Involvement of formyl peptide receptors in the stimulatory effect of crotoxin on macrophages co-cultivated with tumour cells

Crotoxin (CTX) is the main neurotoxic component of Crotalus durissus terrificus snake venom. It inhibits tumour growth and modulates the function of macrophages, which are essential cells in the tumour microenvironment. The present study investigated the effect of CTX on the secretory activity of monocultured macrophages and macrophages co-cultivated with LLC-WRC 256 cells. The effect of the macrophage secretory activities on tumour cell proliferation was also evaluated. Macrophages pre-treated with CTX (0.3 μg/mL) for 2 h were co-cultivated with LLC-WRC 256 cells, and the secretory activity of the macrophages was determined after 12, 24 and 48 h. The co-cultivation of CTX-treated macrophages with the tumour cells caused a 20% reduction in tumour cell proliferation. The production of both H2O2 and NO was increased by 41% and 29% after 24 or 48 h of co-cultivation, respectively, compared to the values for the co-cultures of macrophages of control. The level of secreted IL-1β increased by 3.7- and 3.2-fold after 12 h and 24 h of co-cultivation, respectively. Moreover, an increased level of LXA4 (25%) was observed after 24 h of co-cultivation, and a 2.3- and 2.1-fold increased level of 15-epi-LXA4 was observed after 24 h and 48 h, respectively. Boc-2, a selective antagonist of formyl peptide receptors, blocked both the stimulatory effect of CTX on the macrophage secretory activity and the inhibitory effect of these cells on tumour cell proliferation. Taken together, these results indicate that CTX enhanced the secretory activity of macrophages, which may contribute to the antitumour activity of these cells, and that activation of formyl peptide receptors appears to play a major role in this effect.

Differential effects of undernourishment on the differentiation and maturation of rat enteric neurons

The colocalization, number, and size of various classes of enteric neurons immunoreactive (IR) for the purinergic P2X2 and P2X7 receptors (P2X2R, P2X7R) were analyzed in the myenteric and submucosal plexuses of control, undernourished, and re-fed rats. Pregnant rats were exposed to undernourishment (protein-deprivation) or fed a control diet, and their offspring comprised the following experimental groups: rats exposed to a normal diet throughout gestation until postnatal day (P)42, rats protein-deprived throughout gestation and until P42, and rats protein-deprived throughout gestation until P21 and then given a normal diet until P42. Immunohistochemistry was performed on the myenteric and submucosal plexuses to evaluate immunoreactivity for P2X2R, P2X7R, nitric oxide synthase (NOS), choline acetyltransferase (ChAT), calbindin, and calretinin. Double-immunohistochemistry of the myenteric and submucosal plexuses demonstrated that 100% of NOS-IR, calbindin-IR, calretinin-IR, and ChAT-IR neurons in all groups also expressed P2X2R and P2X7R. Neuronal density increased in the myenteric and submucosal plexuses of undernourished rats compared with controls. The average size (profile area) of some types of neurons in the myenteric and submucosal plexuses was smaller in the undernourished than in the control animals. These changes appeared to be reversible, as animals initially undernourished but then fed a normal diet at P21 (re-feeding) were similar to controls. Thus, P2X2R and P2X7R are present in NOS-positive inhibitory neurons, calbindin- and calretinin-positive intrinsic primary afferent neurons, cholinergic secretomotor neurons, and vasomotor neurons in rats. Alterations in these neurons during undernourishment are reversible following re-feeding

In vivo assessment of specific cytotoxic T lymphocyte killing

The direct killing of target cells by cytotoxic T lymphocytes (CTLs) plays a fundamental role in protective immunity to viral, bacterial, protozoan and fungi infections, as well as to tumor cells. In vivo cytotoxic assays take into account the interaction of target and effector cells in the context of the proper microenvironment making the analysis biologically more relevant than in vitro cytotoxic assays. Thus, the development, improvement and validation of in vivo methods are necessary in view of the importance of the results they may provide. We describe and discuss in this manuscript a method to evaluate in vivo specific cytotoxic T lymphocyte killing. We used as model system mice immunized with human recombinant replication-deficient adenovirus 5 (HAd5) containing different transgenes as the trigger of a CTL-mediated immune response. To these mice, we adoptively transferred syngeneic cells labeled with different vital fluorescent dyes. Donor cells were pulsed (target) or not (control non-target) with distinct CD8 T-cell epitopes, mixed in a 1:1 ratio and injected i.v. into immunized or non-immunized recipient mice. After 18-24h, spleen cells are collected and analysed by flow cytometry. A deviation from the 1:1 ratio of control and target cell populations indicates antigen specific lysis of target cells

Development of quantitative methods for sustainability assessment in process industry

The increasing aversion to technological risks of the society requires the development of inherently safer and environmentally friendlier processes, besides assuring the economic competitiveness of the industrial activities. The different forms of impact (e.g. environmental, economic and societal) are frequently characterized by conflicting reduction strategies and must be holistically taken into account in order to identify the optimal solutions in process design. Though the literature reports an extensive discussion of strategies and specific principles, quantitative assessment tools are required to identify the marginal improvements in alternative design options, to allow the trade-off among contradictory aspects and to prevent the “risk shift”. In the present work a set of integrated quantitative tools for design assessment (i.e. design support system) was developed. The tools were specifically dedicated to the implementation of sustainability and inherent safety in process and plant design activities, with respect to chemical and industrial processes in which substances dangerous for humans and environment are used or stored. The tools were mainly devoted to the application in the stages of “conceptual” and “basic design”, when the project is still open to changes (due to the large number of degrees of freedom) which may comprise of strategies to improve sustainability and inherent safety. The set of developed tools includes different phases of the design activities, all through the lifecycle of a project (inventories, process flow diagrams, preliminary plant lay-out plans). The development of such tools gives a substantial contribution to fill the present gap in the availability of sound supports for implementing safety and sustainability in early phases of process design. The proposed decision support system was based on the development of a set of leading key performance indicators (KPIs), which ensure the assessment of economic, societal and environmental impacts of a process (i.e. sustainability profile). The KPIs were based on impact models (also complex), but are easy and swift in the practical application. Their full evaluation is possible also starting from the limited data available during early process design. Innovative reference criteria were developed to compare and aggregate the KPIs on the basis of the actual sitespecific impact burden and the sustainability policy. Particular attention was devoted to the development of reliable criteria and tools for the assessment of inherent safety in different stages of the project lifecycle. The assessment follows an innovative approach in the analysis of inherent safety, based on both the calculation of the expected consequences of potential accidents and the evaluation of the hazards related to equipment. The methodology overrides several problems present in the previous methods proposed for quantitative inherent safety assessment (use of arbitrary indexes, subjective judgement, build-in assumptions, etc.). A specific procedure was defined for the assessment of the hazards related to the formations of undesired substances in chemical systems undergoing “out of control” conditions. In the assessment of layout plans, “ad hoc” tools were developed to account for the hazard of domino escalations and the safety economics. The effectiveness and value of the tools were demonstrated by the application to a large number of case studies concerning different kinds of design activities (choice of materials, design of the process, of the plant, of the layout) and different types of processes/plants (chemical industry, storage facilities, waste disposal). An experimental survey (analysis of the thermal stability of isomers of nitrobenzaldehyde) provided the input data necessary to demonstrate the method for inherent safety assessment of materials.

Aportaciones al control inverso con modelo de referencia basado en lógica borrosa, redes neuronales y algoritmos genéticos

Programa de doctorado: Ingeniería de Telecomunicación Avanzada.

Development of a field method to assess root parasites of woody plants - ecological analyses of the grapevine-phylloxera interaction

The development and the growth of plants is strongly affected by the interactions between roots, rootrnassociated organisms and rhizosphere communities. Methods to assess such interactions are hardly torndevelop particularly in perennial and woody plants, due to their complex root system structure and theirrntemporal change in physiology patterns. In this respect, grape root systems are not investigated veryrnwell. The aim of the present work was the development of a method to assess and predict interactionsrnat the root system of rootstocks (Vitis berlandieri x Vitis riparia) in field. To achieve this aim, grapernphylloxera (Daktulosphaira vitifoliae Fitch, Hemiptera, Aphidoidea) was used as a graperoot parasitizingrnmodel.rnTo develop the methodical approach, a longt-term trial (2006-2009) was arranged on a commercial usedrnvineyard in Geisenheim/Rheingau. All 2 to 8 weeks the top most 20 cm of soil under the foliage wallrnwere investigated and root material was extracted (n=8-10). To include temporal, spatial and cultivarrnspecific root system dynamics, the extracted root material was analyzed digitally on the morphologicalrnproperties. The grape phylloxera population was quantified and characterized visually on base of theirrnlarvalstages (oviparous, non oviparous and winged preliminary stages). Infection patches (nodosities)rnwere characterized visually as well, partly supported by digital root color analyses. Due to the knownrneffects of fungal endophytes on the vitality of grape phylloxera infested grapevines, fungal endophytesrnwere isolated from nodosity and root tissue and characterized (morphotypes) afterwards. Further abioticrnand biotic soil conditions of the vineyards were assessed. The temporal, spatial and cultivar specificrnsensitivity of single parameters were analyzed by omnibus tests (ANOVAs) and adjacent post-hoc tests.rnThe relations between different parameters were analyzed by multiple regression models.rnQuantitative parameters to assess the degeneration of nodosity, the development nodosity attachedrnroots and to differentiate between nodosities and other root swellings in field were developed. Significantrndifferences were shown between root dynamic including parameters and root dynamic ignoringrnparameters. Regarding the description of grape phylloxera population and root system dynamic, thernmethod showed a high temporal, spatial and cultivar specific sensitivity. Further, specific differencesrncould be shown in the frequency of endophyte morphotypes between root and nodosity tissue as wellrnas between cultivars. Degeneration of nodosities as well as nodosity occupation rates could be relatedrnto the calculated abundances of grape phylloxera population. Further ecological questions consideringrngrape root development (e.g. relation between moisture and root development) and grape phylloxerarnpopulation development (e.g. relation between temperature and population structure) could be answeredrnfor field conditions.rnGenerally, the presented work provides an approach to evaluate vitality of grape root systems. Thisrnapproach can be useful, considering the development of control strategies against soilborne pests inrnviticulture (e.g. grape phylloxera, Sorospheara viticola, Roesleria subterranea (Weinm.) Redhaed) as well as considering the evaluation of integrated management systems in viticulture.

Data management and data analysis in the large European projects GEHA (GEnetics of Healthy Aging) and NU-AGE (NUtrition and AGEing): a bioinformatic approach

The aging process is characterized by the progressive fitness decline experienced at all the levels of physiological organization, from single molecules up to the whole organism. Studies confirmed inflammaging, a chronic low-level inflammation, as a deeply intertwined partner of the aging process, which may provide the “common soil” upon which age-related diseases develop and flourish. Thus, albeit inflammation per se represents a physiological process, it can rapidly become detrimental if it goes out of control causing an excess of local and systemic inflammatory response, a striking risk factor for the elderly population. Developing interventions to counteract the establishment of this state is thus a top priority. Diet, among other factors, represents a good candidate to regulate inflammation. Building on top of this consideration, the EU project NU-AGE is now trying to assess if a Mediterranean diet, fortified for the elderly population needs, may help in modulating inflammaging. To do so, NU-AGE enrolled a total of 1250 subjects, half of which followed a 1-year long diet, and characterized them by mean of the most advanced –omics and non –omics analyses. The aim of this thesis was the development of a solid data management pipeline able to efficiently cope with the results of these assays, which are now flowing inside a centralized database, ready to be used to test the most disparate scientific hypotheses. At the same time, the work hereby described encompasses the data analysis of the GEHA project, which was focused on identifying the genetic determinants of longevity, with a particular focus on developing and applying a method for detecting epistatic interactions in human mtDNA. Eventually, in an effort to propel the adoption of NGS technologies in everyday pipeline, we developed a NGS variant calling pipeline devoted to solve all the sequencing-related issues of the mtDNA.

Non Invasive Tools for Early Detection of Autism Spectrum Disorders

Autism Spectrum Disorders (ASDs) describe a set of neurodevelopmental disorders. ASD represents a significant public health problem. Currently, ASDs are not diagnosed before the 2nd year of life but an early identification of ASDs would be crucial as interventions are much more effective than specific therapies starting in later childhood. To this aim, cheap an contact-less automatic approaches recently aroused great clinical interest. Among them, the cry and the movements of the newborn, both involving the central nervous system, are proposed as possible indicators of neurological disorders. This PhD work is a first step towards solving this challenging problem. An integrated system is presented enabling the recording of audio (crying) and video (movements) data of the newborn, their automatic analysis with innovative techniques for the extraction of clinically relevant parameters and their classification with data mining techniques. New robust algorithms were developed for the selection of the voiced parts of the cry signal, the estimation of acoustic parameters based on the wavelet transform and the analysis of the infant’s general movements (GMs) through a new body model for segmentation and 2D reconstruction. In addition to a thorough literature review this thesis presents the state of the art on these topics that shows that no studies exist concerning normative ranges for newborn infant cry in the first 6 months of life nor the correlation between cry and movements. Through the new automatic methods a population of control infants (“low-risk”, LR) was compared to a group of “high-risk” (HR) infants, i.e. siblings of children already diagnosed with ASD. A subset of LR infants clinically diagnosed as newborns with Typical Development (TD) and one affected by ASD were compared. The results show that the selected acoustic parameters allow good differentiation between the two groups. This result provides new perspectives both diagnostic and therapeutic.

Effects of maternal PETN treatment of spontaneously hypertensive rats on blood pressure in the offspring

Pentaerithrityltetranitrat (PETN) ist ein organisches Nitrat und wird in der Klinik zur Behandlung der Angina Pectoris eingesetzt. PETN hat, wenn direkt verabreicht, kaum Wirkung auf den Blutdruck. Diese Arbeit wurde konzipiert, um einen potentiellen „perinatalen Programmierung“-Effekt von PETN in spontan-hypertensiven Ratten (SHR), einem Rattenmodel der genetischen Hypertonie, zu testen. Die F0-Elterntiere wurden mit PETN (50 mg/kg/Tag) während der Schwangerschaft und der Laktation behandelt; die F1-Nachkommen bekamen nach der Ablaktation normales Haltungsfutter. Der Blutdruck wurde an den Nachkommen vom 3. Monat bis zum 8. Monat nach der Geburt gemessen. Maternale PETN-Behandlung hatte kaum Wirkung auf den Blutdruck in den männlichen SHR-Nachkommen. Dagegen zeigten die weiblichen Nachkommen der PETN-Behandlungsgruppe eine persistente Reduktion des Blutdrucks. Der systolische Blutdruck war in den weiblichen Nachkommen in der PETN-Gruppe etwa 13 mmHg niedriger im 4. Monat und etwa 10 mmHg niedriger im 8. Monat als in den Kontrolltieren. Dieser lang-anhaltende Effekt ging mit einer substanziellen Änderung der Genexpression einher, die auch beim 8. Monat noch nachzuweisen war. In den Aorten der weiblichen F1-Nachkommen wurde Veränderungen an Genexpression der α-adrenergen Rezeptoren sowie Endothelin-Rezeptoren festgestellt, die aber funktionell von minimaler Bedeutung für die PETN-Wirkung waren. Hingegen war eine klare Rolle des StickstoffmoNOXid (NO) zu sehen. Maternale PETN-Behandlung führte zur Heraufregulation der endothelialen NO-Synthase (eNOS) und der GTP-Cyclohydrolase I (GCH-1). GCH-1 ist für die Biosynthese des Tetrahydrobiopterins, eines essentiellen eNOS-Kofaktors, entscheidend, und dadurch auch für die eNOS-Funktionalität. Zusätzlich wurden auch anti-oxidative Enzyme wie die mitochondriale Superoxid-Dismutase (SOD2), die Glutathion-Peroxidase 1 (GPx1) und die Hem-Oxygenase 1 (HO-1) heraufreguliert, und die Superoxid-produzierende NADPH-Oxidase NOX1 herunterreguliert. Dies kann zur Verminderung vom oxidativen Stress und Erhöhung der NO-Bioverfügbarkeit führen. Letztlich wurde auch ~ 74 ~ die Sirtuin 1 (SIRT1) durch maternale PETN-Behandlung heraufreguliert, die auch zur Heraufregulation der SOD2, GPx1 und eNOS beitragen kann. Im Organbad-Experiment wurde die Acetylcholin-induzierte, Endothel-abhängige Vasodilatation in der Aorta der weiblichen Nachkommen der PETN-Gruppe verstärkt. Diese verbesserte Endothelfunktion, was vermutlich aus der Genexpressionsänderung resultiert, stellt sehr wahrscheinlich einen Schlüsselmechanismus der Blutdrucksenkung in den Nachkommen der PETN-behandelten F0-Tiere dar.

Joint lavage for osteoarthritis of the knee

Background Osteoarthritis is the most common form of joint disorder and a leading cause of pain and physical disability. Observational studies suggested a benefit for joint lavage, but recent, sham-controlled trials yielded conflicting results, suggesting joint lavage not to be effective. Objectives To compare joint lavage with sham intervention, placebo or non-intervention control in terms of effects on pain, function and safety outcomes in patients with knee osteoarthritis. Search methods We searched CENTRAL, MEDLINE, EMBASE, and CINAHL up to 3 August 2009, checked conference proceedings, reference lists, and contacted authors. Selection criteria We included studies if they were randomised or quasi-randomised trials that compared arthroscopic and non-arthroscopic joint lavage with a control intervention in patients with osteoarthritis of the knee. We did not apply any language restrictions. Data collection and analysis Two independent review authors extracted data using standardised forms. We contacted investigators to obtain missing outcome information. We calculated standardised mean differences (SMDs) for pain and function, and risk ratios for safety outcomes. We combined trials using inverse-variance random-effects meta-analysis. Main results We included seven trials with 567 patients. Three trials examined arthroscopic joint lavage, two non-arthroscopic joint lavage and two tidal irrigation. The methodological quality and the quality of reporting was poor and we identified a moderate to large degree of heterogeneity among the trials (I2 = 65%). We found little evidence for a benefit of joint lavage in terms of pain relief at three months (SMD -0.11, 95% CI -0.42 to 0.21), corresponding to a difference in pain scores between joint lavage and control of 0.3 cm on a 10-cm visual analogue scale (VAS). Results for improvement in function at three months were similar (SMD -0.10, 95% CI -0.30 to 0.11), corresponding to a difference in function scores between joint lavage and control of 0.2 cm on a WOMAC disability sub-scale from 0 to 10. For pain, estimates of effect sizes varied to some degree depending on the type of lavage, but this variation was likely to be explained by differences in the credibility of control interventions: trials using sham interventions to closely mimic the process of joint lavage showed a null-effect. Reporting on adverse events and drop out rates was unsatisfactory, and we were unable to draw conclusions for these secondary outcomes. Authors' conclusions Joint lavage does not result in a relevant benefit for patients with knee osteoarthritis in terms of pain relief or improvement of function.

The effect of alternative neuronal differentiation pathways on PC12 cell adhesion and neurite alignment to nanogratings

During development and regeneration of the mammalian nervous system, directional signals guide differentiating neurons toward their targets. Soluble neurotrophic molecules encode for preferential direction over long distances while the local topography is read by cells in a process requiring the establishment of focal adhesions. The mutual interaction between overlapping molecular and topographical signals introduces an additional level of control to this picture. The role of the substrate topography was demonstrated exploiting nanotechnologies to generate biomimetic scaffolds that control both the polarity of differentiating neurons and the alignment of their neurites. Here PC12 cells contacting nanogratings made of copolymer 2-norbornene ethylene (COC), were alternatively stimulated with Nerve Growth Factor, Forskolin, and 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic (8CPT-2Me-cAMP) or with a combination of them. Topographical guidance was differently modulated by the alternative stimulation protocols tested. Forskolin stimulation reduced the efficiency of neurite alignment to the nanogratings. This effect was linked to the inhibition of focal adhesion maturation. Modulation of neurite alignment and focal adhesion maturation upon Forskolin stimulation depended on the activation of the MEK/ERK signaling but were PkA independent. Altogether, our results demonstrate that topographical guidance in PC12 cells is modulated by the activation of alternative neuronal differentiation pathways.