In-vitro-Untersuchungen zu antifibrotischen Wirkungen von β-Adrenozeptoragonisten und Glucocorticoiden in primären equinen Bronchialfibroblasten: In-vitro-Untersuchungen zu antifibrotischen Wirkungenvon β-Adrenozeptoragonisten und Glucocorticoidenin primären equinen Bronchialfibroblasten

Einleitung: Die Pathogenese chronisch entzündlicher Atemwegserkrankungen ist mit zunehmender Schädigung und fibrotischen Umbauvorgängen und daraus resultierenden Einschränkungen physiologischer Funktionen verbunden. Bei der Recurrent Airway Obstruction (RAO) des Pferdes sind solche strukturellen Veränderungen häufig assoziiert mit Verdickung der Atemwegswand, subepithelialer Fibrose und Obstruktion der Atemwege. Bei RAO besteht die Standardtherapie darin, die Bronchokonstriktion mit β2-Agonisten und die Entzündung mit Glucocorticoiden aufzuhalten. In den letzten Jahrzehnten konnte jedoch gezeigt werden, dass nicht alle Patienten mit der empfohlenen Therapie ausreichend behandelt werden können und neue Ansatzpunkte der Therapie gefunden werden müssen. Daher konzentrieren sich neuere Forschungsarbeiten auf strukturelle Alterationen in den Atemwegen, das sogenannte Airway-Remodelling. Zielsetzung: In der vorliegenden Arbeit sollen zunächst primäre equine Bronchialfibroblasten (EBF) isoliert, charakterisiert und kultiviert werden. Im nächsten Schritt sollen in diesen Zellen die β2-Rezeptorexpression und –eigenschaften charakterisiert werden. Anschließend soll die Eignung dieser Zellen als Zellmodell zur Untersuchung der zellulären Proliferation und Transformation sowie der Regulation der de-novo-Synthese von extrazellulärer Matrix und deren Beeinflussung durch β2-Agonisten und Glucocorticoide allein oder in Kombination sowie TGF-β überprüft werden. Material und Methoden: Mittels enzymatischer Verdauung mit 0,25 % Trypsin werden die EBF aus den Bronchien von 10 gesunden Schlachtpferden isoliert und in DMEM kultiviert. Diese Zellen werden morphologisch, immunzytochemisch und funktionell in deren Eigenschaften in An- oder Abwesenheit von fetalem bovinem Serum (FBS) oder Pferdeserum (HS) charakterisiert. Die Dichte und Subtypverteilung von β-Adrenozeptoren wird mittels Radioligandbindungsstudien mit [125I]-(-)-Iodocyanopindolol in Gegenwart von Rezeptorsubtyp-selektiven β-Antagonisten (β2: ICI 118,551 und β1: CGP 20712A) in EBF untersucht. Der Einfluss von β2-Agonisten auf die Proliferation und Differenzierung der EBF sowie die Kollagensynthese wird mittels [3H]-Thymidineinbauassay, Bestimmung von Gesamtprotein und α-Smooth Muscle Aktin (α-SMA) mit Lowrymethode und Western Blot-Analyse bzw. [3H]-Prolininkorporationsassay ermittelt. Die statistische Signifikanz wird über einen t-Test für verbundene Stichproben oder eine One-Way-ANOVA mit nachfolgendem Dunnett’s Test ermittelt und das Signifikanzniveau wird P ≤ 0,05 festgelegt. Ergebnisse: EBF können im DMEM-Medium nur in Gegenwart von Serum langfristig wachsen und kultiviert werden. In serum-freiem Medium und unter vorübergehendem Serumentzug können EBF nicht anhaften bzw. lösen sie sich ab. Die Effekte von FBS und HS auf EBF sind allerdings unterschiedlich. FBS fördert die Zellproliferation und -verdopplungsrate sowie die Zellpassage bis zur Passage 20 signifikant besser als HS (max. 9 Passagen). Unter FBS entwickeln EBF eine für Fibroblasten charakteristische spindelförmige Morphologie aber eine schwache α-SMA-Expression, während unter HS die Zellen eine atypische, polygonale Morphologie zeigen, jedoch mit signifikant hohem α-SMA und Proteingehalt. Radioligandenbindungsstudien zeigen, dass EBF lediglich den β2-Adrenozeptorsubtyp mit einer maximalen Rezeptorendichte (Bmax) von 5037 ± 494 Bindungsstellen/Zelle exprimieren. Die Behandlung der EBF mit β-Agonisten (Clenbuterol, Salbutamol, Isoproterenol) führt konzentrationsabhängig zur Abnahme dieser Anzahl der Rezeptoren mit unterschiedlicher Wirkungsstärke (Clenbuterol > Salbutamol > Isoproterenol), wobei Dexamethason diese nicht verändert. Diese Agonisten sowie auch Dexamethason hemmen die Proliferation der EBF, und dies kann in Gegenwart von ICI 118,551 aber nicht von CGP 20712A gehemmt werden, was auf den Einfluss des β2-Adrenozeptors hinweist. β2-Agonisten und Dexamethason gemeinsam resultieren in einer verstärkten Hemmung der EBF-Proliferation. Transforming Growth Factor-β1 (TGF-β1) stimuliert die Transformation von EBF zu Myofibroblasten mit einer erhöhten Expression von α-SMA in EBF, welches eher durch Dexamethason als durch Clenbuterol gehemmt wird. Die Kollagensynthese wird nur durch Dexamethason signifikant gehemmt. Schlussfolgerung: Das erstmalig etablierte EBF-Kulturmodell dient der Erforschung von Signalwegen und neuen Arzneimitteltargets im Zusammenhang mit der Pathogenese der equinen RAO insbesondere dem Atemwegs-Remodelling. So kann gezeigt werden, dass die Verwendung von β2-Agonisten allein oder in Kombination mit Glucocorticoiden eine Hemmung der Proliferation und Transformation von EBF in vitro bewirkt. Dies deutet auf einen zusätzlichen therapeutischen Nutzen von Clenbuterol und Glucocorticoiden hin und impliziert die Notwendigkeit eines frühzeitigen Einsatzes dieser Pharmaka bei der RAO des Pferdes

Rapid action of glucocorticoids in mast cells

Glucocorticoids (GCs) rapidly inhibit mast cells function, but the mechanism of action is still poorly understood. A knowledge of how GCs rapidly suppress allergy and inflammation mediated by these cells will aid in the development of new drugs with a better benefit-risk ratio. The aim of this research work was to study the rapid action of GCs in mast cells and investigate the involvement of the glucocorticoid receptor as a mediator of the rapid effects

Kinetics of the curing reaction of a diglycidyl ether of bisphenol A with a modified polyamine

Differential scanning calorimetry (DSC) was used at different heating rates to study the cure kinetics of the diglycidyl ether of bisphenol A (DGEBA) with a polyaminophenolic product (PAP) derived from the modification of tetraethylenepentamine (TEPA). The activation energy (E-a) based on a single heating rate was compared with E-a based on multiple heating rates (Kissinger, isoconversional method). (C) 2005 Elsevier B.V. All rights reserved

On the stochastic Sine-Gordon model: an interacting field theory approach

We investigate the massive Sine-Gordon model in the finite ultraviolet regime on the two-dimensional Minkowski spacetime $(\mathbb{R}^2,\eta)$ with an additive Gaussian white noise. In particular we construct the expectation value and the correlation functions of a solution of the underlying stochastic partial differential equation (SPDE) as a power series in the coupling constant, proving ultimately uniform convergence. This result is obtained combining an approach first devised in [11] to study SPDEs at a perturbative level with the one discussed in [4] to construct the quantum sine-Gordon model using techniques proper of the perturbative, algebraic approach to quantum field theory (pAQFT). At a formal level the relevant expectation values are realized as the evaluation of suitably constructed functionals over $C^\infty(\mathbb{R}^2)$. In turn, these are elements of a distinguished algebra whose product is a deformation of the pointwise one, by means of a kernel which is a linear combination of two components. The first encompasses the information of the Feynmann propagator built out of an underlying Hadamard, quantum state, while the second encodes the correlation codified by the Gaussian white noise. In our analysis, first of all we extend the results obtained in [3,4] proving the existence of a convergent modified version of the S-matrix and of an interacting field as elements of the underlying algebra of functionals. Subsequently we show that it is possible to remove the contribution due to the Feynmann propagator by taking a suitable $\hbar\to 0^+$-limit, hence obtaining the sought expectation value of the solution and of the correlation functions of the SPDE associated to the stochastic Sine-Gordon model.Comment: 48 page

An Algebraic and Microlocal Approach to the Stochastic Non-linear Schr\"odinger Equation

In a recent work [DDRZ20], it has been developed a novel framework aimed at studying at a perturbative level a large class of non-linear, scalar, real, stochastic PDEs and inspired by the algebraic approach to quantum field theory. The main advantage is the possibility of computing the expectation value and the correlation functions of the underlying solutions accounting for renormalization intrinsically and without resorting to any specific regularization scheme. In this work we prove that it is possible to extend the range of applicability of this framework to cover also the stochastic non-linear Schroedinger equation in which randomness is codified by an additive, Gaussian, complex white noise.Comment: 33 pages. Some typos fixed, Section 1 modifie

Potential of mechanical and physicochemical analysis of human cortical bone for forensic age estimation

Age estimation remains one of the most challenging tasks for forensic practitioners when establishing the biological profile of unknown skeletonised remains. Morphological methods based on developmental markers of bones can provide accurate age estimates. Yet, these methods tend to be unreliable when the individuals are over 35 years of age as all the key developmental markers gradually disappear. Current methods are also highly population- and sex-specific, and individual differences must be considered when interpreting the results. Aspartic acid racemisation, radiocarbon dating and DNA-methylation have all shown to be accurate to only a margin of ± 5 years. A recent method using quantification of biomechanical properties in conjunction with bone microstructure for the mid-femur cortical bone has shown potential and a better accuracy. In the present research, analyses of samples from the 4th rib are detailed and a methodology for estimation of age of individuals is presented. The physical characteristics of ribs are less influenced by mechanical stress compared to weight bearing bones and ribs are relatively accessible from the thoracic cage during autopsy, which increases the applicability of any such rib-based method. This study exploits the changes in the biomechanical properties of bone tissue and matrix. These properties continue to change with age even after skeletal maturity and they are valuable for age estimation. The relationship of 28 variables at the macroscopic, microscopic and structural/compositional level were investigated in 113 ribs retrieved during autopsies. The experimental design comprehensively documents the changes in mineral size and composition, organic matrix quality and their combined effect on mechanical properties. The powder x-ray diffraction (XRD) method is applied to measure the crystal size and strain, and lattice parameters. Further, Fourier-transform infrared (FTIR) spectroscopy is used to investigate the changes in carbonate substitution and crystallinity. This examination allows a dependable evaluation of the increase in mineral to matrix ratio and therefore, the increase in collagen. Subsequent analysis of combined mineral and organic changes with age was done through thermal analysis. The physicochemical characterisation was compared to functional mechanical properties. The study showed that, in contrast to other bones, skeletal maturity for the rib cortical bone is reached between 40 and 50 years of age. The study revealed that with age, there is an increase in crystal size and mineral content that is combined with the decrease in organic matrix amount and quality. It was also possible to confirm the importance of standardisation of the procedures. It was found out that a simple defatting procedure based on consecutive baths of chloroform, methanol and ethanol affects the evaluation of crystal size, which in turn affects the age estimation when using the powder XRD method. The 28 parameters measured using standard biomechanical (nanoindentation and microindentation), physical (DSC/TGA, FTIR and XRD) and histomorphometry (porosity-ImageJ) procedures were used to obtain an age-at-death estimation for unknown skeletal remains. Stepwise regression was employed to develop relations that would produce the best ‘estimates of age-at-death’ vs real age of the cadavers. Seven relationships were proposed: the relationship of primary importance containing 13 parameters and resulting in an R2 = 0.873 with a mean absolute error of ± 4.47. The relationship that performs poorly utilised only five parameters and resulted in an R2 = 0.840, with an average error estimate of ± 4.96 years. To conclude, physicochemical and mechanical characterisation of bone cortical bone shows good potential in accurate age-at-death estimation. Furthermore, the multi-factorial approach allows the combination of different methods to reach the best performance. A further employment of this method may involve histomorphometry and macroscopic examination of the age related degeneration if the sternal rib end, still largely used in forensic anthropology

A microlocal investigation of stochastic partial differential equations for spinors with an application to the Thirring model

On a $d$-dimensional Riemannian, spin manifold $(M,g)$ we consider non-linear, stochastic partial differential equations for spinor fields, driven by a Dirac operator and coupled to an additive Gaussian, vector-valued white noise. We extend to the case in hand a procedure, introduced in [DDRZ20] for the scalar counterpart, which allows to compute at a perturbative level the expectation value of the solutions as well as the associated correlation functions accounting intrinsically for the underlying renormalization freedoms. This framework relies strongly on tools proper of microlocal analysis and it is inspired by the algebraic approach to quantum field theory. As a concrete example we apply it to a stochastic version of the Thirring model proving in particular that it lies in the subcritical regime if $d\leq 2$.Comment: 42 page

Vivencias universitarias en un contexto culturalmente diverso: el caso de los Awajún en la Universidad Nacional de la Amazonía Peruana en Iquitos

La presente investigación plantea como principal objetivo analizar las vivencias de jóvenes estudiantes Awajún que se encuentran al interior del nuevo contexto de la Universidad Nacional de la Amazonía Peruana (UNAP). La etapa universitaria también puede resultar difícil para los jóvenes considerados “occidentales”, ya que es una transición que conlleva muchos cambios; sin embargo existen particularidades en la experiencia universitaria de los jóvenes indígenas. Asimismo, este tema de investigación involucra a la educación, la cual se encuentra entre los derechos esenciales de los individuos.Tesi

Class-Incremental Continual Learning into the eXtended DER-verse

The staple of human intelligence is the capability of acquiring knowledge in a continuous fashion. In stark contrast, Deep Networks forget catastrophically and, for this reason, the sub-field of Class-Incremental Continual Learning fosters methods that learn a sequence of tasks incrementally, blending sequentially-gained knowledge into a comprehensive prediction. This work aims at assessing and overcoming the pitfalls of our previous proposal Dark Experience Replay (DER), a simple and effective approach that combines rehearsal and Knowledge Distillation. Inspired by the way our minds constantly rewrite past recollections and set expectations for the future, we endow our model with the abilities to i) revise its replay memory to welcome novel information regarding past data ii) pave the way for learning yet unseen classes. We show that the application of these strategies leads to remarkable improvements; indeed, the resulting method - termed eXtended-DER (X-DER) - outperforms the state of the art on both standard benchmarks (such as CIFAR-100 and miniImagenet) and a novel one here introduced. To gain a better understanding, we further provide extensive ablation studies that corroborate and extend the findings of our previous research (e.g. the value of Knowledge Distillation and flatter minima in continual learning setups).Comment: 23 pages, 22 figures. To appear in IEEE TPAM