a component associated with the SPI-1 type three secretion system, which is important for virulence in this pathogen.

Cell polarity is an important regulator of cellular processes and is vital in helping to prevent metaplasia and tumorigenesis. There are three many polarity complexes that regulate and maintain epithelial cellular polarity. The Par and Crumbs complexes locate to the apical membrane of the cell, while the Scribble complex is located basolaterally. Of the Scribble complex components, the polarity protein Hugl1, also known as Mgl1 in mice, is especially important in helping to maintain apical basolateral and planar polarity, and is lost in multiple types of cancer. When Hugl1 expression is lost in epithelial cells, it results in a mesenchymal phenotype. We now show that the loss of Hugl1 fundamentally shifts the cellular phenotype and specifically alters EGFR trafficking and signaling. Loss of Hugl1 results in the nuclear translocation of Taz and Slug, increased migration, and the mislocalization of EGFR (Epidermal Growth Factor Receptor), driving cellular growth. Hugl1 regulates the expression of multiple cell identity markers and its loss results in stem cell characteristics, including the increased expression of CD44, and a decrease of CD49f and CD24 expression. The loss of Hugl1 also results in increased growth in soft-agar and prolonged survival when transplanted into NOD-SCID mice; its loss also results in EGF-dependent migration which aids in increasing mammosphere survival. Furthermore, isolated EGFR mislocalization via a point mutation (P667A) also drives these same phenotypes, including activation of Akt and Taz nuclear translocation, indicating the importance of Hugl1 in the regulation of EGFR localization and its signaling. In mice, the loss of total Mgl1 is lethal within days of birth due to hydrocephaly and results in the formation of rosette like structures in the brain that are reminiscent of neuroectodermal tumors. We designed a targeted Mgl1 knockout in the mammary epithelial cells using the Cre/Lox system to evaluate the effects of Mgl1 loss in murine mammary gland development and tumorigenesis. The loss of Mgl1 expression in mice inhibits ductal outgrowth, increases side branching and epithelial layers, and results in the mislocalization of EGFR. While overt mammary tumors did not develop, some individuals did develop hyperplastic nodules that could progress into cancer. The knockdown of Hugl1 in vitro and Mgl1 in vivo reveal how the loss of polarity and presence of Hugl1 results in cancer stem cell characteristics, increased migration, and abnormal signaling due to the mislocalization of EGFR. While these changes result in metaplasia and a potential pre-cancerous state, the loss of Hugl1 alone is not enough to drive the cancer progression, indicating that other mutations or factors are necessary for the development of breast cancer. Because of the key role polarity plays in the prevention of breast cancer development we investigated if the addition of Hugl1 back into breast cancer cells could revert the cancerous cells to a normal epithelial phenotype. Most of the breast cancer cells transfected with Hugl1 expression did not survive, indicating that the re-expression of polarity regulators forces cancer cells to die. The small percentage of cells that did survive re-expression of Hugl1 had retarded growth in soft agar and a decrease in EGFR expression. Together, these data indicate that Hugl1 expression and EGFR activity are closely related and that Hugl1 is required for the proper localization and signaling of EGFR. When Hugl1 is lost, EGFR is mislocalized and fails to be degraded properly, promoting pre-neoplastic changes.

In order for oligodendrocyte progenitor cells (OPCs) to differentiate into fully mature, myelinating oligodendrocytes, they must be specified at the correct times and undergo coordinated changes in both gene expression and morphology. As oligodendrocytes differentiate, they transition from a bipolar morphology into a morphology characterized by a complex network of multiple processes, which will eventually generate membranous structures necessary for myelination of axonal segments. As changes are observed in cellular morphology, oligodendrocytes also undergo changes in their gene expression profile and express genes necessary for both early and later stages of development such as olig1 and myelin basic protein (mbp), respectively. Data from our laboratory demonstrate that autotaxin (ATX), also referred to as phosphodiesterase Iα/autotaxin (PD-Iα/ATX), is involved in all of these processes as a multifunctional protein by regulating lysophospholipid signaling and cell-extracellular matrix interactions. Previously, our laboratory has identified ATX as an oligodendrocyte-secreted factor present in the extracellular environment that via a newly-identified functional domain, named the MORFO domain (modulator of oligodendrocyte remodeling and focal adhesion organization), can regulate adhesion of oligodendrocytes to naturally occurring extracellular matrix (ECM) proteins and ultimately the establishment of the oligodendrocyte’s complex process network. In vitro data presented in this dissertation suggest that lysophosphatidic acid (LPA), via production from ATX’s well characterized lysophospholipase D (lysoPLD) domain, can induce the expression of myelin basic protein (mbp) and the establishment of membranous structures by differentiating oligodendrocytes, both necessary for the initial stages of myelination. Interested in relating these functions to an in vivo model and due to the early embryonic lethality of atx-null mice, we utilized the zebrafish as an in vivo model. The in vivo data presented in this dissertation demonstrate that atx expression in the zebrafish is evolutionarily conserved within vertebrates. Interestingly, in both mouse and the zebrafish, atx was found expressed by cells of the cephalic floor plate in addition to differentiating oligodendrocytes. Functionally the in vivo data presented in this dissertation confirmed ATX’s role in stimulating mbp expression during later stages of oligodendrocyte development. In addition, a novel function for ATX was revealed by the studies undertaken as part of this dissertation that has never been described before. More specifically, based on the timing of atx expression and the phenotype seen upon atx knock-down, the data presented here suggest that ATX, released by the cephalic floor plate, regulates early oligodendrocyte development and/or specification. Taken together, these data identify ATX as a major regulator for early as well as late developmental stages of the oligodendrocyte lineage.

The Phosphatase and actin regulator (Phactr) protein family has been identified as a group of four proteins (Phactr1,2,3,4), interacting with Protein Phosphatase 1 (PP1) and G-actin. G-actin binding to Phactr1 RPEL motifs has been shown to sterically inhibit Phactr1 interaction with PP1 and its nuclear import. Members of the Phactr family are highly expressed in the nervous system and have been implicated in the regulation of the actin cytoskeleton, cell migration and angiogenesis. The molecular mechanism of their signalling is however not well understood. Here I show that Phactr1 functions as a novel regulatory subunit of PP1. Phactr1 contains several binding motifs typical of PP1 regulatory subunits, including an RVxF motif, located in the C-terminal part of Phactr1, partially overlapping with the G-actin binding RPEL motif. Phactr1 binding leaves the PP1 active site accessible for substrates, blocks one of the substrate-binding grooves and creates a highly basic pocket along the PP1 hydrophobic substrate binding groove, thereby defining Phactr1-PP1 specific protein substrates for dephosphorylation. A phosphoproteomic analysis revealed that activation of Phactr1-PP1 complex formation leads to the dephosphorylation of several cytoskeletal proteins (such as IRSp53 and Afadin), leading to rearrangements of the actin cytoskeleton. Induction of Phactr1-PP1 interaction leads to the formation of aberrant actomyosin structures in fibroblasts. The converse phenotype could be induced by non-PP1-binding Phactr1 mutant expression, leading to the formation of long cytoplasmic extensions. In neurons, Phactr1 was enriched in dendritic spines and activation of Phactr1-PP1 complex formation led to changes in dendritic spine morphology. Moreover, I show that Phactr1 L519R mutation, that has been implicated in epilepsy, decreases Phactr1 affinity to G-actin, and thus indirectly activates Phactr1 interaction with PP1. All this suggests that Phactr1 targets PP1 to dephosphorylate specific cytoskeletal proteins, leading to changes in the actin cytoskeleton, which in neurons modulates dendritic spine morphology, leading to changes in neuronal signalling.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma membrane chloride channel expressed in epithelial tissues. While protein kinase A (PKA) is the main activator of CFTR, patch clamp data have suggested a permissive role for PKC in modulating PKA responses. To clarify the role of PKC in CFTR regulation we used PCR mutagenesis to construct a CFTR mutant (9CA) in which the serines or threonines in nine consensus sequences for PKC phosphorylation were replaced by alanines. Its expression in stably transfected baby hamster kidney (BHK) cells was consistently lower than that of wild-type (WT) CFTR. Nevertheless, 9CA cells displayed a robust cAMP-stimulated iodide efflux which was comparable in magnitude to that of WT CFTR cells when normalised for protein expression level. The cpt-cAMP concentration-response relationship in intact cells was similar for WT and 9CA CFTR (EC50 ≈ 100muM), however the onset of iodide efflux from cells expressing the mutant was markedly delayed at all cpt-cAMP concentrations tested. This delay was mimicked by pre-treating WT cells with the PKC inhibitor chelerythrine (10muM), and chelerythrine did not cause further slowing of iodide efflux from 9CA cells. In vitro phosphorylation by PKA and PKC was similar for WT and 9CA CFTR and phosphorylation in the presence of both kinases was additive.
A major impediment to understanding CFTR structure/function has been the difficulty of obtaining sufficient purified CFTR protein for biochemical studies. In an attempt to overcome this problem we expressed a His-tagged CFTR construct in the methylotrophic yeast Pichia pastoris. The final yield of CFTRHis10 following solubilization of Pichia membranes in 0.5% lysophosphatidylglycerol (LPG) and nickel chelate chromatography was ∼20mug/litre. CFTRHis10 was not glycosylated in this yeast system. Both PKA and PKC phosphorylated semi-purified CFTRHis10 in vitro and phosphorylation in the presence of both kinases was additive.

Caulobacter crescentus divides asymmetrically to produce two different progeny, a swarmer progeny that is replication incompetent, and a stalked cell progeny that is replication competent. Upon cell division, a cell cycle regulator protein (CtrA) was identified only in the swarmer cells, and additional circumstantial evidence links this protein to being a repressor of chromosome replication. For example, this response regulator protein binds to five specific sites in the replication origin (Cori) designated [a-e]. We carefully studied the binding characteristics of both phosphorylated (CtrA~P) and unphosphorylated forms of the protein to the five binding sites [a-e] in the replication origin (Chapter 2) and upstream of the ctrA gene (Chapter 3). We showed that phosphorylation significantly enhances binding affinity in the replication origin (Chapter 2) but not upstream of ctrA (Chapter 3). In addition a "pseudo-active" protein form (CtrA D51E) that resembles the phosphorylated form in vivo did not improve the binding characteristics (Chapter 3). These results suggest that enhanced binding on phosphorylation is not the only signal achieved on phosphorylation. In fact, CtrA half-site mutation binding studies shows that phosphorylation stimulates protein/protein interaction and cooperative binding between sites [a] and [b] in the replication origin (Chapter 2). We show that CtrA binding site [b] is the major contributor in the cooperative CtrA binding between [a] and [b] (Chapter 4). We demonstrate that cooperative binding of CtrA~P to sites [a] and [b] repress transcription from a strong promoter (Ps), which in turn blocks plasmid replication. In addition, mutating site [b] to block CtrA binding to [a] and [b] has a deleterious effect on chromosome replication (Chapter 4).
This cooperative CtrA binding at [a] and [b] is independent from the upstream binding sites [c-e] (Chapter 2). CtrA∼P binding in the origin is altered in the presence of the histone-like protein (IHF) that also binds and overlaps CtrA binding site [c] (Chapter 5). In-fact, IHF binds and overlaps binding site [c] (Chapter 5). We propose a replication model in the stalked cell were IHF binding hinders active CtrA binding in the replication origin and regulates cooperative transcription that coincides with replication initiation.

Pseudomonas aeruginosa is a Gram-negative bacillus able to colonize a wide variety of environments. In the human host, P. aeruginosa can establish an acute infection or persist and create a chronic infection. P. aeruginosa is able to establish a niche and persist in human hosts by using a wide array of virulence factors used for: movement, killing host cells, and evading immune cells and antibiotics. Understanding virulence factors and their regulation has proved to be an important means of combating the morbidity and mortality of P. aeruginosa as well as the ever-increasing threat of drug resistance. By targeting virulence factors or their regulators with antivirulence compounds, the bacterium is rendered defenseless and more readily cleared by the immune system. In this study, we examine three different contributors to virulence factor regulation. First, we examined the role of the extracellular sigma factor AlgU and its contribution to regulating a post-transcriptional RsmA. AlgU is most commonly active in chronic infecting strains that produce copious amounts of the virulence factor, alginate. We confirmed that not only was their more RsmA in this background, but that there was a previously unidentified promoter for rsmA regulated by AlgU. In concert with this study, we followed up by studying the effects of AlgR on this unknown promoter. AlgU and AlgR are known to work together, specifically on the alginate operon, and we hypothesized based off of bioinformatics data this was the case with RsmA. Second, due to increased RsmA in this chronic infection strain, we set out to identify potential unknown virulence targets of RsmA. A previously unrevealed target, pasP, was shown to directly interact with RsmA. Third, in an acute infection model strain we identified a new regulatory loop involving the two-component system AlgZ/R. In a pilW strain deficient in the motility virulence factor type IV pili, we showed increased levels of AlgZ/R compared to wildtype, PAO1. The pilW strain produced less pyocyanin, rhamnolipid, and elastase and was attenuated in J774a.1 macrophages. Overall, these studies push the understanding of virulence factor regulation and open the door to potential therapeutic targets in treating P. aeruginosa infections.

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O paclobutrazol (PBZ) é o regulador vegetal utilizado no manejo da produção da mangueira na maioria dos pomares sob as condições semiáridas do Nordeste. Apesar da sua eficiência na produtividade, estudos demonstram que o PBZ permanece ativo no solo por muito tempo e isso pode afetar o desenvolvimento das próximas colheitas, reduzindo o vigor vegetativo. A biodegradação do paclobutrazolem solo não saturado e com histórico de aplicação,foi avaliada durante 49 dias usando glicerol e resíduos industriais (glicerol do biodiesel, torta de amendoim e torta de gergelim) como fontes de adicionais de carbono. Os resíduos agroindustriais (torta de amendoime e torta de gergelim) além de carbono, também são fontes de nitrogênio. Foram realizados estudos cinéticos do consumo de PBZ, além da contagem dos micro-organismos totais, do perfil do pH e da fitotoxicidade no início e final dos experimentos. Adicionalmente, estudos investigativos acerca de produtos da biodegradação foram realizados, com análises de espectroscopia de infravermelho. A torta de amendoim foi o melhor resíduo agroindustrial, sendo usado nas quatro estratégias realizadas na próxima etapa. Nessas quatro estratégias de biodegradação foram realizados experimentos usando 100, 80, 50 e 20 % m/m do solo sem histórico com a adicão de 0, 20, 50 e 80 % m/m de solo com histórico, respectivamente. A adição de solo com histórico (50 ou 80% m/m) e a torta de amendoim favoreceram a biodegradação dosolo sem histórico, cuja biodegradação foi maior que 95%. O resíduoselecionado apresentou-se como uma adequada fonte de carbono e de nitrogênio, e, o solo com histórico um eficiente reservatório de micro-organismos capazes de degradar o PBZ.
Paclobutrazol (PBZ) is a growth regulator used in the management of mango production in most orchards in semi-arid conditions of the Northeast. Despite its efficiency in productivity, studies show that the PBZ remains active in the soil for a long time and this may affect the development of the next crop, reducing plant vigor. The biodegradation of paclobutrazol (PBZ) was evaluated during 49 days using glycerol and agro-industrial wastes (biodieselderived glycerol, peanut cake and sesame cake) as additional carbon sources and unsaturated soil with a history of PBZ application. Kinetic studies were performed of PBZ consumption beyond count of total microorganisms, pH profile and phytotoxicity at the beginning and end of the experiments. Additionally, investigative studies on the caracterization of biodegradation samples were performed with infrared spectroscopy analysis. Biodieselderived peanut cake was the best agro-industrial waste, andthis was used in the following four strategies. Four biodegradation strategies were performed using 100 %, 80 %, 50 % and 20 % of soil without history with the addition of 0 %, 20 %, 50 % and 80 % of soil with history, respectively. The addition of soil with history (50 or 80%) and the selected waste (peanut cake) favored biodegradation of soil without history, with rates about 95 %. The peanut cake waste proved to be an adequate source of carbon and nitrogen, and the soil with history, proved to be an efficient reservoir of microorganisms capable of biodegrading PBZ.

[ES] El contexto de esta Tesis se enmarca en los sistemas de dos componentes (TCS) para comprender el mecanismo de transducción de la señal. Se analizó la especificidad en el reconocimiento de los TCS abarcando estudios a nivel funcional, estructural y evolutivo. Primero se utilizó el sistema HK853-RR468, que al estar previamente caracterizado nos permitió analizar específicamente las regiones de reconocimiento (HK-RR) correspondientes a los Lß3α3 y Lß4α4 de RR468 mutando residuos que determinaran la influencia en la transferencia del grupo fosfato. Los mutantes se caracterizaron de manera bioquímica y se hicieron aproximaciones estructurales pudiendo asignar la reacción de fosfotransferencia a una estructura formada por un complejo entre HK853 y RR468 mutante. Esta estructura nos permitió observar el carácter disociativo de dicha reacción que ha sido descrito previamente y la nula participación del dominio CA. Al mismo tiempo, se analizó la influencia del pH en los residuos catalíticos de la HK y el RR (His y Asp), utilizando un rango de pH de 5 a 8. Los ensayos bioquímicos generados en este rango nos mostraron como la His catalítica perdía su carácter nucleofílico cuando el pH se acercaba y disminuía de 6. Esto se relaciona con el pKa del anillo de imidazol presente en el residuo de His, que se se encuentra en torno a 6 y la pérdida de protonación. También se cristalizó el complejo HK-RR a diferentes pHs donde observamos que la His adquiría un rotámero gauche- que se asignaba a un estado inactivo o de reposo. Por otra parte, se analizó la influencia de la mutación G63V en el RR OmpR, que fue descrita como una mutación relacionada con la resistencia al antibiótico ertapenem. Para esto se generaron mutantes en OmpR en la posición G63, tanto en el dominio REC aislado como en la proteína completa. Los estudios bioquímicos de estas mutaciones demostraron como la mutación en esta posición disminuía la capacidad del RR para fosforilarse e incluso a dimerizar. Esto afectaba a la afinidad de este RR para interaccionar con su ADN correspondiente, las cajas ompF y ompC. Estos efectos se lograron evidenciar con la estructura de OmpRRECG63V, donde se observó como la mutación generaba un cambio conformacional al reducir el tamaño del Lßα3 y generaba un bolsillo hidrofóbico donde quedaba atrapada la cadena lateral de la Val. Finalmente se analizó el aspecto evolutivo de la señalización, para lo que se buscaron organismos endosimbiontes que presentaran una HK y uno o varios RRs. Estas características nos sugerían que la menor presión selectiva nos iba a permitir encontrar organismos con TCSs menos evolucionados cuya especificidad se haya visto reducida. Se analizaron los sistemas de Chlamydia trachomatis, Chlamydia psittaci, Simkania negevensis y Methanobrevibacter sp. Abm4. Solo pudo evidenciarse reacción de fosfotransferencia en el sistema perteneciente a Methanobrevibacter, el cual presenta una HK y 4 RRs. Sin embargo, esta fosfotransferencia presentaba una eficiencia diferenciada, siendo más rápida en RRMet572 y RRMet589-1 mientras que era nula en RRMet589-2. Por su parte las HKs de C. trachomatis y S. negevensis, fueron capaces de fosfotransferir, de manera no selectiva, a RR468, probablemente debido a la alta similitud que presenta la hélice α1 de las HKs con HK853. La aproximación estructural de estos sistemas permitió obtener las estructuras de los RRsMet589-1 y RRMet572, ambos en estado no fosforilado. Las dos estructuras presentaron grandes diferencias conformacionales a partir del Lßα4. Esto sugiere que sus mecanismos de reconocimiento con HKMet y de regulación son diferentes lo que apoya la selectividad diferenciada entre los RRs de este sistema.
[CA] Esta Tesi s'emmarca en l'estudi dels sistemes de dos components (TCS) amb la finalitat d'entendre el seu mecanisme de transducció de senyal basat en l'especificitat de reconeixement a nivell funcional, estructural i evolutiu. Utilitzant el TCS HK853-RR468, analitzarem les regions Lß3α3 y Lß4α4 del regulador de la resposta (RR) RR468, que prèviament s'havien mostrat importants en el reconeixement, generant mutants i determinant la influència en la transferència del grup fosforil. Els mutants foren caracteritzats bioquímicament, observant que afectaven a una reacció específica i permetent-nos captar la reacció de fosfotransferència en una estructura formada per un complex entre HK853 i RR468 mutant. Aquesta estructura va mostrar el caràcter dissociatiu d'aquesta reacció i la nul·la participació del domini CA de la HK. Al mateix tems, s'analitzà l'efecte del pH sobre la transducció del senyal utilitzant els TCS K853-RR468 i EnvZ-OmpR. Els assajos bioquímics generats dins del rang de pH entre 5 i 8 ens mostraren com la His de la HK catalítica perdia el seu caràcter nucleofílic quan el pH s'aproximava i disminuïa de 6, valor del pKa de l'anell d'imidazole de la cadena lateral del residu d'His, indicant que aquesta disminució en l'activitat es correlacionava amb el canvi en la protonació de l'anell. Un exhaustiu estudi estructural del complex HK853-RR468 a diferents pHs mostrà que la His catalítica sempre adquiria un rotàmer gauche- independentment del valor del pH, invalidant el model que proposava que el pH regulava l'activitat de les HKs de la família HisKA induint un canvi en el rotàmer de la His catalítica. D'altra banda, s'analitzà la influència de la mutació G63V en el RR OmpR, que fou descrita com una mutació relacionada amb la resistència a l'antibiòtic ertapenem. Amb aquesta finalitat, es generaren mutants a OmpR a la posició G63 tant al domini REC aïllat com a la proteïna completa. Els estudis bioquímics demostraren com la mutació en aquesta posició disminuïa la capacitat de OmpR per fosforilar-se i per dimeritzar, afectant a la capacitat d'interaccionar amb les seqüències d'ADN palindròmiques diana, corresponents a les caixes ompF i ompC. Aquests efectes es visualitzaren a nivell molecular al resoldre l'estructura del mutant G63V d'OmpRREC, on s'observava com la mutació induïa un canvi conformacional al reduir la mida del Lßα3 generant una butxaca hidrofòbica degut a la presència de la nova Val en posició 63. Aquests canvis es transmeten a la resta de l'estructura d'OmpR produint canvis en Lßα4 i α4 que impedeixen la formació d'una superfície de dimerització competent i impedint la seua interacció amb l'ADN. Finalment, s'analitzà l'aspecte evolutiu de l'especificitat HK-RR. Buscaren organismes endosimbionts que presentaren TCS aïllats consistent en una HK i un o diversos RRs, suggerint que la menor pressió selectiva permetria trobar TCS menys evolucionats, on l'especificitat s'haguera vist reduïda. S'analitzaren HKs i RRs presents en Chlamydia trachomatis, Chlamydia psittaci, Simkania negevensis i Methanobrevibacter sp. Abm4. La reacció de fosfotransferència es va detectar en Methanobrevibacter, que presenta una sola HK i 4 RRs. Aquesta HK mostrà eficiència diferenciada per a la reacció de fosfotransferència, presentant major velocitat per als RRs RRMet572, RRMet589-1 i nul·la per a RRMet589-2. Per la seua banda, les HKs de C. trachomatis i S. negevensis, foren capaces de transferir, de manera no selectiva, a RR468 de Thermotoga maritima, probablement degut a l'alta similitud que presenta l'hèlix α1 de les HKs amb HK853. L'aproximació estructural d'aquests sistemes ens va permetre resoldre les estructures dels RRsMet589-1 i RRMet572 en estat no fosforilat. Les dues estructures presentaren grans diferències conformacionals a partir del Lßα4, els que ens suggereix que els seus mecanismes de reconeixement amb HKMet i de regulació són diferents, cosa que suporta la selectivitat diferenciada entre els RRs d’aquest sistema.
[EN] The context of the Thesis is framed in the two component systems (TCS) to understand the signal transduction mechanism. The specificity in the recognition of TCS was analyzed covering studies at the functional, structural, and evolutionary level. First, the previously characterized HK853-RR468 was used, this allowed us to analyze specific recognition regions corresponding to Lß3α3 and Lß4α4 of RR468 and induce mutations in these regions and understand the recognition between HK and RR and determine the phosphate group transfer's influence. The mutants were characterized biochemically, and structural approximations were prepared, thus assigning the phosphotransfer reaction in a formed structure by an HK8536 and a mutant RR468 complex. This structure allowed us to observe the dissociative character of this reaction that has been previously described and the null participation of the CA domain. Simultaneously, the influence of pH on the catalytic residues of HK and RR (His and Asp) was analyzed, using a pH range of 5 to 8. The biochemical assays generated in this range showed how HK's catalytic His lost the nucleophilic characteristic when pH reached six or below. This is related to the pKa of the imidazole ring present in the His residue that if found around 6 and the loss of protonation. The HK853-RR468 complex was also crystallized at different pHs where we observed that His acquired a gauche- rotamer that was assigned an inactive or resting state. In addition, the influence of mutation G63V in the RR OmpR was analyzed. This mutation was associated with resistance to the antibiotic ertapenem in E. coli. For this, mutants in OmpR were generated in position G63 in both the isolated REC domain and in the whole protein. Biochemical studies of this mutations showed how the mutation in this position reduced the capacity of RR to phosphorylate and even to form a dimer. This affected the affinity of the RR to interact with it's corresponding DNA, the boxes ompF and ompR. These effects were shown with the structure of the REC domain of the OmpR protein mutant G63V. This mutation generated a conformational change by reducing the Lßα3 and generating a hydrophobic pocket that trapped Val's lateral chain. Finally, the evolutive aspect of signaling was analyzed. For this, endosymbiotic organisms that had one HK or many RRs were identified. These characteristics suggested that lower selective pressure would allow us to find organisms with TCSs that showed lower KH-RR specificity. The systems of Chlamydia trachomatis, Chlamydia psittaci, Simkania negevensis, and Methanobrevibacter sp. Abm4 were analyzed. The phosphotransference reaction was only evident in the the Methanobrevigbacter system. This system presents only one KH and four RRs. This HK shows differentiated efficiency in the phosphotranference. It has a higher speed in RRMet572, RRMet589-1 and it is null in RRMet589-2. On the other hand, the HKs of C. trachomatis y S. negevensis were able to transfer in a nonselective manner the RR468 of T. maritima. This is due to the similarity between the α1 helix of the HKs with HK853. The structural approach of there systems allowed us to obtain the structure of RRMet589-1 y RRMet572, both in a non-phosphorylated state. The two structures presented large conformational differences from Lßα4. This suggests that the recognition mechanisms with KHMet and regulation are different. This supports differentiated selectivity between the RRs in this system.
Mideros Mora, C. (2021). Bases moleculares de la especificidad en el mecanismo de transducción de señal en los sistemas de dos componentes bacterianos [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/161920
TESIS

While high power buck-boost regulators have been extensively researched and developed in the academia and industry, low power counterparts have only recently gained momentum due to the advent of different battery powered and remote electronics. The application life-time of such applications, e.g., remote surveillance electronics can be extended tremendously by enabling energy autonomy. While battery powered electronics last long but they must be replenished once the battery is depleted either by replacing the battery or by retrieving the electronics and then recharging. Instead, energy harvesting from available ambient sources on the spot will enable these electronics continuous operation unboundedly, probably even beyond the lifetime of the electronics. Interestingly enough, recent advancements in micro-scale energy transducers compliment these demand [1-13]. Micro-transducers producing energy from different ambient sources have been reported. These transducers produce enough energy to support a wide range of operations of the remote electronics concurrently. These transducers along with an additional storage elements greatly increase the energy autonomy as well as guaranteed operation since harvested energy can then be stored for future use when harvestable energy is temporarily unavailable. Recently several buck-boost regulators with low power and low input operating voltage have been reported both from academia and industry [14-24]. Some of this work focuses on increasing efficiency in the mid-load range (10mA-100mA), while some other focuses on lowering input range. However, so far no one has reported a buck-boost regulator operating with sub-200nW bias power while harvesting energy from sub-500mV input range. This work focuses on the development of a low voltage low bias current buckboost regulator to attain these goals. In this work, complete design of a PFM mode buck-boost regulator has been discussed in details. Basic topology of the regulator and working principle of the implemented architecture along with the advantages of the specific topology over that of the others have been discussed in short to provide an uninterrupted flow of idea. Later, Transistor level design of the basic building blocks of the buck-boost regulator is discussed in details with different design features and how those are attained through transistor level implementation are discussed. Subsequently, the physical layout design technique and considerations are discussed to inform the reader about the importance of the layout process and to avoid pitfalls of design failure due to layout quality issues. Measurement results are presented with the fabricated IC. Different characterization profile of the IC have been discussed with measured data and capture oscilloscope waveforms. Load regulation, line regulation, efficiency, start-up from low voltage, regulation with line and load transient events are measured, presented and discussed. Different characteristics of the prototype are compared with prior arts and are presented in a comparison table. Die micrograph is also presented along with the different issue of the IC testing

Epstein-Barr virus (EBV) is a human herpes virus that, upon primary infection, establishes life-long persistence in B cells (latency). One viral protein, the immediate early lytic protein Zta (also known as BZLF1, ZEBRA, EB1, and Z) plays a significant role in disturbing this latency and inducing a viral productive (lytic) cycle. Expression of Zta, a basic leucine zipper transcription factor, induces a cascade of viral lytic cycle gene expression. The activation of many lytic genes requires the direct binding of Zta to its response elements (ZREs) within proximal promoters of these genes. Much research in recent years has focused on investigating Zta reactivation of latency and its role in lytic viral DNA replication. This has revealed a wealth of knowledge about Zta as a multifunctional transcription factor. However, a complete understanding of Zta transcriptional activation is still missing. Here, utilising ChIP-qPCR, we showed conserved binding patterns for Zta across several EBV lytic gene promoters in two different EBV systems including a non-B cell EBV-infected cell line. Also, using luciferase reporter assays, we show the first functional evidence for a possible role of Zta in controlling transcription regulation at distal regulatory elements (enhancers). Importantly, we identified BNLF2a, an essential viral immune evasion gene, as a direct target for Zta. We identified five ZREs and mapped functional ones within the BNLF2a promoter using mutational analysis and luciferase reporter assays. We also expressed and purified a recombinant GFP-bZIP Zta protein to address Zta binding to BNLF2a ZREs in vitro. Interestingly, using in silico approach, we also identified a conserved sequence in the ZRE flanking region of all five ZREs within BNLF2a promoter and uncovered a role for a possible repressor at ZRE2 flanking region. Our work not only adds to our understanding of Zta transcription regulation but characterises for the first time the regulation of a novel Zta target that has a role in evading the host immune system during EBV pre-latency and lytic cycle.

In this thesis, design of a car door window regulator is presented. This design comprises a mechanism in order that the car door window makes a specified translational motion. First, conceptual design is carried out to obtain the best suitable concept for the design and best suitable concept comes out to be a scissor mechanism. Afterwards, detailed design of the chosen concept is given. In the detail design stage, kinematic synthesis of the mechanism is performed basically using the Cardan motion. Lastly, implementation of the design on a car door is described.

Abstract Adrenomedullin (AM) is a 52-amino acid peptide which is produced in many tissues, including adrenal medulla, lung, kidney and heart. Intravenous administration of AM causes a long-lasting hypotensive effect, accompanied with an increase in the cardiac output in experimental animals. This study was aimed to examine whether AM has any direct effects on myocardial function. In addition to the myocardial contractility, the effects of AM on coronary vascular tone and A-type natriuretic peptide (ANP) release from atria and B-type natriuretic peptide (BNP) gene expression in the ventricles were studied in the perfused rat heart preparation. In spontaneously beating hearts, AM had no effects on the heart rate, but dose-dependently increased the developed tension (DT) with an EC50 of 7 x 10-11 nmol/l, reflecting a potent positive inotropic effect. The lower the initial resting tension, the higher was the elevation in DT. In paced hearts, a protein kinase A inhibitor, H-89, had no effect on AM-induced inotropic effect, and AM did not increase the cAMP content of the ventricular myocardium. In contrast, the inhibitors of sarcoplasmic reticulum Ca2+ stores, ryanodine and thapsigargin, as well as a protein kinase C inhibitor, staurosporine, significantly attenuated the inotropic response to AM. L-type Ca2+ channel blocker, diltiazem, also suppressed the AM-induced elevation in DT. Moreover, AM increased the duration of myocyte action potentials between 10 mV and - 50 mV in isolated rat atria, consistent with an increase in L-type Ca2+ channel current during the plateau. Inotropic effect of endothelin-1 (ET-1), another locally acting peptide, was enhanced by inhibiting the myocardial nitric oxide (NO) synthesis by Nω-nitro-L-arginine methyl ester (L-NAME) in perfused rat heart. The AM-induced inotropic action was unaltered by L-NAME treatment. When AM and ET-1 were administrated in combined infusion, the inotropic response was significantly smaller than that following the infusion of the peptides alone. This attenuated response was more than overcome by infusion of L-NAME, although the individual responses to AM and ET-1 were not modulated by L-NAME at the doses used in the combination. Consistent with its vasodilator action, AM dose-dependently dilated the coronary arteries of the perfused heart. The effect of AM was not dependent on NO under basal conditions or in coronary arteries constricted with ET-1. Furthermore, AM enhanced the stretch-induced release of ANP from the right atrium, but did not affect the ventricular BNP expression induced by ET-1. In conclusion, AM exerts regulatory actions on the heart by increasing cardiac contractility, dilating coronary arteries and modulating stretch-induced ANP release. The inotropic effect of AM was independent of cyclic AMP, but may involve activation of protein kinase C, Ca2+ influx through L-type Ca2+ channels and the release of Ca2+ from the sarcoplasmic reticulum. Endogenous NO production did not modulate the inotropic effect of AM, although the effect of ET-1 was suppressed. Combined administration of AM and ET-1 produced a weak inotropic response most likely because of a potentiated synthesis of NO. Finally, AM had a coronary vasodilator effect and augmented the stretch-induced ANP release in the right atrium.

Axillary bud growth is inhibited by auxin produced at the shoot apex and transported down the stem. Removal of the shoot apex by decapitation can release buds from inhibition, and application of auxin to the cut stump can restore bud inhibition. However auxin action is likely to be indirect because auxin does not accumulate in inhibited buds, and therefore requires a second messenger. The max4 mutant of Arabidopsis has increased b,ud growth that leads to increased branching in mature plants. The axillary buds of isolated nodes are also partially resistant to exogenous auxin applied to the apical cut stump. The max4 mutation also partially rescues the branch!ng of the axr3-1 auxin over-responding mutant. The auxin resistant phenotype of the max4 mutant appears to be specific to bud growth because max4 seedlings were only slightly resistant to exogenous auxin, and the max4 mutation did not rescue other auxin related phenotypes of the Bxr3-1 mutant. The phenotype and auxin physiology of the max4 mutant is reminiscent of the/amosus pea mutants. The ramosus1 mutant regulates a graft transmissible signal that interacts with auxin to inhibit bud growth. The max4 and ramosus1 mutant phenotypes are caused by mutations in orthologous genes, encoding a member of the polyene dioxygenase family. All of the family members characterised to date function around a carbon-carbon double bond of polyene chain compounds with cyclic carbon end groups. MAX4 is most related to animal polyene dioxygenases that cleave carotenoid substrates. Possibly the MAX4/RMS1 proteins cleave a carotenoid to produce a novel mobile signal that iFlhibits bud growth, and may act as an auxin second messenger.

This thesis focuses on the role of the forkhead box factor Foxg1 in the development of the vertebrate telencephalon. Mice mutant for Foxg1 exhibit multiple telencephalic defects demonstrative of an important pleiotropic role of rFoxg1 in telencephalic organogenesis. Severe hypoplasia of the null mutant telencephalon demonstrates that Foxg1 is required for the regulation of telencephalic growth. Experiments descried in this thesis provide the most systematic study to date of the mechanisms by which Foxg1 regulates this process. Broadly, the results demonstrate that Foxg1 is required to maintain progenitor cell fate, at the expense of differentiated cell fate, and to maintain the proliferation rate of progenitors. In addition to telencephalic growth defects, it has been documented that ventral telencephalic cell fates are lost in Foxg1^-/- embryos. This thesis demonstrates that all ventral telencephalic lineages, which give rise to the basal ganglia and neuronal and glial constituents of the cerebral cortex, are completely absent in Foxg1^-/- embryos. The requirement for Foxg1 to repress ectopic dorsal genes and to activate ventral genes is consistent with a role in regulating the response to ventralising signals. The hypothesis that Foxg1 is required for telencephalic cells to respond appropriately to the ventralising morphogen Shh is advanced. Evidence is provided for a role of Foxg1 in the regulation of the expression and activity of Gli3, the major antagonist of the hedgehog signalling pathway. In Foxg1^-/-; Gli3^-/- telencephalon, some aspects of ventral telencephalic fate are recovered. This demonstrates that Foxg1 is required for ventral fate specification, in part through the antagonism of Gli3 action, consistent with the original hypothesis. However, markers of ventro-medial telencephalic fate are not recovered and ectopic expression of dorsal markers persists in double mutants. From these findings it is argued that Foxg1 has hedgehog-independent roles in dorso-ventral patterning of the telencephalon.

CELF1 is an RNA binding protein with regulatory roles in translation, alternative splicing and mRNA degradation. This protein is of particular interest as its upregulation is believed to be involved in the pathogenesis of type 1 myotonic dystrophy. CELF1 functions by binding to a specific sequence in the 3’ untranslated region of its target mRNAs. This sequence has been termed the “EDEN” motif, but the exact requirements for binding of CELF1 were not well defined. In this study we therefore aimed to determine the sequence requirements for an RNA substrate to form a high affinity interaction with CELF1, and characterise the structure of the resulting complex. The CELF1 protein is composed of three structured RNA recognition motifs separated by flexible linkers. Our strategy was to investigate the RNA binding properties of each domain in isolation, and then the requirements for tandem binding of the domains in order to build up the complete “EDEN” motif capable of forming a high affinity complex with the wild type protein. This has been accomplished using NMR spectroscopy to map the chemical shift perturbations in each domain on binding to a range of RNA substrates. ITC was also used to investigate the binding affinities of each domain, and the enhancement of affinity when domains bind in tandem. By these methods we have refined the sequence requirements for simultaneous binding of all domains of CELF1, and designed RNA substrates which will bind with higher affinity than any previously reported. We have also shown the potential involvement of RNA secondary structure in forming the CELF1 binding site, and identified two possible examples of this in natural mRNA targets. CELF1 binding triggers deadenylation of its target mRNAs and this is suspected to be via a mechanism involving recruitment of poly (A) ribonuclease. These two proteins have been shown to interact, but no structural information was available to show which domains were interacting, or whether CELF1 was capable of forming a ternary complex with both RNA and poly(A) ribonuclease. Since the ribonuclease exists as a 146 kDa dimer, the complex of it with CELF1 was an ambitious target for NMR. In this study we demonstrate that high resolution NMR data can be acquired on this key regulatory complex. Using this we go on to confirm the interaction between these two proteins, and that the domains involved in binding suggest a ternary complex is possible.

Introduction Globesity, the worldwide obesity epidemic, represents a major threat and public health burden. An uncontrolled expansion of the adipose tissue followed by a chronic low-grade inflammation leads to the dysfunction of the adipose organ resulting in obesity and its associated metabolic complications. Uncovering the mechanisms of adipogenesis, the development of adipocytes, therefore strikes as a key strategy in combating the disease. MicroRNAs (miRs), a class of small non-coding RNAs, have emerged in recent years as crucial modulators of diverse biological processes such as cell proliferation, differentiation, and signal transduction emphasizing their large potential as targets. Numerous miRs have been associated with the adipose tissue and metabolism and their dysregulation has repeatedly been linked to diseases including diabetes and obesity. This study aimed to investigate the role of miR-34a, an obesity-related miR, in the regulation of pre-adipocyte differentiation. Materials and Methods Mouse 3T3-L1 pre-adipocytes were employed as an in vitro system to study adipogenesis. Oil Red O staining served to evaluate the degree of adipogenesis and the over-expression of miR-34a in adipocytes was achieved by a lentiviral system. MiR and messenger RNA (mRNA) levels were analysed using TaqMan and SYBR Green-based quantitative real time PCR (qPCR) respectively. Results The expression of miR-34a was substantially down-regulated upon treatment of differentiation medium for two days and remained significantly low during the differentiation period compared with undifferentiated pre-adipocytes. Lentivirus-mediated over-expression of miR-34a successfully up-regulated miR-34a. Higher levels of miR-34a in turn mitigated adipogenesis as evidenced by blunted Oil Red O staining. This observation was found to be in good agreement with the qPCR analysis, which showed a down-regulation of several key adipogenic markers. Conclusion The down-regulation of miR-34a is required during pre-adipocyte differentiation for the efficient proceedings of the adipogenic programme. Further investigation is needed to evaluate the potential therapeutic implication of miR-34a-based treatment in managing obesity.
published_or_final_version
Medicine
Master
Master of Medical Sciences