Abstract Acinetobacter baumannii is a prevalent nosocomial where infections are typically secondary infections to patients that already have an infection or other source of being immunocompromised. Like many other infectious bacteria, A. baumannii is increasingly considered a multi-drug resistant pathogen. This eliminates the ability to treat A. baumannii infections with traditional antibiotics, hence the need for another method of treating A. baumannii. This research study was designed to find a way to affect the survival of A. baumannii such that it can be applied to a hospital setting to prevent further infections to immunocompromised patients. One mechanism potentially used by A. baumannii to persist on hospital surfaces is through the use of the bacterial second messenger cyclic-di-GMP (c-di-GMP). This nucleotide signal is regulated in response to environmental conditions, and then activates c-di-GMP-binding proteins that induce phenotypic changes. I hypothesized that by deleting these c-di-GMP-binding proteins that it will produce measurable differences in phenotype like biofilm formation, motility, and desiccation survival. Reducing phenotypes such as these may alter A. baumannii’s ability to persist on hospital surfaces, and potentially lead to future surface eradication. A. baumannii encodes two potential c-di-GMP-binding proteins of particular interest, one that contains a sole PilZ domain and another that pairs a PilZ domain with a hydrolase domain. PilZ domains bind c-di-GMP within a conserved binding site, regulating the conformational structure of the protein, and are named for the first studied PilZ domain within the pilus-associated PilZ protein. Pili are used in pilus-mediated motility and surface attachment, and they are A. baumannii’s primary method of motility due to not having flagellum. I hypothesized that by removing these c-di-GMP-binding proteins, I would interrupt the c-di-GMP signaling that might regulate motility. I am testing two A. baumannii strains: 5075, a recent military hospital isolate and 17978, an older lab strain. A notable difference between these two strains is that 5075 demonstrates twitching motility where it utilizes type IV pili, but 17978 demonstrates swarming motility that has unknown mechanisms. Both c-di-GMP-binding proteins were tested for their role in twitching or swarming motility of the respective strains. I found that swarming motility of 17978 is regulated by both c-di-GMP-binding proteins. While I am still generating the deletion strain for the c-di-GMP-binding hydrolase enzyme, the sole PilZ domain protein is also required for twitching motility in the 5075 strain. These results suggest c-di-GMP regulates both forms of motility in A. baumannii. Future plans include determining the role of the c-di-GMP-binding hydrolase enzyme in twitching motility and identifying the role that these proteins play through binding of c-di-GMP.

Acinetobacter baumannii is an increasingly multidrug-resistant Gram-negative bacterial pathogen and contributes to many hospital-acquired infections. Discovering new treatments against Acinetobacter baumannii infections is necessary as the pathogen adapts to the antimicrobials prescribed by physicians. Cyclic di-GMP (c-di-GMP), a bacterial second messenger, can regulate various phenotypes including biofilm formation, desiccation tolerance, motility, etc.; many of these phenotypes may help A. baumannii better survive a hospital environment, such as dryness on hospital surfaces. Up to twelve c-di-GMP modulating enzymes (CMEs) and two c-di-GMP binding proteins are predicted to be encoded by this pathogen. Diguanylate cyclases (DGCs) produce c-di-GMP, whereas phosphodiesterases (PDEs) degrade c-di-GMP. More c-di-GMP that can bind to its binding proteins means more biofilm formation and less motility. Of the eleven CMEs, 7 are DGCs, 2 are PDEs, and 3 encode both domains (DGCs/PDEs). I hypothesized that biofilm formation, desiccation tolerance, and motility were controlled by c-di-GMP and that we could target these parts of the c-di-GMP signaling network for new treatments. If we disrupt these genes, then we should see a reduction in the regulatory effects of these phenotypes. In this investigation, we generated mutants with a single gene knockout or transposon mutagenesis in two different A. baumannii strains: 17978, a historical laboratory strain that exhibits swarming motility and AB5075, a recent clinical isolate that exhibits twitching motility. To test biofilm formation, we let the mutants grow to their maximum concentration in 96-well plates, stained the plates with crystal violet, and quantified the crystal violet that stained the biofilm. To test for motility, a LB agar plate was stabbed to the plastic surface or dropped on the agar surface with diluted culture to determine the presence of twitching or swarming motility, respectively. To test for desiccation tolerance, we washed the cultures in distilled water to rid the sample of any salt, serially diluted the samples in solution, and plated them out onto LB agar plates. Bacterial counts were quantified before and after desiccation to determine survival of each mutant. From these experiments, 6 DGCs, 1 PDE, and 2 DGCs/PDEs were shown to regulate biofilm formation in AB5075. Furthermore, a PDE and a DGC/PDE were shown to regulate twitching motility in AB5075, while a single DGC was required for tolerating dryness. In strain 17978, we have found a PDE and 4 DGCs that are necessary for swarming motility and are currently conducting biofilm and desiccation tolerance assays. So far, we’ve identified a role for c-di-GMP in A. baumannii biofilm formation, motility, and desiccation survival. Inhibiting the regulation of these pathways could produce novel mechanisms to combat this pathogen in the hospital environment.

Gärningsmannaprofilering (GMP) är en polisiär metod som används vid svårlösta utredningar. I Sverige praktiseras metoden av en central grupp vid Rikskriminalpolisen, den såkallade GMP-gruppen. Studiens övergripande syfte är att förstå och förklara GMP som polisiär metod, samt undersöka metodens effektivitet, korrekthet och vetenskaplighet. Ett särskilt syfte är att testa inlärningsteorin och rutinaktivitetsteorin gentemot GMP-metoden för diskutera eventuella samband. Studien är kvalitativt orienterad och bygger på den hermeneutiska teorin om tolkning av källor. Studien bygger på en omfattande systematisk litteraturstudie i kombination med två intervjuer. Sammanfattningsvis kan man säga att de slutsatser som analysen har är att GMP är en metod som i många fall tycks vara effektiv och som ofta genererar korrekta gärningsmannaprofiler. Dock behövs det mer kunskap om GMPs vetenskapliga förankring. Det har även visat sig att det finns ett samband mellan GMP och både rutinaktvitetsteorin och inlärningsteorin. Sambandet med inlärningsteorin är dock marginellt.

Cyclic di-GMP is a second messenger used by bacteria to regulate motility, extracellular polysaccharide production, and the cell cycle. Recent advances in the measurement of real time cyclic di-GMP levels in single cells have uncovered significant dynamic heterogeneity of second messenger concentrations within bacterial populations. This heterogeneity results in a wide range of phenotypic outcomes within a single population, providing the potential for population survival and adaptability in response to rapidly changing environments. In this chapter, we discuss some of the measurement technologies available for single-cell measurement of cyclic di-GMP concentrations, the resulting discovery of heterogeneous cyclic di-GMP populations, the mechanisms bacteria use to generate this heterogeneity, and the biochemical and functional consequences of heterogeneity on cyclic di-GMP effector binding and the bacterial population.

O diguanilato cíclico (c-di-GMP) é uma molécula de sinalização intracelular que atua na regulação de importantes processos bacterianos como motilidade, formação de biofilme e virulência. As diguanilato ciclases (DGCs), contendo um domínio GGDEF ativo, catalisam a formação de c-di-GMP a partir de duas moléculas de GTP. A bactéria Xanthomonas citri subsp. citri (Xanthomonas axonopodis pv citri; Xac) é o agente causal do cancro cítrico, uma doença que ataca todas as variedades e espécies de citros. O genoma de Xac codifica 31 proteínas contendo domínios GGDEF. Treze destas proteínas possuem também domínios PAS e/ou GAF, que são ubíquos domínios sensores e de sinalização. Para tentar entender melhor o papel na sinalização por c-di-GMP das interações entre domínios GGDEF e domínios PAS e/ou GAF, estudos bioquímicos e funcionais foram realizados com as proteínas XAC0610 e XAC2446. XAC0610 contém um domínio GAF, quatro domínios PAS e um domínio GGDEF conservado. Análises fenotípicas com a linhagem nocaute XacΔ0610 mostraram que XAC0610 atua na regulação da motilidade e sobrevivência de Xac ao tratamento com H2O2. Ensaios de atividade enzimática demonstraram que XAC0610 é uma DGC cataliticamente ativa, e que a mutação sítio-dirigida de um resíduo conservado de lisina (Lys759) provoca uma grande redução na atividade de DGC. Os domínios GAF e PAS de XAC0610 aparentemente não atuam como domínios sensores, entretanto são importantes para a dimerização da proteína, necessária para a obtenção de altos níveis de atividade de DGC. Além disso, várias observações sugerem que XAC0610 não é submetida à inibição alostérica pelo produto, um mecanismo regulatório comumente utilizado para o controle da atividade de DGC. Por outro lado, os dados de cinética enzimática de XAC0610HIS-35-880 revelaram um efeito de cooperatividade positiva para a ligação dos substratos, com uma constante de dissociação para a ligação da primeira molécula de GTP (K1) cerca de 3-5 vezes maior que a constante de dissociação para a ligação da segunda molécula de GTP (K2). A partir deste estudo, nós apresentamos um esquema cinético geral mais apropriado para as análises dos dados cinéticos de enzimas DGCs e propomos que a ligação cooperativa do substrato talvez possa desempenhar um importante papel na regulação in vivo da atividade de algumas DGCs, aumentando sua sensibilidade a pequenas variações nos níveis celulares de GTP. Outra proteína caracterizada neste trabalho, XAC2446 possui um domínio GAF e um domínio GGDEF que, ao contrário do domínio GGDEF de XAC0610, não deve apresentar atividade de DGC. Mesmo assim, análises funcionais mostraram que XAC2446 regula negativamente a formação de biofilme e positivamente a motilidade de Xac. Ensaios de duplo híbrido em leveduras identificaram que XAC2446 interage com XAC2897, contendo um domínio GGDEF potencialmente ativo, e XAC1185, contendo um domínio HD fosfohidrolase de (p)ppGpp. Alguns estudos indicam que altos níveis celulares de c-di-GMP e baixos níveis de (p)ppGpp podem ser necessários durante a formação de biofilme. XAC2446 talvez possa atuar como um inibidor da atividade enzimática de XAC2897 e XAC1185 e influenciar, indiretamente e antagonicamente, tanto os níveis celulares de c-di-GMP quanto de (p)ppGpp. / Cyclic di-GMP is a bacterial second messenger that regulates a range of functions, including cellular motility, biofilm formation and virulence. This molecule is produced from two GTP substrates by the activity of diguanylate cyclases (DGCs) containing a GGDEF domain. The phytopathogenic bacteria Xanthomonas citri subsp. citri (Xanthomonas axonopodis pv citri; Xac) causes citrus canker in a wide variety of citrus species. The Xac genome codes for 31 proteins with GGDEF domains. Thirteen of the 31 Xac GGDEF domain-containing proteins also possess PAS (Per-Arnt-Sim) or GAF (cGMP-specific phosphodiesterases, adenylyl cyclases and FhlA) domains that are ubiquitous signaling and sensory domains. In order to better understand the relationship between these commonly associated domains, biochemical and functional studies were carried out with the XAC0610 and XAC2446 proteins. XAC0610 is a large multi-domain protein containing one GAF domain, four PAS domains and one GGDEF domain. This protein has a demonstrable in vivo and in vitro diguanylate cyclase (DGC) activity. Analysis of a XacΔ0610 knockout strain revealed that XAC0610 plays a role in the regulation of Xac motility and resistance to H2O2. Site-directed mutagenesis of a conserved DGC lysine residue (Lys759 in XAC0610) resulted in a severe reduction in XAC0610 DGC activity. XAC0610 DGC activity was also impaired by removal of the N-terminal GAF and PAS domains, which are probably needed for proper protein dimerization. Furthermore, experimental and in silico analysis suggest that XAC0610 is not subject to allosteric product inhibition, a common regulatory mechanism for DGC activity control. Instead, steady-state kinetics of XAC0610 DGC activity revealed a positive cooperative effect of the GTP substrate with a dissociation constant for the binding of the first GTP molecule (K1) approximately three to five times greater than the dissociation constant for the binding of the second GTP molecule (K2). We present a general kinetics scheme that should be used when analyzing DGC kinetics data and propose that cooperative GTP binding could be a common, though up to now overlooked, feature of these enzymes that may in some cases offer a physiologically relevant mechanism for regulation of DGC activity in vivo. The other characterized protein, XAC2446, has a GAF domain and a degenerated GGDEF domain. Unlike XAC0610, XAC2446 should not present DGC activity. Nevertheless, functional analysis of XAC2446 demonstrated that it plays a role in the regulation of Xac motility and biofilm formation. A yeast two-hybrid screen identifies XAC2897 (a potentially active GGDEF domain-containing protein) and XAC1185 (a (p)ppGpp hydrolase) as specific binding partners of the XAC2446 protein. As indicated by studies in other bacteria, high cellular levels of c-di-GMP and low levels of (p)ppGpp may be both required for biofilm formation. It is possible that XAC2446 might have a role in the antagonistic regulation of c-di-GMP and (p)ppGpp cellular levels by acting as an inhibitor of both XAC2897 and XAC1185 enzymatic activities.

O diguanilato cíclico (c-di-GMP) é uma molécula de sinalização intracelular que atua na regulação de importantes processos bacterianos como motilidade, formação de biofilme e virulência. As diguanilato ciclases (DGCs), contendo um domínio GGDEF ativo, catalisam a formação de c-di-GMP a partir de duas moléculas de GTP. A bactéria Xanthomonas citri subsp. citri (Xanthomonas axonopodis pv citri; Xac) é o agente causal do cancro cítrico, uma doença que ataca todas as variedades e espécies de citros. O genoma de Xac codifica 31 proteínas contendo domínios GGDEF. Treze destas proteínas possuem também domínios PAS e/ou GAF, que são ubíquos domínios sensores e de sinalização. Para tentar entender melhor o papel na sinalização por c-di-GMP das interações entre domínios GGDEF e domínios PAS e/ou GAF, estudos bioquímicos e funcionais foram realizados com as proteínas XAC0610 e XAC2446. XAC0610 contém um domínio GAF, quatro domínios PAS e um domínio GGDEF conservado. Análises fenotípicas com a linhagem nocaute XacΔ0610 mostraram que XAC0610 atua na regulação da motilidade e sobrevivência de Xac ao tratamento com H2O2. Ensaios de atividade enzimática demonstraram que XAC0610 é uma DGC cataliticamente ativa, e que a mutação sítio-dirigida de um resíduo conservado de lisina (Lys759) provoca uma grande redução na atividade de DGC. Os domínios GAF e PAS de XAC0610 aparentemente não atuam como domínios sensores, entretanto são importantes para a dimerização da proteína, necessária para a obtenção de altos níveis de atividade de DGC. Além disso, várias observações sugerem que XAC0610 não é submetida à inibição alostérica pelo produto, um mecanismo regulatório comumente utilizado para o controle da atividade de DGC. Por outro lado, os dados de cinética enzimática de XAC0610HIS-35-880 revelaram um efeito de cooperatividade positiva para a ligação dos substratos, com uma constante de dissociação para a ligação da primeira molécula de GTP (K1) cerca de 3-5 vezes maior que a constante de dissociação para a ligação da segunda molécula de GTP (K2). A partir deste estudo, nós apresentamos um esquema cinético geral mais apropriado para as análises dos dados cinéticos de enzimas DGCs e propomos que a ligação cooperativa do substrato talvez possa desempenhar um importante papel na regulação in vivo da atividade de algumas DGCs, aumentando sua sensibilidade a pequenas variações nos níveis celulares de GTP. Outra proteína caracterizada neste trabalho, XAC2446 possui um domínio GAF e um domínio GGDEF que, ao contrário do domínio GGDEF de XAC0610, não deve apresentar atividade de DGC. Mesmo assim, análises funcionais mostraram que XAC2446 regula negativamente a formação de biofilme e positivamente a motilidade de Xac. Ensaios de duplo híbrido em leveduras identificaram que XAC2446 interage com XAC2897, contendo um domínio GGDEF potencialmente ativo, e XAC1185, contendo um domínio HD fosfohidrolase de (p)ppGpp. Alguns estudos indicam que altos níveis celulares de c-di-GMP e baixos níveis de (p)ppGpp podem ser necessários durante a formação de biofilme. XAC2446 talvez possa atuar como um inibidor da atividade enzimática de XAC2897 e XAC1185 e influenciar, indiretamente e antagonicamente, tanto os níveis celulares de c-di-GMP quanto de (p)ppGpp. / Cyclic di-GMP is a bacterial second messenger that regulates a range of functions, including cellular motility, biofilm formation and virulence. This molecule is produced from two GTP substrates by the activity of diguanylate cyclases (DGCs) containing a GGDEF domain. The phytopathogenic bacteria Xanthomonas citri subsp. citri (Xanthomonas axonopodis pv citri; Xac) causes citrus canker in a wide variety of citrus species. The Xac genome codes for 31 proteins with GGDEF domains. Thirteen of the 31 Xac GGDEF domain-containing proteins also possess PAS (Per-Arnt-Sim) or GAF (cGMP-specific phosphodiesterases, adenylyl cyclases and FhlA) domains that are ubiquitous signaling and sensory domains. In order to better understand the relationship between these commonly associated domains, biochemical and functional studies were carried out with the XAC0610 and XAC2446 proteins. XAC0610 is a large multi-domain protein containing one GAF domain, four PAS domains and one GGDEF domain. This protein has a demonstrable in vivo and in vitro diguanylate cyclase (DGC) activity. Analysis of a XacΔ0610 knockout strain revealed that XAC0610 plays a role in the regulation of Xac motility and resistance to H2O2. Site-directed mutagenesis of a conserved DGC lysine residue (Lys759 in XAC0610) resulted in a severe reduction in XAC0610 DGC activity. XAC0610 DGC activity was also impaired by removal of the N-terminal GAF and PAS domains, which are probably needed for proper protein dimerization. Furthermore, experimental and in silico analysis suggest that XAC0610 is not subject to allosteric product inhibition, a common regulatory mechanism for DGC activity control. Instead, steady-state kinetics of XAC0610 DGC activity revealed a positive cooperative effect of the GTP substrate with a dissociation constant for the binding of the first GTP molecule (K1) approximately three to five times greater than the dissociation constant for the binding of the second GTP molecule (K2). We present a general kinetics scheme that should be used when analyzing DGC kinetics data and propose that cooperative GTP binding could be a common, though up to now overlooked, feature of these enzymes that may in some cases offer a physiologically relevant mechanism for regulation of DGC activity in vivo. The other characterized protein, XAC2446, has a GAF domain and a degenerated GGDEF domain. Unlike XAC0610, XAC2446 should not present DGC activity. Nevertheless, functional analysis of XAC2446 demonstrated that it plays a role in the regulation of Xac motility and biofilm formation. A yeast two-hybrid screen identifies XAC2897 (a potentially active GGDEF domain-containing protein) and XAC1185 (a (p)ppGpp hydrolase) as specific binding partners of the XAC2446 protein. As indicated by studies in other bacteria, high cellular levels of c-di-GMP and low levels of (p)ppGpp may be both required for biofilm formation. It is possible that XAC2446 might have a role in the antagonistic regulation of c-di-GMP and (p)ppGpp cellular levels by acting as an inhibitor of both XAC2897 and XAC1185 enzymatic activities.

Orientador: Edson Antunes / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T23:29:22Z (GMT). No. of bitstreams: 1 Monteiro_PriscilaFukumura_M.pdf: 903494 bytes, checksum: 9d17a9657d72fedec2f6efc40cc3b082 (MD5) Previous issue date: 2013 / Resumo: As plaquetas desempenham uma função fisiológica importante no sistema hemostático, em resposta a lesão vascular através da prevenção da hemorragia. A adesão ou agregação plaquetária são eficazes na contribuição sinérgica de várias interações de múltiplos receptores, que transmitem sinais de ativação que iniciam uma série de respostas bioquímicas e morfológicas, associadas à remodelação do citoesqueleto, a secreção granular e a geração e liberação de agonistas endógenos solúveis, tais como ADP e tromboxano A2 (TXA2). O NO derivado da célula endotelial exerce um efeito inibitório na função da plaquetaria através da ativação de cGMP / PKG, a qual, por sua vez leva a uma redução na concentração de Ca2 + prevenindo assim a adesão e agregação de plaquetas à parede vascular. No entanto, a disfunção endotelial, presente em certas condições patológicas é caracterizada por uma diminuição da biodisponibilidade de NO que leva a ativação anormal das plaquetas conduzindo a trombose vascular À disfunção plaquetária é considerada uma fase final de complicações cardiovasculares no diabetes mellitus tipo II, obesidade, aterosclerose, levando ao resultado clínico, tais como enfarte do miocárdio, acidente vascular cerebral e doença arterial periférica. A obesidade é um importante problema de saúde pública, atingindo todas as idades e grupos socioeconômicos elevando a incidência de doenças cardiovasculares e endócrino-metabólica. Um estado crônico de stress oxidativo e inflamação são a marcados pela adiposidade que desempenha um papel crucial nos eventos fisiopatológicos desta desordem. Estes efeitos pró-inflamatórios e pró-oxidante estão associados com o aumento de ERO com diminuição da biodisponibilidade, o que aumenta o risco de eventos trombóticos aterosclerose. No entanto, os mecanismos pelos quais a adiposidade induz disfunção plaquetária são pouco esclarecidos. Além disso, a maioria dos eventos cardiovasculares fatais como consequência de complicação trombótica não estão associadas à estenose vascular completa, mas sim com as alterações de biomarcadores pró-inflamatórios e pró-oxidantes, o que pode prever futuros eventos cardiovasculares. Nossa hipótese é que a produção de ERO intraplaquetário causada pela adiposidade contribui para eventos trombóticos e distúrbios endocrinometabólico. Assim, investigou-se a reatividade plaquetária ex-vivo em resposta ao ADP e trombina, em ratos alimentados com dieta hiperlipídica, e o envolvimento de ERO e via do NO-cGMP na modulação da reatividade de plaquetária / Abstract: Platelets play an important physiological function in haemostasis system in response to vascular injury by preventing hemorrhage. Effective platelet adhesion and aggregation require the synergistic contribution of multiple receptor-ligand interactions that transmit activating signals initiating a range of platelet biochemical and morphological responses, linked to cytoskeleton remodeling, granule secretion and the generation and release of endogenous soluble agonists, such as ADP and thromboxane A2 (TXA2). Endothelial cell-derived nitric oxide (NO) exerts an inhibitory effect in the platelet function by activation of cGMP/PKG pathway, which in turn leads to reduction in concentration of Ca2+, thus preventing adhesion and aggregation of platelets to the vascular wall. Nonetheless, endothelium dysfunction, present in certain pathological conditions is characterized by a decreased NO bioavailability which incites abnormal platelet activation leading to vascular thrombosis. Platelet dysfunction is considered an end stage of cardiovascular complications in type II diabetes mellitus, obesity and atherosclerosis that results in clinical outcomes such as myocardial infarction, stroke and peripheral artery disease. Obesity is an important public health problem affecting all ages and socioeconomic groups greatly elevating the incidence of cardiovascular and endocrine-metabolic disorders. A chronic state of oxidative stress and inflammation are the hallmark of adiposity that plays a pivotal role in the physiopathological events in this disorder. These proinflammatory and pro-oxidant effects are associated with increased reactive-oxygen species (ROS) production and decreased NO bioavailability, which increases the risk of athero thrombotic events. Nonetheless, the exact mechanisms by which adiposity induces platelet dysfunction remain poorly investigated. In addition, most of fatal cardiovascular events as consequence of thrombotic complication are not associated with complete vascular stenosis, but rather with alterations of pro-inflammatory and pro-oxidant biomarkers, which can predict future cardiovascular events. We hypothesized that intraplatelet ROS production in adiposity contributes to thrombotic events in endocrinemetabolic disorders. Therefore, we have investigated the ex-vivo platelet reactivity in response to ADP and thrombin in high fat-fed rats, and the involvement of platelet-derived ROS and NO-cGMP pathway in modulating the platelet reactivity / Mestrado / Farmacologia / Mestra em Farmacologia

Des conditions de carence entrainent une mort cellulaire développementale chez le protiste Dictyostelium discoideum. Dans un système in vitro, des cellules de Dictyostelium sont mises en conditions de carence, puis l'addition des inducteurs DIF-1 ou c-di-GMP conduit à une mort cellulaire vacuolaire. DIF-1 est un polyketide produit par Dictyostelium et induisant la différenciation des cellules pré-tiges. Le dinucléotide cyclique c-di-GMP était connu comme un second messager chez les procaryotes, et comme un déclencheur de l'immunité innée dans des cellules de mammifères. Il a été montré par d'autres que des cellules de Dictyostelium peurent produire et détecter c-di-GMP.Pour analyser la signalisation par c-di-GMP chez Dictyostelium, nous avons utilisé la mutagénèse aléatoire et la mutagénèse ciblée. En utilisant des mutants inactivant stlB ou dmtA, nous avons démontré que DIF-1 endogène ou exogène est nécessaire pour la signalisation par c-di-GMP dans Dictyostelium. En conséquence, nous avons amélioré l'étape de sélection dans une mutagenèse aléatoire en utilisant c-di-GMP et un peu de DIF-1 comme inducteurs, ce qui a produit plusieurs mutants. Par ailleurs j’ai testé par mutagenèse ciblée des hypothèses basées sur les informations connues dans Dictyostelium ou d'autres types de mort cellulaire. Trois molécules ont été essayées, DDX41 comme récepteur putatif de c-di-GMP, l' uniport mitochondrial pour le Ca2+(MCU) et la Na+/K+ATPase (IonA).En résumé, au cours de ma thèse, nous avons démontré une relation entre la signalisation c-di-GMP et a signalisation DIF-1 dans Dictyostelium et identifié plusieurs nouvelles molécules de la mort cellulaire par mutagenèse aléatoire. / The protist Dictyostelium discoideum undergoes development cell death when under starvation. To investigate the molecular mechanism of Dictyostelium cell death, an in vitro system has been used. Dictyostelium cells were starved and then cell death was induced by DIF-1 or c-di-GMP. About 40h after induction, cells underwent vacuolar cell death. DIF-1 is a polyketide, produced by Dictyostelium prespore cells, which induces prestalk cell differentiation. c-di-GMP was well known not only as a second messenger produced and sensed by bacteria but also as a trigger of innate immunity in mammalian cells. Dictyostelium was recently found by another laboratory to produce and sense c-di-GMP. To analyze c-di-GMP signaling in Dictyostelium cell death, we used random mutagenesis and targeted mutagenesis. By using the knockout mutants stlB- and dmtA-, we demonstrated that endogenous or exogenous DIF-1 is required for c-di-GMP signaling in Dictyostelium. In contrast, endogenous c-di-GMP is not necessary for exogenous DIF-1 signaling. As a consequence, we improved the selection step in random mutagenesis by using c-di-GMP and a little DIF-1 as inducers, which produced several mutants. Another part of my project was to test by targeted mutagenesis some hypotheses, based on known information in Dictyostelium or other similar cell death types. Three molecules have been tested, the c-di-GMP putative receptor DDX41, the mitochondrial Ca2+ uniporter (MCU) and the Na+/K+-ATPase (IonA).In summary, during my thesis, we have demonstrated a relation between c-di-GMP signaling and DIF-1 signaling in Dictyostelium and identified several new cell death molecules by random mutagenesis.

T cells modified for expression of Chimeric Antigen Receptors (CARs) were the first genemodified cell products approved for use in cancer immunotherapy. CAR-T cells engineered with gammaretroviral or lentiviral vectors (RVs/LVs) targeting B-cell lymphomas and leukemias have shown excellent clinical efficacy and no malignant transformation due to insertional mutagenesis to date. Large-scale production of RVs/ LVs under good-manufacturing practices for CAR-T cell manufacturing has soared in recent years. However, manufacturing of RVs/LVs remains complex and costly, representing a logistical bottleneck for CAR-T cell production. Emerging gene-editing technologies are fostering a new paradigm in synthetic biology for the engineering and production of CAR-T cells. Firstly, the generation of the modular reagents utilized for gene editing with the CRISPR-Cas systems can be scaled-up with high precision under good manufacturing practices, are interchangeable and can be more sustainable in the long-run through the lower material costs. Secondly, gene editing exploits the precise insertion of CARs into defined genomic loci and allows combinatorial gene knock-ins and knock-outs with exciting and dynamic perspectives for T cell engineering to improve their therapeutic efficacy. Thirdly, allogeneic edited CAR-effector cells could eventually become available as “off-the-shelf” products. This review addresses important points to consider regarding the status quo, pending needs and perspectives for the forthright evolution from the viral towards gene editing developments for CAR-T cells.

Evaluating aerodynamic noise from aircraft engines is a design stage process, so that it conform to regulations at airports. Aerodynamic noise is also a principal source of structural vibration and internal noise in short/vertical take off and landing and rocket launches. Acoustic loads may be critical for the proper functioning of electronic and mechanical components. It is imperative to have tools with capability to predict noise generation from turbulent flows. Understanding the mechanism of noise generation is essential in identifying methods for noise reduction. Lighthill (1952) and Lighthill (1954) provided the first explanation for the mechanism of aerodynamic noise generation and a procedure to estimate the radiated sound field. Many such procedures, known as acoustic analogies are used for estimating the radiated sound field in terms of the turbulent fluid flow properties. In these methods, the governing equations of the fluid flow are rearranged into two parts, the acoustic sources and the propagation terms. The noise source terms and propagation terms are different in different approaches. A good description of the turbulent flow field and the noise sources is required to understand the mechanism of noise generation. Computational aeroacoustics (CAA) tools are used to calculate the radiated far field noise. The inputs to the CAA tools are results from CFD simulations which provide details of the turbulent flow field and noise sources. Reynolds-Averaged Navier Stokes (RANS) solutions can be used as inputs to CAA tools which require only time-averaged mean quantities. The output of such tools will also be mean quantities. While complete unsteady turbulent flow details can be obtained from Direct Numerical Simulation (DNS), the computation is limited to low or moderate Reynolds number flows. Large eddy simulations (LES) provide accurate description for the dynamics of a range of large scales. Most of the kinetic energy in a turbulent flow is accounted by the large-scale structures. It is also the large-scale structures which accounts for the maximum contribution towards the radiated sound field. The results from LES can be used as an input to a suitable CAA tool to calculate the sound field. Numerical prediction of turbulent flow field, the acoustic sources and the radiated sound field is at the focus of this study. LES based on explicit filtering method is used for the simulations. The method uses a low-pass compact filter to account for the sub-grid scale effects. A one-parameter fourth-order compact filter scheme from Lele (1992) is used for this purpose. LES has been carried out for four different flow situations: (i) round jet (ii) plane jet (iii) impinging round jet and (iv) impinging plane jet. LES has been used to calculate the unsteady flow evolution of these cases and the Lighthill’s acoustic sources. A compact difference scheme proposed by Hixon & Turkel (1998) which involves only bi-diagonal matrices are used for evaluating spatial derivatives. The scheme provides similar spectral resolution as standard tridiagonal compact schemes for the first spatial derivatives. The scheme is computationally less intensive as it involves only bi-diagonal matrices. Also, the scheme employs only a two-point stencil. To calculate the radiated sound field, the Helmholtz equation is solved using the Green’s function approach, in the form of the Kirchhoff-Helmholtz integral. The integral is performed over a surface which is present entirely in the linear region and covers the volume where acoustic sources are present. The time series data of pressure and the normal component of the pressure gradient on the surface are obtained from the CFD results. The Fourier transforms of the time series of pressure and pressure gradient are then calculated and are used as input for the Kirchhoff-Helmholtz integral. The flow evolution for free jets is characterised by the growth of the instability waves in the shear layer which then rolls up into large vortices. These large vortical structures then break down into smaller ones in a cascade which are convected downstream with the flow. The rms values of the Lighthill’s acoustic sources showed that the sources are located mainly at regions immediately downstream of jet break down. This corresponds to the large scale structures at break down. The radiated sound field from free jets contains two components of noise from the large scales and from the small scales. The large structures are the dominant source for the radiated sound field. The contribution from the large structures is directional, mainly at small angles to the downstream direction. To account for the difference in jet core length, the far field SPL are calculated at points suitably shifted based on the jet core length. The peak value for the radiated sound field occurs between 30°and 35°as reported in literature. Convection of acoustic sources causes the radiated sound field to be altered due to Doppler effect. Lighthills sources along the shear layer were examined in the form of (x, t) plots and phase velocity pattern in (ω, k) plots to analyse for their convective speeds. These revealed that there is no unique convective speeds for the acoustic sources. The median convective velocity Uc of the acoustic sources in the shear layer is proportional to the jet velocity Uj at the center of the nozzle as Uc ≈ 0.6Uj. Simulations of the round jet at Mach number 0.9 were used for validating the LES approach. Five different cases of the round jet were used to understand the effect of Reynolds number and inflow perturbation on the flow, acoustic sources and the radiated sound field. Simulations were carried out for an Euler and LES at Reynolds number 3600 and 88000 at two different inflow perturbations. The LES results for the mean flow field, turbulence profiles and SPL directivity were compared with DNS of Freund (2001) and experimental data available in literature. The LES results showed that an increase in inflow forcing and higher Reynolds number caused the jet core length to reduce. The turbulent energy spectra showed that the energy content in smaller scale is higher for higher Reynolds number. LES of plane jets were carried out for two different cases, one with a co-flow and one without co-flow. LES of plane jets were carried out to understand the effect of co-flow on the sound field. The plane jets were of Mach number 0.5 and Reynolds number of 3000 based on center-line velocity excess at the nozzle. This is similar to the DNS by Stanley et al. (2002). It was identified that the co-flow leads to a reduction in turbulence levels. This was also corroborated by the turbulent energy spectrum plots. The far field radiation for the case without co-flow is higher over all angles. The contribution from the low frequencies is directional, mainly towards the downstream direction. The range of dominant convective velocities of the acoustic sources were different along shear layers and center-line. The plane jet results were also used to bring out a qualitative comparison of flow and the radiation characteristics with round jets. For the round jet, the center-line velocity decays linearly with the stream-wise distance. In the plane jet case, it is the square of the center-line velocity excess which decays linearly with the stream-wise distance. The turbulence levels at any section scales with the center-line stream-wise velocity. The decay of turbulence level is slower for the plane jet and hence the acoustic sources are present for longer distance along the downstream direction. Subsonic impinging jets are composed of four regions, the jet core, the fully developed jet, the impingement zone and the wall jet. The presence of the second region (fully developed free jet) depends on the distance of the wall from the nozzle and the length of the jet core. In impinging jets, reflection from the wall and the wall jet are additional sources of noise compared to the free jets. The results are analysed for the contribution of the different regions of the flow towards the radiated sound field. LES simulations of impinging round jets and impinging plane jet were carried out for this purpose. In addition, the results have been compared with equivalent free jets. The directivity plots showed that the SPL levels are significantly higher for the impinging jets at all angles. For free jets, a typical time scale for the acoustic sources is the ratio of the nozzle size to the jet velocity. This is ro/Uj for round jets and h/Uj for plane jets. For impinging jets, the non-dimensionlised rms of Lighthill’s source indicates that the time scale for acoustic sources is the ratio of the height of the nozzle from the wall to the jet velocity be L/Uj. LES of impinging round jets was carried out for two cases with different inflow perturbations. The jets were at Reynolds number of 88000 and Mach number of 0.9, same as the free jet cases. The impingement wall was at a distance L = 24ro from the nozzle exit. For impinging round jets, the SPL levels are found to be higher than the equivalent free jets. From the SPL levels and radiated noise spectra it was shown that the contribution from the large scale structures and its reflection from the wall is directional and at small angles to the wall normal. The difference in the range of angles where the radiation from the large scale structures were observed shows the significance of refraction of sound waves inside the flow. The rms values of the Lighthill’s sources indicate two dominant regions for the sources, just downstream of jet breakdown and in the impingement zone. The LES of impinging plane jet was done for a jet of Mach number 0.5 and Reynolds number of 6000. The impingement wall was at a distance L = 10h from the nozzle exit. The radiated sound field appears to emanate from this impingement zone. The directivity and the spectrum plots of the far field SPL indicate that there is no preferred direction of radiation from the impingement zone. The Lighthill’s sources are concentrated mainly in the impingement zone. The rms values of the sources indicate that the peak values occur in the impingement zone. The results from the different flow situations demonstrates the capability of LES with explicit filtering method in predicting the turbulent flow and radiated noise field. The method is robust and has been successfully used for moderate Reynolds number and an Euler simulation. An important feature is that LES can be used to identify acoustic sources and its convective speeds. It has been shown that the Lighthill source calculations, the calculated sound field and the observed radiation patterns agree well. An explanation for these based on the different turbulent flow structures has also been provided.