Tesis sobre el tema "LHCSR"

La sfingosina-1 fosfato (S1P) è un lisosfingolipide presente nel liquido follicolare ovarico insieme alle gonadotropine glicoproteiche. L'ormone luteinizzante (LH) e l'ormone follicolo-stimolante (FSH) sono necessari per garantire la steroidogenesi, la gametogenesi e la riproduzione. La gonadotropina corionica umana (hCG) agisce durante la gravidanza sullo stesso recettore di LH, LHCGR, per stimolare la produzione di progesterone da parte del corpo luteo e mantenere la gravidanza. Inoltre, le gonadotropine sono fattori di crescita e differenziazione, che modulano la proliferazione cellulare, la sopravvivenza e l'apoptosi. Sia S1P che le gonadotropine esercitano le loro funzioni fisiologiche legandosi a recettori accoppiati a proteine G (GPCR). S1P si lega e attiva cinque recettori specifici, S1P1-5, modulando diverse vie di segnalazione. S1P1 e S1P3 sono altamente espressi nelle cellule primarie della granulosa luteina umana (hGLC). Questo studio mira a caratterizzare il ruolo della segnalazione intracellulare indotto da S1P e gonadotropine nel determinare lo sviluppo del follicolo ovarico in vitro. A questo scopo sono state utilizzate granulose umane e linee cellulari che esprimono stabilmente FSHR e LHCGR sotto il controllo di un promotore inducibile. Sono state valutate l’eterodimerizzazione di S1PR1 con LHCGR/FSHR e GPER e la cinetica delle proteine G e il reclutamento della β-arrestina 2 con LHCGR mediate da LH e hCG, così come l'attivazione di secondi messaggeri e l’internalizzazione di LHCGR in vitro. hGLC e hGL5 sono state trattate con dose fissa (0,1 μM) di S1P e con specifici agonisti di S1P1 e S1P3, SEW2871 e CYM5541. Nelle cellule della granulosa, S1P e, in maniera minore, SEW2871 e CYM5541, hanno indotto pCREB. Non è stata rilevata alcuna produzione di cAMP e l'attivazione di pCREB è avvenuta anche in presenza dell'inibitore della PKA H-89. Inoltre, pCREB S1P-dipendente è stata attenuata dall'inibitore di MEK U0126 e da un bloccante dei canali Ca2+ di tipo L, il verapamil. La completa inibizione di pCREB si è verificata bloccando S1P2 o S1P3 con specifici antagonisti. pCREB S1P-dipendente ha indotto l’espressione di FOXO1 ed EREG, confermando il ruolo esclusivo delle gonadotropine e delle interleuchine in questo processo, ma non ha influenzato la steroidogenesi. hCG induce l'accoppiamento Gαs-, Gq e β-arrestin 2 con LHCGR in modo più efficace rispetto a LH. In presenza di Dynasore, un inibitore dell'internalizzazione, hCG mantiene una cinetica simile, ma non LH, che necessita dell'endocitosi di LHCGR per indurre l'accoppiamento del recettore. Questi dati riflettono la cinetica ormone-specifica dell'attivazione dell'effettore a valle correlata alle proteine G e alla β -arrestina 2. LH ha indotto un rapido aumento di cAMP ed è più potente di hCG nell'attivazione di pERK1/2. È interessante notare che la cinetica dell'aumento di Ca2+ intracellulare indotto da hCG dipende dall'internalizzazione di LHCGR rispetto a LH che non riesce a indurre un aumento di Ca2+ intracellulare. È stata valutata l'interazione tra LHCGR e specifici marcatori degli endosomi per studiare l'internalizzazione di LHCGR mediata dalle gonadotropine. LH è più potente di hCG nel promuovere il riciclaggio di LHCGR. Questo studio ha dimostrato che S1P può indurre un'attivazione di pCREB indipendente da cAMP nelle cellule della granulosa, sebbene ciò non sia sufficiente per indurre la sintesi del progesterone. L'espressione genica di FOXO1 ed EREG indotta da S1P suggerisce che l'attivazione dell'asse S1P-S1PR può cooperare con le gonadotropine nel modulare lo sviluppo del follicolo. L'internalizzazione di LHCGR è fondamentale per modulare i segnali specifici di LH e hCG che influenzano le proteine G e l'accoppiamento β -arrestina 2 e le cascate di segnalazione a valle.
Sphingosine-1 phosphate (S1P) is a lysosphingolipid present in the ovarian follicular fluid together with glycoprotein hormone gonadotropins. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are necessary to ensure steroidogenesis, gametogenesis and reproduction. Human chorionic gonadotropin (hCG) acts during pregnancy via the same receptor for LH, the LHCGR, to stimulate progesterone production by the corpus luteum and maintaining pregnancy. In addition, gonadotropins are growth and differentiation factors, modulating cell proliferation, survival and apoptosis. Both S1P and gonadotropins exert their physiological functions by binding cognate G protein-coupled receptors (GPCRs). At nanomolar concentrations, S1P binds and activates five specific receptors, known as S1P1-5, modulating different signaling pathways. S1P1 and S1P3 are highly expressed in human primary granulosa lutein cells (hGLC). This study aims to characterize the role of S1P- and gonadotropins-induced signaling in determining ovarian follicle development in vitro. To this purpose were used human granulosa, cell lines stably expressing FSHR and LHCGR under the control of an inducible promoter, treated with gonadotropins and S1P. S1PR1 heterodimerization to LHCGR/FSHR and GPER and the kinetics of LH- and hCG-mediated G proteins and β-arrestin 2 coupling to LHCGR were evaluated, such as the activation of related second messengers and kinases, and the role of gonadotropins-induced LHCGR internalization in vitro. hGLC and hGL5 cells were treated with a fixed dose (0.1 μM) of S1P, or by S1P1- and S1P3-specific agonists SEW2871 and CYM5541. In granulosa cells, S1P and, at a lesser extent, SEW2871 and CYM5541, potently induced pCREB. No cAMP production was detected and pCREB activation occurred even in the presence of the PKA inhibitor H-89. Moreover, S1P-dependent pCREB was dampened by MEK inhibitor U0126 and by the L-type Ca2+ channel blocker verapamil. The complete inhibition of pCREB occurred by blocking either S1P2 or S1P3 with the specific receptor antagonists, or under PLC/PI3K depletion. S1P-dependent pCREB induced FOXO1 and EREG, confirming the exclusive role of gonadotropins and interleukins in this process, but did not affect steroidogenesis. The kinetics of LH and hCG-mediated G proteins and β-arrestin 2 coupling to their receptor, and the activation of related second messengers and kinases were evaluated by BRET. hCG induces Gαs-, Gq and β-arrestin 2 coupling to LHCGR more effectively than LH. Under receptor internalization blockade by Dynasore, hCG maintains similar kinetics, but not LH, which needs LHCGR endocytosis for inducing receptor coupling. These data reflect hormone-specific kinetics of downstream effector activation related to G proteins and β-arrestin 2. LH induced a rapid cAMP increase and is more potent than hCG in activating pERK1/2. Interestingly, the kinetic of hCG-induced intracellular Ca2+ increase depends on LHCGR internalization than LH that fails in inducing intracellular Ca2+ increase, consistently with weak Gq recruitment. The interaction between LHCGR and specific markers of endosomes were evaluated to estimate LHCGR internalization mediated by gonadotropins. Indeed, LH is more potent than hCG in promoting LHCGR recycling. This study demonstrated that S1P may induce a cAMP-independent activation of pCREB in granulosa cells, although this is not sufficient to induce progesterone synthesis. S1P-induced FOXO1 and EREG gene expression suggests that the activation of S1P-S1PR axis may cooperate with gonadotropins in modulating follicle development. LHCGR internalization is fundamental for modulating LH- and hCG-specific signals impacting G proteins and β-arrestin 2 coupling, and the downstream signaling cascades.

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Made available in DSpace on 2018-11-30T12:45:47Z (GMT). No. of bitstreams: 1 Marina Platzeck Chaves.pdf: 891914 bytes, checksum: 43d527b64ad46e92f25496042406c5e9 (MD5) Previous issue date: 2018-05-30
Steroids and gonadotrophins are essential for the regulation of antral follicular development and the late stages of preantral development. Although the luteinizing hormone receptor (LHR) has been detected in the preantral follicles of rats, rabbits, and pigs, the expression of this receptor in bovine fetal ovary has not been demonstrated. The present study aimed to quantify the expression of the LHR and the mRNA abundance of the genes LHR binding protein (LRBP), STAR, HSD3B1, CYP17A1, and CYP19A1 during the development of bovine fetal ovary. In addition, we aimed to identify and quantify the expression of bta-miR-222 (a regulatory microRNA of the LHCGR gene). In summary, LHR expression was observed in the preantral follicle in bovine fetal ovary, from oogonias to primordial, primary and secondary stages, and the mRNA abundance was lower on day 150 than day 60. However, the mRNA abundance of LRBP followed the opposite pattern. The LHR protein was detected in oogonia, primordial, primary, and secondary follicles. Moreover, both oocytes and granulosa cells showed positive immunostaining for LHR. Similar to LRBP, the abundance of bta-miR-222 was higher on day 150 than day 60 or 90 of gestation. With regard to the gene expression of steroidogenic enzymes; only the mRNA abundance of STAR was higher on day 150 than on day 60. In conclusion, these results suggested the involvement of LHCGR/LRBP regulation with mechanisms related to the development of preantral follicles, especially during the establishment of secondary follicles. Furthermore, the present data reinforced that the reduced expression of LHR mRNA in bovine fetal ovaries on day 150 was related to the higher expression of LRBP and bta-miR-222.
Esteroides e gonadotrofinas são essenciais para a regulação do desenvolvimento folicular antral e os estágios finais do desenvolvimento pré-antral. Embora o receptor do hormônio luteinizante (LHR) tenha sido detectado nos folículos pré-antrais de ratos, coelhos e porcos, a expressão deste receptor no ovário fetal bovino não foi demonstrada. O presente estudo teve como objetivo quantificar a expressão do LHR e a abundância de mRNA da proteína de ligação LHR (LRBP), STAR, HSD3B1, CYP17A1 e CYP19A1 durante o desenvolvimento do ovário fetal bovino. Além disso, objetivamos identificar e quantificar a expressão de bta-miR-222 (microRNA regulador do gene LHCGR). Em resumo, a expressão de LHR foi observada no folículo pré-antral no ovário fetal de bovino e a abundância de mRNA foi menor no dia 150 do que no dia 60. No entanto, a abundância de mRNA da LRBP seguiu o padrão oposto. Semelhante a LRBP, a abundância de bta-miR-222 foi maior no dia 150 do que no dia 60 ou 90. Com relação à expressão gênica de enzimas esteroidogênicas; apenas a abundância de mRNA de STAR foi maior no dia 150 do que no dia 60. A proteína LHR foi detectada em oogônia, folículos primordiais, primários e secundários. Além disso, ambos os oócitos e células da granulosa apresentaram imunolocalização positiva para LHR. Em conclusão, estes resultados sugeriram o envolvimento da regulação do LHCGR / LBPB com mecanismos relacionados ao desenvolvimento de folículos pré-antrais, especialmente durante o estabelecimento de folículos secundários. Além disso, os presentes dados reforçaram que a expressão reduzida de mRNA de LHR em ovários fetais bovinos no dia 150 estava relacionada à maior expressão de LRBP e bta-miR-222.

Classicamente, la segnalazione dipendente dall’attivazione del cAMP mediata dal recettore dell'ormone follicolo-stimolante (FSHR) e dal recettore dell'ormone luteinizzante (LH) (LHCGR) favorisce la crescita del follicolo ovarico umano e la maturazione degli ovociti. Tuttavia, esistono dati in vitro contraddittori che suggeriscono un diverso significato fisiologico della segnalazione di cAMP mediata da FSHR, mostrando allo stesso tempo l'attivazione di eventi steroidogenici e pro-apoptotici. Questi segnali possono essere alterati dagli estrogeni che inducono eventi anti-apoptotici attraverso i loro recettori nucleari e all'azione non genomica di un recettore degli estrogeni accoppiato a proteine G (GPER). Lo scopo del progetto è chiarire il ruolo degli estrogeni/gonadotropine e dei loro recettori di membrana nella regolazione della fisiologia ovarica e nella selezione del follicolo dominante. In questo studio è stato dimostrato che GPER forma dimeri sia con FSHR che con LHCGR sulla superficie cellulare di cellule HEK293 che sovraesprimono i due recettori e di cellule primarie della granulosa luteina umana (hGLC). Gli eteromeri FSHR/GPER riprogrammano i segnali del cAMP e di morte, in stimoli proliferativi fondamentali per sostenere la sopravvivenza degli ovociti. Nelle cellule della granulosa umana, i segnali di sopravvivenza mancano quando è presente un rapporto FSHR:GPER elevato, che influisce negativamente sulla maturazione del follicolo e si correla con l'accoppiamento preferenziale alla proteina Gαs, l’attivazione della via cAMP/PKA e la capacità di risposta ad FSH dei pazienti sottoposti a stimolazione ovarica controllata. Gli eteromeri FSHR/GPER, invece, mediano segnali anti-apoptotici e proliferativi dipendenti da FSH tramite il dimero Gβγ e la compromissione della formazione degli eteromeri o il knockdown GPER aumenta la morte cellulare e la steroidogenesi FSH-dipendenti. Al contrario, il complesso GPER/LHCGR non influenza la produzione di cAMP indotta da LH e hCG e non compromette l'attivazione della via cAMP/PKA. Ciò si evince dalla simile fosforilazione di CREB ed ERK1/2 e dalla stessa produzione di progesterone in hGLC trattate con siRNA contro GPER e quelle trattato con mock. È interessante notare che GPER riduce l’accoppiamento LHCGR/Gαq e di conseguenza impedisce il rilascio intracellulare di Ca2+ e l'accumulo di IP1, dopo stimolazione con LH e hCG, in cellule HEK293 che co-esprimono LHCGR e GPER rispetto alle cellule che esprimono solo LHCGR. Inoltre, è stato dimostrato che in presenza di GPER la cinetica dell'internalizzazione di FSHR attraverso endosomi precoci e tardivi è ridotta, suggerendo la sua capacità di bloccare FSHR a livello intracellulare e riducendo il riciclaggio di FSHR sulla membrana. Infatti, l'internalizzazione di FSHR è necessaria affinché GPER inibisca la risposta del cAMP indotta da FSH. Secondo i nostri risultati, gli estrogeni sono selettivamente coinvolti nella regolazione dei segnali pro e anti-apoptotici, nell'internalizzazione dei recettori attraverso i complessi FSHR/GPER e nella modulazione della cascata di segnali mediata da LHCGR. I nostri risultati indicano come la maturazione degli ovociti dipenda dalla capacità del GPER di modulare i segnali selettivi di FSHR e LHCGR, indicando che gli eteromeri dei recettori delle gonadotropine possono essere un marker della proliferazione cellulare.
Classically, follicle-stimulating hormone receptor (FSHR) and luteinizing hormone (LH) receptor (LHCGR) -driven cAMP-mediated signaling boosts human ovarian follicle growth and oocyte maturation. However, contradicting in vitro data suggest a different view on physiological significance of FSHR-mediated cAMP signalling, showing at the same time the activation of steroidogenic and pro-apoptotic events. These signals can be impaired by estrogens inducing anti-apoptotic events via nuclear receptors and non-genomic action of a G protein-coupled estrogen receptor (GPER). The aim of the project is to better understand the role of estrogens/gonadotropins and their membrane receptors in regulating ovarian physiology and the selection of the dominant follicle. In this study it was demonstrated that GPER heteromerizes both with FSHR and LHCGR at the cell surface of HEK293 cells overexpressing the two receptors as well as human primary granulosa lutein cells (hGLC). FSHR/GPER heteromers reprogram cAMP/death signals into proliferative stimuli fundamental for sustaining oocyte survival. In human granulosa cells, survival signals are missing at high FSHR:GPER ratio, which negatively impacts follicle maturation and strongly correlates with preferential Gαs protein/cAMP-pathway coupling and FSH responsiveness of patients undergoing controlled ovarian stimulation. In contrast, FSHR/GPER heteromers triggered anti-apoptotic/proliferative FSH signaling delivered via the Gβγ dimer, whereas impairment of heteromer formation or GPER knockdown enhanced the FSH-dependent cell death and steroidogenesis. On the other hand, GPER/LHCGR complex does not affect the LH and hCG-induced cAMP production and do not compromise the activation of the cAMP/PKA pathway, as it is indicated by similar CREB and ERK1/2 phosphorylation and same progesterone production in hGLC treated with siRNA against GPER, and the mock-treated one. Interestingly, GPER displace the LHCGR/Gαq coupling and consequently impedes the intracellular Ca2+ release and IP1 accumulation in LHCGR-GPER co-expressing HEK293 cells upon LH and hCG treatment compared to LHCGR-expressing cells. Also, it was demonstrated that in presence of GPER the kinetic of FSHR internalization through early and late endosomes is reduced, suggesting its ability to blockade FSHR at the intracellular level and reduce FSHR recycling on cell membrane. Indeed, FSHR internalization is necessary for GPER to inhibit FSH-induced cAMP response. According to our results, estrogens are selectively involved in the regulation of pro- and anti-apoptotic signals and receptor internalization through FSHR/GPER complexes and in modulation of LHCGR-mediated signaling cascade. Our findings indicate how oocyte maturation depends on the capability of GPER to shape FSHR and LHCGR selective signals, indicating hormone receptor heteromers may be a marker of cell proliferation.

Orientador: Anthony César de Souza Castilho
Resumo: A superestimulação ovariana é uma biotecnologia amplamente empregada na espécie bovina para a obtenção de múltiplas ovulações. Com este objetivo diversos protocolos superestimulatórios surgiram, dentre eles o protocolo P-36 e sua variação, o protocolo P-36/eCG. Ambos os tratamentos utilizam o hormônio folículo estimulante (FSH) na indução do crescimento folicular. Como é acreditado que no último dia do tratamento, as células da granulosa folicular possuam receptores do hormônio luteinizante (LH; LHR), duas últimas doses de FSH foram substituídas pela administração de gonadotrifina coriónica equina (eCG; P-36/eCG). A molécula de eCG possui atividade tanto LH quanto FSH por se ligar a ambos receptores, aumentando a resposta ovulatória. Os dois tratamentos têm demonstrado eficácia quanto ao desenvolvimento de oócitos competentes para a produção embrionária, no entanto pouco se sabe sobre seus efeitos na diferenciação celular no folículo ovariano. Por isso, o presente estudo investigou os efeitos da superestimulação ovariana com FSH (P-36) ou FSH combinado com eCG (P-36/eCG) sobre aspectos bioquímicos e a produção de hormônios esteroides. Adicionalmente, quantificou-se a abundância de miRNAs reguladores da expressão do mRNA do LHR e outros miRNAs relacionados com o desenvolvimento folicular ovariano. Os resultados obtidos mostram que os tratamentos superestimulatórios alteram o perfil bioquímico intrafolicular e a concentração de estradiol no plasma. Aliado a isso, também alteram... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Ovarian overstimulation is a biotechnology widely used in the bovine species to obtain multiple ovulations. With this objective, several protocols were introduced, including the P-36 protocol and its variation, the P-36/eCG protocol. Both treatments use follicle stimulating hormone (FSH) to induce the follicular growth. As it is believed that on the last day of treatment, follicular granulosa cells have luteinizing hormone (LHR) receptors, two last doses of FSH have been replaced by administration of equine chorionic gonadotrifine (eCG; P-36/eCG). The eCG molecule has LH and FSH activity by binding to both receptors, increasing the ovulatory response. Both treatments has demonstrated efficacy in the development of oocytes competent for embryo production, however little is known about their effects on cell differentiation in the ovarian follicle. Therefore, the present study investigated the effects of ovarian superstimulation using FSH (P-36) or FSH combined with eCG (P-36/eCG) on biochemical aspects and production of steroid hormones. In addition, the abundance of miRNAs regulating the expression of LHR mRNA and other miRNAs related to ovarian follicular development. Results demonstrated that superstimulatory treatments alter the intrafollicular biochemical profile and the plasma estradiol concentration. In addition, they also alter the expression of LHR and miRNAs regulating LHR mRNA expression, possibly modulating ovulatory capacity in superstimulated ovarian follicles.
Mestre

Polycystic ovary syndrome (PCOS) is the leading cause of anovulatory infertility and the most common endocrinopathy of women of reproductive age. Genome-wide association studies (GWAS) identified a number of loci associated PCOS in different ethnic populations, including women with Asian and European ancestry. Replication studies have confirmed some of these associations. Among the loci identified are those located near the LH receptor gene (LHCGR), a clathrin-binding protein gene (DENND1A) that also functions as a guanine nucleotide exchange factor, and the gene encoding RAB5B, a GTPase and protein involved in vesicular trafficking. The functional significance of one of these GWAS candidates (DENND1A) was supported by our discovery that a truncated protein splice variant of DENND1A termed DENND1A.V2, is elevated in PCOS theca cells, and that forced expression of DENND1A.V2 in normal theca cells increased CYP11A1 and CYP17A1 expression and androgen synthesis, a hallmark of PCOS. We previously proposed that the PCOS GWAS loci could be assembled into a functional network that contributes to altered gene expression in ovarian theca cells, resulting in increased androgen synthesis. Here we demonstrate the localization of LHCGR, DENND1AV.2 and RAB5B proteins in various cellular compartments in normal and PCOS theca cells. hCG and forskolin stimulation affects the distribution and co-localization of DENND1A.V2 and RAB5B in various cellular compartments This cytological evidence supports our PCOS gene network concept, and raises the intriguing possibility that LHCGR activation, via a cAMP-mediated process, promotes the translocation of DENND1A.V2 and RAB5B-containing vesicles from the PCOS theca cell cytoplasm into the nucleus, resulting in increased transcription of genes involved in androgen synthesis.

BPS/IC (Bladder Pain Syndrome/Interstitial Cystitis) ist ein sehr schweres und noch weitgehend unverstandenes Krankheitsbild in der Urologie. Viele Frauen sind im Alltag durch den ständigen Harndrang und die Schmerzen stark eingeschränkt und von Depressionen betroffen. Die Aufklärung der Pathogenese ist deshalb sehr wichtig, um eine adäquate Therapie für die Betroffenen zu entwickeln und die Krankheit möglichst frühzeitig diagnostizieren zu können. Das Schwangerschaftshormon hCG (humanes Choriongonadotropin) besitzt differenzierende und wachstumsfördernde Eigenschaften und eine Rolle in der Urothelregeneration und – stabilisierung scheint möglich. Daher ist das Ziel dieser Arbeit seinen Rezeptor, den LHCGR (Luteinizing-Hormone/Choriogonadotropin Rezeptor), in der Harnblase nachzuweisen und die urothelialen und muskulären Charakteristika zwischen gesunden und an BPS/IC erkrankten Harnblasen zu vergleichen. Die Darstellung des LHCGR erfolgte auf Proteinebene mittels indirekter Immunfluoreszenz und auf mRNA-Ebene durch Standard-PCR. Es zeigten sich im Urothel von Harnblase und Ureter 5 unterschiedliche Verteilungsmuster des Rezeptors hinsichtlich seiner Expression in verschiedenen Zellschichten und seiner subzellulären Lokalisation. Je nach Urothelzustand und zwischen den Entitäten Kontroll- bzw. BPS/IC-Harnblase variierten diese Muster in ihrer Häufigkeit. In anderen Epithelien, wie dem Vaginalepithel, änderte sich die zelluläre Verteilung des LHCGR in Abhängigkeit vom Differenzierungsgrad der Zellen. Es scheint möglich, dass auch die Rezeptorexpression in Urothelzellen deren verschiedene Differenzierungszustände widerspiegelt. Dies unterstützt den für hCG vermuteten Einfluss auf die Epithelregeneration. Ein Vergleich der urothelialen Fluoreszenzintensitäten zwischen weiblichen Kontroll – und BPS/IC-Harnblasen zeigte eine signifikant stärkere Expression des Rezeptors bei erkrankten Patienten. Dem gegenüber war kein Unterschied im Detrusor, weder zwischen Kontroll – und BPS/IC-Harnblasen noch im geschlechtsspezifischen Vergleich, festzustellen. Damit scheint der Rezeptor seine Hauptaufgabe vorrangig im Urothel zu entfalten. Die Korrelationsanalysen ergaben keinen signifikanten Zusammenhang zwischen dem Erkrankungsalter (Zeitpunkt der Diagnosestellung und Biopsieentnahme) und der LHCGR-Immunfluoreszenz. Ein endokrinologischer Einfluss auf die Rezeptorexpression wurde dadurch unwahrscheinlich und unterstützt die immer akzeptiertere Auffassung, dass BPS/IC nicht mehr mit der Menopause assoziiert ist. Neben dem Urothel und Detrusor zeigten auch Lamina propria und Gefäße von Harnblase und Ureter die Expression des LHCGR in der Immunhistochemie. Unterschiedliche Clustermuster des Rezeptors im Detrusor ließen auf die Oligomerisierung des Rezeptors schließen. Die Bedeutung dieser Zusammenschlüsse ist jedoch noch unklar, wobei unterschiedliche funktionelle Zustände des Rezeptors vermutet werden. Orientierung bieten andere Rezeptoren, die durch Dimerisierung verschiedener Rezeptorvarianten ihre Funktionalität verbessern oder verschlechtern konnten. Obwohl für keine bisher entdeckte Variante des LHCGR eine definitive Aufgabe ermittelt werden konnte, scheinen doch viele Varianten auch unterschiedliche Funktionen wahrnehmen zu können. Besonders auf der Regulierbarkeit des Rezeptors mittels interagierender Splicevarianten sollte das Augenmerk zukünftiger Studien liegen. Ob durch Komplexbildung verschiedener Varianten oder Bildung nichtfunktioneller trunkierter Rezeptoren, die Kontrollmöglichkeiten sind vielfältig und können auch auf Liganden wirken. Letztlich ließ der Nachweis des LHCGR in allen Schichten von Harnblase und Ureter eher eine globale Rolle des Rezeptors im Harntrakt des Menschen vermuten. Dazu passten auch die bereits nachgewiesenen Einflüsse seiner Liganden auf die Blasenfunktion von Hunden. Die hier vorgelegte Arbeit untersuchte zum ersten Mal die Expression des LHCGR mittels PCR und Immunhistochemie in humanen Harnblasen und Ureteren. Dabei löste sie sich von den sonst üblichen Vorstellungen einer Beziehung des Rezeptors zu Blasentumoren, Schwangerschaft oder Inkontinenz. Diagnose und Therapie von BPS/IC sind zur Zeit noch ständigen Wandlungen unterworfen und dabei entgehen viele Patienten der (frühen) Diagnosestellung und einer adäquaten Behandlung. Diese Studie sollte dazu beitragen neue Einblicke in die Pathophysiologie der Erkrankung zu erlangen, um eine kausale Therapie entwickeln zu können. Zukünftig könnten diese Ergebnisse dabei helfen die Anwendung einer sensitiven und vor allem spezifischen Diagnostik auf molekularer Ebene (mRNA - oder Proteinnachweis) zu ermöglichen.

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.

L’endométriose est une pathologie chronique, bénigne, caractérisée par la présence de tissu endométrial (glande et stroma) en dehors de l’utérus. La forte prévalence de cette maladie, sa symptomatologie invalidante et son coût annuel considérable en font un véritable enjeu de santé publique. Le traitement de l’endométriose comprend un volet chirurgical, potentiellement délabrant, et un volet médical, basé sur des produits anti-gonadotropes en première intention. L’endométriose est d’origine multifactorielle. Sa physiopathologie demeure mal connue. Alors que le reflux menstruel via les trompes semble être le primum movens, plusieurs étapes sont nécessaires à la formation des lésions d’endométriose : l’adhésion et l’implantation des cellules endométriales au mésothélium péritonéal, la prolifération cellulaire soutenue par un phénomène d’angiogenèse et enfin, la réaction inflammatoire. Ces étapes sont dépendantes de facteurs génétiques, immunologiques et environnementaux. L’inflammation joue un rôle clef dans la pathogénie de l’endométriose. Nous avons montré dans un premier temps, à l’aide d’une double approche de PCR quantitative et d’immunohistochimie, la surexpression du récepteur de la LH (LHCGR) dans les tissus endometriosiques ectopiques. A l’aide d’un modèle in vitro, nous avons démontré que la stimulation du LHCGR par l’hCG activait les MAPK (avec une augmentation du rapport pERK/ERK), exerçait une effet prolifératif et enfin, induisait la surexpression de nombreux gènes cibles : CYP19A1, NR5A1, INSL3, VEGFA et PTGS2. Ensuite, nous avons étudié les principaux acteurs de la voie des prostaglandines. Nous avons montré une nette perturbation de cette voie en faveur d’une augmentation de l’inflammation avec une surexpression de la PTGS2 ainsi que des récepteurs des prostaglandines PTGER2, 3 et 4 dans l’endomètre des patientes endométriosiques. Nous avons ensuite étudié la voie des sphingosines en analysant l’expression de ses acteurs clef (SPHK1-2, SGPP1-2, SGPL1, SPHAKAP, S1PR1-5). Nous avons mis en évidence pour la première fois l’existence d’une profonde dérégulation de l’expression des enzymes et des récepteurs de cette voie en faveur d’une diminution du catabolisme du Sphingosine-1phosphate. Cette perturbation est à l’origine de la réaction inflammatoire qui participe à l’entretien de la prolifération et de la croissance des cellules endométriosiques. Dans un deuxième temps, nous avons exploré le retentissement systémique de l’endométriose à l’aide du modèle d’une interleukine pro-inflammatoire et fibrosante, l’IL-33, et de deux interleukines anti-inflammatoires l’IL-19 et l’IL-22. L’IL-33 est significativement plus élevée dans le sérum des patientes endométriosiques en particulier en cas d’endométriose profonde. Nous avons également mis en évidence l’existence d’une corrélation significative avec le nombre et la sévérité des lésions profondes. En opposition, les interleukines anti-inflammatoires IL-19 et IL-22 sont significativement diminuées dans le sérum des patientes endométriosiques. En conclusion, nous avons montré l’existence d’une perturbation des voies inflammatoires : la voie de la PTGS2 et de celle des sphingosines ainsi que le rôle pro-inflammatoire du LCGHR. Nous avons également mis en évidence le déséquilibre de la balance des cytokines systémiques inflammatoires et anti-inflammatoires dans l’endométriose. Ainsi, ces médiateurs de l’inflammation pourraient être considérés comme de potentiels marqueurs évolutifs de l’endométriose. Leur utilisation pourrait permettre d’effectuer un diagnostic plus précoce, et d’envisager de nouvelles thérapeutiques ciblées
Pas de résumé en anglais

Attraverso la fotosintesi le piante usano l’energia solare per produrre composti ridotti dalla CO2 e alla fine biomassa. I fotosistemi (PSI e PSII) sono complessi che legano i pigmenti responsabili della raccolta della luce, separazione di carica e hanno un ruolo essenziale nel trasporto di elettroni dall’acqua al NADPH. Accoppiato al trasporto elettronico c’è la formazione di un gradiente protonico che sostiene l’attività della ATPasi per produrre ATP. PSI e PSII rappresentano straordinarie macchine per l’utilizzo dell'energia solare, eppure hanno un punto debole in quanto sono riducenti monovalenti che portano alla produzione di specie reattive dell'ossigeno (ROS) in un ambiente, oggi, ricco di ossigeno creato dagli organismi fotosintetici. Inoltre, lo stato eccitato di tripletto della clorofilla reagisce con l'ossigeno molecolare per produrre ossigeno singoletto danneggiando proteine, lipidi e pigmenti presenti nei cloroplasti. Questo è il motivo per cui la luce in eccesso è dannosa e le alghe hanno evoluto meccanismi di fotoprotezione, che le piante hanno esteso e migliorato per la sopravvivenza in un ambiente terrestre ancora più stressato. Tra questi meccanismi di fotoprotezione, di particolare interesse è il Non-Photochemical Quenching (NPQ), che rapidamente (in pochi secondi) reagisce all’ incremento degli stati eccitati della clorofilla. Il quenching porta alla dissipazione termica dell'energia assorbita in eccesso, è scatenato dal gradiente ΔpH generato attraverso la membrana tilacoidale e richiede una specifica famiglia di proteine, i Complessi di Raccolta della Luce (LHC) . LHC formano una grande superfamiglia di proteine che legano xantofille/clorofille e sono associate al PSII e PSI avendo un ruolo diretto nella raccolta della luce e/o nel quenching. Due proteine LHC-simili, PSBS e LHCSR, sono indispensabili per il NPQ rispettivamente nelle piante vascolari e alghe verdi insieme alle xantofille luteina e/o zeaxantina, che sono i ligandi delle proteine LHC. Di particolare interesse è la zeaxantina perché viene sintetizzata in eccesso di luce a partire dalla violaxantina nel cosiddetto ciclo delle xantofille. La zeaxantina svolge un ruolo centrale nella fotoprotezione da detossificazione dei ROS, smorzando gli stati di tripletto della clorofilla (3Chl *) e, molto interessante per il mio lavoro, aumentando NPQ. Durante il mio dottorato, ho usato il muschio Physcomitrella patens come organismo modello per studiare il meccanismo di NPQ con particolare riferimento al ruolo di zeaxantina. P. patens ha una posizione strategica nell’albero della vita in quanto si trova tra le alghe verdi e le piante superiori; inoltre, è stato tra i primi organismi ad emergere dall'acqua per colonizzare l'ambiente terrestre, caratterizzazione da diverse condizioni stressanti, attraverso l'evoluzione di nuovi meccanismi di fotoprotezione. Le proteine PSBS, che è apparsa per prima in P. patens, ma anche LHCSR sono ancora attive in questo organismo, offrendo così la possibilità di studiare il NPQ sia delle alghe che delle piante nello stesso background genetico e biochimico. Questa possibilità può essere sfruttata grazie ad un ulteriore caratteristica, unica, di P. patens tra gli organismi fotosintetici eucarioti, ossia la sua capacità di fare ricombinazione omologa (HR) ad alta efficienza, rendendo il “gene targeting” una procedura standard. Comprendere la modulazione del NPQ durante l’acclimatazione a stress abiotici è essenziale per la piena comprensione del suo ruolo. Ho iniziato il mio lavoro dopo aver osservato che P. patens risponde a moderato stress salino e osmotico aumentando la sua attività NPQ. Sorprendentemente, l’aumento di NPQ non era dovuto sovra-accumulo delle proteine PSBS e LHCSR come nel caso dell’acclimatazione ad alta luce e al freddo. Ho potuto correlare l’aumento di NPQ, in seguito questi stress, con l’accumulo di zeaxantina. Per verificare il ruolo della zeaxantina, abbiamo identificato un unico gene VDE nel genoma di P. patens e abbiamo fatto dei mutanti che non esprimevano questo gene (knock out, KO). Le piante vde KO non erano capaci di produrre zeaxantina e mostravano una drammatica riduzione dell’NPQ così come una aumentata fotoinibizione in seguito a stress da alta luce. Abbiamo anche introdotto la mutazione VDE in genotipi che esprimevano solo la proteina LHCSR o PSBS mostrando che l’NPQ dipendente da LHCSR è, a sua volta, molto dipendente dalla zeaxantina rispetto all’NPQ dipendente da PSBS, con un rapporto vicino a 10. In questo lavoro, per la prima volta, ho isolato LHCSR nella sua forma nativa, di proteina che lega clorofilla a/b e xantofille. Inoltre, ho mostrato che in LHCSR, a differenza di PSBS, l’aumento di NPQ avviene attraverso il legame diretto della zeaxantina. Lo spettro di assorbimento e le caratteristiche dei pigmenti legati a LHCSR nativa combaciano con i dati riportati per LHCSR3 ricombinante di Chlamydomonas reinhardtii con l’eccezione che LHCSR di C. reinhardtii è zeaxantina-indipendente. Precedenti studi hanno identificato due funzioni essenziali per le proteine scatenanti NPQ : i) la funzione di sensori del pH (trovato anche in PSBS) e ii) la funzione di quenching (che si trova anche in altre proteine LHCB ) come LHCB4. Nelle piante queste due funzioni sono svolte da subunità proteiche diverse, rendendo così difficile studi in vitro. La recente scoperta di LHCSR ha reso la prospettiva di chiarire le basi molecolari del NPQ possibile: infatti, questa proteina è l'unica finora conosciuta per comprendere l'insieme delle funzioni necessarie per NPQ nella stessa unità strutturale. Nell’ultima parte di dottorato, ho provato a fare chiarezza nel meccanismo d'azione di LHCSR concentrandomi da un lato nella localizzazione sub - organello di questa proteina insieme allo studio della localizzazione di PSBS nelle membrane tilacoidali. Le membrane tilacoidi di P. patens sono organizzate in grana, ben definiti, e membrane stromatiche e sono differenzialmente esposti al compartimento stromale solubile come nelle piante vascolari. Ho sfruttato la possibilità di frazionare le membrane grana e stroma-lamelle per verificare la loro localizzazione con detergenti e frazionamento meccanico. Sorprendentemente , ho trovato che PSBS è localizzata nelle membrane granali mentre LHCSR è localizzato in membrane stromatiche: ciò suggerisce un meccanismo d'azione diverso di NPQ . Con queste informazioni ottenute, proponiamo un modello sperimentale per l'attivazione del quenching LHCSR-dipendente: LHCSR è ricco di residui acidi nella superficie esposta al lumen; in seguito ad eccesso di luce l’acidificazione potrebbe neutralizzare queste cariche e permettere la diffusione di LHCSR verso i grana grazie ad una ridotta repulsione con i PSII-LHCII supercomplessi. L’isolamento di LHCSR e la sua localizzazione, mi hanno incoraggiata ad ottimizzare e sfruttare queste preparazioni. Sebbene fossi conscia della difficoltà di questo lavoro, ho così deciso di provare a purificare LHCSR con (+) e senza (-) zeaxantina legata usando P. patens WT in quanto lo studio di LHCSR nella sua conformazione “quenchata” e non è un ambizioso ma essenziale target per la ricerca sulla fotosintesi. Come per qualsiasi progetto a lungo termine, ho concepito diverse strategie per l’isolamento di LHCSR sia dalla pianta WT di P. patens sia da creando versioni “taggate” della proteina che contengono una coda di istidina per facilitare la sua purificazione. Inoltre, ho anche sovra-espresso la proteina in tabacco. I potenziali vantaggi e le insidie di questo progetto sono descritte e discusse in questa tesi insieme a risultati preliminari.
Through photosynthesis plants use solar energy for producing reduced compounds from CO2 and finally biomass. Photosystems (PSI and PSII) are multisubunit pigment-binding complexes responsible for light harvesting, charge separation and play an essential role in electron transport from water to NADPH. Coupled to photosynthetic electron transport is the formation of a transmembrane pH gradient that sustains ATPase activity to produce ATP. PSI and PSII represent extraordinary machines for solar energy exploitation and yet they have a weak point in being univalent reductants which leads to production of reactive oxygen species (ROS) in the present day oxygen-rich environment that photosynthetic organisms have been creating. Moreover, chlorophyll is an excellent sensitizer and its triplet excited state reacts with molecular oxygen to yield singlet oxygen. This is why excess light is harmful and algae have evolved photoprotective mechanisms, which plants have extended and improved for survival in the even more challenging land environment. Of particular interest is Non-Photochemical Quenching (NPQ) of chlorophyll fluorescence which rapidly (within seconds) reacts to enhancement of the chlorophyll excited states. Quenching leads to the thermal dissipation of the energy absorbed in excess, is triggered by the ΔpH gradient generated across thylakoid membrane and requires specific members of the Light Harvesting Complexes (LHCs) protein family. LHCs form a large superfamily of chlorophyll-xanthophyll-binding proteins associated to PSII and PSI playing a direct role in light harvesting and/or energy quenching. Two LHC-like proteins, PSBS and LHCSR, are indispensable for NPQ respectively in vascular plants and green algae together with the xanthophylls lutein and/or zeaxanthin which are ligands for LHC proteins. Of particular interest is zeaxanthin because it is synthesized in excess light only from pre-existing violaxanthin in the so called xanthophyll cycle. Zeaxanthin plays a central role in photoprotection by scavenging of ROS quenching triplet states of chlorophyll (3Chl*) and, most interesting for my work, enhancing NPQ. During my PhD, I used the moss Physcomitrella patens as model organism to study the mechanism of NPQ with particular reference to the role of zeaxanthin. P. patens has a strategic position in the tree of life: it is an evolutionary intermediate between green algae and higher plants and was among the first organisms emerging from water to colonize the stressful land environment through the evolution of new photoprotective mechanisms. PSBS first appeared in P. patens and yet LHCSR proteins are still active yielding the possibility of studying both algal and plant NPQ in the same genetic and biochemical background. This opportunity can be exploited due to a further unique property of P. patens among eukaryotic photosynthetic organisms, i.e. its ability to perform Homologous Recombination (HR) at high efficiency, making gene targeting a standard procedure. Understanding the modulation of NPQ during acclimation to abiotic stress is essential for the full comprehension of its role. I started my work after the observation that P. patens responds to moderate salt and osmotic stress by increasing its NPQ activity. Surprisingly, NPQ enhancement was not due to over-accumulation of PSBS and/or LHCSR proteins as in the case of high light and cold acclimation. I could correlate NPQ enhancement under salt and osmotic stress with the over accumulation of zeaxanthin. When trying to verify the role of zeaxanthin we identified the unique VDE gene in P. patens genome and we knocked it out. vde KO plants were unable to produce zeaxanthin and showed a dramatic reduction in NPQ as well as an enhanced photoinhibition under excess light conditions. The introduction of the VDE mutation into LHCSR-only and PSBS-only genotypes showed that LHCSR-dependent NPQ is far more dependent on zeaxanthin than the PSBS-dependent NPQ with an activation ratio close to 10. In this work for the first time, I isolated LHCSR in the form of native chlorophyll a/b–xanthophyll-binding protein and found that the NPQ enhancement actually occurs through the direct binding of zeaxanthin to the LHCSR protein, different from the case of PSBS. Absorption spectrum and pigment binding properties of native LHCSR closely fit previously data reported for recombinant Chlamydomonas reinhardtii LHCSR3 whose activity, however, is zeaxanthin independent. Previous studies have identified two essential functions associated to essential proteins triggering NPQ: i) the pH detection function (also found in PSBS) and ii) the quenching function (also found in other LHCB proteins) such as LHCB4. In plants these two functions are carried out by distinct proteic subunits, thus making difficult in vitro studies. The recent finding of LHCSR protein has made the perspective of elucidating the molecular basis of NPQ possible: in fact, this protein is the only protein so far known to comprise the whole set of functions needed for NPQ into the same structural unit. Along the last part of my PhD work, I decided to move new steps towards the understanding of the mechanism of action of LHCSR by focusing on one side on the sub-organelle localization of this protein together with the study of the localization of PSBS in thylakoid membranes. P. patens thylakoid membranes are organized into well-defined grana stacks and stroma membranes which are differentially exposed to the stromal soluble compartment as in vascular plants. I exploited the possibility to fractionate grana and stroma-lamellae membranes to verify their localization using detergents and by mechanical fractionation. Surprisingly, I found that PSBS is localized in grana membranes while LHCSR is localized in stroma exposed membranes suggesting a different action mechanism on NPQ. Here on these basis I am proposing a tentative model for the activation of LHCSR-dependent quenching, specifically located at the periphery of grana stacks. LHCSR is rich in acidic residues in its lumen-exposed surface, acidification under excess light conditions would neutralize these charges and allow diffusion towards the grana partition domains thanks to a reduced repulsion with PSII-LHCII supercomplexes. The results reported in Chapter 2 (isolation of zeaxanthin-binding LHCSR) and Chapter 3 (localization of LHCSR in the margins/stroma fraction of thylakoid membranes) encouraged me to initiate the ambitious task of optimizing and scaling up these preparations. Although I was conscious about the difficulty of this work, I decided to try the purification of LHCSR +/- zeaxanthin from WT P. patens because the differential study of LHCSR in its quenched vs unquenched conformation is an ambitious but essential target for photosynthesis research. As for any long term project, I have conceived several strategies for the isolation of LHCSR from either WT P. patens or overexpressed using WT sequence or tagged versions of the protein using a poly-Histidine tail (His-tag) to facilitate its purification. Alternatively I also have attempted overexpressing LHCSR in tobacco. The potential advantages and pitfalls of this project are described and discussed in PhD thesis together with preliminary results.

Photosynthetic organisms harvest light and convert energy into biomass through the process of photosynthesis. Antenna systems are responsible for light harvesting and transfer the energy towards the Reaction Centres (RCs). However, too much light can be damaging and therefore the excess energy is dissipated as heat through a process called Non-Photochemical Quenching (NPQ). NPQ is activated by decreased pH on the luminal side of the thylakoid membranes by either PSBS or LHCSR found in higher plants and green algae respectively. When the antenna systems receive too much light the singlet Chl excited states produced by photon absorption cannot be quenched by photochemistry fast enough, leading to intersystem crossing producing Chl triplet states, which react with O2 and results in the synthesis of Reactive Oxygen Species (ROS). ROS accumulation causes photoinhibition of photosynthesis, drastically limiting growth. Therefore, dissipating light when in excess and using even the last photon under low light is a difficult exercise which is essential to ensure maximal growth. However, plants are more “interested” in surviving stress and reproducing than in growing big. Therefore, they have developed a hysteretic response to light: in order to avoid damage, plants over-regulate energy dissipation thus growing less than could be afforded under farming conditions, indicating that there is large room for engineering energy dissipation and increase crop production. Furthermore, besides changes in light intensity, plants experience changes in spectral composition as well. However, PSII and PSI have slightly different absorbance spectra and for an efficient linear electron flow between the two photosystems, it is essential that the excitations between the two photosystems are balanced. This is regulated by so-called state transitions a shuttling of antenna proteins between PSII and PSI. In Chapter 1 the differences and similarities between a variety of oxygenic photosynthetic organisms is reviewed. The focus lies on the different sets of antenna systems that evolved during the evolution and how the antenna systems that we currently find in plants and green algae have such an important role in photoprotection. In Chapter 2, PSBS in A. thaliana has been replaced with LHCSR1 from the moss P. patens, an evolutionary intermediate both expressing functional PSBS and LHCSR. The complemented A. thaliana lines showed a partial recovery of NPQ. The partial recovery of NPQ was mainly caused by the reduced capacity to convert violaxanthin into zeaxanthin in A. thaliana in comparison with P. patens. In chapter 3, several different A. thaliana lines lacking one or more PSII-antenna complexes were complemented with LHCSR1 form P. patens in order to identify a possible interaction partner of LHCSR1, where Lhcb5 (CP26) has been identified as the most likely interaction partner of LHCSR1. In chapter 4, the complemented lines from Chapter 2 were grown in different fluctuating light conditions to see whether LHCSR1 could increase the biomass production. However, in all cases WT grew the same or better than the complemented lines. In specific cases the presence of LHCSR1 could partly improve growth in comparison to npq4. In Chapter 5, we looked at the locations of interaction of LHCII with PSI. This is especially interesting since LHCII is the most important protein to induce NPQ, and does perform state-transitions, a process which is essential for an even energy distribution between the two photosystems and therefore necessary to grow properly. A second LHCII-PSI interaction site has been confirmed by looking at the energy transfer in isolated stroma membranes in State I or State II of WT and a mutant devoid of the PSI antenna. We show that the presence of the PSI-antenna (Lhca1-4) increase the rate of energy transfer from LHCII to PSI by 4 times and thus are essential for a proper binding of LHCII to PSI.

Photosynthetic organisms can use solar energy to produce organic biomass starting from simple elements as CO2 and water, releasing oxygen as side product. Algae are characterized by high growth rate, extremely rapid life cycle and intrinsic high photosynthetic efficiency. Moreover, microalgae can also be cultivated in a mixed autotrophic/heterotrophic condition, using reduced carbon sources. Several algal strains are characterized by high lipid accumulation or production of high value compounds. Thus, algae not only represent a valid alternative to plants, but they also play a central role considering the sustainability related to their cultivation. Wastewaters and flue gas can be used to ensure nutrients and CO2 for carbon fixation, and, after biomass harvesting, water can be reused leading to a far lower consumption with respect to plants (especially in closed photobioreactor in which the evaporation is low). Unfortunately, algae evolved in conditions extremely different compared to actual industrial ones which involves 24/24 hours of high irradiance, strong shaking as well as high CO2 concentration: all these elements ensure high photosynthetic rate and thus high biomass accumulation but make necessary a domestication of strains. Since this need became evident, engineers, biologists and biotechnologists had tried to overcome algae cultivation limitations in order to became it feasible and economically useful. From a biotechnological point of view several targets could be pointed. Optimization of absorption/dissipation of light energy is one of the most interesting and explored. This thesis reports the use of several approaches to investigate the heat dissipation mechanisms (NPQ) in green algae, mainly focusing on the model organism Chlamydomonas reinhardtii. The results obtained reveal the molecular mechanisms of energy conversion from excitation energy into heat by the activity of specific pigment binding proteins called LHCSR (Light Harvesting Stress Related), going deep into details of the protein domains and pigments involved in the quenching process and the protein interaction network necessary for NPQ. In particular, the regulation of the accumulation of LHCSR proteins in Chlamydomonas reinhardtii revealed to be a successful genetic engineering strategy to improve biomass productivity. Among the possible application of microalgae, one of the most promising one is their use as green factories to produce high value products: here, we report the metabolic engineering of Chlamydomonas reinhardtii as a bio-factory for ketocarotenoids production. The use of microalgae as host to produce high value metabolites, represents, indeed, an effective way to break down costs related to their cultivation with a potential high impact into the market. Astaxanthin is, currently, produce using Haematococcus. lacustris (recently renamed from Haematococcus pluvialis) in which, its accumulation causes a stop in growth. For that reason, in this thesis effects of astaxanthin accumulation of H. lacustris was investigated. This thesis presents, with different approaches, a leap forward in microalgae domestication both trough enrichment of knowledge about NPQ and trough application of metabolic engineering to develop green bio-factories.

Non-photochemical quenching (NPQ) of chlorophyll fluorescence is a process essential for the regulation of photosynthesis and plant protection from light stress. In vascular plants this process is triggered by a luminal pH sensor, the PSBS protein, which transduces chloroplast lumen acidification, induced by excess light, into a quenching reaction occurring within specific interacting chromophore-bound lightharvesting proteins (LHC). In algae, such as Chlamydomonas reinhardtii, stress-related light-harvesting proteins (LHCSR) fulfill both pH sensing and quenching reactions, due to their capacity of binding chlorophylls and xanthophylls. The moss Physcomitrella patens, an evolutionary intermediate between algae and plants, has both PSBS and LHCSR active in quenching with LHCSR working in a direct zeaxanthin-dependent manner. Plants and mosses have a very similar organization of thylakoid membranes thus suggesting LHCSR might be active in plants. To verify this hypothesis, we overexpressed lhcsr1 gene into Arabidopsis thaliana PSBS mutant, npq4, and screened transformants by fluorescence video-imaging, resulting to the isolation of A. thaliana plants, which accumulate a pigment-binding, NPQ-active LHCSR1 in thylakoid membranes. In the context of functional and structural analysis of LHCSR1 protein, a series of in vivo transformations was performed using A. thaliana mutants altered in xanthophyll content or lacking specific LHC subunits. For this reason the double mutant npq1npq4 - unable to convert violaxathin into zeaxanthin - was complemented in order to verify the direct dependence of LHCSR1 on zeaxanthin, mutant lut2npq4 was used due to its complete lack of lutein and antenna mutants NoMnpq4 and ch1lhcb5 were used due to their lack of either minor antennas or the complete antenna system respectively; all of them overexpressing LHCSR1 in different levels. Finally, a first approach for the in vivo mutational analysis of P. patens LHCSR1 has been initiated, using A. thaliana as a tool for heterologous protein expression.

BPS/IC (Bladder Pain Syndrome/Interstitial Cystitis) ist ein sehr schweres und noch weitgehend unverstandenes Krankheitsbild in der Urologie. Viele Frauen sind im Alltag durch den ständigen Harndrang und die Schmerzen stark eingeschränkt und von Depressionen betroffen. Die Aufklärung der Pathogenese ist deshalb sehr wichtig, um eine adäquate Therapie für die Betroffenen zu entwickeln und die Krankheit möglichst frühzeitig diagnostizieren zu können. Das Schwangerschaftshormon hCG (humanes Choriongonadotropin) besitzt differenzierende und wachstumsfördernde Eigenschaften und eine Rolle in der Urothelregeneration und – stabilisierung scheint möglich. Daher ist das Ziel dieser Arbeit seinen Rezeptor, den LHCGR (Luteinizing-Hormone/Choriogonadotropin Rezeptor), in der Harnblase nachzuweisen und die urothelialen und muskulären Charakteristika zwischen gesunden und an BPS/IC erkrankten Harnblasen zu vergleichen. Die Darstellung des LHCGR erfolgte auf Proteinebene mittels indirekter Immunfluoreszenz und auf mRNA-Ebene durch Standard-PCR. Es zeigten sich im Urothel von Harnblase und Ureter 5 unterschiedliche Verteilungsmuster des Rezeptors hinsichtlich seiner Expression in verschiedenen Zellschichten und seiner subzellulären Lokalisation. Je nach Urothelzustand und zwischen den Entitäten Kontroll- bzw. BPS/IC-Harnblase variierten diese Muster in ihrer Häufigkeit. In anderen Epithelien, wie dem Vaginalepithel, änderte sich die zelluläre Verteilung des LHCGR in Abhängigkeit vom Differenzierungsgrad der Zellen. Es scheint möglich, dass auch die Rezeptorexpression in Urothelzellen deren verschiedene Differenzierungszustände widerspiegelt. Dies unterstützt den für hCG vermuteten Einfluss auf die Epithelregeneration. Ein Vergleich der urothelialen Fluoreszenzintensitäten zwischen weiblichen Kontroll – und BPS/IC-Harnblasen zeigte eine signifikant stärkere Expression des Rezeptors bei erkrankten Patienten. Dem gegenüber war kein Unterschied im Detrusor, weder zwischen Kontroll – und BPS/IC-Harnblasen noch im geschlechtsspezifischen Vergleich, festzustellen. Damit scheint der Rezeptor seine Hauptaufgabe vorrangig im Urothel zu entfalten. Die Korrelationsanalysen ergaben keinen signifikanten Zusammenhang zwischen dem Erkrankungsalter (Zeitpunkt der Diagnosestellung und Biopsieentnahme) und der LHCGR-Immunfluoreszenz. Ein endokrinologischer Einfluss auf die Rezeptorexpression wurde dadurch unwahrscheinlich und unterstützt die immer akzeptiertere Auffassung, dass BPS/IC nicht mehr mit der Menopause assoziiert ist. Neben dem Urothel und Detrusor zeigten auch Lamina propria und Gefäße von Harnblase und Ureter die Expression des LHCGR in der Immunhistochemie. Unterschiedliche Clustermuster des Rezeptors im Detrusor ließen auf die Oligomerisierung des Rezeptors schließen. Die Bedeutung dieser Zusammenschlüsse ist jedoch noch unklar, wobei unterschiedliche funktionelle Zustände des Rezeptors vermutet werden. Orientierung bieten andere Rezeptoren, die durch Dimerisierung verschiedener Rezeptorvarianten ihre Funktionalität verbessern oder verschlechtern konnten. Obwohl für keine bisher entdeckte Variante des LHCGR eine definitive Aufgabe ermittelt werden konnte, scheinen doch viele Varianten auch unterschiedliche Funktionen wahrnehmen zu können. Besonders auf der Regulierbarkeit des Rezeptors mittels interagierender Splicevarianten sollte das Augenmerk zukünftiger Studien liegen. Ob durch Komplexbildung verschiedener Varianten oder Bildung nichtfunktioneller trunkierter Rezeptoren, die Kontrollmöglichkeiten sind vielfältig und können auch auf Liganden wirken. Letztlich ließ der Nachweis des LHCGR in allen Schichten von Harnblase und Ureter eher eine globale Rolle des Rezeptors im Harntrakt des Menschen vermuten. Dazu passten auch die bereits nachgewiesenen Einflüsse seiner Liganden auf die Blasenfunktion von Hunden. Die hier vorgelegte Arbeit untersuchte zum ersten Mal die Expression des LHCGR mittels PCR und Immunhistochemie in humanen Harnblasen und Ureteren. Dabei löste sie sich von den sonst üblichen Vorstellungen einer Beziehung des Rezeptors zu Blasentumoren, Schwangerschaft oder Inkontinenz. Diagnose und Therapie von BPS/IC sind zur Zeit noch ständigen Wandlungen unterworfen und dabei entgehen viele Patienten der (frühen) Diagnosestellung und einer adäquaten Behandlung. Diese Studie sollte dazu beitragen neue Einblicke in die Pathophysiologie der Erkrankung zu erlangen, um eine kausale Therapie entwickeln zu können. Zukünftig könnten diese Ergebnisse dabei helfen die Anwendung einer sensitiven und vor allem spezifischen Diagnostik auf molekularer Ebene (mRNA - oder Proteinnachweis) zu ermöglichen.

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014
Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) is an autosomal dominant syndrome resulting from a mutation in the Fumarate Hydratase enzyme gene which consists of leiomyomas of the skin and uterus and Renal Cell Carcinoma. The exact mechanism of tumor formation is still unknown. However, it is thought to relay on the activation of hypoxia inducible factors with promotion of angiogenesis and anaerobic glycolysis. Renal carcinomas in these patients have specific features that distinguish them from the ones found in other familial syndromes and which are important to their diagnosis and management. Although renal tumors have an incomplete penetrance in these patients, they are extremely aggressive and can metastize early, which is why annual screening with abdominal Magnetic Ressonance Imaging is recommended, as well as immediate surgical excision once detected. The following work presents a case of HLRCC and aims to review the main features and management of Renal Cell Carcinoma in these patients.
A Leiomiomatose Hereditária e Carcinoma de Células Renais (LHCCR) é um síndrome de transmissão autossómica dominante resultante de uma mutação no gene da enzima Fumarato Hidratase e caracterizado pela presença de leiomiomas cutâneos e uterinos e Carcinoma de Células Renais. Embora o exato mecanismo de génese tumoral permaneça desconhecido, pensa-se que a sua origem esteja na ativação de fatores indutores de hipoxia com promoção da angiogénese e glicólise anaeróbia. Os carcinomas renais destes doentes têm características específicas que os distinguem dos encontrados noutros síndromes familiares e que são importantes para o seu diagnóstico e abordagem terapêutica. Apesar dos tumores renais terem uma penetrância incompleta nestes doentes, são extremamente agressivos e metastizam precocemente, pelo que se aconselha a realização de rastreio anual com Ressonância Magnética abdominal e, uma vez detetados, atuação cirúrgica imediata. O trabalho apresentado reporta um caso de LHCCR e tem como objetivo rever as principais características e abordagem terapêutica do Carcinoma de Células Renais nestes doentes.