Estudio de la regulación dinámica de la expresión génica en respuesta a estrés osmótico en levadura

[EN] Cells respond to environmental stimuli by fine tuned regulation of gene expression. In this thesis we investigate the dose dependent modulation of the genetic response upon nutrient and stress signals in yeast. A destabilized version of firefly luciferase was used in living yeast cells as a real-time reporter for gene expression. This highly sensitive and non-invasive system can be simultaneously used upon many different experimental conditions in small culture aliquots. This allows the dose-response behaviour of gene expression driven by any yeast promoter to be reported and can be used to quantify important parameters, such as the threshold, sensitivity, response time, maximal activity and synthesis rate for a given stimulus. We applied the luciferase assay to the nutrient-regulated GAL1 promoter and the stress-responsive GRE2 promoter. We find that luciferase expression driven by the GAL1 promoter responds dynamically to growing galactose concentrations, with increasing synthesis rates determined by the light increment in the initial linear phase of activation. The GAL1 gene is activated with continuously increasing synthesis rates in a well defined range of galactose concentrations, correlating with a dynamic increase of histone remodeling and subsequent association of the RNAPII complex. Dose dependent chromatin remodeling appears to be the basis for the dynamic GAL1 expression since mutants with impaired histone dynamics show severely truncated dose response profiles. In the case of the GRE2 promoter, we demonstrate that the very short-lived version of luciferase used here is an excellent tool to quantitatively describe transient transcriptional activation. The luciferase expression controlled by the GRE2 promoter responds dynamically to a gradual increase of osmotic or oxidative stress stimuli, which is mainly based on the progressive increase of the time the promoter remains active. In contrast, the GRE2 promoter operates like an off/on switch in response to increasing osmotic stress with almost constant synthesis rates and exclusively temporal regulation of histone remodeling and RNAPII occupancy. The Gal3 inducer and the Hog1 MAP kinase seem to determine the different dose response strategies at the two promoters. Our analysis reveals important differences in the way dynamic signals create dose sensitive gene expression outputs. Taken together, the luciferase assay described here is an attractive tool to rapidly and precisely determine and compare kinetic parameters of gene expression. Additionally, the function of the specific transcriptor factor Smp1 involved in the yeast osmostress response was investigated. Location analyses upon osmotic stress reveal that Smp1 associates preferentially with the whole transcribed regions (ORFs) upon stress as opposed to other transcriptional activators involved in the osmostress response. However, Smp1 seems to be important for stress-activated gene expression only in the presence of the natural induced gene and not of artificial promoter fusions. This highlights the possible role of Smp1 in regulating gene expression from ORF sequences rather than promoter regions.[ES] Las células responden a los estímulos ambientales a través de una regulación precisa de la expresión génica. En este trabajo se investigó la modulación dosis dependiente de la expresión de genes activados en respuesta a estrés y por nutrientes. Se utilizó una versión desestabilizada de luciferasa de luciérnaga en células vivas de levadura como reportero para la detección de la expresión génica en tiempo real. Este sistema altamente sensible y no invasivo puede ser utilizado simultáneamente en diferentes condiciones experimentales a través de pequeñas alícuotas de cultivo. Esto permite la caracterización dosis-respuesta de la regulación de los promotores de levadura y puede ser utilizado para cuantificar parámetros importantes como el umbral, la sensibilidad, el tiempo de respuesta, la actividad máxima y el ratio de síntesis provocado por un determinado estímulo. Se aplicó el ensayo luciferasa al promotor GAL1 regulado por nutrientes y al promotor GRE2 activado en respuesta a estrés. Se observó que la expresión de la luciferasa activada por el promotor GAL1 responde de forma dinámica a las crecientes concentraciones de galactosa, con un incremento del ratio de síntesis determinado por el aumento de luz en la fase lineal inicial de la activación, en función de una gama de concentraciones de galactosa bien definidas. Este mecanismo de regulación depende de un aumento en la remodelación de las histonas y la consecuente asociación del complejo ARN pol II. La remodelación de la cromatina dosis dependiente parece ser la base de la expresión dinámica de GAL1, pues los mutantes relacionados con la dinámica de las histonas muestran perfiles dosis respuesta severamente afectados. En el caso del promotor GRE2, se demostró que una versión de una luciferasa desestabilizada es una herramienta excelente para describir de forma cuantitativa la activación transcipcional transitoria. La expresión de la luciferasa controlada por el promotor GRE2 responde de forma dinámica al aumento gradual de estímulo de estrés osmótico u oxidativo. La activación se observa principalmente en el incremento progresivo del tiempo en que el promotor permanece activo. Diferentemente de GAL1, el promotor GRE2 opera a través de un cambio apagado/encendido en respuesta a un aumento de estrés osmótico a través de ratios de síntesis prácticamente constantes y cuya regulación solamente depende de la remodelación de la cromatina y de la permanencia de la ARN pol II. Finalmente, se puede especular que el inductor Gal3 y la MAPK Hog1 son las moléculas determinantes para las diferentes estrategias de respuesta dinámica para los dos promotores. En este trabajo se identifican importantes diferencias en la señalización dinámica determinada por la dosis de estímulo en la expresión génica. En conjunto, el ensayo de luciferasa presentado en este trabajo puede ser una herramienta interesante para determinar y comparar de forma rápida y precisa los parámetros de la expresión génica. Adicionalmente se investigó la función del factor de transcripción Smp1 involucrado en la respuesta a osmoestrés en levadura. Un análisis de la asociación a la cromatina in vivo bajo estrés osmótico demostró que Smp1 se une preferentemente a regiones transcritas (ORFs) lo que refleja un comportamiento diferente comparando con otros activadores transcripcionales de la respuesta a estrés osmótico. Sin embargo, Smp1 parece ser importante para la expresión génica activada por estrés osmótico sólo en la presencia del gen natural inducido y no de fusiones artificiales del promotor. Esto evidencia el posible papel de Smp1 en la regulación de la expresión génica desde secuencias ORF y no en las regiones promotoras.[CA] Les cèl·lules responen als estímuls ambientals a través d'una regulació precisa de l'expressió gènica. A aquest treball es va investigar la modulació dosi dependent de l'expressió de gens activats en resposta a estrès i per nutrients. Es va utilitzar una versió desestabilitzada de luciferasa de cuca de llum en cèl·lules vives de llevat com a reporter per a la detecció de l'expressió gènica a temps real. Aquest sistema altament sensible i no invasiu pot ser utilitzat simultàniament en diferents condicions experimentals a través de xicotetes alíquotes de cultiu. Això permet la caracterització dosi-resposta de la regulació dels promotors de llevat i pot ser utilitzat per a quantificar paràmetres importants com el llindar, la sensibilitat, el temps de resposta, l'activitat màxima i el rati de síntesi provocat per un determinat estímul. L'assaig luciferasa es va aplicar al promotor GAL1 regulat per nutrients i al promotor GRE2 activat en resposta a estrès. Es va observar que l'expressió de la luciferasa activada pel promotor GAL1 respon de forma dinàmica a les creixents concentracions de galactosa, amb un increment del rati de síntesi determinat per l'augment de llum en la fase lineal inicial de l'activació, en funció d'una gama de concentracions de galactosa ben definides. Aquest mecanisme de regulació depèn d'un augment en la remodelació de les histones i la conseqüent associació del complex ARN pol II. La remodelació de la cromatina dosi dependent sembla ser la base de l'expressió dinàmica de GAL1, ja què els mutants relacionats amb la dinàmica de les histones mostren perfils dosi-resposta severament afectats. En el cas del promotor GRE2, es va demostrar que una versió d'una luciferasa desestabilitzada és una eina excel·lent per a descriure de forma quantitativa l'activació transcripcional transitòria. L'expressió de la luciferasa controlada pel promotor GRE2 respon de forma dinàmica a l'augment gradual d'estímul d'estrès osmòtic o oxidatiu. L'activació s'observa principalment a l'increment progressiu del temps al qual el promotor roman actiu. De forma diferent de GAL1, el promotor GRE2 opera a través d'un canvi apagat/encès en resposta a un augment d'estrès osmòtic a través de ratis de síntesi pràcticament constants i als quals la seua regulació només depèn de la remodelació de la cromatina i de la permanència de l'ARN pol II. Finalment, es pot especular que l'inductor Gal3 i la MAPK Hog1 són les molècules determinants per a les diferents estratègies de resposta dinàmica per als dos promotors. A aquest treball s'identifiquen importants diferències a la senyalització dinàmica determinada per la dosi d'estímul a l'expressió gènica. En conjunt, l'assaig luciferasa presentat a aquest treball pot ser una eina interesant per a determinar i comparar de forma ràpida i precisa els paràmetres de l'expressió gènica. Addicionalment, es va investigar la funció del factor de transcripció Smp1 involucrat en la resposta a osmoestrès en llevat. Una anàlisi de l'associació a la cromatina in vivo sota l'estrès osmòtic va demostrar que Smp1 s'uneix preferentment a regions transcrites (ORFs), el qual reflecteix un comportament diferent comparant amb altres activadors transcripcionals de la resposta a estrès osmòtic. Tot i així, Smp1 sembla ser important per a l'expressió gènica activada per estrès osmòtic només en la presència del gen natural induït i no de fusions artificials del promotor. Això evidencia el possible paper de Smp1 en la regulació de l'expressió gènica des de seqüències ORF i no a les regions promotores.Rienzo, A. (2016). Estudio de la regulación dinámica de la expresión génica en respuesta a estrés osmótico en levadura [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/62160TESI

LE INFEZIONI DI CRANIOPLASTICA: BASI SPERIMENTALI PER LA SOSTITUZIONE IMMEDIATA DEI SOSTITUTI CRANICI INFETTI IN RELAZIONE ALLE DIFFERENTI SPECIE BATTERICHE ED ALL'EVIDENZA CLINICA

Le infezioni di cranioplastica rappresentano una delle pià complesse sfide della moderna neurochirurgia. Il loro trattamento presuppone un complesso equilibrio fra strategia di trattamento medico, revisione chirurgica, decisioni in merito alla possibilitò in caso di recidive multiple, di optare per procedure non convenzionali. Il miglioramento delle terapie antibiotiche in questo campo non ha fornito i risultati sperati, poichè si è accompagnato ad una progressiva comparsa di germi ad elevata resistenza in grado di formare biofilm . Scopo di questo lavoro è stato quello di investigare le affinità di proliferazione di diversi germi multiresistenti sulle quattro tipologie principali di materiali da cranioplastica usati, per poi confrontare i risultati del trattamento in laboratorio e sull'umano. Abbiamo considerato 4 germi, la Klebsiella Pneumoniae, l'Acinetobacter Baumanii, la Pseudomonas Aeruginosa e lo Stafilococco Aureo, come già detto tutti multiresistenti, con senbilità ad una sola famiglia antibiotica. Siamo partiti per lo Stafilococco dal materiale di crescita maggiore (idrossiapatite), dal quale abbiamo poi provveduto ad infettare gli altri materiali (resina acrilica, PEEK, Titanio) e ancora una volta l'idrossiapatite stessa, testata senza e con terapia antibiotica. Lo stafilococco ha mostrato una affinità specifica per l'idrossiapatite, modificata solo dalla terapia antibiotica. La Klebsiella, nelle stesse condizioni, ha mostrato di preferire il PEEK, seguito dal titanio, con scarsa affinità al contrario per l'idrossiapatite. La Pseudomonas ha mostrato una tendenza ad affinità verso il PEEK ed il titanio, sebbene non costante, laddove al contrario l'Acinetobacter è risultato essere in grado di crescere aspecificamente su qualunque terreno. In clinica, questi dati sono stati confermati, mostrando come nelle infezioni da Stafilococco e Klebsiella la sostituzione immediata del materiale infetto a maggiore affinità con uno a bassa affinità, supportata dalla terapia antibiotica, abbia permesso di non lasciare il paziente privo di copertura cranica, indipendentemente dalla severità dell'infezione. Al contrario, Pseudomonas ed Acinetobacter si sono mostrati particolarmente resistenti, lasciando scarse ( nel caso di Penudomonasd) e nessuna (per Acinetobacter) possibilità di effettuare la stessa procedura con successo. Questi risultati rappresentano solo una fase preliminare, basata sulla numerificazione delle colonie sviluppate, pertanto necessiteranno di essere sostanziati da un ulteriore approfondimento con coloranti per l'attività metabolica ed una eventuale valutazione di microscopia confocale per identificare le modalità di crescita batterica.Cranioplasty infections are one of the major challenges of modern Neurosurgery. Their treatment requires a complex balance between medical and surgical strategies, especially in case of frequent relapses, requiring unconventional procedures. The improvement of antibiotic therapy did not meet operators’ expectations, due to the appearance of multidrug resistant germs, able to form biofilm. Aim of this work was to investigate the growth preferences of some MDR species on the 4 more common cranioplasty materials, to be then compared to human results. Four germs were selected, Klebsiella Pneumoniae, Acinectobacter Baumanii, Pseudomonas Aeruginosa, Staphylococcus Aureus, sensitive to treatment with only one antibiotic. Staphylococcus showed preference for growth in Hydroxyapathite and when allowed to grow on the same material and on acrylic resin, PEEK and titanium, its preference remained unchanged. Klebsiella behaved similarly , but preferring PEEK and titanium. On the counterside, Pseudomonas, although showing a propensity for growth on PEEK, was also effective on titanium and hydroxyapathite, where Acinetobacter was able to grow similarly everywhere. We wanted to know if patients presenting with an active infection of their cranioplasty could successfully undergo its immediate replacement. Our results suggested that this could happen with Stph Aureus and Klebsiella, in some case with Pseudomonas, never with Acinetobacter. Data from our clinical series confirmed these results. Although the capacity of surgery and antibiotics to inferfere with bacterial growth on cranioplasty can be confirmed only for some species, further studies requiring a metabolic-based evaluation and confocal microscopy observation of bacterial growth will be needed to substantiate these data

Leptin activates the anandamide hydrolase promoter in human T lymphocytes through STAT3

Physiological concentrations of leptin stimulate the activity of the endocannabinoid-degrading enzyme anandamide hydrolase (fatty acid amide hydrolase, FAAH) in human T lymphocytes up to approximately 300% over the untreated controls. Stimulation of FAAH occurred through up-regulation of gene expression at transcriptional and translational levels and involved binding of leptin to its receptor with an apparent dissociation constant (K(d)) of 1.95 +/- 0.14 nm and maximum binding (B(max)) of 392 +/- 8 fmol x mg protein(-1). Leptin binding to the receptor triggered activation of STAT3 but not STAT1 or STAT5 or the mitogen-activated protein kinases p38, p42, and p44. Peripheral lymphocytes of leptin knock-out (ob/ob) mice showed decreased FAAH activity and expression (approximately 25% of the wild-type littermates), which were reversed to control levels by exogenous leptin. Analysis of the FAAH promoter showed a cAMP-response element-like site, which is a transcriptional target of STAT3. Consistently, mutation of this site prevented FAAH activation by leptin in transient expression assays. Electrophoretic mobility shift and supershift assays further corroborated the promoter activity data. Taken together, these results suggest that leptin, by up-regulating the FAAH promoter through STAT3, enhances FAAH expression, thus tuning the immunomodulatory effects of anandamide. These findings might also have critical implications for human fertility

Progesterone activates fatty acid amide hydrolase (FAAH) promoter in human T lymphocytes through the transcription factor Ikaros. Evidence for a synergistic effect of leptin.

Physiological concentrations of progesterone stimulate the activity of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH) in human T lymphocytes, up to a ∼270% over the untreated controls. Stimulation of FAAH occurred through up-regulation of gene expression at transcriptional and translational level and was specific. Indeed, neither the activity of the anandamide-synthesizing N-acyltransferase and phospholipase D, nor the activity of the anandamide transporter, nor the binding to cannabinoid receptors were affected by progesterone under the same experimental conditions. The activation of FAAH by progesterone was paralleled by a decrease (down to 60%) of the cellular levels of anandamide and involved increased nuclear levels of the transcription factor Ikaros. Analysis of the FAAH promoter showed an Ikaros binding site, and mutation of this site prevented FAAH activation by progesterone in transient expression assays. Electrophoretic mobility shift and supershift assays further corroborated the promoter activity data. Furthermore, the effect of progesterone on FAAH promoter was additive to that of physiological amounts of leptin, which binds to a cAMP response element-like site in the promoter region. Taken together, these results suggest that progesterone and leptin, by up-regulating the FAAH promoter at different sites, enhance FAAH expression, thus tuning the immunomodulatory effects of anandamide. These findings might also have critical implications for human fertility

A Novel and Robust Security Approach for Authentication, Integrity, and Confidentiality of Lithium-ion Battery Management Systems

Battery management systems (BMSs) play a critical and crucial role in ensuring the safety and the efficiency of the batteries. The increasing BMS complexity, the expanding interconnections between batteries and applications, and the introduction of cloud-based energy storage system structures have led to growing concerns about battery cybersecurity. For instance, the data exchange between the local and remote BMS parts can be exposed to cybersecurity attacks. Classic BMSs are not equipped with security mechanisms that are instead essential to protect their integrity and reliability and prevent serious consequences such as loss of data, equipment damage, and counterfeiting of battery components. This work highlights the importance of securing BMSs against cyber threats and discusses the current state of the art of cybersecurity in BMSs. The main outcome is the proposal of a novel and robust security approach to design a BMS able to prevent misuse and undesired manipulation of battery equipment and data. The proposed design approach can be used as enabling technology to support the application to the BMSs of the most diffused security mechanisms adopted by the state of the art as cybersecurity protections

Retention of nativelike conformation by proteins embedded in high external electric fields.

In this Communication, we show that proteins embedded in high external electric fields are capable of retaining a nativelike fold pattern. We have tested the metalloprotein azurin, immobilized onto SiO2 substrates in air with proper electrode configuration, by applying static fields up to 106–107V∕m. The effects on the conformational properties of protein molecules have been determined by means of intrinsic fluorescence measurements. Experimental results indicate that no significant field-induced conformational alteration occurs. Such results are also discussed and supported by theoretical predictions of the inner protein fields

Evaluation of prognostic preoperative factors in patients undergoing surgery for spinal metastases: Results in a consecutive series of 81 cases

Background: Surgical treatment of spinal metastases should be tailored to provide pain control, neurological deficit improvement, and vertebral stability with low operative morbidity and mortality. The aim of this study was to analyze the predictive value of some preoperative factors on overall survival in patients undergoing surgery for spinal metastases. Methods: We retrospectively analyzed a consecutive series of 81 patients who underwent surgery for spinal metastases from 2015 and 2021 in the Clinic of Neurosurgery of Ancona (Italy). Data regarding patients’ baseline characteristics, preoperative Karnofsky Performance Status Score (KPS), and Frankel classification grading system, histology of primary tumor, Tokuhashi revised and Tomita scores, Spine Instability Neoplastic Score, and Epidural Spinal Cord Compression Classification were collected. We also evaluated the interval time between the diagnosis of the primary tumor and the onset of spinal metastasis, the type of surgery, the administration of adjuvant therapy, postoperative pain and Frankel grade, and complications after surgery. The relationship between patients’ overall survival and predictive preoperative factors was analyzed by the Kaplan–Meier method. For the univariate and multivariate analysis, the log-rank test and Cox regression model were used. P ≤ 0.05 was considered as statistically significant. Results: After surgery, the median survival time was 13 months. In our series, the histology of the primary tumor (P < 0.001), the Tomita (P < 0.001) and the Tokuhashi revised scores (P < 0.001), the preoperative KPS (P < 0.001), the adjuvant therapy (P < 0.001), the postoperative Frankel grade (P < 0.001), and the postoperative pain improvement (P < 0.001) were significantly related to overall survival in the univariate analysis. In the multivariate analysis, the Tomita (P < 0.001), Tokuhashi revised scores (P < 0.001), and the adjuvant therapy were confirmed as independent prognostic factors. Conclusion: These data suggest that patients with limited extension of primitive tumor and responsive to the adjuvant therapy are the best candidates for surgery with better outcome

A computational approach to investigate TDP-43 C-terminal fragments aggregation in Amyotrophic Lateral Sclerosis

Many of the molecular mechanisms underlying the pathological aggregation of proteins observed in neurodegenerative diseases are still not fully understood. Among the diseases associated with protein aggregates, for example, Amyotrophic Lateral Sclerosis (ALS) is of relevant importance. Although understanding the processes that cause the disease is still an open challenge, its relationship with protein aggregation is widely known. In particular, human TDP-43, an RNA/DNA binding protein, is a major component of pathological cytoplasmic inclusions described in ALS patients. The deposition of the phosphorylated full-length TDP-43 in spinal cord cells has been widely studied, and it has been shown that the brain cortex presents an accumulation of phosphorylated C-terminal fragments (CTFs). Even if it is debated whether CTFs represent a primary cause of ALS, they are a hallmark of TDP-43 related neurodegeneration in the brain. Here, we investigate the CTFs aggregation process, providing a possible computational model of interaction based on the evaluation of shape complementarity at the interfaces. To this end, extensive Molecular Dynamics (MD) simulations were conducted for different types of fragments with the aim of exploring the equilibrium configurations. Adopting a newly developed approach based on Zernike polynomials, for finding complementary regions of the molecular surface, we sampled a large set of exposed portions of the molecular surface of CTFs structures as obtained from MD simulations. The analysis proposes a set of possible associations between the CTFs, which could drive the aggregation process of the CTFs.Comment: 9 pages, 4 figures, 1 tabl

Satellite Communication for the Adaptable Railway Communication System, Lab-Test Results and Field Test Preparation

This paper reports the activities of the X2Rail-5 WP3 work done on an adaptable communication system (ACS) in relation to satellite communication (SatCom). The ACS connects trackside applications with on-board side. Since coverage along the lines with a dedicated system (railway operator owned) is expensive, it can be supplemented using available public networks or other technologies. The paper presents the resutls of a SatCom prototype and the ACS field test preparation

Azurin for Biomolecular Electronics: a Reliability Study

The metalloprotein azurin, used in biomolecular electronics, is investigated with respect to its resilience to high electric fields and ambient conditions, which are crucial reliability issues. Concerning the effect of electric fields, two models of different complexity agree indicating an unexpectedly high robustness. Experiments in device-like conditions confirm that no structural modifications occur, according to fluorescence spectra, even after a 40-min exposure to tens of MV/m. Ageing is then investigated experimentally, at ambient conditions and without field, over several days. Only a small conformational rearrangement is observed in the first tens of hours, followed by an equilibrium state