Using Hebb’s (1961) paradigm, it has been shown that older adults (OAs) fail to learn recurrent visuospatial supra-span sequence information (Turcotte, Gagnon, & Poirier, 2005); a deficit which has not been demonstrated on verbal versions of the same task or in younger adults (YAs). Since the Hebb paradigm is thought to rely on working memory and thus attention (Conway & Engle, 1996), one interpretation concerns an OA’s capacity to allocate the necessary attentional resources to carry out the various components of the task. Five studies investigated this proposal. The first three (Article 1) examined attention in a general manner by reducing the amount of attentional resources that a YA could devote to carrying out the visuospatial Hebb supra-span sequence learning task through the implementation of a verbal dual task (DT) procedure. The fourth (Article 2) further investigated the role of attention by using a DT induced at retrieval that overlapped extensively with the requirements (spatial and response features) of the visuospatial Hebb task. The final study (Article 3) aimed to use our previous findings to demonstrate learning among OAs in a visuospatial Hebb learning paradigm in which the motor response was replaced by a verbal response. Our findings confirm that attentional resources employed at the retrieval phase of the task appear to be particularly important for the demonstration of visuospatial sequence learning. The inclusion of a spatial and motor based DT at recall eliminated learning of the repeated sequence in YAs. Interestingly, the learning deficit of OAs was partially eliminated when the motor and spatial requirements at retrieval were reduced. Our findings offer strong support to the contention that supra-span learning of the Hebb type is not altered by the effect of age. However, learning deficits can be observed among OAs when the retrieval component of the task overly taxes attention-related processes. In the case of the visuospatial sequences, the basis of the deficit likely concerns an individual’s capacity to discriminate between responses made to previously presented sequences versus those that need to be made in reaction to the just seen sequence.

The purpose of this work is to measure the degree of intelligibility of native and non-native English speech as well as analyse the major sources of intelligibility failure when speakers of these varieties of English interact. British and American English (henceforth BrE and AmE) and Cameroon English (hereafter CamE) are used as a case study with focus on segmental and supra segmental features. The study was motivated by a number of concerns, several of which are more prominent: First, it was motivated by the trepidation scholars like Gimson (1965, 1980); Prator (1968); etc. nursed that the unprecedented spread of English across the globe and the emergence of non-native varieties would cause English to disintegrate into mutually unintelligible languages, in the way Romance languages devolved from their Latin ancestor. The second motivation was that previous researchers (Bansal 1969, Tiffen 1974) on intelligibility have often concentrated their efforts on the traditional approach, which sees intelligibility from a one-sided perspective. To them, the non-native varieties of English are deficient and not different varieties from the native varieties. They were seen as substandard, incorrect, and unintelligible and thus needed remediation at all costs. The native varieties were seen as prestigious, correct, intelligible and the sole norm that must be emulated by non-native English speakers. In this way any interaction between a native speaker and a non-native speaker should be characterised by the non-native speaker making all the efforts to be understood as well as to understand the native English-speaking partner. This explains in large part why these researchers concentrated on measuring the intelligibility of non-native speech to native speakers and never vice versa. It was as if it was treasonable to measure the intelligibility of native speech to non-native speakers. Even if some researchers managed to do this, the comments that followed such data still showed that the aim was not to test the intelligibility of native speakers but to find out how efficient the non-native speakers were in understanding the native speaker. Another aim could also be to reinforce the teaching of the native norm, which was seen as “correct” against non-native features, which were seen as “incorrect”, to measuring intelligibility. While accepting that these studies reflected the conventional wisdom of the time, this study aims to move the debate forward by looking at intelligibility from a two-sided perspective. It sees communication between speakers of different varieties as a game of give and take, where both participants “tune in” to make the process successful rather than one participant being obliged to make all the efforts because s/he speaks a new English variety. That explains why we are testing not only the intelligibility of non-native speakers to native speakers but also native speakers to non-native speakers. / Gegenstand der vorgelegten Promotionsarbeit ist die Untersuchung der gegenseitigen Verständlichkeit von muttersprachlichem und nicht-muttersprachlichem Englisch. Im besonderen werden die Hauptquellen und Ursachen des Scheiterns von Verständlichkeit in einer empirischen Studie bestimmt, klassifiziert und analysiert. Die Untersuchung wird exemplarisch anhand des Kamerunischen Englisch einerseits und des Britischen und Amerikanischen Englisch anderseits vorgenommen. Motiviert ist diese Arbeit vor allem durch folgende Punkte. Erstens bedarf es der Auseinandersetzung mit den durch eine Reihe von Autoren geäußerten Befürchtungen (z.B. Gimson 1965, 1980 und Prator 1968), daß die Herausbildung und Entwicklung neuer Varianten des Englischen letztlich zu einer Auflösung des Englischen in gegenseitig nicht mehr verständliche Sprachen führt, ein Prozeß, wie er sich historisch bei der Entstehung der romanischen Sprachen aus dem Lateinischen vollzog. Derartige Befürchtungen werden genährt durch die bisher ohnegleichen fortschreitende Verbreitung des Englischen über den gesamten Globus. Hier ergibt sich die dringende Notwendigkeit vergleichender Studien zur gegenseitigen Verständlichkeit zwischen den bestehenden Varianten. Zweitens folgen die meisten zu diesem Thema vorliegenden Untersuchungen im wesentlichen einer traditionellen, überkommenen Grundperspektive: die nicht-muttersprachlichen Varianten des Englischen werden als „abweichend“ bzw. sogar „defizitär“ aus Sicht der muttersprachlichen betrachtet, nicht aber als eigenständige Sprachformen (z.B. Bansal 1969; Tiffen 1974). Dies führt nach Auffassung des Autors zu einer einseitigen Betrachtung und Bewertung. Im besonderen wird in der sprachlichen Interaktion bei einem solchen Zugang die Last zu verstehen und für den Kommunikationspartner verständlich zu sein einseitig dem nicht-muttersprachlichen Sprecher übertragen. Auf diesem Hintergrund untersuchen die vorliegenden Studien anderer Autoren primär die Verständlichkeit nicht-muttersprachlicher Sprachformen für den muttersprachlichen Sprecher, nicht jedoch die umgekehrte Konstellation. Wenn die umgekehrte Perspektive überhaupt berücksichtigt wird, so zeigen die Kommentare, daß nicht die Verständlichkeit muttersprachlicher Sprecher für den Nichtmuttersprachler eigentliches Ziel und Gegenstand der Untersuchung war, sondern vielmehr die Frage, wie effizient sich Nichtmuttersprachler beim Verstehen muttersprachlicher Äußerungen zeigten. Des weiteren stehen diese Studien oft im Kontext des Bestrebens, im institutionalisierten Spracherwerb die muttersprachlichen Normen gegen die nicht-muttersprachlichen Merkmale durchzusetzen, die als „nicht korrekt“ angesehen werden. Diese Positionen, der zugrundeliegende Zugang und die einseitige Ausrichtung bedürfen einer kritischen Auseinandersetzung.

“La vie ne produit pas de corps symétrique”L. Pasteur. Briser la symétrie-miroir est un sujet fascinant qui revêt un rôle crucial en physique, en chimie et en biologie.Le but ultime de cette étude est de démontrer expérimentalement la conjecture de Curie de-Gennes. Cette hypothèse a été proposée pour la première fois par P. Curie 1894 et plus tard développée par De-Gennes. Elle indique qu’il est possible de générer une dissymétrie (excès énantiomérique (e.e.)) en soumettant un mélange racémique à l'influence combinée de champs magnétique et électrique colinéaires. Une telle influence modifie la cinétique du système : selon l'orientation relative des champs magnétique et électrique, l'un des énantiomères est formé plus rapidement que l'autre en raison d’une énergie d'activation inférieure (Ea). Une telle influence a été ultérieurement appelée « faussement chirale » par Barron. Pour démontrer expérimentalement cette conjecture, deux expériences différentes ont été développées :1) La cristallisation énantiosélective d'un réseau de coordination chiral sous des influences physiques externes (champ magnétique ou force de rotation) par diffusion lente. Un petit e.e. est supposé être créé puis amplifié par la croissance des cristaux à l'interface solvant/vapeur d'éthanol. Dans cette expérience, nous avons constaté que la démonstration de conjecture de Curie de Gennes est difficile à atteindre en raison de la forte dispersion de la moyenne des valeurs du e.e. obtenues et de la faiblesse de l'effet attendu.2) La création d'un e.e. dans un atropisomère racémique d’un cristal liquide à base de biphényle en appliquant des champs magnétique et électrique (anti) parallèles. Un arrangement supramoléculaire hélicoïdal des molécules de biphényles dans la phase nématique augmentera le signal lié à la présence d’un e.e. à un niveau mesurable. Au cours de cette seconde expérience, nous avons observé expérimentalement un e.e des dérivés à base de biphényle induit par une combinaison (anti)parallèle de champs magnétique et électrique. Cet excès dépend linéairement du produit des champs magnétique et électrique et l’ordre de grandeur des résultats observés expérimentalement correspond aux estimations théoriques. Compte tenu des résultats obtenus, sous réserve d’ultimes vérifications, cette expérience constitue une démonstration expérimentale convaincante de la conjecture Curie de-Gennes. / “La vie ne produit pas de corps symétrique” L. Pasteur Mirror symmetry breaking is an ever-fascinating topic that plays a crucial role in physics, chemistry, and biology. The ultimate goal of this study is to demonstrate experimentally the so-called Curie de-Gennes conjecture. It was proposed for the first time by P. Curie (1894) and later developed by de-Gennes. It stipulates that it is possible to generate dissymmetry (enantiomeric excess (e.e.)) by submitting a racemic mixture to the combined influence of collinear magnetic and electric fields. Such influence modifies the kinetics of the system: depending on the relative orientation of the magnetic and electric fields, one of the enantiomers is formed faster than the other because of a lower activation energy (Ea),. This influence was later named by Barron as « falsely chiral ». To demonstrate this conjecture, two different experiments were developed:1) The enantioselective crystallization of a chiral metal-organic framework under external physical influence (magnetic field or rotation force) by slow diffusion. The small e.e. is expected to be created and amplified by crystal growth at the interface aqueous solution / ethanol-saturated vapors. In this experiment, we have found that the Curie de-Gennes conjecture demonstration is difficult to achieve due to high dispersion of the average of the observed e.e. and the weakness of the expected effect. 2) Creating an e.e. in a racemic atropisomeric biphenyl based liquid crystal by applying (anti)parallel magnetic and electric fields. The helical twisting power of biphenyls molecules in the nematic phase will enhance the dichroic signal related to the expected e.e. to a measurable level. During this second experiment we have experimentally observed an e.e. in biphenyl-based nematic liquid crystals induced by (anti)parallel combination of magnetic and electric fields. This excess was linearly dependent on the product of magnetic and electric fields and, within one order of magnitude, its absolute value corresponds to the theoretical estimation. Ultimate checks shall confirm that these results constitute a convincing experimental demonstration of the Curie de Gennes conjecture.

Les prairies supra-forestières sont des écosystèmes bien représentés dans les milieux de montagne et qui s’avèrent vulnérables aux changements climatiques et aux modalités d’utilisation des terres. Dans ce contexte, l’objectif principal de cette thèse a été d’évaluer l’effet des forçages climatiques et du pastoralisme sur la végétation des prairies supra-forestières à différentes échelles spatio-temporelles. En particulier, nous avons cherché à comprendre les réponses des prairies à la variabilité interannuelle du climat (température et précipitation), aux tendances sur le long terme (réchauffement) et aux évènements extrêmes (vagues de chaleur et sécheresses) ; ainsi qu’aux modalités de la gestion pastorale (charges et calendriers de pâturage). Pour cela, nous avons réalisé des analyses sur un nombre de métriques phénologiques, météorologiques et pastorales. Les premières, dérivées principalement de l’indice de différence normalisée de la végétation (NDVI) à partir d’images satellite de moyenne (MODIS) et de haute résolution (Landsat et SPOT), ainsi que des données acquises au sol à la volée et en continu sur cinq alpages. Les deuxièmes, calculées à partir des ré-analyses du modèle climatique SAFRAN de Météo-France. Les troisièmes, estimées à partir des données des Enquêtes Pastorales régionales (1996-1997 et 2012-2014), et des cahiers d’alpage du dispositif Alpages Sentinelles. Concernant la végétation des prairies supra-forestières, nos résultats mettent en évidence : (i) des tendances au verdissement assez généralisées, (ii) des tolérances plus importantes aux vagues de chaleur qu’aux périodes de sécheresse estivales, (iii) des sensibilités plus importantes aux facteurs climatiques qu’aux pressions de pâturage, (iv) des réponses légères mais positives aux modalités de gestion pastoral, et (v) un potentiel de repousse en fin de saison. L’originalité de nos résultats a été de montrer l’existence de relations plus fortes entre les facteurs climatiques et la végétation des prairies, qu’entre cette dernière et le pâturage. Ils ont également éclairé les apports de l’utilisation de la télédétection dans l’étude de ces milieux. / Mountain grasslands are wide distributed ecosystems which are particularly vulnerable to climate and land use changes. In this context, the main objective of this thesis was to assess the effect of climate and pastoral drivers on mountain grasslands vegetation at different spatiotemporal scales. In particular, we wanted to understand plant responses to annual variations of climate (temperature and precipitation), long term warming and extreme events (heat waves and droughts); as well as the land management impact (grazing intensity and calendars). Hence, we performed several analyses using phenological, meteorological and pastoral metrics. First, we derived phenological metrics mainly from the Normalized Difference Vegetation Index (NDVI) extracted from two sources: satellite remote sensing images at moderate (MODIS) and high (Landsat and SPOT) resolution and field remote sensing measures acquired with portable and fixed sensors over five mountain grasslands. Second, we estimated meteorological metrics from reanalysis of the climatic model SAFRAN provided by Météo France. Third, we estimated pastoral metrics from two regional pastoral surveys (1996-1997 and 2012-2014) and several local documents (“cahiers d’alpage”) produced within the project “Alpages Sentinelles”. Regarding mountain grasslands vegetation, our results showed: (i) widely distributed greening trends, (ii) higher tolerance to heat waves than to droughts, (iii) higher sensibility to climate factors than to grazing pressures, (iv) weak but positive responses to grazing, and (v) a regrowth potential at the end of the season. The originality of our results was to exhibit the stronger relationships between climate factors and vegetation phenology, than between the latter and grazing pressures. Moreover, we highlighted the important contributions of remote sensing data to study mountain ecosystems.

Southern Turkey’s Amuq Valley has been described as a point of convergence bridging distant regions within the ancient Near East. Through an in depth techno-typological and chemical characterization study of 290 obsidian artefacts, this research details changes in deep-time patterns of obsidian use from the Late Neolithic to Early Bronze Age (6000 BCE – 2400 BCE), arguing that shifting traditions of consumption reflect socio-economic developments both within and beyond the Northern Levant. These artefacts come from the three sites of Tell al-Judaidah, Tell Dhahab and Tell Kurdu, the material excavated during the 1930’s by the University of Chicago’s Oriental Institute. Methodologically raw material sourcing was achieved using energy dispersive X-ray fluorescence spectroscopy (EDXRF) in the well-established McMaster XRF Lab [MAX Lab]. With these artefacts’ raw materials all being exotic to the Amuq Valley, originating from various outcrops in Cappadocia, the Lake Van region and Transcaucasia (Turkey and Armenia), over 1000km away, this study not only offers new insight into how Amuq Valley communities engaged in long-distance relations, but also contributes to a larger, deep-time regional study of obsidian consumption as a proxy for understanding significant shifts in Near Eastern socio-economics, from hunter-gatherers to the earliest states. In turn, this study, by employing an Annales school framework to consider practice over deep time at the local and supra-regional level further contributes to an ‘archaeology of the long-term’. / Thesis / Master of Arts (MA) / This research involved the chemical analysis of 290 artefacts of archaeological obsidian – a naturally occurring substance made of crystallized lava - as a means of studying ancient exchange systems in the Near East. More specifically, this study covers archaeological periods from 6000 B.C.E. (Late Neolithic) to 2400 B.C.E. (Early Bronze Age) in the Amuq Valley region of southern Turkey. These artefacts were procured during excavations under the Oriental Institute Museum (University of Chicago) beginning in the 1930s. All artefacts are exotic to the Amuq Valley from several known obsidian outcrops in Anatolia (Turkey), some over 1000km away. Analysis was conducted using X-ray fluorescence (XRF) to match each artefact to its geological origin thereby identifying the range of exotic materials were exchanged across long-distances. The goal of this research was to uncover social and/or economic dynamics of the Amuq Valley through deep-time with regards to the greater obsidian trade network of the Near East.

This work will explore the musical ideas and philosophy discussed by George Russell in his book “The Lydian Chromatic Concept of Tonal Organization” (2001). Through the lens of a performing musician, improviser and composer/arranger, it will explore how the ideas of unity (vertical) and duality (horizontal), interact with each other through Tonal Gravity, and how they can be practically implemented as tools used to expand the scope of vertical (harmonic) consciousness and individual expression. Through my studies of the LCC, I have composed new music, in addition to contextualizing familiar jazz repertoire, in new and refreshing ways. Russell describes the Lydian Chromatic scale and its eleven member scales, as well as three different coexistent levels of Tonal Gravity in his River Trip analogy (Vertical, Horizontal and Supra-Vertical), which together opens up both ingoing and outgoing tonal resources. I have been exploring ways of integrating these elements in my expression. George Russell’s 50 years of work on The Concept was explicitly made to inspire future innovators to develop “[…] intellectual brilliance, intuitive perception, emotional fire, and spiritual depth.” (Russell, 2001, p. 98). Studying the Concept has helped me find my own expression more believable. By avoiding mechanical playing and rather developing my ears and fluency on the instrument, it allows for harmonic color and expression to be felt and created, instead of prepared licks and patterns. / <p><strong>For my exam concert I chose to play my own compositions in a quartet with the following contributing musicians:</strong></p><p>Trumpet and compositions - Øyvind Solheim</p><p>Piano - Rasmus Mannervik</p><p>Double bass - Anton Berndts</p><p>Drums - Richard Andersson Rasheed</p><p>The concert took place at the Royal College of Music in Stockholm on the 26th of March 2024. The set list consisted of six pieces, some of which had seamless transitions in-between each other. Since we had rehearsed with an open approach, we went in to the performance with the same attitude of playing something new.</p><p><strong>Repertoire (with timestamps from the recording)</strong></p><p>1. [00:00:40 – 00:05:40] October March - Øyvind Solheim</p><p>2. [00:06:35 – 00:15:40] Reaching - Øyvind Solheim</p><p>3. [00:16:30 – 00:25:40] Acrimony - Øyvind Solheim</p><p>4. [00:25:40 – 00:36:32] Wheelez - Øyvind Solheim</p><p>5. [00:38:02 – 00:47:40] Acending, Decending - Øyvind Solheim</p><p>6. [00:49:29 – 00:58:47] Lam-ent - Øyvind Solheim</p>

Cette thèse est centrée sur la mesure et l'interprétation du rayonnement X mou ([1 keV; 15 keV] environ) dans les Tokamaks. Le chapitre 2 montre que ce rayonnement véhicule des informations sur la température et la densité du plasma, sur sa configuration magnétique, et sur son contenu en impuretés. Malheureusement les mesures effectuées sont intégrées spatialement et spectralement et résultent des contributions de tous les ions présents.Le diagnostic X mou de Tore Supra s'articule autour de diodes semi-conductrices présentée dans le chapitre 3 aux côté d'un détecteur à gaz testé avec succès. Une nouvelle méthode de détermination de la réponse spectrale d'un photodétecteur n'utilisant qu'un tube X mou portable est également décrite.Les inversions tomographiques, qui permettent d'accéder au champ d'émissivité reconstruit dans une section poloidale, font l'objet du chapitre 4. Les améliorations apportées à un algorithme particulier sont détaillées.Une comparaison systématique entre les positions horizontales du maximum d'émissivité et de l'axe magnétique est présentée au chapitre 5.Le chapitre 6 décrit une hypothèse concernant la résilience de la fonction de rayonnement X mou d'une impureté vis-à-vis du transport de cette impureté. Cette hypothèse permet de déduire la densité d'une impureté de son émissivité X mou. Les processus physiques justifiant cette hypothèse, ainsi que leur domaine de validité sont analysés avec soin.Le chapitre 7 présente les asymétries poloidales d'émissivité X mou. Les premiers résultats d'expériences mises en oeuvres à ASDEX-U pour vérifier les dépendences paramétriques de deux types particuliers d'asymétries sont détaillés. / This thesis focuses on measuring and interpreting the Soft X-Ray (SXR) radiation (approximately [1 keV; 15 keV]) in Tokamaks.As explained in Chapter 2, this radiation conveys information about the plasma density, temperature, magnetic equilibrium and impurity content. However, the measured data is spectrally and spatially-integrated and results from several physical phenomena affecting every ion species. Tore Supra's SXR diagnostics is based on semiconductor diodes presented in Chapter 3, along with a new gas detector successfully tested in laboratory and on Tore Supra. A new methodology for absolute spectral characterisation of photo detectors using a portable SXR tube is presented. Tomographic inversion algorithms, that grant access to reconstructions of the SXR emissivity field in a poloidal cross-section, are presented in Chapter 4. Improvements implemented on one particular algorithm are detailed with examples of application. A comparison between the position of the SXR emissivity maximum and the magnetic axis reconstructed by an equilibrium code is presented in Chapter 5.Chapter 6 presents an approach used to derive an impurity density from its SXR emissivity using the robustness of its SXR cooling factor with respect to impurity transport. The physics accounting for this robustness is studied and a first map of the domain of validity of this method is provided. Chapter 7 addresses poloidal asymmetries of the SXR emissivity field. Two types of asymmetries are presented as well as experiments conducted on ASDEX-U to verify their parametric dependences. A new type of SXR asymmetry, observed on Tore Supra is introduced.

The purpose of this study is to analyse the role of the Organisation of African Unity (OAU) as an international governmental organisation in regional co-operation and stability from 1963 until 2000. It is also aims to evaluate the OAU's success or failure as the initiator of African unity and the driver of regional co-operation and stability in Africa within political, economic and social spheres. As background, the motivation for the study is discussed and the problem is stated. From this, research questions are formulated, and objectives identified. Three hypotheses are formed, which the study aimed to prove. The range of core theoretical foundations, concepts, characteristics, theories, approaches and classifications are examined in detail as foundations for an understanding and evaluation of the role of the OAU. Regional organisations as a phenomenon are also studied in-depth with reference to their nature, meaning and historical origins. Inter-state relations in the international community are theoretically explored, as well as the position and potential of regionalism within international public administration. Concepts, characteristics, types and functions of regional organisations and the role of co-operation, sovereignty and supra-nationalism in regional co-operation are covered. Following this, a study is made of the historical origins, nature and character of Pan-Africanism and the evolution of the OAU. Based on the structural-functional approach, the nature and essential characteristics of the organisation are analysed, with reference to its structures, and the tasks of the Secretary-General and its various commissions. In addition, the former relationship between the OAU and the UN is also highlighted. The role of the OAU is evaluated as a regional organisation involved in the establishment of regional co-operation in Africa in the political, economic, cultural and social spheres. The study concludes with an evaluative synthesis of its findings, proposals and conclusions. The OAU is found to have been successful in certain regards, but in others, it failed to attain its primary purpose: to forge unity on the continent and to create co-operation among states. Its successor, the African Union could learn some valuable lessons from the OAU's history. / Public Administration / (D.P.A.)

Introdução: A patogênese molecular dos tumores adrenocorticais é heterogênea e ainda pouco compreendida. A hiperexpressão do gene do fator de crescimento semelhante à insulina II (IGF-II) tem sido demonstrada na maioria dos carcinomas adrenocorticais em adultos. Os efeitos mitogênicos do IGF-II são mediados pela interação com o receptor de IGF-I (IGF-IR). Adicionalmente, o fator esteroidogênico 1 (SF-1) e o fator codificado por uma região crítica do cromossomo X associada ao sexo reverso e à hipoplasia adrenal congênita (DAX-1), ambos envolvidos no desenvolvimento e na esteroidogênese adrenal, também têm sido implicados na tumorigênese adrenocortical. Objetivos: Analisar a expressão gênica e a imunorreatividade dos fatores IGF-II, IGF-IR, SF-1 e DAX-1 em tumores adrenocorticais de crianças e adultos. Avaliamos ainda os efeitos de um inibidor seletivo do IGF-IR (NVP-AEW541) na proliferação celular e apoptose de linhagens celulares de tumores adrenocorticais. Métodos: Neste estudo, a expressão gênica foi determinada por PCR quantitativa em tempo real em 57 tumores adrenocorticais (37 adenomas e 20 carcinomas). Vinte e três pacientes tinham idade inferior ou igual a 15 anos. A análise de imunohistoquímica foi realizada em 109 tumores adrenocorticais (71 adenomas e 38 carcinomas). Os efeitos do tratamento com NVP-AEW541 (0,3 a 30 M) na proliferação celular e apoptose foram avaliados nas células NCI H295 de carcinoma adrenocortical humano e em uma nova linhagem celular estabelecida a partir de um adenoma adrenocortical pediátrico da nossa casuística. Resultados: A hiperexpressão do gene IGF-II foi evidenciada nos tumores adrenocorticais benignos e malignos de crianças (média ± EPM, 50,8 ± 18,5 vs. 31,2 ± 3,7, respectivamente; p= 0,23). Em adultos, a expressão do gene IGF-II foi significativamente mais elevada nos carcinomas adrenocorticais quando comparada com os adenomas (270,5 ± 130,2 vs. 16,1 ± 13,3; p= 0,0001). O percentual das células neoplásicas imunorreativas para o IGF-II não foi significativamente diferente entre os adenomas e carcinomas adrenocorticais pediátricos (14,1 ± 2,8% vs. 31,1 ± 13,1%, respectivamente; p= 0,32). Em adultos, o percentual das células neoplásicas positivas para o IGF-II foi significativamente maior nos carcinomas adrenocorticais em relação aos adenomas (34,4 ± 5,8% vs. 14,2 ± 3,2, respectivamente; p= 0,03). Os valores de RNAm do IGF-IR foram significativamente mais elevados nos carcinomas adrenocorticais pediátricos em relação aos adenomas (9,1 ± 1,2 vs. 2,6 ± 0,3; p= 0,0001), enquanto a expressão deste receptor foi similar nos tumores adrenocorticais benignos e malignos de adultos (1,6 ± 0,3 vs. 1,8 ± 0,5, respectivamente; p= 0,75). Os valores de RNAm do IGF-IR [risco relativo (RR) 2,0, intervalo de confiança (IC) de 95% 1,2 a 3,1; p= 0,004] e os critérios histopatológicos de Weiss (RR 1,8, IC de 95% 1,2 a 2,7; p= 0,003) foram marcadores independentes de metástases em crianças e adultos, respectivamente. O NVP-AEW541 inibiu a proliferação celular estimulada por IGF-II de forma dose e tempo dependentes nas 2 linhagens celulares de tumores adrenocorticais através de uma significativa indução da apoptose. Adicionalmente, a hiperexpressão do gene SF-1 foi identificada em 13% e 15% dos tumores adrenocorticais diagnosticados em crianças e adultos, respectivamente. O percentual das células neoplásicas com imunorreatividade nuclear para SF-1 foi significativamente maior nos tumores adrenocorticais pediátricos em relação aos tumores diagnosticados em adultos (29,1 ± 5,4% vs. 8,3 ± 2,3%, respectivamente; p= 0,0001). Estes dados indicam que o aumento da expressão do SF-1 nos tumores adrenocorticais pediátricos ocorre em nível pós-traducional. A hiperexpressão do gene DAX-1 foi identificada em 39% dos tumores adrenocorticais, com uma prevalência semelhante em crianças e adultos. De forma similar, o aumento da expressão da proteína DAX-1 foi identificado em 36% e 27% dos tumores adrenocorticais diagnosticados em crianças e adultos, respectivamente. A imunorreatividade nuclear para DAX- 1 foi semelhante nos adenomas e carcinomas adrenocorticais (29,2 ± 3,8% vs. 21,4 ± 5,8% das células neoplásicas, respectivamente; p= 0,12). Conclusões: A hiperexpressão do IGF-II tem um papel relevante na tumorigênese adrenocortical. A hiperexpressão do gene IGF-IR foi um marcador biológico independente do carcinoma adrenocortical metastático em crianças. Os efeitos anti-tumorais in vitro do NVP-AEW541 sugerem que o IGF-IR constitui um potencial alvo terapêutico para o carcinoma adrenocortical humano. Adicionalmente, o aumento da expressão do SF-1 foi evidenciado predominantemente nos tumores adrenocorticais pediátricos. A hiperexpressão do DAX-1 constitui um evento importante na patogênese molecular dos tumores adrenocorticais benignos e malignos / Introduction: The molecular pathogenesis of adrenocortical tumors is heterogeneous and incompletely understood. Insulin-like growth factor II (IGF-II) overexpression has been demonstrated in adult adrenocortical carcinomas. IGF-II exerts its mitogenic effects through interaction with IGF-I receptor (IGF-IR). In addition, steroidogenic factor 1 gene (SF-1) and dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome gene (DAX-1), which regulate adrenal development and steroidogenesis, have been also involved in adrenocortical tumorigenesis. Objectives: To analyze gene and protein expression of IGF-II, IGF-IR, SF-1 and DAX-1 in pediatric and adult adrenocortical tumors. We also evaluated the effects of a selective IGF-IR kinase inhibitor (NVP-AEW541) on adrenocortical tumor cell lines. Methods: Gene expression was determined by quantitative real-time PCR in 57 adrenocortical tumors (37 adenomas and 20 carcinomas) from 23 children and 34 adults. Twenty and three patients were younger than 15 years. A tissue microarray analysis was performed on a large cohort of 109 ACT (71 adenomas and 38 carcinomas; 39 children and 70 adults) In addition, the effects of NVP-AEW541 treatment (0.3 to 30M) on proliferation and apoptosis were investigated in the NCI H295 cell line and in a new cell line established from a pediatric adrenocortical adenoma of our cohort. Results: IGF-II transcripts were overexpressed in pediatric adrenocortical carcinomas and adenomas (mean ± SE, 50.8 ± 18.5 vs. 31.2 ± 3.7, respectively; p= 0.23). IGF-II gene expression was significantly higher in adult adrenocortical carcinomas than in adenomas (270.5 ± 130.2 vs. 16.1 ± 13.3; p= 0.0001). The percentual of neoplastic cells immunostaining for IGF-II was not statistically different between pediatric adrenocortical adenomas and carcinomas (14.1 ± 2.8% vs. 31.1 ± 13.1%, respectively; p= 0.32). Otherwise, the percentual of positive neoplastic cells for IGF-II was significantly higher in adult adrenocortical carcinomas than in adenomas (34.4 ± 5.8% vs. 14.2 ± 3.2, respectively; p= 0.03). IGF-IR mRNA levels were significantly higher in pediatric adrenocortical carcinomas than in adenomas (9.1 ± 1.2 vs. 2.6 ± 0.3; p= 0.0001), whereas similar IGF-IR expression levels were identified in adult adrenocortical carcinomas and adenomas (1.6 ± 0.3 vs. 1.8 ± 0.5, respectively; p= 0.75). In a Cox multivariate analysis, IGF-IR gene expression [hazard ratio (HR) 2.0, 95% confidence interval (CI) 1.2 to 3.1; p= 0.004] and Weiss score (HR 1.7, 95% CI 1.2 to 2.7; p= 0.003) were independent biomarkers of metastasis in pediatric and adult adrenocortical tumors, respectively. Furthermore, NVP-AEW541 blocked cell proliferation in a dose- and time-dependent manner in both NCI H295 and pediatric adrenocortical cell lines through a significant increase of apoptosis. Additionally, SF-1 gene overexpression was identified in 13% and 15% of pediatric and adult adrenocortical tumors, respectively. The percentual of neoplastic cells with nuclear immunoreactivity for SF-1 was significantly higher in pediatric than in adult adrenocortical tumors (29.1 ± 5.4% vs. 8.3 ± 2.3%, respectively; p= 0.0001). These findings suggest that SF-1 overexpression occurs at the translational level in pediatric adrenocortical tumors. DAX-1 gene overexpression was identified in 39% of adrenocortical tumors with a similar frequency in children and adults. Similarly, DAX-1 protein overexpression was identified in 36% and 27% of pediatric and adult adrenocortical tumors, respectively. DAX-1 immunostaining on nuclei was not statistically different in benign and malignant adrenocortical tumors (29.2 ± 3.8% vs. 21.4 ± 5.8% of neoplastic cells, respectively; p= 0.12). Conclusion: IGF-II overexpression has a pivotal role to adrenocortical tumorigenesis. IGFIR overexpression was a potential biomarker of metastases in children with adrenocortical carcinoma. We demonstrated that a selective IGF-IR kinase inhibitor had anti-tumor effects in adult and pediatric ACT cell lines, suggesting that IGF-IR inhibitors represent a promising therapy for human adrenocortical carcinoma. In addition, SF-1 overexpression might be mainly involved in pediatric adrenocortical tumorigenesis. DAX-1 overexpression has an important role to molecular pathogenesis of benign and malignant adrenocortical tumors

Introdução: Uma elevada incidência de tumores adrenocorticais pediátricos e de adultos é observada nas regiões sul e sudeste do Brasil. Hiperexpressão dos genes IGF1R, SF1 e FGFR4 tem sido descrita em tumores adrenocorticais. Apesar de hiperexpressão ser um evento comum em diversas neoplasias, ainda não são claros os mecanismos moleculares que seriam responsáveis por essa falha na regulação da expressão. Objetivos: Determinar o número de cópias dos genes IGF1R, SF1 e FGFR4 em tumores adrenocorticais diagnosticados em crianças e adultos. Adicionalmente correlacionaremos os dados de expressão gênica e/ou protéica de IGF1R, SF1 e FGFR4 com o diagnóstico histológico e evolutivo dos tumores adrenocorticais. Pacientes e métodos: Sessenta e quatro pacientes com tumores adrenocorticais foram selecionados para o estudo. Todos os pacientes foram submetidos à avaliação clínica e tratamento cirúrgico. Oito glândulas adrenais normais obtidas em cirurgias renais ou autópsias foram utilizadas como controles. DNA genômico extraído dos tecidos normais e tumorais da glândula suprarrenal foram utilizados como substrato nas reações de multiplex ligation-dependent probe amplification (MLPA) com o intuito de se determinar o número de cópias dos genes IGF1R, SF1 e FGFR4. PCR em tempo real (SYBR Green) foi realizado para confirmar os dados de MLPA para os genes IGF1R e SF1. Resultados: Amplificação do gene IGF1R foi detectada por MLPA e confirmada por PCR em tempo real SYBR Green em apenas um carcinoma adrenocortical. Adicionalmente, amplificação gênica de outros loci (IGFBP3, FGFR4 e NSD1) bem como de sondas controles foi observada, sugerindo uma condição aneuplóide neste tumor maligno. Amplificação de SF1 foi detectada em 10 tumores adrenocorticais (8 pediátricos e 2 de adultos). Os valores de expressão gênica foram significantemente maiores em tumores associados com amplificação gênica quando comparados com tumores sem amplificação. Além disso, imunorreatividade para SF-1 foi detectada nos tumores com aumento no número de cópias. Doze amplificações do locus FGFR4 (3 pediátricos e 9 de adultos) foram demonstradas por MLPA. A amplificação do locus FGFR4 e hiperexpressão deste gene foram significantemente mais relacionados a carcinomas. Conclusões: Amplificação do gene IGF1R é um evento raro nos tumores adrenocorticais pediátricos e de adultos. A hiperexpressão de IGF1R em tumores adrenocorticais pediátricos não foi secundária à amplificação gênica. Amplificação do gene SF1 foi evidenciada predominantemente em tumores adrenocorticais pediátricos e se correlacionou com hiperexpressão gênica e protéica. Amplificação do locus FGFR4 foi demonstrada predominantemente em tumores adrenocorticais malignos de adultos. Amplificação de oncogenes representa um mecanismo molecular relevante na tumorigênese adrenocortical / Introduction: A high incidence of adrenocortical tumors in children and adults has been observed in Southern and Southeastern regions of Brazil. Overexpression of IGF1R, SF1 and FGFR4 genes have been described in adrenocortical tumors. Despite of overexpression be a common event in several neoplasias, the molecular mechanism implicated in this upregulation remains unknown. Objectives: To determine the copy number of IGF1R, SF1 and FGFR4 genes in pediatric and adult adrenocortical tumors. Additionally, correlate with IGF1R, SF1 and FGFR4 gene and/or protein expression data as well as with the histological diagnosis and evolution of the adrenocortical tumors. Patients and methods: Sixty and four patients with adrenocortical tumors were selected for this study. All patients were submitted to clinical evaluation and surgical treatment. Eight normal adrenal glands obtained in renal surgery or autopsies were used as controls. The MLPA reactions were performed with the DNA extracted from adrenal gland tissues in order to determine the copy number of IGF1R, SF1 and FGFR4 genes. SYBR Green real-time PCR was carried out to confirm MLPA data for IGF1R and SF1 genes. Results: IGF1R amplification was detected by MLPA and confirmed by SYBR green real-time PCR in only one adrenocortical carcinoma. Additionally, other loci amplification was detected (IGFBP3, FGFR4 and NSD1) as well as for control probes, suggesting aneuploidy in this malignant tumor. SF1 amplifications were shown in 10 adrenocortical tumors (8 from children and 2 from adults). The SF1 mRNA levels were significantly higher in adrenocortical tumors associated with increased SF1 gene copies when compared with adrenocortical tumors without gene amplification. Moreover, all adrenocortical tumors with SF1 gene amplification showed a strong SF1 staining. Twelve FGFR4 locus amplifications (3 from children and 9 from adults) were demonstrated by MLPA. FGFR4 locus amplification and overexpression of this gene were significantly more related to carcinomas. Conclusions: IGF1R amplification is a rare event in adrenocortical tumors and it was not responsible for the IGF1R overexpression of pediatric and adult adrenocortical tumors. SF1 gene amplification was detected predominantly in pediatric adrenocortical tumors and was associated with gene and protein overexpression. FGFR4 locus amplification was demonstrated mainly in adult maligant adrenocortical tumors. FGFR4 amplification and upregulation were more associated to adrenocortical carcinomas. Oncogenes amplification represents an important molecular mechanism in adrenocortical tumorigenesis