Mitochondrial hyperfusion via metabolic sensing of regulatory amino acids

The relationship between nutrient starvation and mitochondrial dynamics is poorly understood. We find that cells facing amino acid starvation display clear mitochondrial fusion as a means to evade mitophagy. Surprisingly, further supplementation of glutamine (Q), leucine (L), and arginine (R) did not reverse, but produced stronger mitochondrial hyperfusion. Interestingly, the hyperfusion response to Q + L + R was dependent upon mitochondrial fusion proteins Mfn1 and Opa1 but was independent of MTORC1. Metabolite profiling indicates that Q + L + R addback replenishes amino acid and nucleotide pools. Inhibition of fumarate hydratase, glutaminolysis, or inosine monophosphate dehydrogenase all block Q + L + R-dependent mitochondrial hyperfusion, which suggests critical roles for the tricarboxylic acid (TCA) cycle and purine biosynthesis in this response. Metabolic tracer analyses further support the idea that supplemented Q promotes purine biosynthesis by serving as a donor of amine groups. We thus describe a metabolic mechanism for direct sensing of cellular amino acids to control mitochondrial fusion and cell fate

The impact of AMPK signalling and clinical therapeutics on cancer metabolism

Cancer metabolism is intricately re-wired to support tumour growth. Despite the initial discovery that cancers depend on aerobic glycolysis, it is well appreciated that cancer cells exploit numerous metabolic pathways, including those linked to mitochondrial metabolism, to help fuel tumorigenesis. Metabolic gene programs are controlled by transcriptional complexes, such as the peroxisome proliferator-activated gamma coactivator 1 (PGC-1) / estrogen-related receptor alpha (ERR) axis, which act as master orchestrators of metabolism. The activity of the PGC-1α/ERRα axis is upregulated by the AMP-activated protein kinase (AMPK), a central metabolic regulator that is triggered in response to energetic stress. The work in this thesis demonstrates that the AMPK/PGC-1α/ERRα axis increases the bioenergetic functions of cancer cells, but inhibits one-carbon metabolism and purine biosynthesis, resulting in improved sensitivity to methotrexate (MTX), a chemotherapeutic drug widely used in the clinic. MTX treatment promotes bioenergetic functions and has antiproliferative effects that are dependent on AMPK. As a result, the combination of MTX with AMPK activators can improve chemotherapeutic response. Recently, there is increased interest in repurposing metabolic drugs to treat cancer. We show that the antidiabetic drug canagliflozin decreases cancer cell proliferation and reduces the activity of the citric acid cycle through perturbation of glutamine metabolism. The work in this thesis unravels the role of the AMPK/PGC-1α/ERRα pathway in controlling antifolate response and reinforces the premise of pharmacologically targeting cancer metabolism to impede tumourigenesis.Le métabolisme du cancer s'adapte de manière complexe pour soutenir la croissance tumorale. Outre leur dépendance à la glycolyse aérobie, tel que découvert initialement, les cellules cancéreuses exploitent de nombreuses autres voies métaboliques, incluant celle du métabolisme mitochondrial, afin d'alimenter la tumorigenèse. Les gènes métaboliques sont contrôlés par des complexes transcriptionnels tels que l'axe du peroxisome proliferator-activated gamma coactivator 1 (PGC-1) / estrogen-related receptor alpha (ERR), des facteurs de transcription bien connus dans l'orchestration du métabolisme. La régulation de cet axe est activée par l'AMPK, un acteur métabolique central qui est induit en réponse à un stress énergétique. Les travaux présentés dans cette thèse montrent que l'activation de l'axe AMPK/PGC-1α/ERRα augmente la fonction bioénergétique tout en diminuant le métabolisme du folate et la biosynthèse des purines, rendant ainsi ces cellules cancéreuses sensibles au méthotrexate (MTX), un agent chimiotherapeutique communément utilisé chez des patients atteints de cancer. Les mécanismes d'action du MTX se traduisent par une promotion des fonctions bioénergétiques tout en ayant des effets antiprolifératifs dépendants de l'AMPK. Par conséquent, la combinaison du MTX et d'un activateur de l'AMPK pourrait améliorer la réponse chimiothérapeutique, rendant ainsi le traitement plus efficace. L'utilisation de médicaments métaboliques suscite de plus en plus un grand intérêt dans le traitement du cancer. En effet, nous avons mis en évidence que la canagliflozine, un médicament anti-diabétique, induit une diminution de la prolifération des cellules cancéreuses et une réduction de l'activité du cycle de Krebs, due à la perturbation du métabolisme de la glutamine. Ainsi les travaux présentés dans cette thèse mettent en lumière le rôle de la voie AMPK/PGC-1α/ERRα dans le contrôle de la réponse anti-folate et le potentiel de la thérapie pharmacologique ciblant le métabolisme du cancer comme stratégie pour bloquer la tumorigenèse

Non-Metallic Inclusion Changes in Si-Mn Killed Steels

Steel must be deoxidized during refining to avoid the formation of gas porosity during solidification, and many alloys. In the case of Si-Mn killed steels, Si and Mn are added to form low melting point inclusions that can prevent nozzle clogging and that are vitreous upon solidification, so they can deform upon mechanical processing. Depending on the activity of SiO2 of the slags used during secondary refining, the steel-slag reactions can further deoxidize the steel and reduce oxides from the slag and refractories such as MgO, Al2O3 and CaO. These reduced elements can increase the melting point and hardness of the inclusions. Some of these impurities can enter the steelmaking process from the ferroalloys themselves because of their manufacturing process.In this work, the process variables that influence the thermodynamics and kinetics of the non-metallic inclusion formation, transformation and removal are reviewed. It is found that the gas stirring rate is found to have a significant impact on the kinetics of steel-slag reactions and inclusion generation and removal. It is also found that basic slags will cause pick-up of Al, Mg and Ca and promote the formation of undesired inclusions in the steel melt. During solidification, inclusions can precipitate at different chemical compositions depending on the remaining dissolved oxygen content in the melt. Ferroalloys used for deoxidizing the steel such as FeSi typically contains Ca that will impact steel cleanliness and can be used to the advantage of the process, i.e. the treatment of alumina inclusions.A one-parameter kinetic model for steel-slag-inclusion reactions is developed using FactSage. The potential effect of three different slag chemistries on the trajectory of inclusion modification are tested using the model for 1.25%Mn-0.25%Si steel. It is seen that Al, Mg and Ca pick-up are expected for all tested systems. The SiO2 activity, directly influenced by slag binary basicity, controls the reduction of the oxides. For high basicity, alumina-containing slags, the inclusions will transform from liquid Mn-silicates to alumina to spinels to Ca, Mg-aluminates to liquid slag. For low basicity, low alumina slags, the inclusions will remain liquid. For CaO-SiO2-MgO-CaF2 slags, Mn-silicates are predicted to become 2CaO.SiO2 and MgO.Inclusions in lollipop samples of degassed Si-killed steels with CaO-SiO2-MgO-CaF2 slags are analyzed using ASPEX automated feature analysis and inclusion extraction. The inclusions found to be MgO and CaO-rich after degassing. After degassing, CaO-rich inclusions are found both containing and not containing fluorine, indicating Ca pick-up from slag and slag emulsification. After trimming, primary inclusions are mostly Ca-silicates and do not contain fluorine - their origin is discussed. It is observed that Mg content in steel increases during a heat while Ca content decreases.Induction furnace experiments are done with MgO and CaO crucibles for different slag compositions. The one-parameter kinetic model predicts the inclusion composition over time. It is found that more basic slags in the CaO-Al2O3-MgO-SiO2 will drive more Al and Mg pick-up than less basic slags. The addition of 5%MnO and 10ÊF2 to CaO-Al2O3-MgO-SiO2 slags did not significantly change the steel-slag reaction kinetics and non-metallic inclusion composition. It is found that CaO-SiO2-MgO-CaF2 are strongly desulfurizing and can modify Mn-silicate inclusions to forsterite and MgO. There is no observable CaO precipitation when the melt is reoxidized after equilibration. One experiment with CaO-SiO2 -CaF2 slags in a CaO-3%ZrO2 crucible yielded CaO-containing inclusions, with estimated [Ca] before reoxidation of 0.3 ppm. It is found that Ca solubility in the steel is significantly lower than predicted by FactSage. Experiments using FeSi containing 2Ê to deoxidize the steel and treat Al-killed and Si-killed steels showed that the Ca yield is higher than the typically observed for CaSi2 powder addition in the same experimental setup. When [O] is higher (for Si-killed steels), the Ca yield is higher, and inclusions reach equilibrium faster than for Al-killed steels which have very low [O]. The inclusion-inclusion reactions in Ca-treated Al-killed steel took several minutes more than expected from kinetic models. It was found that the mass transfer coefficient for the configuration is controlled by convection in the melt, and there should not be significant differences when using 600g or 1000g melts for the experiments.Confocal Laser Scanning Microscopy was used to observe the reoxidation behavior of a steel sample taken from a Si-killed degassed heat, where [Ca] and [Mg] was expected to be high. The steel sample was oxidized using ungettered argon. The gas phase mass transfer coefficient for a CSLM sample was measured by evaporation of Mn. The oxidation experiments could precipitate Mg-rich inclusions but there was no observable increase in the CaO content of these inclusions. <br

An efficient approach based on geometrical analysis to optimize AM process

New methodologies of Design for Additive Manufacturing (DFAM) and relative CAx tools are the key enabling technologies allowing to get the major benefits from AM. It is evident that an increased integration of all CAx phases would lead to a more efficient design and engineering workflow. One of the major bottlenecks of such a holistic approach, which affects AM product time-to-market and restricts its commercial exploitation, is the need of a high amount of time and human resources for simulation, modelling and postprocessing of all the engineering activities. That is not always manageable, especially during a preliminary stage of concept design or feasibility analysis. Modern mathematical approaches are facing, with the ambition to contribute to some of the most challenging engineering tasks to fulfil strict requirements of structural resistance, lightness, noise, static and dynamic stiffness. The effectiveness of analytical and geometrical tools and methods for the study and the optimization of shapes were already demonstrated. As an example, it is well known that small fillet radius are stress concentrators and must be avoided for a robust design of structural parts. In the same way, we can demonstrate that poorly uniform temperature distribution of the material during additive layer manufacturing can be correlated with geometrical section areas and variations along the growing axis, as well as with supports shape configurations. In this paper, a novel approach is presented which identifies optimal orientation and support configurations, uniquely based on geometrical criteria. The benefits are avoiding long structures to minimise the amount of waste material, accounting for the distribution of the piece weight, effectively draining the thermal field from all the areas of the piece to the build platform. With FE based macroscale process simulations, it is possible to evidence improved thermal strain distributions of the optimised configuration. As part of the R&D activity within the “STAMP” project financed by Regione Piemonte, an innovative function was developed and successfully applied to minimize distortions and residual stresses of SLM parts. Within the same project, a software interface was developed for setting user parameters and individual jobs. The interface provides detailed information of the laser path and physical variables, which can be used for off-line or in-line monitoring and control and provides a model for process simulation. It is also possible to view the entire laser path and the detail of each individual trait, and to generate a file containing structured data as a FEM model, for setting boundary conditions and process parameters. The algorithms are included in the integrated platform for AM developed by ITACAe, named AMTOP®. Starting from geometrical, functional and structural product specifications, the platform includes a topological optimization phase, new geometry construction, FEM discretization, FEA validation, optimal orientation and supports creation, so representing a fully integrated product & process engineering workflow

A numerical study on automatic mesh refinement for the simulation of Edney shock-shock interactions

The growing interest in hypersonic flight has motivated several research efforts devoted to the study of shock-shock interactions which can dramatically influence the heat load on different components of hypersonic aircraft. In the present work the use of an adaptive numerical scheme for the study of Edney type IV and type VI interactions is investigated. The numerical approach is based on a discontinuous Galerkin finite element method in which an h-adaptive algorithm is used to automatically refine the mesh in the regions where the most interesting flow structures are present. Several adaptation criteria are investigated. Furthermore, it is shown that the gradients obtained by the internal element reconstruction are not suitable for the computation of indicators because they are strongly affected by the shock capturing stabilisation. An alternative approach, based on the use of gradients obtained from a least-squared reconstruction which involves the neighbouring elements, is proposed

Knowledge and practices regarding antibiotics use: Findings from a cross-sectional survey among Italian adults

Background and objectives: This study aimed to assess the knowledge on antibiotics and antimicrobial resistance (AMR) and the antibiotic use among the general public in Southern Italy and to analyze whether sociodemographic characteristics could be associated with poor knowledge and improper practices.Methodology: From March to November 2019, a face-to-face interview was conducted with adult subjects attending the waiting room of 27 randomly selected general practitioners (GPs) in Southern Italy. The questionnaire covered sociodemographic characteristics, knowledge on antibiotics and AMR and practices regarding the consumption of and self-medication with antibiotics.Results: The response rate was 89.7%. In the sample, 29.2% thought that antibiotics are effective for viral infections, and 49.5% correctly recognized the definition of AMR. Predictors of good knowledge about antibiotics and AMR were female gender and a higher education level. Almost half of the respondents had used antibiotics in the previous year and 23.6% took antibiotics to treat a common cold and/or fever. Among participants, 25.5% reported to have bought antibiotics without a prescription, and 30.6% were classified as antibiotic self-medication users. Use of antibiotics in the previous 12 months and having taken an antibiotic after a phone consultation with the GP were positively associated with both antibiotic use for a common cold and/or fever and self-medication with antibiotics.Conclusions and implications: The findings of this study highlighted a considerable antibiotic consumption in the adult population of Southern Italy together with misconceptions regarding the correct indication for antibiotic use that could foster indiscriminate antibiotic use. Lay Summary: The findings of this study highlighted a considerable antibiotic consumption in the adult Italian population together with misconceptions regarding the correct indication for antibiotic use that could foster indiscriminate antibiotic use. Almost a quarter of the respondents took antibiotics to treat a common cold and/or fever and reported to have bought antibiotics without a prescription

Quality in perinatal care: applying performance measurement using joint commission on accreditation of healthcare organizations indicators in Italy

Abstract Background Maternal and child health are internationally considered to be among the best measures for assessing health-care quality. The study was carried out with the following aims: 1) to assess the quality of perinatal care (PC) by measuring the frequencies of the five PC indicators developed by the Joint Commission on Accreditation of Healthcare Organizations (JCAHO) and comparing results with international standards; 2) to examine whether maternal, pregnancy care and neonatal characteristics could be factors associated with the quality of perinatal care hospital performance, measured through these indicators. Methods We retrospectively reviewed medical charts of women over the age of 18 who experienced delivery in Gynecology/obstetrics wards between January–December 2016, and those of their newborns hospitalized in the Neonatology or Neonatal Intensive Care Unit (NICU) of a public non-teaching hospital in Catanzaro (Italy). Indicators were calculated according to the methodology specified in the manual for JCAHO measures. Univariate and multivariate analyses were performed to test the independent association of maternal, pregnancy care and neonatal characteristics on the adherence to JCAHO PC indicators. Results The records of 1943 women and 1974 newborns were identified and reviewed in order to be included in at least one of the PC indicators. Elective/early-term delivery, was performed in 27.6% of eligible women, far from the recommended goal (0%); cesarean section in nulliparous women with a term, singleton baby in a vertex position exceeded the suggested target of < 24% and the adherence to antenatal steroids administration was suboptimal (87%). Results of the exclusive breastfeeding indicator achieved a better performance (81%) and compliance with the PC-04 indicator was satisfactory with only 0.4% healthcare-associated bloodstream infection developed in eligible newborns. Conclusions This is the first study performed in Italy that has evaluated the quality of PC by using all the five JCAHO indicators. The application of this feasible set of indicators allowed us to measure several aspects of PC for which there is no standardized monitoring system in Italy. Our findings revealed significant deficiencies in the adherence to recommended processes of PC and suggest that there is still substantial work required to improve care

Antibiotic prescriptions to adults with acute respiratory tract infections by Italian general practitioners

Aida Bianco, Rosa Papadopoli, Valentina Mascaro, Claudia Pileggi, Maria Pavia Department of Health Sciences, Medical School, University of Catanzaro &ldquo;Magna Gr&aelig;cia&rdquo;, Catanzaro,&nbsp;Italy Purpose: The overuse, misuse, and underuse of antimicrobial agents often lead to the spread of antibiotic-resistant microorganisms. The aim of our study was to describe the pattern of antibiotic prescriptions for acute respiratory tract infections (RTIs) among the adult population and the factors associated with antibiotic prescribing. Patients and methods: The study involved patients who visited a general practitioner with suspected acute RTI. Patients with diagnosis of acute sinusitis, acute pharyngitis, acute bronchitis, and influenza were included in the study. We evaluated the presence of an indication for antibiotic therapy for selected diseases according to international guidelines. The appropriateness of any prescribed molecule was also evaluated. Results: A total of 1,979 cases of acute RTIs were included: 1,196 (60.4%) pharyngitis, 359 (18.2%) bronchitis, 234 (11.8%) influenza, and 190 (9.6%) sinusitis. An antibiotic prescription was given in 67.3% of the consultations and was not indicated by the guidelines in 66.5% of the total RTIs. Macrolides were the most frequently prescribed antibiotics accounting for 32.5% of all those prescribed, followed by amoxicillin with clavulanic acid (31.1%) and fluoroquinolones (14.2%). The highest overprescription was associated with pharyngitis (65.9%) and the lowest with influenza (4.9%). A throat swab was performed only in 11 of all the patients with a diagnosis of acute pharyngitis. Conclusion: The present study showed a very high frequency of nonevidence-based prescription of antibiotics at the primary care level. Future improvement programs should focus on development of evidence-based guidelines, access to postgraduate training, and better availability of diagnostic tools. Keywords: antibiotic prescribing, appropriateness, general practitioner, primary care, respiratory tract infections, Ital