Purificazione e caratterizzazione enzimatica dell'anaphase promoting complex, un regolatore essenziale del ciclo cellulare

L’ubiquitinazione è uno dei meccanismi regolatori fondamentali del ciclo cellulare. La progressione del ciclo cellulare è infatti finemente regolata dalla degradazione controllata, nel tempo e nello spazio, di specifiche proteine che, modificate covalentemente da catene di ubiquitina, diventano target di degradazione per il proteasoma. L’ubiquitinazione è pertanto un processo controllato ed avviene grazie a complessi proteici multienzimatici con specificità diversa per vari targets. Il sistema di ubiquitinazione è composto almeno da tre enzimi chiave - E1, E2, E3 - che sono richiesti per l’iniziale attivazione e coniugazione dell’ubiquitina (E1 – ubiquitin activating enzyme – ed E2 – ubiquitin conjugating enzyme - , rispettivamente) e per il suo legame covalente alla proteina target (E3 – ubiquitin ligase). Durante lo svolgimento della tesi di laurea, ho focalizzato la mia attenzione verso una E3 ubiquitin ligase, l’anaphase promoting complex/cyclosome (APC/C), coinvolta nella degradazione di proteine regolatorie del processo mitotico. L’APC/C è un complesso enzimatico di 1.5-MDa composto da almeno 12 subunità che lavora in concerto con specifci E1 ed E2 e che necessita di un coattivatore (Cdc20 o Cdh1) per il riconoscimento e il binding di specifici substrati. Obiettivo del lavoro di tesi è la comprensione della regolazione di questo enzima attraverso la ricostituzione in vitro della attività ubiquitinante APC/C mediata. A tale scopo, avvalendomi di tecniche di biologia molecolare e biochimica, ho prodotto e purificato diversi componenti necessari per la corretta attività enzimatica; tra questi l’E2 – ubiquitin conjugating enzyme - UbcH10 e il substrato dell’APC/C Cyclin B1. Ho inoltre purificato l’APC/C tramite immunoprecipitazione da cellule HeLa. Al fine di garantire la più alta specificità possibile nel segnale derivante dal saggio in vitro, il substrato Cyclin B1 è stato preventivamente iodinato (125I) per permetterne la detezione attraverso autoradiografia. Il saggio che ho messo a punto con queste componenti purificate è assai efficace, in quanto permette una completa ubiquitinazione del substrato in vitro in circa 30 minuti di reazione. Il saggio serve adesso come base per caratterizzare le proprietà enzimatiche dell’APC/C. In particolare, gli studi si concentrano sulla dipendenza della attività dell’APC/C da1) presenza del coattivatore (Cdc20 o Cdh1); 2) stato di modificazione (fosforilazione in particolare); e 3) presenza di binding partners, in particolare i regolatori negativi del checkpoint mitotico (spindle assembly checkpoint). Inoltre, durante questi mesi, ho seguito la produzione di anticorpi (mono e policlonali) contro una subunità dell’enzima, Apc7, avendone preventivamente espresso e purificato un frammento, al fine di poterli utilizzare per l’immunoprecipitazione del complesso ed eventualmente per la sua localizzazione mediante tecniche di immunofluorescenza

An RNAi screen identifies TLR2/6 as mediators of a novel inflammatory pathway for rapid hepcidin-independent hypoferremia

Systemic iron homeostasis is essential for human health. Its maintenance critically depends on the interaction between the hepatic hormone hepcidin and the sole known iron exporter ferroportin (FPN) predominantly expressed in hepatocytes, duodenal enterocytes and macrophages. Hepcidin binding leads to FPN internalization and degradation resulting in cellular iron retention. Iron is an essential nutrient also for pathogens and plays a central role in host-pathogen interactions. The innate immune system fights infections by sequestration of iron in macrophages of the reticuloendothelial system. The resulting hypoferremia represents a major host defence strategy. A current model posits that hepcidin is the crucial effector of this response, as its release from macrophages and hepatocytes provokes FPN protein decrease and, consequently, tissue iron retention. The aim of my PhD project was to identify novel cellular regulators of hepcidin-mediated ferroportin (FPN) degradation, a fundamental process that controls systemic iron homeostasis. To reach this aim I generated a HeLa cell line expressing a hFPN-renilla fusion protein, which was used for a focused high-throughput RNAi screen targeting kinases and related proteins. Out of 779 genes tested, the screen identified 71 putative regulators of FPN protein stability. Validation experiments confirmed the phenotype of 24 genes. Interestingly, most validated regulators of FPN expression conferred hepcidin-independent FPN regulation. From these I selected 14 genes associated with immune processes for further characterization in murine bone marrow-derived macrophages (BMDMs). Finally, my studies focused on Toll-like receptor 6 (TLR6) as an effective regulator of FPN expression in BMDMs and I investigated how the TLR6 activation pathway modulates iron regulation in the inflammatory context. TLR2/6 ligation by the synthetic lipoprotein derived from Mycoplasma: FSL1 triggered a profound decrease in FPN mRNA and protein expression in BMDMs as well as in the liver and the spleen of mice. Unexpectedly hepcidin expression remained unchanged. Hepcidin-independent FPN down regulation was a conserved response to different microbial lipopeptides and elicited a fast, hepcidin-independent hypoferremia pathway. These findings were further confirmed in C326S FPN knock-in mice with a disrupted hepcidin/FPN regulatory circuitry. This work challenges the prevailing role of hepcidin in inflammatory hypoferremia and suggests that rapid hepcidin-independent FPN down regulation may represent the first line response to restrict iron access to pathogens

A redução da maioridade penal e os preceitos da inimputabilidade penal de crianças e adolescentes no ordenamento jurídico brasileiro

Trata-se de monografia apresentada no Curso de Direito da Faculdade de Ciências Jurídicas e Sociais do Centro Universitário de Brasília como requisito parcial para obtenção do título de Bacharela em Direito. O foco do trabalho é analisar os aspectos constitucionais e jurídicos das propostas de emenda constitucional em trâmite no Congresso Nacional que visam à redução da maioridade penal. A monografia está estruturada em três capítulos: inicialmente, expõem-se as nuances da evolução histórica do estabelecimento de idade mínima para imputabilidade penal no ordenamento jurídico brasileiro; a seguir, aborda a estruturação do direito e justiça da infância e juventude com fundamento doutrina da proteção integral prevista na Constituição Federal de 1988 e no Estatuto da Criança e do Adolescente; e, por fim, examina a polarização jurídica e doutrinária acerca das principais justificativas que subjazem à pauta reformista do Congresso Nacional para consolidar a redução da maioridade penal. A monografia foi desenvolvida mediante a metodologia bibliográfica qualitativa

DEVELOPMENT OF THE LIFE SKILLS FOR PROMOTION OF HEALTH WITH ART-THERAPY

Individuals, who work in an organization, develop a shared perception that influences their behavior and emotions. This perception guides operators in the interpretation of the main business processes and in the modes of decision-making. The Italian Ministry of Public Administration in 2004 issued a directive to improve the organizational well-being and the emotional state of the environment in the workplace. This law identifies the necessity of an organizational climate that fosters creativity at the workplace, for the development and the efficiency of public administration. Several studies have shown that the development of creativity in the operators becomes a resource for the organization to facilitate the adaptation to change and to the solution of problems. So the techniques of creativity can be used as a training strategy for the quality management and human resources, optimizing services. The following pilot study evaluates the effectiveness of a training course for veterinary staff of ASL Napoli 1 Centre The aim of the course has been promoting the well-being, the development of life skills and the resilience of the learners using techniques of creativity and art therapy

Evaluation of hearing protection used by police officers in the shooting range

AbstractIntroductionImpact noise is characterized by acoustic energy peaks that last less than a second, at intervals of more than 1s.ObjectiveTo quantify the levels of impact noise to which police officers are exposed during activities at the shooting range and to evaluate the attenuation of the hearing protector.MethodsMeasurements were performed in the shooting range of a military police department. An SV 102 audiodosimeter (Svantek) was used to measure sound pressure levels. Two microphones were used simultaneously: one external and one insertion type; the firearm used was a 0.40 Taurus® rimless pistol.ResultsThe values obtained with the external microphone were 146dBC (peak), and a maximum sound level of 129.4dBC (fast). The results obtained with the insertion microphone were 138.7dBC (peak), and a maximum sound level of 121.6dBC (fast).ConclusionThe findings showed high levels of sound pressure in the shooting range, which exceeded the maximum recommended noise (120dBC), even when measured through the insertion microphone. Therefore, alternatives to improve the performance of hearing protection should be considered

JUAN MANTOVANI: GESTIÓN EDUCATIVA Y POLÍTICA CULTURAL EN LA PROVINCIA DE SANTA FE (1938-1941)

Analizamos la gestión del intelectual santafesino Juan Mantovani (San Justo, provincia de Santa Fe, 1898 - Alemania, 1961) entre 1938 y 1941como Ministro de Instrucción Pública y Fomento (en adelante MIPyF) de laprovincia de Santa Fe (Argentina) durante la gobernación del conservadorManuel de Iriondo (1937 - 1943). Mantovani impulsó en ese período un ambicioso programa tendiente a crear y consolidar instituciones culturales y de formación de artistas. Además, es conocido el apoyo brindado a Olga Cossettini que lo llevó a participar de diversas actividades realizadas por ella desde la dirección de la Escuela N° 69 “Dr. Gabriel Carrasco”.En este estudio consideramos como fuentes, los discursos de Juan Mantovani vinculados a su función ministerial, pronunciados y publicados en ese marco. Estos documentos ofrecen una vía de acceso a las ideas de Mantovani asociadas a la difusión de las artes y la cultura, en el despliegue de una política cultural, artística y educativa.Para el análisis aplicamos las claves metodológicas de la historia intelectual que permiten articular los rasgos de una política educativa más amplia con las líneas de los discursos que marcaron la trayectoria de Mantovani como funcionario, educador e intelectual, y su impronta en la definición de los campos pedagógico, artístico y cultural

Shanghai Fever: Not Only an Asian Disease

Objectives: To describe a case of Shanghai fever disease and to analyze other published reports in non-Asiatic countries, defining clinical characteristics and highlighting that this is not only an Asian disease. Study design: A computerized search without language restriction was conducted using PubMed and Scopus; all references listed were hand-searched to identify any other relevant literature. An article was considered eligible for inclusion in the systematic review if it reported cases with Shanghai fever described in non-Asiatic countries. Our case was also included in the analysis. Results: Ten articles reporting 10 cases of Shanghai fever disease were considered. Fever, diarrhea and ecthyma gangrenosum were the most frequent symptoms observed. Blood was the most common site of isolation for Pseudomonas aeruginosa. Three patients underwent surgery due to necrotizing enteritis and intestinal perforation. Meningitis was documented in one case. None of the patients received antipseudomonal antibiotics within 24 h of admission. The outcome was good in nine cases; only one patient died due to multiple organ failure from Pseudomonas sepsis. No common primary immune deficiency was identified in these patients. Extremely young age (<1 year) was the only host factor predisposing to Shanghai fever. Conclusions: It is important to shed light on this disease in non-Asiatic countries and take into account that it can also affect healthy children. Pediatricians, therefore, should consider Shanghai fever among diagnoses in children with community-onset diarrhea, fever and skin lesions suggestive of ecthyma gangrenosum to start an appropriate treatment sooner and to reduce the mortality in these children

Elective cesarean section on maternal request: ethical and legal considerations

Cesarean is defined as the delivery of a fetus through surgical incisions made through the uterine wall. Primary cesarean sections often lead to repeat cesareans, which may lead to placenta accreta spectrum disorders. The ethic of exposing mothers to these risks, as well as performing surgery on what is otherwise a healthy patient, become entangled with the demand for patient choice, as well as the increasing financial strain on our healthcare system. The main question to be examined is whether it is ethical to allow women to opt for cesarean delivery in the absence of obstetric indication (i.e. cesarean delivery on maternal request), taking into account the increased risk to the mother and her future offspring

Identification of a high affinity binding site for abscisic acid on human lanthionine synthetase component C-like protein 2

Lanthionine synthetase component C-like protein 2 (LANCL2) has been identified as the mammalian receptor mediating the functional effects of the universal stress hormone abscisic acid (ABA) in mammals. ABA stimulates insulin independent glucose uptake in myocytes and adipocytes via LANCL2 binding in vitro, improves glucose tolerance in vivo and induces brown fat activity in vitro and in vivo. The emerging role of the ABA/LANCL2 system in glucose and lipid metabolism makes it an attractive target for pharmacological interventions in diabetes mellitus and the metabolic syndrome. The aim of this study was to investigate the presence of ABA binding site(s) on LANCL2 and identify the amino acid residues involved in ABA binding. Equilibrium binding assays ([3H]-ABA saturation binding and surface plasmon resonance analysis) suggested multiple ABA-binding sites, prompting us to perform a computational study that indicated one putative high-affinity and two low-affinity binding sites. Site-directed mutagenesis (single mutant R118I, triple mutants R118I/R22I/K362I and R118I/S41A/E46I) and equilibrium binding experiments on the mutated LANCL2 proteins identified a high-affinity ABA-binding site involving R118, with a KD of 2.6 nM ± 1.2 nM, as determined by surface plasmon resonance. Scatchard plot analysis of binding curves from both types of equilibrium binding assays revealed a Hill coefficient >1, suggesting cooperativity of ABA binding to LANCL2. Identification of the high-affinity ABA-binding site is expected to allow the design of ABA agonists/antagonists, which will help to understand the role of the ABA/LANCL2 system in human physiology and disease