No description available.

The glycoprotein hormone family comprising of Luteinizing Hormone (LH), Chorionic Gonadotropin (hCG), Follicle Stimulating Hormone (FSH) and Thyroid Stimulating Hormone (TSH) plays important role in reproduction and overall physiology of the organism. These hormones are heterodimeric molecules consisting of an identical α subunit non-covalently associated with the hormone-specific β subunit. Both subunits of all these hormones are N-glycosylated. In addition, hCGβ subunit also has four O-linked oligosaccharides located at the C-terminus of the polypeptide(1). The α and β subunits of all these hormones contain five and six disulfide bonds respectively and the crystal structures of hCG and hFSH indicate that both subunits of the hormones belong to the cystine knot family of proteins(2-4). Although the β subunits are hormone specific, there are distinct similarities in these subunits with the 12 cysteines conserved in all these subunits (1). These hormones, because of their unique structural features have proved to be important models for structure–function relationship studies of complex dimeric glycoproteins. Folding of subunits during biosynthesis, role of glycosylation in folding pathways and in vitro and in vivo bioactivity of the hormone, as well as, identification of domains important for subunit association, receptor binding and subsequent signal transduction have been topics of active investigations. The receptors of these hormones belong to the family of G-protein coupled receptors (GPCR) and have unique hormone specific exodomain not present in other members of the GPCR family and characteristic seven transmembrane domains followed by a C terminal domain(5). Primary structure analysis of Glycoprotein hormone receptors family revealed sequence conservation, maximum homology being observed in the transmembrane domain (TMD)(6). Significant homologies could be observed in the hormone specific extracellular domains (ECD) also (7). Despite these homologies, the receptors exhibit exquisite specificity with very low cross reactivity with other members of the hormone family (8). Elucidation of the molecular details of the contacts between the hormone and the receptors has not been achieved so far. Various approaches have been employed to delineate the residues or domains of both hormone and receptors involved in interaction. These include testing of chimeras or mutants of hormones or receptors for changes in activity (9-12), chemical modifications(13) and competition with peptides from either hormones (14) or receptors (15). Polyclonal and monoclonal antibodies against glycoprotein hormones and various fragments of their receptors have been used to determine the role of different domains of both in binding and response (6, 16, 17). However, till date there is no consensus on the specific mechanisms by which the glycoprotein hormone docks onto its receptor. It was proposed that the initial contact between the hormone and the receptor occurs through high affinity binding of the hormone specific β subunit to the Leucine rich regions of the ECD that results in conformational changes in both hormone, as well as, the receptor and brings hormone/ECD complex closer to the TMD of the receptor. The secondary, relatively lower affinity interactions between the hormone and the receptor then take place through common α subunit and exoloops of TMD of the receptor resulting in signal generation (18, 19). Recently a different kind of model has been proposed which suggests that the hormone does not make any direct contacts with the TMD of the receptor. The signal is transduced by the change in contacts between ECD and TMD brought about by hormone’s interaction with ECD(8, 20). The present study was initiated with an overall objective of understanding the molecular details of the hormone receptor interactions of this family, particularly hCG- LH receptor interactions. Two different approaches were employed for this purpose; the first, direct approach being structure elucidation of the members of the glycoprotein hormone family while the second approach uses antibodies against hCG as tools to probe into hormone-receptor interactions. The results obtained using these two approaches have been consolidated in the present thesis and are organized as follows. Chapter 1 is an extensive review of the literature and it builds background for the present work while the exact aim and scope of the present work have been defined in Chapter 2. Chapter 3 describes cloning, expression and purification of recombinant glycoprotein hormones hLH, hCG and single chain derivative of hCG. The Chapter 4 gives details of the molecular aspects of hCG-LH receptor interaction dissected using hCG monoclonal antibodies (MAbs). Chapter 5 discusses implications of the observations made in the present study and states the future directions envisaged. There are a number of endocrinopathies associated with abnormal levels of glycoprotein hormones and treatments of such disorders often demand large quantities of either agonists or antagonists of the hormones. The structure-function relationship studies should help in identifying domains/residues important for subunit interaction, receptor binding, and signal transduction, which would also allow engineering of agonists and antagonists of hormone action. However, structure determination of the glycoprotein hormone family using X-ray crystallography has proved to be a difficult task and it is believed that the heterogeneity in glycosylation is the primary reason for this low success rate in the process of crystallization. The first crystal structure of hCG was that of completely deglycosylated hCG but such a molecule displays antagonistic behavior(2, 3). Use of NMR spectroscopy, the alternate method commonly used for structure determination is often limited by the availability of large quantities of biologically active hormones free of any contaminants. Large quantities of LH, hCG and FSH are also required for treatment of infertile patients suffering from gonadotropin deficiency. The first goal of the present study was thus to produce and purify biologically active recombinant hCG and hLH. Owing to the inherent features of glycoprotein hormones and their potential therapeutic applications, the recombinant expression of these hormones is an important goal from both basic research, as well as, commercial point of view. Considering the above mentioned features it is clear that the expression system used for the hyperexpression of these glycoprotein hormones should also serve as a model system for investigating structure–function relationships and folding of subunits during biosynthesis, in addition to providing sufficient quantities of the hormones for clinical applications. It has been demonstrated that N-linked glycosylation during biosynthesis facilitates protein folding and conformational maturation of glycoprotein hormone subunits into an assembly-competent, biologically active form (21). Therefore, the ideal recombinant expression system should also be able to glycosylate the protein during biosynthesis. The Pichia pastoris yeast expression system was chosen for hyperexpression of glycoprotein hormones as it blends the advantages of both bacterial and mammalian expression systems. Earlier, expression of biologically active hCG and the subunits of hCG and bovine FSH using Pichia pastoris expression system has been reported from the laboratory (22, 23). Chapter 3 (section 3.3.1) of the thesis describes hyperexpression of hLH. The expression of these heterologous proteins was scaled up using fermentation procedures to fulfill the requirements of large quantities of hormones for various applications. Purification of Pichia expressed hormones turned out to be a complex task as large quantity of the hormone was secreted out in the fermentation medium (10litre volume) that was of high ionic strength. Of several different strategies attempted for concentration and partial purification of recombinant hCG, hydrophobic interaction chromatography (HIC) using Phenyl Sepharose matrix emerged as the most efficient technique as a first step of purification. Subsequently, cation exchange chromatography using SP- Sepharose matrix yielded completely purified biologically active recombinant hCG (section 3.3.2). The preliminary data also suggested that Pichia cells express a biologically active form of hCG which appeared to be less glycosylated and of lower molecular weight. Using the same protocol purification of hLH, as well as, single chain derivative of hCG, phCGαβ was achieved (section 3.3.3). These recombinant proteins were characterized extensively using various biochemical, as well as, immunological criteria and were shown to be similar to their natural counterparts with respect to their ability to bind LH receptor and to transduce signal as judged by radioreceptorassays and in vitro bioassays respectively. The hydrophobic interaction chromatography proved an important starting point for purification of all the other members of the glycoprotein hormone family expressed using Pichia pastoris expression system. With the availability of purified, biologically active recombinant hCG in large quantities it was now possible to make attempts towards structure elucidation using NMR spectroscopy. The structure determination of such complex proteins by NMR spectroscopy is made relatively easier by labeling the proteins with magnetically more active, stable isotopes of carbon and nitrogen, 13C and 15N respectively however the cost is often prohibitively high. The Pichia pastoris expression system offers simple means of labeling the proteins as the cells can be grown on simple salts of carbon and nitrogen such as 13C labeled methanol, 15N labeled ammonium chloride or ammonium sulphate. The Chapter 3 also gives a brief account of the preliminary attempts made to label the recombinant hCG with 15N and the structural studies carried out with the carbohydrate moieties of the recombinant hCG using solution NMR spectroscopy. This work was carried out in collaboration with the laboratory of Prof. J.P Kamerling of the University of Utrecht, Netherlands and the efforts are currently underway to elucidate the complete structure of the Pichia expressed hCG. The common feature of receptors and antibodies against the ligand is that both display very specific, high affinity binding towards the ligand. Hence, it is logical to speculate that the antigen binding regions of the antibodies that inhibit hormone binding and/or response, exhibit homology with distinct domains of the receptor. By identifying the epitopes recognized by such antibodies, it should be possible to predict contact points between the hormone and the receptor. In the present study, this hypothesis has been tested using monoclonal antibodies (MAbs) against hCG recognizing different epitopes in the hormone molecule and having different effects on hormone binding and response (Chapter 4). These MAbs were classified as α subunit specific, β subunit specific or heterodimer specific depending on their abilities to bind either subunit in addition to the hormone itself. Interestingly, it was observed that the hCGβ subunit specific MAbs, as well as, heterodimer specific MAbs inhibited hCG receptor binding and hence the response generated by hCG, while the hCGα subunit specific MAbs inhibited only response to the hormone without interfering in binding (Section 4.3.1). To dissect out these interactions further the epitopes recognized by these antibodies on hCG molecule were determined (Section 4.3.2), single chain fragment variable (ScFv) were generated from each of these antibodies and it was shown that these ScFv retain the functionality of the original antibody (Section 4.3.3). Further, the amino acid sequence of each antibody was determined (Section 4.3.4) and finally shown that the antigen binding domains of antibodies show homology to the distinct regions of the LH receptor on sequence alignments between the two using three different programs (4.3.5). The hCGβ subunit specific MAb 52/28' displayed distinct homology with the ECD of LH receptor while the α subunit specific MAb C10 showed regions homologous to TMD of the receptor and the heterodimer specific MAb E12 was found to be similar to the hinge region of the receptor. This clearly indicates that the β subunit of hCG is in close contact with ECD of the receptor while the α subunit makes contacts with the TMD of receptor. The present study thus supports the existing model of hormone receptor interactions, which states that the hormone first binds to the exodomain of the receptor mainly through its β subunit while the integrity of the α subunit is critical for signaling. (24, 25). Also, the observations made in the present study exhibit an interesting possibility of antigen antibody complexes being used as surrogate models for gaining insights into hormone receptor complex. Further, it has been reported that hCG has immunocontraceptive potential(26). Active and passive immunization studies with hCG in primates and humans have demonstrated the possibility of controlling fertility by the antibodies capable of neutralizing hCG. This forms the basis for female contraceptive vaccine that has undergone Phase II clinical trials in India. The MAb E12 characterized in the present study displayed highly specific binding to heterodimeric hCG exclusively without showing any cross reactivity with hLH (Section 4.3.1). The epitope mapping analysis revealed that this antibody recognizes a unique discontinuous epitope present only in the heterodimeric hCG and is distinct from the unique C-terminal extension of hCGβ absent in hLHβ (Section 4.3.2). The MAb, IgG or its recombinant single chain fragment variable (ScFv), inhibited response to hCG, but not to hLH (4.3.3). Thus, the epitope recognized by this MAb is an ideal candidate antigen for immunocontraception. The MAb E12 can also be used for passive immunization in case of emergency contraception. Another potential application of hCG specific antibodies is in homing and the treatment of tumors that secrete hCGβ subunit. The hCGβ subunit specific MAbs used in the present study 52/12 and 52/28' that inhibited hCG receptor binding as well as response generated by hCG can be used in treating such tumors. The functional ScFvs generated from these MAbs in the present study can be made use of on humanization. Thus, the present study has yielded some important molecules for therapeutic applications besides providing a new platform for structure-function relationship studies of the complex glycoprotein hormones.

Die vorliegende Arbeit widmet sich der Darstellung des Kunstmarktgeschehens und der Entscheidungsfindung von Galeristen. Ziel ist es aufzuzeigen, welche Auswirkungen Erfahrung und Expertise sowie ein begrenzter Informationszugang auf die angewandte Entscheidungslogik und das Entscheidungsverhalten von Galeristen haben. Sie behandelt die Thematik der kausalen und effektualen Entscheidungslogik und der unterschiedlichen Entscheidungsansätze von Novizen und Experten. Gemäß Sarasvathy (2001) tendieren Novizen zu kausaler und Experten zur effektualer Logik. Sie unterscheiden sich durch ihren Grad an Expertise, welche auf Deliberate Practice, Erfahrung und kontinuierlich erbrachter überragender Leistungserbringung basiert (Ericsson 2006; Mitchell et al. 2005:3, Dew et al. 2009: 289). Gegenstand der Untersuchung war die Beantwortung der Fragen, ob sich die Berufserfahrung, das Geschlecht und der akademische Werdegang des Galeristen auf die angewandte Entscheidungslogik auswirken. Die Ergebnisse belegen, dass die Berufserfahrung einen signifikanten Einfluss auf die angewandte Entscheidungslogik der Galeristen hat: Novizen-Galeristen präferieren die kausale Entscheidungslogik, Experten-Galeristen die effektuale. In Bezug auf das Geschlecht ist nachweisbar, dass Galeristinnen am häufigsten die kausale Entscheidungslogik anwenden. Dasselbe Bild stellt sich bei Galeristen – ungeachtet ihres Geschlechts – ohne akademische Ausbildung ein. Die Arbeit setzt zudem ihren Fokus auf die experimentelle Untersuchung des Preisbildungsverfahrens durch Galeristen, wobei insbesondere der etwaige Einfluss der Overconfidence im Mittelpunkt steht. Die Studienergebnisse lassen darauf schließen, dass ein signifikantes Maß an Overconfidence dazu führt, dass die Preise für Kunstwerke niedriger gesetzt werden. Zudem konnte ein Wissenseffekt festgestellt werden: Je versierter ein Galerist im Kunstmarkt ist, desto höher setzt er den Preis für ein als „ausstellungswürdig" deklariertes Kunstwerk. / The objective of this dissertation is to shed more light on the primary art market and the decision-making processes of its protagonists, the gallery owners. The doctoral thesis focuses on the potential impact of experience and expertise on the gallerists‘ applied decision-making logic and the consequences of limited access to information for the gallery owners‘ decision-making behaviour. In particular, the distinction between novices and experts and their decision-making is addressed. According to Sarasvathy (2001), novices tend to use a predictive decision-making logic (causation), whereas experts apply a non-predictive logic (effectuation). They differ in their level of expertise, which is based on deliberate practice, experience and continuous outstanding and superior performance in a particular domain (Ericsson 2006; Mitchell et al. 2005:3, Dew et al. 2009: 289). The studies conducted examined whether the professional experience, gender and academic career of the gallery owner affects the applied decision-making logic. The results show that professional experience has a significant influence on the applied decision-making logic of the gallery owner: novice-gallerists prefer the causal approach, expert gallery owners favour the effectual decision-making logic. With regard to gender and the academic career, it can be proven that female gallery owners and gallerists without academic training most often apply the causal decision-making logic. This dissertation also focuses on the experimental analysis of the influence and impact of overconfidence on the price setting processes of gallery owners. The results indicate that a significant level of overconfidence leads to lower prices for works of art. In addition, a knowledge effect could be observed: the more sophisticated the gallery owners are, the higher will be the price they set for an art work they consider to be suitable for an exhibition.

No description available.

Die über 600 Gemälde, Miniaturen und Reliefplastiken umfassende Sammlung des Berliner Verlegerehepaars Franz und Frieda von Lipperheide ist Teil eines umfangreichen und einzigartigen Quellenkonvoluts, welches die unterschiedlichen ständischen, regionalen und geschlechterspezifischen Ausformungen von Mode, Tracht und Kostüm und ihrer Zeichenhaftigkeit abbildet. Die Werke der Sammlung, visuelle Zeugnisse europäischer und außereuropäischer Kleidermoden aus der Zeit von ca. 1430 bis 1900, changieren zwischen künstlerischen Entwürfen und dokumentarischen Zeugnissen. Sie waren erstmals 1906 als Teil der „Lipperheideschen Kostümbibliothek“, einer Schenkung von Franz und Frieda von Lipperheide an das Kunstgewerbemuseum Berlin, für die Öffentlichkeit zugänglich, seit 1934 jedoch ohne Domizil, später geteilt und deponiert. Der erstmaligen Aufarbeitung von Genese und Geschichte der Sammlung, ihrem Kontext als Wissensspeicher für die verlegerischer Tätigkeit der Eheleute, dem ursprünglichen Umfang – die private Sammlung umfasste Textilien, Bildzeugnisse, Textquellen und Sekundärliteratur –, der Sammlungsstrategie und dem Quellenverständnis widmet sich die vorliegende Arbeit. Im Kontext des 19. Jahrhunderts, zeitgenössischer Museumsgründungen und einer kunstgewerblichen Neuausrichtung, repräsentiert die Gemäldesammlung den methodischen Ansatz einer frühen Kostümgeschichtsschreibung. / The collection of the Berlin publishers Franz and Frieda von Lipperheide, comprising more than 600 paintings, miniatures and reliefs, is part of an extensive and unique compendium of sources depicting the various forms of fashion, traditional costume and dress, referencing their specifics to social class, region and gender. As visual evidence of European and non-European dress fashions from around 1430 to 1900, the pieces in the collection alternate between artistic designs and documentary evidence. They were first made accessible to the public in 1906 as part of the Lipperheide Costume Library, donated to the Berlin Museum of Decorative Arts by Franz and Frieda von Lipperheide. Since 1934, however, they have been without a residence, and were later divided up and deposited. The present study is the first complete analysis of the collection, looking at its origin and history, its context as a repository of knowledge for the spouses’ publishing activities, its original scope, its strategy and the conception of sources. Furthermore, it highlights the original scope of the private collection, which comprised textiles, pictorial evidence, text sources and secondary literature. In the context of the 19th century, informed by museum foundations and a rebirth of the arts and crafts, the Lipperheide collection of paintings represents a methodological approach of an early costume historiography.

No description available.

Die vorliegende Dissertation untersucht gewaltsam verschwundene Welten der deutschen (donauschwäbischen) und jüdischen Minderheiten im serbischen Südostbanat, einst Teil der Militärgrenze der Habsburgermonarchie, heute als geographisches Dreieck zwischen Ungarn, Rumänien und Serbien aufgeteilt. Denkmäler und Grabsteine mit deutschen Inschriften im Südostbanat zeugen von Verflechtungen innerhalb der Geschichte von donauschwäbischen und jüdischen Minderheiten. Sie sind heute in der Region nicht fast mehr anzutreffen und sind wie abwesende Nachbar:innen. Vor diesem Hintergrund folgt die Arbeit den damaligen und gegenwärtigen Konvivenzräumen. Damit einhergehend nimmt die vorliegende Analyse eine erzähltheoretische Perspektive ein, indem sie Friedhöfe/ Denkmäler/ Gedenkstätten und Archivquellen als “narrative Artefakte” versteht, die “Weltlichkeit” (worldness) enthalten. Es wird danach gefragt, wie Menschen in den heutigen Banater Nachbarschaften unterschiedliche und doch nah beieinander liegende Erzählwelten (storyworlds) hinsichtlich der abwesenden Minderheiten schaffen. Auf wessen Vergangenheit gehen die Erinnerungen zurück? Wer bewohnt diese Gedächtnisräume? Die Forschung widmet sich den wandelnden Ausgrenzungen und Eingrenzungen entlang den konstruierten vielstimmigen, ineinander verschränkten Erzählwelten. Abwesenheit dient als poetisch-analytisches Konzept, wodurch die theoretischen Felder der Narratologie und Anthropology des Todes und der Gewalt – durch Friedhofsforschung – zusammentreffen. / This thesis follows “German cemeteries” that still exist in Southeast Banat in Serbia, once part of the Habsburg Military Frontier, today a borderland between Serbia, Romania and Hungary. I am interested in how abandoned, demolished, forgotten, and reconstructed cemeteries with German-inscribed gravestones mirror rhythms of the past and present. These very inscriptions convey entangled stories about Germans and Jews. Nowadays, they are primarily absent neighbors. For this reason, I adopt a narratological perspective in which cemeteries, monuments and memorials, archives and oral interviews act as narrative instances bearing worldness. In attempting to reconstruct the worlds behind, the study asks how remaining people narratively create distinct, often conflictual and yet close storyworlds about absent Jews and Germans and their past in the region, the Holocaust and forced displacement after the Second World War (Vertreibung). With whose memories are the new realities filled? Who inhabits these? Whose history counts? Combining empirical work with concepts bridging history/ memory, the anthropology of death and violence, narratology, and literary fictional texts and images, the thesis explores stories retold and memories silenced, repressed, and haunting.

Am Beispiel der Kunsthistorischen Institute Breslau und Posen sowie der dort tätigen Kunsthistoriker und Kunsthistorikerinnen wird dargelegt, wie sich in der Zeit des Nationalsozialismus die Disziplin Kunstgeschichte an der sog. Ostforschung beteiligte. Die "Ostforschung" zielte nicht auf eine Erforschung von Kunst und Kultur der osteuropäischen Nachbarländer ab, sondern auf die Ermittlung des "deutschen" Anteils an deren Herausbildung. Mit Breslau und seinem Direktor Dagobert Frey stehen einerseits ein Institut und ein Protagonist an der Ostgrenze des Deutschen Reiches und mit Posen andererseits ein im besetzten Polen an der Reichsuniversität gegründetes Institut und sein Leiter Otto Kletzl im Fokus. Der Schwerpunkt wird auf deren Forschungen zum Nachbarland Polen gelegt. Die Autorin zeigt auf, wie politische Prämissen Eingang in die Lehre und in die Publikationen fanden und wie diese Positionen durch Vorträge und Ausstellungsmitarbeit auch im außeruniversitären Bereich vermittelt und verbreitet wurden. Die Direktoren beider Institute waren zudem in weiteren außeruniversitären Forschungsprojekten, Vereinen sowie Institutionen der Ostforschung aktiv. Sowohl Dagobert Frey (Breslau) als auch Otto Kletzl (Posen) beteiligten sich zudem für eine begrenzte Zeit als Kunstgutachter am Kunstraub im besetzten Polen. Neben diesen beiden Hauptprotagonisten werden die Karriereverläufe von Nachwuchswissenschaftlern und Nachwuchswissenschaftlerinnen dargelegt, eröffnete der Krieg doch für eine begrenzte Zeit auch Frauen die Möglichkeit, in akademische Positionen zu gelangen. Die Arbeit kombiniert auf der Basis einer umfassenden Quellenrecherche institutionengeschichtliche, biographische und handlungstheoretische Ansätze, um die Handlungsspielräume im Spannungsfeld von Wissenschaft und Politik herauszuarbeiten. Die Autorin kommt zu dem Ergebnis, dass sich die untersuchten Kunsthistoriker an der Legitimierung und Unterstützung der deutschen Okkupationspolitik in Osteuropa beteiligten. / Focussing the institutes of art history in Breslau und Posen as well as male and female arthistorians working there the author shows how the discipline arthistory participated in the so-called Ostforschung (Eastern research) in the time of nationalsocialism. "Eastern research" was not interested in the research of art and culture of the Easteuropean neighbourcontries, but in the "German" role in their development. Two places, persons and institutes are the center (focus): Breslau and its director Dagobert Frey and his institute, a protagonist from the Eastern border of the German Reich on the one hand and Otto Kletzl - leader of an institute in Posen at the so-called ReichsuniversitŠt (Reichs-University) on the other hand. The main focus lies on their research concerning Poland. The author shows how political topics found their way in the teaching: in lectures as well as in the publications. She also makes clear how their positions were spread in the field beyond universiy by lectures and their participation in exhibitions. Both directors have further been very active in non-university research projects, associations and institutes of the Eastern research. Dagobert Frey (Breslau) and also Otto Kletzl (Posen) participated for a certain time as experts in the art robbery in occupied Poland. Next to these main protagonists the career of young researchers, men and women, are presented. For a short time the war opens women the opportunity to get into academic positions. Based on a broad archival research the author combines institutional, biographical and action theory approaches to show the scope of activities in the tension zone between science and politics. She comes to the result that the art historians participated in the legitimization and support of the German occupation politics in Eastern Europe.

Introdução: Os receptores do peptídeo insulinotrópico dependente de glicose (GIPR) e do hormônio luteinizante (LHCGR) são receptores acoplados à proteína G com amplo padrão de expressão tecidual. A expressão anômala destes receptores tem sido descrita em casos de hiperplasia adrenal macronodular independente de ACTH (AIMAH) e em alguns adenomas, resultando em aumento da secreção hormonal (cortisol, andrógenos e aldosterona) pelo cortex adrenal. O papel destes receptores em outras formas de hiperplasia, como a doença adrenocortical nodular pigmentosa primária (PPNAD), aumento da adrenal associado à neoplasia endócrina múltipla tipo 1 (MEN1), e em carcinoma do córtex adrenal tem sido pouco investigado; sendo assim, considera-se relevante estudar a expressão destes receptores nos pacientes com tumores adrenocorticais esporádicos, nos pacientes com AIMAH, PPNAD e aumento adrenal associado à MEN1. Objetivos: 1) Caracterização molecular dos casos de neoplasia endócrina múltipla tipo 1 e PPNAD: pesquisa de mutações dos genes MEN1 e PRKAR1A e análise da perda de heterozigose (LOH) destes genes no tecido adrenal destes pacientes. 2) Quantificar a expressão do GIPR e do LHCGR em tecido adrenocortical normal, tumoral, hiperplásico e correlacionar a expressão destes com a classificação histológica dos tumores adrenocorticais. Pacientes: 55 pacientes (30 adultos) com tumores adrenocorticais (37 adenomas e 18 carcinomas); 7 pacientes com AIMAH, 4 com MEN1, 1 com PPNAD e tecidos controles (adrenal; testículo e pâncreas). Métodos: extração de DNA genômico, RNA e síntese de DNA complementar (cDNA); amplificação por PCR das regiões codificadoras dos genes MEN1 e PRKAR1A seguida por seqüenciamento automático. Pesquisa de LOH pela amplificação de microssatélites por PCR e análise pelo programa GeneScan. Quantificação da expressão do GIPR e do LHCGR por PCR em tempo real pelo método TaqMan e estudo de imunohistoquímica para GIPR nos tumores adrenocorticais. Resultados: identificação de 3 mutações (893+ 1G>A, W183X e A68fsX118) e dois polimorfirmos (S145S e D418D) no gene MEN1 e uma mutação (Y21X) no PRKAR1A. Ausência de LOH nos tecidos adrenais estudados. A expressão do GIPR e do LHCGR foi identificada em tecidos adrenais normais, tumorais e hiperplásicos. O nível de expressão do GIPR foi mais elevado nos tumores adrenocorticais malignos que nos benignos tanto no grupo pediátrico (mediana= 18,1 e 4,6, respectivamente; p <0,05), quanto no grupo adulto (mediana = 4,8 e 1,3 respectivamente; p <0,001). O nível de expressão do LHCGR, no grupo pediátrico, foi elevado tanto nos tumores benignos quanto nos malignos (mediana= 6,4 e 4,3, respectivamente). No grupo adulto os níveis de expressão deste receptor foram extremamente baixos nos tumores malignos em relação aos benignos (mediana= 0,06 e 2,3, respectivamente; p <0,001). A imunohistoquímica para o GIPR foi variável e não correlacionada à expressão do gene GIPR. Não houve diferença nos níveis de expressão do GIPR e do LHCGR nas hiperplasias do córtex adrenal. Conclusões: a presença de LOH e mutação em heterozigose composta do gene MEN1 e do PRKAR1A foram afastadas como mecanismos responsáveis pelo aumento adrenal tanto nos pacientes com MEN1 como no paciente com PPNAD. A hiperexpressão de GIPR está associada a malignidade nos tumores adrenocorticais nos grupos adulto e pediátrico e a baixa expressão de LHCGR está associada a malignidade nos tumores adrenocorticais somente no grupo adulto. / Introduction: The glucose- dependent insulinotropic peptide receptor (GIPR) and luteinizing hormone receptor (LHCGR) are G-protein coupled receptors with a wide tissue expression pattern. The aberrant expression of these receptors has been described in cases of ACTH-independent macronodular adrenal hyperplasia (AIMAH) and in some adenomas, resulting in the increase of adrenal cortex hormonal secretion (cortisol, androgens and aldosterone). The role of these receptors in other forms of adrenocortical hyperplasia, such as primary pigmented nodular adrenocortical disease (PPNAD), adrenal enlargement associated with multiple endocrine neoplasia type 1 (MEN1), and adrenocortical carcinoma has been scarcely investigated. Thus, the study of the expression of these receptors in patients with sporadical adrenocortical tumors, AIMAH, PPNAD and adrenal enlargement associated to MEN1 was considered important. Objectives: 1) Molecular study in patients with multiple endocrine neoplasia type 1 and PPNAD: mutation screening of MEN1 and PRKAR1A genes and analysis of the loss of heterozygosis (LOH) of these genes in the adrenal lesions of these patients. 2) To quantify the GIPR and LHCGR expression, in normal, tumor and hyperplasic tissue and to correlate the expression of these receptors with the adrenocortical tumor histology. Patients: 55 patients (30 adults) with adrenocortical tumors (37 adenomas and 18 carcinomas); 7 patients with AIMAH, 4 with MEN1, 1 with PPNAD and control tissue (adrenal, testis and pancreas). Methods: Extraction of genomic DNA, RNA and synthesis of complementary DNA (cDNA); PCR-amplification of the coding regions of MEN1 and PRKAR1A, followed by direct sequencing. LOH study using polymorphic marker amplification by PCR and GeneScan software analysis. Quantification of GIPR and LHCGR expression using realtime PCR -TaqMan method and GIPR immunohistochemistry study in adrenocortical tumors. Results: Identification of 3 mutations (893+ 1G>A, W183X and A68fsX118) and two polymorphic alterations (S145S and D418D) in MEN1 and a mutation (Y21X) in the PRKAR1A gene; LOH was not identified in adrenal tissue. The GIPR and LHCGR expression was identified in normal, tumor and hyperplasic adrenal tissues; the GIPR expression level was more elevated in malignant tumors compared to benign tumors in pediatric (median = 18.1 and 4.6, respectively; p <0.05) and adult patients (median = 4.8 and 1.3 respectively; p <0.001). The LHCGR expression in pediatric patients was elevated in benign as well as in malignant tumors (median = 6.4 and 4.3, respectively). In the adult group, the expression level of these receptors was extremely low in malignant tumors in relation to benign ones (median = 0.06 and 2.3, respectively; p <0.001). The GIPR immunohistochemistry was variable and did not correlate with GIPR gene expression. No difference between GIPR and LHCGR expression levels was observed in the different forms of hyperplasia. Conclusions: The presence of LOH and mutations in compound heterozygosis of MEN1 and PRKAR1A genes were ruled out as the mechanisms responsible for the adrenal enlargement in patients with multiple endocrine neoplasia type 1. GIPR overexpression is associated with malignant adrenocortical tumors in the adult and pediatric patients and low LHCGR expression is associated with malignant adrenocortical tumors only in the adult patients.

Introdução: Os receptores do peptídeo insulinotrópico dependente de glicose (GIPR) e do hormônio luteinizante (LHCGR) são receptores acoplados à proteína G com amplo padrão de expressão tecidual. A expressão anômala destes receptores tem sido descrita em casos de hiperplasia adrenal macronodular independente de ACTH (AIMAH) e em alguns adenomas, resultando em aumento da secreção hormonal (cortisol, andrógenos e aldosterona) pelo cortex adrenal. O papel destes receptores em outras formas de hiperplasia, como a doença adrenocortical nodular pigmentosa primária (PPNAD), aumento da adrenal associado à neoplasia endócrina múltipla tipo 1 (MEN1), e em carcinoma do córtex adrenal tem sido pouco investigado; sendo assim, considera-se relevante estudar a expressão destes receptores nos pacientes com tumores adrenocorticais esporádicos, nos pacientes com AIMAH, PPNAD e aumento adrenal associado à MEN1. Objetivos: 1) Caracterização molecular dos casos de neoplasia endócrina múltipla tipo 1 e PPNAD: pesquisa de mutações dos genes MEN1 e PRKAR1A e análise da perda de heterozigose (LOH) destes genes no tecido adrenal destes pacientes. 2) Quantificar a expressão do GIPR e do LHCGR em tecido adrenocortical normal, tumoral, hiperplásico e correlacionar a expressão destes com a classificação histológica dos tumores adrenocorticais. Pacientes: 55 pacientes (30 adultos) com tumores adrenocorticais (37 adenomas e 18 carcinomas); 7 pacientes com AIMAH, 4 com MEN1, 1 com PPNAD e tecidos controles (adrenal; testículo e pâncreas). Métodos: extração de DNA genômico, RNA e síntese de DNA complementar (cDNA); amplificação por PCR das regiões codificadoras dos genes MEN1 e PRKAR1A seguida por seqüenciamento automático. Pesquisa de LOH pela amplificação de microssatélites por PCR e análise pelo programa GeneScan. Quantificação da expressão do GIPR e do LHCGR por PCR em tempo real pelo método TaqMan e estudo de imunohistoquímica para GIPR nos tumores adrenocorticais. Resultados: identificação de 3 mutações (893+ 1G>A, W183X e A68fsX118) e dois polimorfirmos (S145S e D418D) no gene MEN1 e uma mutação (Y21X) no PRKAR1A. Ausência de LOH nos tecidos adrenais estudados. A expressão do GIPR e do LHCGR foi identificada em tecidos adrenais normais, tumorais e hiperplásicos. O nível de expressão do GIPR foi mais elevado nos tumores adrenocorticais malignos que nos benignos tanto no grupo pediátrico (mediana= 18,1 e 4,6, respectivamente; p <0,05), quanto no grupo adulto (mediana = 4,8 e 1,3 respectivamente; p <0,001). O nível de expressão do LHCGR, no grupo pediátrico, foi elevado tanto nos tumores benignos quanto nos malignos (mediana= 6,4 e 4,3, respectivamente). No grupo adulto os níveis de expressão deste receptor foram extremamente baixos nos tumores malignos em relação aos benignos (mediana= 0,06 e 2,3, respectivamente; p <0,001). A imunohistoquímica para o GIPR foi variável e não correlacionada à expressão do gene GIPR. Não houve diferença nos níveis de expressão do GIPR e do LHCGR nas hiperplasias do córtex adrenal. Conclusões: a presença de LOH e mutação em heterozigose composta do gene MEN1 e do PRKAR1A foram afastadas como mecanismos responsáveis pelo aumento adrenal tanto nos pacientes com MEN1 como no paciente com PPNAD. A hiperexpressão de GIPR está associada a malignidade nos tumores adrenocorticais nos grupos adulto e pediátrico e a baixa expressão de LHCGR está associada a malignidade nos tumores adrenocorticais somente no grupo adulto. / Introduction: The glucose- dependent insulinotropic peptide receptor (GIPR) and luteinizing hormone receptor (LHCGR) are G-protein coupled receptors with a wide tissue expression pattern. The aberrant expression of these receptors has been described in cases of ACTH-independent macronodular adrenal hyperplasia (AIMAH) and in some adenomas, resulting in the increase of adrenal cortex hormonal secretion (cortisol, androgens and aldosterone). The role of these receptors in other forms of adrenocortical hyperplasia, such as primary pigmented nodular adrenocortical disease (PPNAD), adrenal enlargement associated with multiple endocrine neoplasia type 1 (MEN1), and adrenocortical carcinoma has been scarcely investigated. Thus, the study of the expression of these receptors in patients with sporadical adrenocortical tumors, AIMAH, PPNAD and adrenal enlargement associated to MEN1 was considered important. Objectives: 1) Molecular study in patients with multiple endocrine neoplasia type 1 and PPNAD: mutation screening of MEN1 and PRKAR1A genes and analysis of the loss of heterozygosis (LOH) of these genes in the adrenal lesions of these patients. 2) To quantify the GIPR and LHCGR expression, in normal, tumor and hyperplasic tissue and to correlate the expression of these receptors with the adrenocortical tumor histology. Patients: 55 patients (30 adults) with adrenocortical tumors (37 adenomas and 18 carcinomas); 7 patients with AIMAH, 4 with MEN1, 1 with PPNAD and control tissue (adrenal, testis and pancreas). Methods: Extraction of genomic DNA, RNA and synthesis of complementary DNA (cDNA); PCR-amplification of the coding regions of MEN1 and PRKAR1A, followed by direct sequencing. LOH study using polymorphic marker amplification by PCR and GeneScan software analysis. Quantification of GIPR and LHCGR expression using realtime PCR -TaqMan method and GIPR immunohistochemistry study in adrenocortical tumors. Results: Identification of 3 mutations (893+ 1G>A, W183X and A68fsX118) and two polymorphic alterations (S145S and D418D) in MEN1 and a mutation (Y21X) in the PRKAR1A gene; LOH was not identified in adrenal tissue. The GIPR and LHCGR expression was identified in normal, tumor and hyperplasic adrenal tissues; the GIPR expression level was more elevated in malignant tumors compared to benign tumors in pediatric (median = 18.1 and 4.6, respectively; p <0.05) and adult patients (median = 4.8 and 1.3 respectively; p <0.001). The LHCGR expression in pediatric patients was elevated in benign as well as in malignant tumors (median = 6.4 and 4.3, respectively). In the adult group, the expression level of these receptors was extremely low in malignant tumors in relation to benign ones (median = 0.06 and 2.3, respectively; p <0.001). The GIPR immunohistochemistry was variable and did not correlate with GIPR gene expression. No difference between GIPR and LHCGR expression levels was observed in the different forms of hyperplasia. Conclusions: The presence of LOH and mutations in compound heterozygosis of MEN1 and PRKAR1A genes were ruled out as the mechanisms responsible for the adrenal enlargement in patients with multiple endocrine neoplasia type 1. GIPR overexpression is associated with malignant adrenocortical tumors in the adult and pediatric patients and low LHCGR expression is associated with malignant adrenocortical tumors only in the adult patients.