Morphologische Charakterisierung der BRAF mutierten Melanome mittels Immunhistochemie, Dermatoskopie und konfokaler Laserscanmikroskopie

Background Since the advent of molecular targeted therapies, mutational profiling of malignant melanoma has become of outmost importance. Melanomas can be distinguished based on the presence of BRAF mutations. Estimated 40-60% of melanomas harbour a BRAF mutation, most of them V600E. Current guidelines recommend testing every advanced melanoma for BRAF mutation, since BRAF inhibitors are available for both adjuvant and therapeutic setting. BRAF Exon 15 PCR amplification and sequencing of genomic DNA now represents the gold standard for BRAF analysis in melanoma samples. Molecular genetic tests are however expensive and time consuming. Since different melanoma subtypes can be distinguished based on genetic mutations such as BRAFV600E, corresponding morphological patterns might be hypothesized. Aim of this study was to further characterize BRAF mutated melanomas compared to their wild type counterpart based on alternative methods such as in-vivo confocal microscopy (RCM), dermoscopy and immunohistochemistry (IHC). Methods Eight BRAFV600E mutated melanomas (six primary and two metastases), paired with age-, sex- and tumour thickness- matched wild-type controls were analysed with dermoscopy and in-vivo confocal microscopy. On the other side, eighteen melanomas with known BRAF mutational status (BRAFV600E, V600K, V600R and wild-type) were additionally evaluated through immunohistochemistry with anti-BRAF antibodies. Results Most common dermoscopic features in BRAFV600E mutated melanomas were irregularly distributed dots and globules and gray-blue blotches (62%), followed by irregular vessels, white regression, and peppering (50%). Most common RCM patterns were pleomorphic pagetoid cells, disarrangement at the dermoepidermal junction (DEJ), decohesed junctional nests, and bright particles at the dermal–epidermal junction (75%). Peppering in dermoscopy and plump bright cells in RCM were more frequently found in BRAFV600E mutated primary melanomas compared to wild-type ones (63% and 37%, respectively). In IHC, V600E-specific antibody stained all melanomas harbouring V600E and V600R mutation, but was not able to recognize BRAF V600K-mutated melanomas. Conclusions Dermoscopy and confocal microscopy are related to each other and might provide useful information in the non-invasive initial categorization of melanoma patients potentially harbouring a BRAFV600E mutation. At the same time, IHC might be a useful tool for the initial diagnosis of a BRAFV600E mutation in subjects with high risk or metastatic melanomas potentially benefiting from a systemic therapy with BRAF inhibitors. After the IHC screening, molecular techniques shall be used in V600E wild type melanomas, in the search for less frequent non-V600E BRAF mutations.Hintergrund Seit dem Aufkommen molekularer zielgerichteter Therapien ist die Mutationsprofilierung von malignen Melanomen von größter Bedeutung geworden. Melanome können anhand des Vorhandenseins von BRAF-Mutationen unterschieden werden. Schätzungsweise 40-60% der Melanome weisen eine BRAF-Mutation auf, die meisten davon V600E. Aktuelle Richtlinien empfehlen, jedes fortgeschrittene Melanom auf BRAF-Mutationen zu testen, da BRAF-Inhibitoren sowohl im adjuvanten als auch im therapeutischen Setting verfügbar sind. Molekularpathologische Techniken sind der Goldstandard für den Nachweis von BRAF-Mutationen, insbesondere die Ampflizierung und Sequenzierung von BRAF Exon 15 aus genomischer DNA mittels PCR. Solche Tests sind jedoch teuer und zeitaufwändig. Da verschiedene Melanom-Subtypen anhand genetischer Mutationen wie BRAFV600E unterschieden werden können, können entsprechende morphologische Muster angenommen werden. Ziel dieser Studie war es, BRAF-mutierte Melanome im Vergleich zu ihrem Wildtyp-Korrelat auf der Grundlage alternativer Methoden wie konfokaler in-vivo-Mikroskopie (RCM), Dermatoskopie und Immunhistochemie (IHC) weiter zu charakterisieren. Methoden Acht mutierte BRAFV600E-Melanome (sechs primäre und zwei Metastasen), gepaart mit alters-, geschlechts- und tumourdickenangepassten Wildtyp-Kontrollen, wurden mit Dermoskopie und konfokaler in-vivo-Mikroskopie analysiert. Auf der anderen Seite wurden 18 Melanome mit bekanntem BRAF-Mutationsstatus (BRAFV600E, V600K, V600R und Wildtyp) zusätzlich durch Immunhistochemie mit Anti-BRAF-Antikörpern bewertet. Ergebnisse Die häufigsten dermatoskopischen Merkmale bei mutierten BRAFV600E-Melanomen waren unregelmäßig verteilte Punkte und Schollen sowie grau-blaue homogene Areale (62%), gefolgt von unregelmäßigen Gefäßen, weißer Regression und Peppering (50%). Die häufigsten RCM-Muster waren pleomorphe pagetoide Zellen, Störungen der dermo-epidermalen Junktion (DEJ), atypische junktionale Melanozytennester und helle Partikel an der DEJ (75%). Peppering in der Dermatoskopie und plumpe helle Zellen in RCM wurden häufiger in mutierten primären BRAFV600E-Melanomen als in Wildtyp-Melanomen gefunden (63% vs. 37%). In der IHC färbte der V600E-spezifische Antikörper alle Melanome, die eine V600E- und V600R-Mutation enthielten, konnte jedoch keine BRAF V600K-mutierten Melanome erkennen. Schlussfolgerungen Dermatoskopie und konfokale Mikroskopie zeigen eine gute Korrelation zueinander und könnten nützliche Informationen für das nicht-invasive, preliminäre Screening und die Charakterisierung von BRAFV600E-mutierten Melanomen liefern. Gleichzeitig könnte die Immunhistochemie als erster Schritt zum Nachweis der BRAFV600E-Mutation bei der Auswahl von Patienten mit fortgeschrittenen Melanomen als Kandidaten für eine systemische Therapie mit BRAF-Inhibitoren wirksam eingesetzt werden. IHC sollte von molekularen Techniken bei p.V600E-negativen Melanomen gefolgt werden, um p.V600K und andere seltene Nicht-V600E-BRAF-Mutationen zu entdecken

The Forgotten Homerist: Reassessing William Ewart Gladstone's Role in the Victorian Reception of Homer (1872-1884)

This thesis uses William Gladstone’s Homeric research to reassess the relationship between nineteenth-century Britain and the ancient past. Gladstone (1809-1898), who served as Prime Minister four times during the Victorian period, has often been dismissed as a scholar of Homer: too enthusiastic, too much of a dilettante, too ready to cast aside evidence. But, through a careful examination of unstudied archival evidence, it is possible to build a very different picture. During the 1870s, Gladstone embarks on a Homeric campaign which changes his contemporaries’ understanding of time and history. By carefully exploiting recent archaeological discoveries – particularly in the case of Schliemann’s discovery of Troy – Gladstone works to bring both Homer and Troy out of the world of myth and into that of history. As this thesis will demonstrate, for many Victorians, Gladstone, not Schliemann, brought Homer’s Troy to light, in the ruins of Hissarlik. Working behind the scenes, over the course of many years, Gladstone revolutionises his contemporaries’ understanding of the study of Homer. He pioneers the study of what he calls ‘Homerology’: a new approach to the poems. Gladstone’s Homerology sees the epics as vital sources for the scientific investigation of the ancient past. Gladstone presents Victorian Britain with a new model of time and history, where myth becomes a historical reality. Consequently, for Gladstone, it is the Homerist who, above all, has the right to write about the ancient past of man. Through a series of case studies - which have been unnoticed or unrecognised by previous scholarship, this thesis demonstrates that Gladstone’s Homer shaped many key Victorian discourses about the earliest history of mankind: from archaeology to evolution. In so doing, it makes the case for a granular, archive-driven methodology for classical reception, one which is equipped to capture the nuances, complications, and complexities of relationships with the ancient past

A note on Nordhaus-Gaddum-type inequaliies for the automorphic H-chromatic index of graphs

The automorphic H-chromatic index of a graph G is the minimum integer m for which G has a proper edge-coloring with m colors which is preserved by a given automorphism H of G. We consider the sum and the product of the automorphic H-chromatic index of a graph and its complement. We prove upper and lower bounds in terms of the order of the graph when H is chosen to be either a cyclic group of prime order or a group of order four

Ab initio Simulation of Optical Limiting: The Case of Metal-Free Phthalocyanine

We present a fully ab initio, non-perturbative description of the optical limiting properties of a metal-free phthalocyanine, by simulating the effects of a broadband electric field of increasing intensity. The results confirm reverse saturable absorption as leading mechanism for optical limiting phenomena in this system and reveal that a number of dipole-forbidden excitations are populated by excited-state absorption, at more intense external fields. The excellent agreement with the experimental data supports our approach as a powerful tool to predict optical limiting, in view of applications

Morphologische Charakterisierung der BRAF mutierten Melanome mittels Immunhistochemie, Dermatoskopie und konfokaler Laserscanmikroskopie

Background Since the advent of molecular targeted therapies, mutational profiling of malignant melanoma has become of outmost importance. Melanomas can be distinguished based on the presence of BRAF mutations. Estimated 40-60% of melanomas harbour a BRAF mutation, most of them V600E. Current guidelines recommend testing every advanced melanoma for BRAF mutation, since BRAF inhibitors are available for both adjuvant and therapeutic setting. BRAF Exon 15 PCR amplification and sequencing of genomic DNA now represents the gold standard for BRAF analysis in melanoma samples. Molecular genetic tests are however expensive and time consuming. Since different melanoma subtypes can be distinguished based on genetic mutations such as BRAFV600E, corresponding morphological patterns might be hypothesized. Aim of this study was to further characterize BRAF mutated melanomas compared to their wild type counterpart based on alternative methods such as in-vivo confocal microscopy (RCM), dermoscopy and immunohistochemistry (IHC). Methods Eight BRAFV600E mutated melanomas (six primary and two metastases), paired with age-, sex- and tumour thickness- matched wild-type controls were analysed with dermoscopy and in-vivo confocal microscopy. On the other side, eighteen melanomas with known BRAF mutational status (BRAFV600E, V600K, V600R and wild-type) were additionally evaluated through immunohistochemistry with anti-BRAF antibodies. Results Most common dermoscopic features in BRAFV600E mutated melanomas were irregularly distributed dots and globules and gray-blue blotches (62%), followed by irregular vessels, white regression, and peppering (50%). Most common RCM patterns were pleomorphic pagetoid cells, disarrangement at the dermoepidermal junction (DEJ), decohesed junctional nests, and bright particles at the dermal–epidermal junction (75%). Peppering in dermoscopy and plump bright cells in RCM were more frequently found in BRAFV600E mutated primary melanomas compared to wild-type ones (63% and 37%, respectively). In IHC, V600E-specific antibody stained all melanomas harbouring V600E and V600R mutation, but was not able to recognize BRAF V600K-mutated melanomas. Conclusions Dermoscopy and confocal microscopy are related to each other and might provide useful information in the non-invasive initial categorization of melanoma patients potentially harbouring a BRAFV600E mutation. At the same time, IHC might be a useful tool for the initial diagnosis of a BRAFV600E mutation in subjects with high risk or metastatic melanomas potentially benefiting from a systemic therapy with BRAF inhibitors. After the IHC screening, molecular techniques shall be used in V600E wild type melanomas, in the search for less frequent non-V600E BRAF mutations.Hintergrund Seit dem Aufkommen molekularer zielgerichteter Therapien ist die Mutationsprofilierung von malignen Melanomen von größter Bedeutung geworden. Melanome können anhand des Vorhandenseins von BRAF-Mutationen unterschieden werden. Schätzungsweise 40-60% der Melanome weisen eine BRAF-Mutation auf, die meisten davon V600E. Aktuelle Richtlinien empfehlen, jedes fortgeschrittene Melanom auf BRAF-Mutationen zu testen, da BRAF-Inhibitoren sowohl im adjuvanten als auch im therapeutischen Setting verfügbar sind. Molekularpathologische Techniken sind der Goldstandard für den Nachweis von BRAF-Mutationen, insbesondere die Ampflizierung und Sequenzierung von BRAF Exon 15 aus genomischer DNA mittels PCR. Solche Tests sind jedoch teuer und zeitaufwändig. Da verschiedene Melanom-Subtypen anhand genetischer Mutationen wie BRAFV600E unterschieden werden können, können entsprechende morphologische Muster angenommen werden. Ziel dieser Studie war es, BRAF-mutierte Melanome im Vergleich zu ihrem Wildtyp-Korrelat auf der Grundlage alternativer Methoden wie konfokaler in-vivo-Mikroskopie (RCM), Dermatoskopie und Immunhistochemie (IHC) weiter zu charakterisieren. Methoden Acht mutierte BRAFV600E-Melanome (sechs primäre und zwei Metastasen), gepaart mit alters-, geschlechts- und tumourdickenangepassten Wildtyp-Kontrollen, wurden mit Dermoskopie und konfokaler in-vivo-Mikroskopie analysiert. Auf der anderen Seite wurden 18 Melanome mit bekanntem BRAF-Mutationsstatus (BRAFV600E, V600K, V600R und Wildtyp) zusätzlich durch Immunhistochemie mit Anti-BRAF-Antikörpern bewertet. Ergebnisse Die häufigsten dermatoskopischen Merkmale bei mutierten BRAFV600E-Melanomen waren unregelmäßig verteilte Punkte und Schollen sowie grau-blaue homogene Areale (62%), gefolgt von unregelmäßigen Gefäßen, weißer Regression und Peppering (50%). Die häufigsten RCM-Muster waren pleomorphe pagetoide Zellen, Störungen der dermo-epidermalen Junktion (DEJ), atypische junktionale Melanozytennester und helle Partikel an der DEJ (75%). Peppering in der Dermatoskopie und plumpe helle Zellen in RCM wurden häufiger in mutierten primären BRAFV600E-Melanomen als in Wildtyp-Melanomen gefunden (63% vs. 37%). In der IHC färbte der V600E-spezifische Antikörper alle Melanome, die eine V600E- und V600R-Mutation enthielten, konnte jedoch keine BRAF V600K-mutierten Melanome erkennen. Schlussfolgerungen Dermatoskopie und konfokale Mikroskopie zeigen eine gute Korrelation zueinander und könnten nützliche Informationen für das nicht-invasive, preliminäre Screening und die Charakterisierung von BRAFV600E-mutierten Melanomen liefern. Gleichzeitig könnte die Immunhistochemie als erster Schritt zum Nachweis der BRAFV600E-Mutation bei der Auswahl von Patienten mit fortgeschrittenen Melanomen als Kandidaten für eine systemische Therapie mit BRAF-Inhibitoren wirksam eingesetzt werden. IHC sollte von molekularen Techniken bei p.V600E-negativen Melanomen gefolgt werden, um p.V600K und andere seltene Nicht-V600E-BRAF-Mutationen zu entdecken

Distributed Control for Collective Behaviour in Micro-unmanned Aerial Vehicles

Full version unavailable due to 3rd party copyright restrictions.The work presented herein focuses on the design of distributed autonomous controllers for collective behaviour of Micro-unmanned Aerial Vehicles (MAVs). Two alternative approaches to this topic are introduced: one based upon the Evolutionary Robotics (ER) paradigm, the other one upon flocking principles. Three computer simulators have been developed in order to carry out the required experiments, all of them having their focus on the modelling of fixed-wing aircraft flight dynamics. The employment of fixed-wing aircraft rather than the omni-directional robots typically employed in collective robotics significantly increases the complexity of the challenges that an autonomous controller has to face. This is mostly due to the strict motion constraints associated with fixed-wing platforms, that require a high degree of accuracy by the controller. Concerning the ER approach, the experimental setups elaborated have resulted in controllers that have been evolved in simulation with the following capabilities: (1) navigation across unknown environments, (2) obstacle avoidance, (3) tracking of a moving target, and (4) execution of cooperative and coordinated behaviours based on implicit communication strategies. The design methodology based upon flocking principles has involved tests on computer simulations and subsequent experimentation on real-world robotic platforms. A customised implementation of Reynolds’ flocking algorithm has been developed and successfully validated through flight tests performed with the swinglet MAV. It has been notably demonstrated how the Evolutionary Robotics approach could be successfully extended to the domain of fixed-wing aerial robotics, which has never received a great deal of attention in the past. The investigations performed have also shown that complex and real physics-based computer simulators are not a compulsory requirement when approaching the domain of aerial robotics, as long as proper autopilot systems (taking care of the ”reality gap” issue) are used on the real robots.EOARD (European Office of Aerospace Research & Development), euCognitio

Excitons in carbon nanotubes: an ab initio symmetry-based approach

The optical absorption spectrum of the carbon (4,2) nanotube is computed using an ab-initio many-body approach which takes into account excitonic effects. We develop a new method involving a local basis set which is symmetric with respect to the screw symmetry of the tube. Such a method has the advantages of scaling faster than plane-wave methods and allowing for a precise determination of the symmetry character of the single particle states, two-particle excitations, and selection rules. The binding energy of the lowest, optically active states is approximately 0.8 eV. The corresponding exciton wavefunctions are delocalized along the circumference of the tube and localized in the direction of the tube axis.Comment: 4 pages, 1 LaTex file + 4 eps figure

Manifold Spines and Hyperbolicity Equations

We give a combinatorial representation of compact connected orientable 3-dimensional manifolds with boundary and their special spines by a class of graphs with extrastructure which are strictly related to o-graphs defined and studied in [3] and [4]. Then we describe a simple algorithm for constructing the boundary of these manifolds by using a list of 6-tuples of non-negative integers. Finally we discuss some combinatorial methods for determining the hyperbolicity equations. Examples of hyperbolic 3- manifolds of low complexity illustrate in particular cases the constructions and algorithms presented in the paper

Chitosan based biomaterials: soft tissue engineering applications

In recent years, considerable attention has been given to chitosan (CS)-based biomaterials and their applications in the field of soft tissue engineering (TE). CS is a glycosaminoglycan derived from chitin, the primary structural polymer in crustacean exoskeletons. CS is biocompatible, biodegradable, easily formed into various structures (i.e. sponges, nanofibers and films) under mild processing conditions and can be chemically modified through graft copolymerization and crosslinking. However, the rapid degradation of CS and its low mechanical strength are concerns that may limit its use in clinical applications. In the first part of the thesis, different non cytotoxic crosslinkers were used aiming at improving the structural properties of CS. Genipin (GP), γ-glycidoxypropyltrimethoxysilane (GPTMS), dibasic sodium phosphate (DSP) were selected as biocompatible CS crosslinkers as reported in literature. After a preliminary physico-chemical and mechanical characterization, the proper crosslinking compounds were selected for the development of different typologies of CS scaffolds for both human and veterinary applications. CS- based scaffolds were developed as nerve guidance channels (NGCs) and internal fillers fabrication to promote peripheral nerve regeneration in humans. Two CS based hollow NGCs were prepared and tested in vitro and in vivo (coded as CS flat membrane and bi-layer CS membrane) and a CS based nanostructured internal filler was optimized and characterized in vitro. i. CS flat membranes were prepared by solvent casting. According to the results obtained in the first part of the thesis, DSP alone (CS/DSP) or in association with the GPTMS (CS/GPTMS_DSP) were used as crosslinkers. CS crosslinked membranes showed permeation to nutrients and did not exert any cytotoxic effect on RT4-D6P2T. The higher mechanical stability of CS/GPTMS_DSP under wet state allowed to confirm the RT4-D6P2T attachment and proliferation as well as the neurite outgrowth of dorsal root ganglia (DRG) on CS substrates. Before in vivo implantation in rats, CS/GPTMS_DSP and CS/DSP membranes were easily rolled up to form a NGC. Then, membranes were used to bridge median nerve defects in rats. After 12 week post-operative CS/GPTMS_DSP tubes were found to be detached from the distal suturing site and functional recovery did not occurred. On the other hand, crushed nerve encircled with CS/DSP membranes, allowed nerve fibre regeneration and functional recovery, showing similar results to autografts. ii. Bi-layer CS membranes were developed using a two-step coating technique. CS/DSP and CS/GPTMS_DSP flat membranes were combined to produce scaffold structures with good biocompatibility in the inner layer (CS/DSP) and with the desired mechanical strength imparted by the outer (CS/GPTMS_DSP, GPTMS 25% wt./wt.). Gradual water uptake and permeation to small molecules was observed compared to single layers. From in vivo tests, median nerves treated with bi-layer tubes displayed regenerated and aligned fibres at the injury site. iii. CS crosslinked electrospun nanofibres were fabricated by electrospinning solutions containing CS, polyethylene oxide (PEO), and dimethylsulphoxide (DMSO). PEO and DMSO were introduced to allow the spinnability of CS solutions at high polymer concentration with controllable fiber size and increase fiber yields by relaxing CS chain entanglement. Optimization of the process and solution parameters allowed to obtain CS nanofibres with size of 128±17 nm. To increase CS stability in aqueous media, DSP was used as crosslinker After DSP crosslinking fibre size decreased to 109±17 nm while an increase in the mechanical strength (E, from 63±10 MPa to 113±8 MPa) was observed compared to uncrosslinked nanofibrous matrices. In the third part of the thesis, CS porous membranes with improved antimicrobial properties were prepared for veterinary application. The developed scaffolds were fabricated by freeze-drying to promote the wound healing process and to reduce the bacterial proliferation in chelonian shell injury site. Different ratios of silver nanoparticles (AgNPs, 5%, 10% and 15% wt. /wt.) and gentamicin sulphate (GS, 3.5 mg/ml) were loaded into the CS/GPTMS_DSP membranes to impart the proper antibacterial properties and to favor drug release avoiding the risk of systemic toxicity. After a preliminary in vitro characterization, CS/GPTMS_DSP loaded with AgNPs at a concentration of 10% wt./wt (CS/GPTMS_DSP_AgNP10) was selected as ideal candidate for this application field. GS release profile from CS/GPTMS_DSP_GS evidenced high burst release of the antibiotics in the first day (about 70%). Finally, GS and AgNPs (10 % wt./wt.) effect on bacterial inhibition was evaluated and confirmed against Gram+ and Gram-. The results reported in this thesis work demonstrate that CS is a promising candidate for applications in human and veterinary soft TE. Mechanical and physico-chemical properties of CS scaffolds can be tuned by using different crosslinking methods. By the in vitro characterization, GPTMS and DSP were selected as ideal compounds to the development of scaffolds for peripheral nerve regeneration (in human) and wound healing (in animals). Four different morphologies (3 for peripheral nerve regeneration and 1 for wound healing application) were obtained by varying the fabrication methods and the final composition. All membranes were found to satisfy the requirements for the application of interest. CS based membranes developed for peripheral nerve regeneration were found to be biocompatible, and successful functional recovery was observed in case of CS/DSP and bi-layer membranes. Porous membranes with improved antimicrobial properties were prepared to enhance wound healing in chelonians and were found to be effective against a broad spectrum of bacteria following the release of two different investigated antimicrobial agents (AgNPs and GS)