SPHINGOLIPID SIGNALING AND DISEASE: THE KEY ROLE OF CERAMIDE TRAFFIC IN CELL FATE REGULATION

Cancer and diabetes are among the most common diseases in western societies. Sphingolipids, a class of lipids ubiquitously present in eukaryotic membranes, play a key role in the regulation of different signal transduction pathways involved in the modulation of many cellular functions [1, 2]. The past two decades have seen increased interest in the bioactive sphingolipids ceramide (Cer) and sphingosine-1 phosphate (S1P). Cer, a central molecule of sphingolipid metabolism, is involved in the control of many cell-stress responses, including growth arrest, senescence and cell death [3]. On the other hand, several studies have proposed a crucial role of S1P in cell growth and survival, cell migration, angiogenesis, and inflamma\uaction [3]. Thus, Cer and S1P can differentially regulate cell death and survival by controlling opposing signaling pathways [4]. So far, a large amount of studies has clarified the complexity of the interplay between S1P, Cer and sphingolipid metabolism and these implications in the etiology of several human diseases. Effectively, deregulation of sphingolipid metabolism is implicated in numerous diseases, and accumulating evidence has shown a clear indication that sphingolipids have important role in the pathogenesis of diabetes and cancer [5]. Very recently, it is emerging a pivotal role of Cer traffic from the Endoplasmic Reticulum (ER) to the Golgi apparatus in the regulation of sphingolipid metabolism. In fact, Cer transport is a highly regulated step in the sphingolipid biosynthesis. Two main mechanisms are involved in Cer transport from ER to the Golgi apparatus: a protein-mediated transport, operated by CERT [6, 7] and a CERT-independent vesicular transport. Moreover, several studies reported that ER is a critical intracellular organelle involved in the control of cell fate [8, 9]. In this light, Cer accumulation in the ER appears to be a key element in the promotion of cell death in different human diseases, as well as in glioblastoma and diabetes. In light of these findings, in my Ph.D. course I wanted to evaluate in cellular models of glioblastoma (GBM) and type 2 diabetes (T2D) the role of Cer transport in the regulation of cell fate. Part 1: Glucolipotoxicity impairs ceramide flow from the ER to the Golgi apparatus in INS-1 \uf062-cells T2D is the most common form of diabetes characterized by insulin resistance and \u3b2-cell dysfunction. The etiology of T2D is not well established but it is know that loss of insulin secretion is directly linked to a loss of function and death of pancreatic \u3b2-cells [10]. Glucolipotoxicity is a condition determined by the combined action of elevated glucose and free fatty acids (FFAs) levels that exerts deleterious effects on pancreatic \uf062-cell function and survival. Several mechanisms have been proposed for glucolipotoxicity-induced \u3b2-cell dysfunction, and, among them, ER stress and elevations of the proapoptotic sphingolipid Cer appear to play key roles. Moreover, Cer accumulation due to glucolipotoxicity can be associated to the induction of ER stress. As in other cells, in the INS-1 insulinoma cell line, two main mechanisms are involved in Cer transport from the ER to the Golgi apparatus: a CERT-mediated transport [6, 11], and a CERT-independent vesicular transport. Glucolipotoxic conditions, obtained with 0.4 mM palmitate and 30 mM glucose administration, strongly affected both of them by reducing CERT protein synthesis and activity, and the rate of Cer vesicular traffic, thus promoting Cer accumulation in the ER. [11, 12]. The two Cer transport mechanisms are effectively involved in the control of sphingolipid metabolism, participating in the regulation of Cer accumulation in the ER involved in pancreatic \uf062-cell function and death during T2D. Part 2: The stimulation of Cer traffic from the ER to the Golgi apparatus S1P-dependent as a survival factor in T98G glioma cells. GBM is the most common malignant primary brain tumor in adults and one of the most lethal human cancers [13]. S1P and Cer have emerged as bio-effectors molecules, involved in both glioblastoma development and resistance [3, 14-17]. Several lines of evidence indicated that, different anticancer drugs, including etoposide, exert cytotoxic effects also promoting accumulation of Cer in the ER. Thus, the transport of Cer from ER to Golgi apparatus could be represent a key pathway for limiting Cer accumulation in the ER and escape from cell death. Moreover, we recently demonstrated that vesicular-mediated Cer transport is positively regulated by the pro-survival pathway phosphatidylinositol 3-phosphate kinase (PI3K)/Akt [18], known effectors of extracellular S1P. In T98G glioma cells, S1P treatment was able to increase vesicular Cer transport from the ER to the Golgi apparatus by PI3K/Akt pathway activation, inducing Cer conversion to SM and glycosphingolipids (GSL). In these cells, the cytotoxic Cer accumulation in the ER promoted by the anticancer drug etoposide was strongly reduced after S1P administration. S1P, by enhancing Cer traffic, act as pro-oncogenic factor, favoring both the reduction of the pro-apoptotic Cer at the ER and the synthesis of complex sphingolipids. By this mechanism S1P also stimulates the generation of new membranes, functional to cell growth and consequently to the tumor survival and progression

Phosphatidylinositol 3-Kinase/AKT pathway regulates the endoplasmic reticulum to Golgi traffic of ceramide in glioma cells : a link between lipid signaling pathways involved in the control of cell survival

Different lines of evidence indicate that both aberrant activation of the phosphatidylinositol 3-OH kinase (PI3K)/Akt survival pathway and down-regulation of the death mediator ceramide play a critical role in the aggressive behavior, apoptosis resistance, and adverse clinical outcome of glioblastoma multiforme. Furthermore, the inhibition of the PI3K/Akt pathway and the up-regulation of ceramide have been found functional to the activity of many cytotoxic treatments against glioma cell lines and glioblastomas as well. A reciprocal control between PI3K/Akt and ceramide signaling in glioma cell survival/death is suggested by data demonstrating a protective role of PI3K/Akt on ceramide-induced cell death in glial cells. In this study we investigated the role of the PI3K/Akt pathway in the regulation of the ceramide metabolism in C6 glioma cells, a cell line in which the PI3K/Akt pathway is constitutively activated. Metabolic experiments performed with different radioactive metabolic precursors of sphingolipids and microscopy studies with fluorescent ceramides demonstrated that the chemical inhibition of PI3K and the transfection with a dominant negative Akt strongly inhibited ceramide utilization for the biosynthesis of complex sphingolipids by controlling the endoplasmic reticulum (ER) to Golgi vesicular transport of ceramide. These findings constitute the first evidence for a PI3K/Akt-dependent regulation of vesicle-mediated movements of ceramide in the ER-Golgi district. Moreover, the findings also suggest the activation of the PI3K/Akt pathway as crucial to coordinate the biosynthesis of membrane complex sphingolipids with cell proliferation and growth and/or to maintain low ceramide levels, especially as concerns those treatments that promote ceramide biosynthesis in the ER

A TWO-DIMENSIONAL THERMODYNAMIC MODEL TO PREDICT HEART THERMAL RESPONSE DURING OPEN CHEST PROCEDURES

In this work, the temperature distribution of the heart in an open chest surgery scenario is studied. It is also evaluated the cardiac thermal effects of the injection of a cooling liquid in the aorta root, which is used in infrared thermography. The finite element method was used to develop a model that predicts the temperature distribution modification in a 2-dimensional slice of the heart. This thermodynamic model allows the computational simulation of the thermal cardiac response to open chest procedures, which are required by cardiac surgery. The influence of several operating parameters (e.g., coronary flow rate, temperature) on the resulting thermal distribution is analyzed. Therefore, this analysis allows the identification of parameters that could be controlled to minimize the loss of energy, and consequently, avoiding the hazardous thermal distribution that could put the heart in danger during cardiac surgery

INFRARED IMAGING AND COMPUTERIZED TOMOGRAPHY IN BREAST CANCER: CASE STUDY

This work presents a case study of a 75-year-old woman breast withcancer. The investigation process used infrared image, mammography,computerized tomography (CT) and ultrasound guided biopsy toassess, stage and final diagnostic of the tumor. Each one of theseevaluations brings an isolated piece of information that results in thecorrect diagnostic, and treatment. As early diagnostic of breast cancergoes towards improvement in diagnostics and better therapeutics, it isreasonable to state that breast cancer diagnostics must be achieved asearly as possible. An association between infrared image abnormalitiesand computerized tomography is acknowledged and is assumed that acorrelation could exist. The technical literature demonstrated thattumor depth could be inferred from infrared images, but criticalinformation such as breast perfusion for accurate predictions are notavailable yet. Considering that a mathematical model could modelbreast perfusion, this study proposes that tumor morphology and depthin breast cancer could be adequately determined using mathematicalmodeling, infrared imaging, and computerized tomography incomplementary actions

SURGERY BY INFRARED VISION

This paper is about the development, uses and advantages of infrared thermography. Its principles had already been used by old civilizations as a manner of diseases diagnosis. The discovery of the infrared waves and its heating power happened on 1800, but its use in modern Medicine as a diagnosis method was only possible because of the scientific and technological development demanded by the Great Wars, in the 20th century. Here the authors present some news and promising surgery applications

DEVELOPMENT OF AN AIR HEATING AND HUMIDIFYING SYSTEM FOR MECHANICAL VENTILATION OF INTENSIVE CARE UNIT PATIENTS

The mechanical ventilators, also named artificial ventilators, are devices used in Intensive Care Units (ICU) for clinically critical patients. The equipment provides artificial breathing for the patient, creating favorable conditions for the patient recovery. It works as a system that is connected to the electric grid and two other networks, one supplies compressed air and the other supplies pure oxygen. Inside the equipment the gases are mixed, and then warmed up and humidified to be finally delivered to the patient. The existing system for treatment of the mixture is not effective, since it delivers the breathing air to the patient out of the normal physiology recommended range, i.e., temperature ~32-34°C and relative humidity ~80-95%. The objective of this work was to develop a new air heating and humidifying system for mechanical ventilation. First, it was built an air feeding system, composed by a compressor and an electronically controlled valve to simulate an actual mechanical ventilation system in the laboratory. A device for conditioning the air (temperature and humidity) was conceived and built for treating the air feeding system output air. The results of the laboratory measurements show that the developed equipment was capable of making the breathing air to reach the recommended ranges by the normal physiology

A proposito di un caso di ricostruzione della mano mediante uso di sostituto dermico

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Soluble trivalent engagers redirect cytolytic T cell activity toward tumor endothelial marker 1.

Tumor endothelial marker 1 (TEM1) is an emerging cancer target with a unique dual expression profile. First, TEM1 is expressed in the stroma and neo-vasculature of many human carcinomas but is largely absent from healthy adult tissues. Second, TEM1 is expressed by tumor cells of mesenchymal origin, notably sarcoma. Here, we present two fully human anti-TEM1 single-chain variable fragment (scFv) reagents, namely, 1C1m and 7G22, that recognize distinct regions of the extracellular domain and possess substantially different affinities. In contrast to other, well-described anti-TEM1 binders, these fragments confer cytolytic activity when expressed as 2 <sup>nd</sup> generation chimeric antigen receptors (CARs). Moreover, both molecules selectively redirect human T cell effector functions toward TEM1 <sup>+</sup> tumor cells when incorporated into experimental soluble bispecific trivalent engagers that we term TriloBiTEs (tBs). Furthermore, systemic delivery of 1C1m-tB prevents the establishment of Ewing sarcoma tumors in a xenograft model. Our observations confirm TEM1 as a promising target for cancer immunotherapy and illustrate the prospective translational potential of certain scFv-based reagents

Detection of Synaptic Proteins in Microglia by Flow Cytometry.

A growing body of evidence indicates that microglia actively remove synapses in vivo, thereby playing a key role in synaptic refinement and modulation of brain connectivity. This phenomenon was mainly investigated in immunofluorescence staining and confocal microscopy. However, a quantification of synaptic material in microglia using these techniques is extremely time-consuming and labor-intensive. To address this issue, we aimed to quantify synaptic proteins in microglia using flow cytometry. With this approach, we first showed that microglia from the healthy adult mouse brain contain a detectable level of VGLUT1 protein. Next, we found more than two-fold increased VGLUT1 immunoreactivity in microglia from the developing brain (P15) as compared to adult microglia. These data indicate that microglia-mediated synaptic pruning mostly occurs during the brain developmental period. We then quantified the VGLUT1 staining in microglia in two transgenic models characterized by pathological microglia-mediated synaptic pruning. In the 5xFAD mouse model of Alzheimer's disease (AD) microglia exhibited a significant increase in VGLUT1 immunoreactivity before the onset of amyloid pathology. Moreover, conditional deletion of TDP-43 in microglia, which causes a hyper-phagocytic phenotype associated with synaptic loss, also resulted in increased VGLUT1 immunoreactivity within microglia. This work provides a quantitative assessment of synaptic proteins in microglia, under homeostasis, and in mouse models of disease