Rozprawy doktorskie na temat „Receptor”

A Doença de Parkinson é uma doença altamente incapacitante e de grande prevalência. Pouco se sabe sobre sua etiologia e os tratamentos atuais consistem na diminuição dos sintomas, uma vez que ainda não foi encontrada uma maneira de reverter o déficit de neurônios dopaminérgicos observados nos pacientes acometidos. Sabe-se que os receptores purinérgicos são encontrados por todo o sistema nervoso central, não só no indivíduo adulto como também em diferentes estágios do desenvolvimento embrionário e estão envolvidos com proliferação e diferenciação celular. Este trabalho estudou a participação dos receptores purinérgicos em modelo animal de doença de Parkinson por lesão dos neurônios dopaminérgicos da via nigroestriatal com 6-OH dopamina (6-OHDA). Realizamos a análise do perfil de expressão gênica dos diferentes receptores após a lesão e subsequente modulação. Observamos expressão gênica alterada dos receptores P2X7 e P2Y6 até 5 semanas após a lesão. O uso do antagonista do receptor P2X7 Brilliant Blue G (BBG) induziu a regeneração da via nigroestriatal e o uso do antagonista do receptor P2Y6 MRS2578 preveniu a morte dos neurônios. Como esses efeitos foram acompanhados pela inativação de células microgliais, supõe-se que o controle do microambiente neuroinflamatório causado pela injeção de 6-OHDA seja a principal causa do efeito antiparkinsoniano observado pelo tratamento com BBG e MRS2578. Além disso, o transplante celular com células precursoras neuraisnão foi capaz de reverter o comportamento hemiparkinsoniano dos animais lesionados. Apesar do uso concomitante com BBG reduzir o comportamento, parece que esse efeito deve-se ao BBG per se, uma vez que o tratamento somente com o antagonista de P2X7 foi mais eficaz. De maneira geral, a modulação da atividade dos receptores purinérgicos se mostrou uma ferramenta promissora na pesquisa de cura e compreensão das bases moleculares da Doença de Parkinson
Parkinson\'s disease is a highly disabling and prevalent disease. Little is known about its etiology and the current treatments consist in the reduction of the symptoms, since there is no known method to reverse the dopaminergic neurons deficit observed in patients. Purinergic receptors are found throughout the central nervous system, not only in the adult individual but also at different stages of embryonic development, and are involved in proliferation and differentiation. This work investigated the role of purinergic receptors in the animal model of Parkinson\'s disease induced by 6-OH dopamine (6-OHDA) injection and consequent death of dopaminergic neurons of the nigrostriatal pathway. Patterns of purinergic receptors gene expression after the lesion and subsequent modulation were analyzed. We observed altered gene expression of P2X7 and P2Y6 receptors within 5 weeks of injury. The use of the P2X7 receptor antagonist Brilliant Blue G (BBG) induced the regeneration of the nigrostriatal pathway and treatment with P2Y6 receptor antagonist MRS2578 prevented the death of the neurons. Since these effects were accompanied by the inactivation of microglial cells, it is assumed that the control of neuroinflammatory milieu caused by the 6-OHDA injection is the main cause of the antiparkinsonian effect observed by the treatment with BBG and MRS2578. In addition, transplantation with neural precursor cells was not able to reverse the hemiparkinsonian behavior of injured animals. Although concomitant use with BBG improved cell engraftment, it appears that this effect is due to BBG per se, since treatment with only this P2X7receptor antagonist was more effective. In general, modulation of purinergic receptor activity showed to be a promising tool in the research of cure and understanding of the molecular bases of Parkinson\'s Disease.

The sigma-1 receptor (σ-1R) is an endoplasmic reticulum (ER) protein that modulates a number of ion channels. It is hypothesized that σ-1Rs activated with agonist translocate to the plasma membrane. The σ-1R potentiates N-methyl-D-aspartate Receptors (NMDARs), important constituents of synaptic plasticity. NMDARs are anchored in the plasma membrane by Postsynaptic Density Protein-95 (PSD-95). The mechanism behind σ-1R modulation of NMDARs is not known. The results of my investigation confirm that σ-1Rs localize extrasomatically. Following σ-1R activation, σ-1R localization to dendrites and postsynaptic densities (PSDs) is upregulated. Unpublished work from our lab has shown that σ-1Rs associate with PSD-95 and NMDARs. Furthermore, immunocytochemistry (ICC) showed σ-1R colocalization with PSD-95 and NMDAR subunits. After σ-1R activation there was significantly increased colocalization between σ-1R, PSD-95, and GluN2B. Overall, this study may have provided insight into the molecular mechanism behind σ-1R modulation of NMDARs, which could have implications in the understanding of synaptic plasticity.

Cell membrane receptors help to mediate communication between the cell and its environment. The largest group of these membrane receptors belong to the family of G protein-coupled receptors (GPCRs). GPCRs contain seven transmembrane (TM) helices and signal predominantly through heterotrimeric G proteins in response to diverse extracellular stimuli. Previously, three levels of amino acid conservation were proposed to understand the structure and function of a GPCR. This includes “signature” amino acids, “group –conserved” amino acids and amino acids conserved only within a specific subfamily. The group-conserved residues in class A GPCR family involve amino acid conservation of up to 99% when considered as a group of small and weakly polar residues (Ala, Gly, Ser, Cys and Thr). These group-conserved residues have been proposed as key determinants in helix-helix interactions. Therefore, I selected these residues for structure-function analysis in the amine and the prostanoid receptor sub-families of class A GPCRs. Molecular and biochemical assays clearly demonstrate the importance of group-conserved residues in β2-adrenergic receptor and thromboxane A2 receptor (TP) structure and function. These studies led to the identification of a non-synonymous single nucleotide polymorphic variant (nsSNP) A160T in TP to be a constitutively active mutant (CAM). Further, the TP-CAM was used as a pharmacological tool that enabled classification of well-known TP-blockers, into neutral antagonists and inverse agonists. The role of TP-A160T in prostanoid receptors, TP- Prostacyclin receptor (IP) heterodimerization and signaling was investigated. Activation of a GPCR ultimately leads to structural changes in its intracellular loops (ICLs), which in turn activates G-protein. TP activates its cognate G protein (Gαq), while IP mediates signaling, through Gαs. Using TP-IP chimeric receptors, molecular modelling, and site directed mutagenesis studies I determined the specific ICL regions required for G protein coupling in TP and IP. Significant challenges exist in expressing and purifying GPCR-CAMs in amounts required to pursue biophysical studies. Using tetracycline inducible HEK293S system, A160T was expressed at high-levels and CD spectropolarimetry studies were successfully pursued on the purified A160T. The CD spectra showed that the loss of thermal stability of the A160T mutant is due to the subtle changes in the secondary structure of the A160T protein. These studies involving molecular, biochemical and pharmacological approaches provide novel insights into the structure and function of prostanoid receptors TP and IP.

Low affinity Fcg receptor III (FcgRIII, CD16) triggers a variety of cellular events upon binding to the Fc portion of IgG. A real-time flow cytometry method was developed to measure the affinity and kinetics of such low affinity receptor/ligand interactions, which was shown as an easily operated yet powerful tool. Results revealed an unusual temperature dependence of reverse rate of CD16aTM dissociating from IgG. Except for a few studies using mammalian cell CD16s, most kinetics analyses use purified aglycosylated extracellular portion of the molecules, making it impossible to assess the importance of the receptor anchor and glycosylation on ligand binding. We used a micropipette adhesion frequency assay to demonstrate that the anchor length affects the forward rate and affinity of CD16s for IgG in a species specific manner, most likely through conformational changes. Receptor glycosylation dramatically reduced ligand binding by 100 folds. T cell receptor (TCR) is arguably the most important receptor in the adaptive human immune system. Together with coreceptor CD4 or CD8, TCR can discriminate different antigen peptides complexed with major histocompatibility complex (MHC) molecule (pMHC), which differ by as few as only one amino acid, and trigger different T cell responses. When T cell signaling was suppressed, TCR had similar affinity and kinetics for agonist and antagonist pMHC whose binding to CD8 was undetectable. TCR on activated T cell had a higher affinity for pMHCs, suggesting that TCRs organize themselves differently on activated T cells than on naïve T cells. In the absence of inhibitors for signaling, TCR binds agonist pMHC with several orders of magnitude higher affinity than antagonist pMHC. In addition, engagement of TCR by pMHC signals an upregulation of CD8 binding to pMHC, which is much stronger than the TCR-pMHC binding. The transition from weak TCR binding to the strong CD8 binding takes place around 0.75 second after TCR in contact with pMHC and can be reduced by several inhibitors of tyrosine and lipid phosphorylation, membrane rafts, and actin cytoskeleton. These results provide new insights to understanding T cell discrimination.

Os receptores nucleares compreendem uma superfamília de proteínas intracelulares reguladas relacionados estruturalmente, capazes de reconhecer sequências específicas de DNA e regulam a transcrição de genes alvos respondendo a sinais metabólicos, hormônios e outras moléculas regulatórias integrando muitas vias de sinalização. Os receptores ativadores da proliferação de peroxissomos (PPARs) são receptores nucleares que regem a transcrição de vários genes envolvidos principalmente no metabolismo de ácidos graxos e energia. A ativação do PPARY possui um amplo aspecto de funções biológicas, regulando o metabolismo, reduzindo a inflamação, influenciando o equilíbrio das células imunes, inibindo a apoptose e o estresse oxidativo e melhorando a função endotelial. Estes efeitos parecem ser benéficos não apenas em diabetes e aterosclerose, mas também em várias outras condições. Os agonistas do PPARY são utilizados como sensibilizadores de insulina para o tratamento da diabetes II, sendo um alvo molecular dos fármacos tiazolidinadionas. Diversos efeitos colaterais severos associados ao uso dos fármacos desta classe e à importância do PPARY no metabolismo de glicose e na sensibilização da insulina, o presente trabalho justifica-se como um esforço para avançar na compreensão da interação entre ligantes sintéticos com o receptor PPARY e a proposição de moléculas mais seguras e mais eficazes para a manutenção de níveis euglicêmicos. Foi realizada a expressão, a purificação, seguida de estudos cristalográficos em cinco ligantes selecionados a partir de etapas de docking realizados anteriormente pelo nosso grupo de Biotecnologia Molecular do Instituto de Física de São Carlos. Os ensaios de cristalização do PPARY complexado a ligantes sintéticos resultaram em duas estruturas cristalográficas que apresentaram uma conformação em que os ligantes não interagiram diretamente na hélice 12 como descritos para agonistas totais do PPARY, adotando características de agonistas parciais. Esses ligantes apresentaram interações hidrofóbicas que estabilizam as fitas-β. Este conjunto de informações estruturais apresentados neste trabalho para o PPARY proporcionou um entendimento das interações que esse receptor é capaz de fazer na presença de um ligante, além de que poderão ser úteis no desenvolvimento de novos moduladores seletivos do PPARY semelhante ao que já se encontram no mercado, porém com efeitos colaterais reduzidos.
Nuclear receptors comprise a superfamily of structurally-related regulated intracellular proteins capable of recognizing specific DNA sequences and regulating the transcription of target genes responding to metabolic signals, hormones and other regulatory molecules integrating many signaling pathways. Peroxisome proliferator-activating receptors (PPARs) are nuclear receptors that govern the transcription of several genes involved primarily in fatty acid and energy metabolism. Activation of PPARY has a broad aspect of biological functions, regulating metabolism, reducing inflammation, influencing immune cell balance, inhibiting apoptosis and oxidative stress, and improving endothelial function. These effects appear to be beneficial not only in diabetes and atherosclerosis, but also in several other conditions. PPARY agonists are used as insulin sensitizers for the treatment of diabetes II, being a molecular target of the thiazolidinediones drugs. A number of severe side effects associated with the use of drugs of this class and the importance of PPARY in glucose metabolism and insulin sensitization, the present work is justified as an effort to advance the understanding of the interaction between synthetic ligands with the PPARY receptor and proposing safer and more effective molecules for the maintenance of euglycemic levels. The expression, purification, followed by crystallographic studies in five ligands selected from docking steps previously performed by our Molecular Biotechnology group of the Physics Institute of São Carlos. The crystallization assays of PPARY complexed to synthetic ligands resulted in two crystallographic structures that exhibited a conformation in which the ligands did not interact directly in helix 12 as described for total PPARY agonists, adopting characteristics of partial agonists. These ligands showed hydrophobic interactions that stabilize the β-ribbons. This set of structural information presented in this work for the PPARY was of great value for the understanding of the interactions that this receptor is able to make in the presence of a ligand, besides that they could be useful in the development of new selective modulators of the PPARY similar to that are already on the market, but with reduced side effects.

O sistema olfatório de mamífero pode discriminar milhares de odores presentes no meio ambiente. Aproximadamente 1000 diferentes receptores olfatórios (ORs) são expressos no epitélio olfatório (OE) do nariz, Os ORs detectam os odores e transmitem os sinais resultantes para o bulbo olfatório (OB) no cérebro. Os ORs pertencem a super família dos receptores acoplados a proteína G (GPCR) e apresentam sete domínios transmembrânicos putativos. Por razões desconhecidas, os ORs são retidos no retículo endoplasmático quando expressos em linhagens de células de mamíferos heterólogas. Provavelmente, proteínas acessórias sejam requeridas para o endereçamento dos Ors para a superficie celular. No presente estudo, utilizamos o OR M93 para estudar os mecanismos de expressão de um ORo A dissertação teve como objetivos específicos: (l) construção de um vetor para expressão do OR M93 em fusão com GFP em levedura e análise de sua localização celular; (2) identificar proteínas expressas no epitélio olfatório de camundongo que interajam com os ORs. A análise por microscopia de fluorescência revelou que a expressão do OR M93 fusionado a GFP demonstrou um padrão de fluorescência que sugere a retenção do OR M93 no retículo endoplasmático. Nós utilizamos o sistema de duplo híbrido em levedura para varrer uma biblioteca de cDNA de epitélio olfatório de camundongo com uma isca correspondente à região N-terminal do OR M93. Quatro proteínas candidatas foram identificadas: HLA-B associado ao transcrito 3 (BAT-3/ Scythe), superfamília transmembrana 4 (membro CD82), superfamília transmembrana 4 (membro OAP-I) e sindecan (membro SDC2) (\"GenBank accession numbers\": BC026647, D14883, BC0430n e BC047144). A análise da hibridação in situ destas proteínas, revelou que a proteína OAP-1 é a melhor candidata a interação com OR M93. Dessa maneira, nós indicamos a proteína OAP-1 como possível proteína candidata a auxiliar o OR a ser expresso de maneira funcional em sistemas heterólogos.
The mammalian olfactory system can discrim inate thousands of odorants present in the environrnent. Approximately 1000 different olfactory receptors (ORs) are expressed in the olfactory epithelium (OE) of the nose. The ORs detect odorants and transmit the resulting signals to the olfactory bulb (OB) of the brain. ORs belong to the G-protein-coupled receptor (GPCR) super family and have seven putative transmembrane domains. For unknown reasons, the ORs are retained in the endoplasmatic reticulum when expressed in heterologous mammalian cell lines. Probably accessory proteins are required for the sorting of the ORs to the cell surface. In the present work, we used the OR M93 to study the mechanisms of OR expression. Our goals were to (1) construct an expression vector for OR M93 in fusion with GFP in yeast and (2) to identify proteins expressed in the mouse OE that interact with ORs. The analysis by fluorescence microscopy suggested that OR M93 in fusion with GFP was retained in the endoplasmic reticulum (ER) of yeast. We used the yeast two-hybrid system to screen a mouse OE cDNA library with a bait corresponding to the N-terminal region ofthe üR M93. Four potential candidates were identified: HLA-B associated transcript 3 (BAT-3/Scythe), transmembrane 4 superfamily (CD82 member), transmembrane 4 superfamily (TSPN-3 member) and syndecan (SDC2). In situ hybridization analysis suggests that OAP-l protein represents the best candidate for interaction with OR M93. We suggest the OAP-l protein could be an accessory protein required for the sorting of the ORs to the cell surface in heterologous cell lines.

Chez les mammiferes, la lipogenese ou synthese de novo des acides gras joue un rôle essentiel a l'homeostasie energetique. Elle est particulierement active dans le foie. Le Liver X Receptor (LXR) est un recepteur nucleaire de classe II qui est implique dans la regulation de l'expression de genes importants dans cette voie metabolique. Au niveau hepatique, LXR regule directement l'expression de certains genes de la lipogenese et aussi l'expression des facteurs de transcription SREBP-1c et ChREBP intervenant respectivement dans la reponse hepatique a l'insuline et au glucose. Les ligands naturels de LXR sont les oxysterols, des derives oxygenes du cholesterol. Aussi, LXR est avant tout considere et connu comme un senseur du cholesterol. Au cours de ces travaux nous nous sommes interesses in vivo au role de LXR dans la regulation transcriptionnelle de la lipogenese hepatique en fonction de differents stimuli: pharmacologiques, inflammatoires et nutritionnels. Par une approche pharmacologique, nous avons etudie la regulation croisee avec entre LXR et le recepteur active par les proliferateurs de peroxisome (PPAR . Nous avons aussi montre que l'inflammation intestinale est un puissant inhibiteur de la lipogenese hepatique. Enfin, nous avons mis en evidence le role de LXR dans la regulation de la lipogenese en reponse a une carence en acides gras essentiels et a un regime riche en fructose
In mammals, lipogenesis or de novo fatty acid synthesis plays an essential part in energy homeostasis. It is particularly active in the liver. The Liver X Receptor (LXR) is a class II nuclear receptor that regulates the expression of important genes involved in this pathway. In the liver, LXR directly controls the expression of lipogenic genes and also the expression of transcription factors such as SREBP-1c and ChREBP required for the hepatic response to insulin and glucose respectively. Natural ligands for LXR are oxysterols, which are oxygenated derivatives of cholesterol. Therefore, LXR is primarily considered and known as a cholesterol sensor. In this work, we were interested in the role of LXR in the transcriptional control of hepatic lipogenesis in vivo in response to distinct stimuli: pharmacological agonists, gut inflammation and changes in diet composition. Through a pharmacological study, we highlighted the cross-talk between LXR signaling and the regulation of the Peroxisome Proliferator Activated Receptor (PPAR ). We have also evidenced that experimentally induced colitis induces a potent inhibition of hepatic lipogenesis. Finally, we have shown that LXR is involved in the regulation of lipogenesis in response to essential fatty acid deficiency and to high fructose

Thesis (Ph. D.)--Medical College of Ohio, 2004.
"In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Sciences." Major advisor: Manohar Ratnam. Includes abstract. Document formatted into pages: iii, 293 p. Title from title page of PDF document. Includes bibliographical references (p. 175-281).

Los receptores acoplados a proteína G (GPCR) conforman la familia de receptores de membrana más grande. El numeroso y variado tipo de señales que detectan han otorgado a estos receptores un alto interés farmacológico. Además, las interacciones entre diferentes tipos de GPCR formando complejos oligoméricos dan lugar a complejos con características bioquímicas diferenciadas de los protómeros que los forman. En esta Tesis Doctoral se han estudiado diferentes aspectos derivados este tipo de interacciones, centrando estos experimentos alrededor del receptor A2A de adenosina (A2AR), un importante neuromodulador del Sistema Nervioso Central. Por una parte, mediante la combinación de las técnicas de transferencia de energía resonante bioluminiscente (BRET) y de complementación bimolecular fluorescente (BiFC) se ha podido detectar in vivo que A2AR forma oligómeros con más de dos protómeros, tanto de tipo homomérico (Gandía et al., 2008) como heteromérico. En este último caso, se ha estudiado en concreto el oligómero de A2AR con el receptor D2 de dopamina y el receptor metabotrópico 5 de glutamato (Cabello et al., 2009). A continuación, se ha aplicado una variante de la técnica de doble híbrido específica para proteínas de membrana (MYTH), con la intención de detectar proteínas interaccionantes con A2AR. Gracias a esta aproximación, se han encontrado nuevas proteínas candidatas a interaccionar con nuestro receptor, destacando entre ellas un GPCR huérfano, GPR37. Mediante técnicas físicas y funcionales en modelos de cultivo celular y animales se ha podido validar la interacción A2AR/GPR37 y se ha comprobado que la presencia de GPR37 modifica la funcionalidad del receptor A2A de adenosina. Finalmente, para profundizar en las características estructurales de GPR37, poco conocidas hasta el momento, se ha estudiado la cola C-terminal del receptor. Así, se ha visto que existe una región rica en residuos de cisteína que regula el tráfico del receptor hacia la membrana plasmática. Además, este dominio rico en cisteínas modula el estrés de retículo endoplasmático generado al sobreexpresar GPR37 en cultivo celular y también la inducción de vías apoptóticas (actividad de caspasa-3) en estas mismas condiciones (Gandía et al., 2013).
G protein-coupled receptors (GPCR) consitute the biggest family of membrane receptors. Since they detect a large and diverse number of signals, they have a growing pharmacological interest. Furthermore, the interactions between different types of GPCR form oligomeric complexes that show different biochemical properties than the protomers they are made of. Different aspects of these interactions have been studied in this Doctoral Thesis, focusing the experiments around the adenosine A2A receptor, being adenosine an important modulator of the Central Nervous System. Firstly, by means of the combination of the bioluminescent ressonant energy transfer (BRET) and bimolecular fluorescent combination (BiFC) techniques we have detected in vivo that A2AR is able to form oligomers made up of more than two protomers, leading to homomeric complexes (Gandía et al., 2008) as well as others of heteromeric nature. In this latter case, we have studied the oligomer of A2AR with the dopamine D2 and glutamate metabotropic 5 receptors (Cabello et al., 2009). Following these experiments, we have applied a modified version of the yeast two-hybrid technique set up for membrane proteins (MYTH) in order to detect A2AR-interacting proteins. Thanks to this approach, we have found new potential interactors, and among them an orphan GPCR has stood out: GPR37. By means of physical and functional techniques in cell culture and animal models we have validated the A2AR/GPR37 interaction and we have demonstrated that the presence of GPR37 modifies the functionality of A2AR. Finally, in order to better understand the rather less studied structural characteristics of GPR37, we have studied its C-terminal tail. Thus, we have observed the presence of a cysteine-rich region that regulates the trafficking of the receptor to the plasma membrane. Furthermore, this cystein-rich domain modulates the GPR37-dependent endoplasmic reticulum stress, as well as the induction of apoptotic pathways (capase-3 activity) (Gandía et al., 2013).

Reversible protein phosphorylation plays a key role in cell signalling during neural development and thus controls cell proliferation, survival, differentiation and function. Kinases and their counter-partners the phosphatases tightly regulate protein phosphorylation. In the developing nervous system the neurotrophin receptor family of protein tyrosine kinases (TrkA, B and C) are major players in this signalling network during normal neuron development and also in several diseases such as neuropathies, degenerative disorders and cancers. Recently, receptor-type protein tyrosine phosphatases (RPTPs) were suggested to be possible regulators of Trks. Thus understanding the relationships between RPTPs and Trks may help to develop new therapeutics to control aberrant neurotrophin signalling in disease. In this study I investigated the relationship between RPTPs and Trks in murine embryonic sensory neurons from dorsal root ganglia (DRGs), a primary cell model. The expression and coexpression of RPTPs and Trks was extensively studied during critical stages of DRG maturation using qPCR arrays at Merck-Serono, Geneva, and fluorescent in-situ hybridization and immunohistochemical techniques. This revealed a relatively high expression of several candidate RPTPs, which were expressed in particular TrkA+, TrkB+ and/or TrkC+ subpopulations of sensory neurons, indicating a potential relationship in their signalling functions. To further analyze a potential direct interaction between candidate RPTPs with Trk proteins, a bimolecular-fluorescent complementation assay (BiFc) was tested. However, this particular assay, when used with type I transmembrane proteins, suffered from high, unspecific protein interactions. In the main experimental approach, a lentiviral-mediated shRNAi-induced knockdown system in primary cell cultures was set-up and the effects of the knockdowns of Ptprf, Ptprs and Ptpro on endogenous Trk gene expression and Trk phosphorylation and activation were analysed. These results suggest a potential role of the encoded proteins LAR and RPTPσ in Trk function and of RPTP-BK in the differentiation and specification of Trk+ neurons.

Thesis (Ph. D.)--University of Kentucky, 2003.
Title from document title page. Document formatted into pages; contains xix, 74p. : ill. Includes abstract. Includes bibliographical references (p. 62-72).

The Class C subfamily of the G protein coupled receptor (GPCR) family is one of the most important in the central nervous system (CNS), and includes the glutamatergic and GABAergic metabotropic receptors (mGlu and GABAB). The mGlu receptors are fundamental in modulating the efficiency of chemical neurotransmission in the CNS and, as a consequence, they are also involved in several neurological and neurodegenerative disorders. Recently, dimerization of these receptors has become an important topic of investigation and it has been proposed to be crucial to physio-pathological processes in the CNS, as well as to the impact of therapeutics in patients. In this thesis, I will discuss the basic properties of Class C GPCRs focusing on the first and second group of mGlu receptors (Group I and II) and their possible dimerization and/or cross talk with other receptors. I will focus on recent findings concerning these processes that have been mainly obtained by using purified isolated nerve endings (here referred to as synaptosomes), a subcellular preparation of choice for studying presynaptic release regulating receptors. Starting from the pharmacological characterization of Group I and II mGlu receptors, I will then discuss different examples of cross-talk linking these receptors to other ones (i.e. the GABAB and the 5HT2A receptors). I will also show results obtained using electrophysiology to study the role of these receptor subtypes in the modulation of synaptic transmission in hippocampal slices. The resulting picture is undoubtedly complex and highlights how the cross-talk and dimerization of these receptors represent a new frontier in neuropharmacological studies.

Os receptores nucleares constituem uma superfamília de fatores de transcrição regulados pela interação com hormônios. Esta superfamília inclui, por exemplo, os receptores de hormônio tireoidiano, estrogênio, androgênio, glicocorticóide e mineralocorticóide. Neste trabalho, empregamos técnicas de biologia estrutura e bioinformática para estudar as interações entre alguns dos membros da família de receptores nucleares e seus respectivos ligantes. Para o receptor de hormônio tireoidiano, foi demonstrado, através da análise das estruturas cristalográficas das duas isoformas do receptor ligados aos tiromimético Triac, que os componentes entálpicos visíveis nas estruturas não explicam a seletividade do ligante. Dados de dinâmica molecular confirmaram que a seletividade do hormônio tem um importante componente entrópico. Empregando a técnica de dinâmica molecular, estudamos a ligação do receptor de mineralocorticóide humano à aldosterona, ao cortisol, à espironolactona e à cortisona e simulamos ainda o efeito da mutação S810L, conhecida por converter a atividade antagonista da cortisona e da espironolactona em agonista. A análise das simulações revelou um perfil de ligações de hidrogênio similar na ligação do receptor selvagem ao cortisol e à aldosterona. A cortisona perde, por conta da inserção de uma hidroxila na posição 11, uma ligação de hidrogênio importante com a Asn770 e, por isso, tem menor energia potencial de ligação. A espironolactona perde a mesma ligação de hidrogênio ao mesmo tempo em que aumenta o número de contatos de van der Waals pela inserção do grupo tioacetil na posição 7. A mutação S810L simulada no complexo com cortisona, cortisol e espironolactona não interfere no padrão de ligações de hidrogênio estabelecidas entre o receptor e os ligantes, mas altera a mobilidade de uma das regiões propostas como rota de dissociação. Propomos, portanto, que a mutação interfere na cinética de dissociação dos ligantes e não no padrão de interações estabelecidas no equilíbrio. Simulações de dissociação induzida do ligante confirmam esta proposição. Na última etapa, utilizamos os modelos experimentalmente determinados para o receptor ativado por proliferadores peroxissomais gama para a busca de novos ligantes através da técnica de docking molecular. Neste trabalho, utilizamos uma base de dados com aproximadamente um milhão de compostos. Destes, quatro foram selecionados após o docking molecular e testados experimentalmente. Um dos compostos testados se mostrou ativo neste receptor, apresentando uma atividade de 60-70% da atividade da rosiglitazona, conhecido agonista total do PPARg.
Nuclear receptors are a superfamily of hormone-regulated transcriptional factors. This superfamily includes, for example, the receptor for thyroid hormone, estrogen, androgen, gluco and mineralocorticoid. In this work, we used structural biology and bioinformatic tools to study the interactions between some members of the nuclear receptor superfamily and its respective ligands. We showed by the analysis of the crystal structures of both thyroid hormone receptor isoforms bound to the thyromimetic Triac that the enthalpic components visible in the structures do not explain the ligand selectivity. Molecular dynamics simulation data confirmed later that the hormone selectivity has an important entropic component. Using the molecular dynamics simulation, we studied, in a second stage, the interaction between the human mineralocorticoid receptor bound to aldosterone, cortisol, spironolactone and cortisone and also simulated the effects of the mutations S810L, known to convert the antagonist properties of spironolactone and cortisone in an agonist activity. The analysis of the simulations showed a similar profile in hydrogen bonds established between the wild type receptor bound to cortisol and aldosterone. Cortisone looses an important hydrogen bond with Asn770 because of the insertion of a carbonyl group in the 11 position and shows a decreased binding potential energy. Spironolactone loses the same interaction but has an increased number of van der Waals contacts because of the insertion of a tioacetyl group in the 7 position. The mutant S810L simulated in complex with cortisol, cortisone and spironolactone showed that the mutation do not interfere with the hydrogen bond profile established between the receptor and the ligands but changes the mobility of a region in the receptor previously proposed as a ligand dissociation route. Ligand unbinding simulations through steered molecular dynamics (SMD) confirm that aldosterone and cortisol unbind differentially and the mutation S810L alters the unbinding profile. We then propose that the mutation changes the kinetics of ligand association/dissociation without changing the profile of the interactions established in the equilibrium. In the last stage, we used the experimentally determined structural model of the peroxissome proliferator-activated receptor gamma to search for novel ligands using the molecular docking technique. For this work, we used a database containing about 1 million compounds. Among those, four compounds were selected after the docking computation and experimentally tested. One of these compounds was found to be active in the receptor, showing about 60-70% of the agonistic activity of rosiglitzone, a known PPARg total agonist.

Thesis (Ph. D.)--University of Florida, 2004.
Typescript. Title from title page of source document. Document formatted into pages; contains 202 pages. Includes Vita. Includes bibliographical references.

Os tumores malignos apresentam um aumento da expressão dos receptores de lipoproteínas, devido ao aceleramento da proliferação celular com consequente aumento da necessidade de lípides para a síntese das membranas celulares. Esse aumento da expressão dos receptores de LDL no câncer pode ser utilizado para concentrar fármacos de ação antineoplásica em tecido tumoral, utilizando lipoproteínas ou nanoemulsões semelhantes a lipoproteínas como veículo. No presente estudo, foram investigados os efeitos da quimioterapia convencional na expressão dos receptores de LDL e LRP-1 em 16 pacientes com carcinoma de mama estádios II ou III, não candidatas à cirurgia conservadora e com indicação de tratamento quimioterápico neoadjuvante. A expressão dos receptores LDLR e LRP-1 foi avaliada por imunoistoquimica em tecido mamário normal e em tecido neoplásico antes e depois da quimioterapia neoadjuvante. Quatro pacientes que apresentaram resposta completa à quimioterapia foram retiradas da análise da expressão de receptores por não existir tumor no fragmento cirúrgico. Em relação ao LDLR, a expressão desse receptor no tecido neoplásico foi maior em comparação ao tecido normal em 8 das 11 pacientes. Após a quimioterapia, a expressão do receptor de LDL diminuiu em 6, aumentou em 4 e não se alterou em 2 pacientes. Do mesmo modo, a expressão do receptor LRP-1 no tecido tumoral estava aumentada em relação ao tecido normal em 4 pacientes das 12 avaliadas. Em comparação com o tecido tumoral antes da quimioterapia, a expressão do receptor LRP-1 diminuiu em 6, aumentou em 4 e permaneceu inalterada em 2 pacientes após a quimioterapia. Esses dados mostram que o efeito da quimioterapia na expressão dos receptores de lipoproteínas foi heterogêneo. A redução da expressão dos receptores não foi o padrão observado, o que indica que o uso de sistemas de carreamento de fármacos via receptores de LDL para o tratamento do câncer pode ser de grande importância. Esses resultados podem contribuir para o desenho de futuros estudos clínicos
Proliferative tumor cells present a high expression of LDL receptors due to accelerated mitosis rates which takes to increased need of lipids internalization for building new membranes. Upregulation of LDL receptors may be used as a gate to deliver anticancer drugs to tumor tissues using lipoproteins or artificial nanoemulsions as vehicle. This study investigated the effects of conventional chemotherapy on the expression of LDL and LRP-1 receptors in 16 patients with breast cancer in stage II or III who were not candidates to conservative surgery and with indication of neo-adjuvant chemotherapy. Expression of LDL and LRP-1 receptor was evaluated by immunohistochemistry in normal and neoplastic breast tissue before and after chemotherapy. For absence of tumor in the surgical fragments, 4 patients who presented complete response to chemotherapy were excluded from this analysis. In relation of LDLR, the expression in neoplastic tissue was higher than in normal tissue in 8 of 11 patients. After chemotherapy, LDL receptor expression diminished in 6, increased in 4 and unchanged in 2 patients. Expression of LRP-1 in tumor tissue was higher in 4 of 12 patients when compared to normal tissue. After chemotherapy, the expression of LRP-1 diminished in 6, increased in 4 and showed no difference in 2 patients. These data show that the chemotherapy effects on the tumor expression of LDL receptors were very heterogeneous. The diminution of the receptor expression is not the post-chemotherapy pattern, allowing the use of drug carrier systems that target cancer cells via the LDL receptor pathway. These results may contribute for the design of future clinical assays

Orientador: Aparecida Machado de Moraes
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: A estria atrófica cutânea ou striae distensae (SD) é uma afecção muito comum, sendo causa freqüente de procura por consultas dermatológicas. Ainda que não representem qualquer risco à saúde física, produzem impacto emocional e induzem busca por tratamentos trabalhosos, caros e dolorosos e, com freqüência, inadequados. Além disso, o surgimento de striae distensae pode refletir alteração do tecido conjuntivo e indicar condições patológicas locais e sistêmicas. Alguns autores descrevem as estrias cutâneas como uma condição de estiramento ou distensão da pele, com perda ou ruptura das fibras elásticas na região acometida. Entretanto, vários autores observam que as estrias não surgem com frequência sobre a pele acima de tumores abdominais, ascites, hemorragias extensas ou grandes hérnias. Atualmente admite-se que sua etiopatogenia é multifatorial, englobando aspectos mecânicos, bioquímicos e genéticos. No entanto, considerando-se a multiplicidade de fatores envolvidos, a literatura é divergente e inconclusiva. Portanto, através do estudo de fatores clínicos associados às SD e dos receptores hormonais (estrógeno, andrógeno e glicocorticóide), pretendeu-se entender melhor a participação dos hormônios na fisiopatogênese das estrias. Para o estudo clínico foram selecionados pacientes com queixa de estrias cutâneas e a comparação foi feita com grupo controle de número semelhante, atendido aleatoriamente por outras queixas no Ambulatório Geral de Dermatologia do HC, FCM/UNICAMP. O estudo da expressão dos receptores hormonais foi realizado por Western blot em oito amostras de pele de estrias recentes, com menos de um ano de evolução, comparando com a avaliação de pele sem lesão de região palpebral de oito pacientes que se submenteram a blefaroplastia. Observou-se que fatores como adolescência, gestação e obesidade estão significativamente relacionados ao surgimento das SD. Constatou-se ainda que a idade materna jovem e o ganho ponderal durante a gestação são importantes fatores associados ao desenvolvimento das lesões. Além disso, a localização das lesões correlaciona-se ao fator causador das estrias. Em relação ao estudo dos receptores hormonais, observou-se que na SD recentes há duas vezes mais expressão de receptores de estrógeno e 1,7 vezes mais expressão receptores de andrógeno e glicocorticóide. Alguns autores interpretam SD como cicatrizes. Após influência hormonal, haveria uma reação inflamatória inicial que determinaria a destruição de fibras elásticas e colágenas. O processo seria seguido de regeneração das fibras, um fenômeno de remodelação dinâmica,ou seja, um balanço entre síntese de colágeno e sua quebra, o qual reestrutura o tecido para acomodar as forças que agem sobre ele, resultando na formação das SD. O balanço entre a expressão de receptores de estrógeno, andrógeno e glicocorticóide poderia induzir as modificações específicas da matriz extracelular, o que levaria a esse fenômeno de remodelação. Em concordância com outros estudos, observamos que as estrias surgem em situação de grande modificação sistêmica, como adolescência e gestação. Através das observações morfológicas e moleculares, nota-se que as SD estão correlacionadas com intensas alterações do tecido conectivo. Os resultados mostram-se relevantes e representam mais um passo na compreensão do mecanismo fisiopatológico da estrias cutâneas e abrem espaço para novas linhas de pesquisa, relacionadas às SD e a outras alterações do tecido conectivo.
Abstract: Stretch marks or striae distensae (SD) are a very common condition which often results in persons searching dermatological treatment. While not presenting any risk to one's physical health, the emotional impact often induces those so affected to demand medical treatments, which are often laborious, expensive and painful, and frequently ineffective. Beyond this, the appearance of SD may indicate other alterations in conjunctive tissues, including both local and systemic pathologies. Some authors described cutaneous strias as a condition of stretching or distension of the skin which results in the loss or rupture of elastic fibers in the affected areas. However, many authors have also observed that SD do not occur frequently associated with abdominal tumors, ascites, extensive hemorrhages or large hernias. Current medical opinion is that its etiopathogenesis is multifactored, englobing mechanical, biochemical and genetic aspects. Nevertheless, in view of the multiplicity of the factors involved, the literature is divergent and inconclusive. This study was proposed to look for better understanding of the role played by hormones in the physiopathology of SD studying clinical factors associated with SD and hormone receptors (estrogen, androgen and glycocorticoid). Patients complaining of SD were selected for inclusion in this clinical study, and comparisons were made with a control group of a similar number of persons who had been treated for other medical conditions at the General Ambulatory Dermatology Care Facility at HC, FCM/UNICAMP. The expression of hormone receptors was undertaken of the Western Blot testing of recent skin striations, in comparison with lesion-free skin taken from the eyelids of patients who had undergone blepharoplasty. The study revealed that factors such as adolescence, pregnancy and obesity are significantly related to the appearance of SD. It was further established that age (the younger the gravid, the greater the possibility of SD) and significant weight gain during pregnancy are important factors associated with the development of SD lesions. In addition, there was a positive correlation with the location of the lesions. In relation to the study of hormone receptors, it was observed that recently-formed SD have two times more expression of estrogen receptors, and 1,7 times more expression of androgen and glycocorticoid receptors. Some authors classify SD as scars. Under hormonal influence, there is an initial inflammatory reaction which results in the destruction of elastic fibers and collagens. This is followed by the regeneration of the destroyed fibers, a phenomenon of dynamic remodeling, a balance between the synthesis and destruction of collagens, which restructures the tissue in order to accommodate the forces acting upon it, resulting in the formation of SD. The balance between the expression of estrogen, androgen and glycocorticoid may elicit the specific modifications of the extracellular matrix which leads to the phenomenon of remodalation. In agreement with other studies, we observed that the strias arise under conditions of significant system modification, such as adolescence and pregnancy. The observation of morphological and molecular changes showed that there is a correlation between SD and with intense alterations in connective tissue. The results of this study are relevant and represent an important step in the understanding of physiopathological mechanisms in stretch marks, opening new horizons for new directions in research of SD and other alterations in connective tissues as well.
Doutorado
Clinica Medica
Doutor em Clínica Médica

Dopamine receptors belong to the G protein-coupled receptor (GPCR) superfamily and are classified into two families: D1-like (D1R and D5R) and D2-like (D2R, D3R and D4R), based on their ability to stimulate or inhibit adenylyl cyclase (AC). Classically, GPCRs (including D2R and D3R) are desensitized by the activation of the serine/threonine protein kinase C (PKC) upon phorbol-12-myristate-13-acetate (PMA) treatment. Previous studies demonstrate that while human D5R (hD5R) is also strongly desensitized upon PMA treatment, the human D1R (hD1R) undergo a robust PMA-induced sensitization. The aim of this PhD thesis was to explore how the canonical PKC- or phorbol ester-linked pathway can control the responsiveness of two similar GPCRs like hD1R and hD5R in an opposite fashion. Our data indicate that hD1R sensitization and hD5R desensitization are not mediated by a direct modulation of AC activity by PKC. Using a chimeric approach, we identified the third intracellular loop (IL3) as the key structural determinant controlling in an opposite manner the PMA-mediated regulation of hD1R and hD5R. To delineate the potential PKC phosphorylation sites, a series of mutation of serine (Ser) and threonine (Thr) located into IL3 of hD1R and hD5R were used. No hD1R mutation decreased the PMA-mediated sensitization. This suggests that hD1R phosphorylation is not required for PMA-induced sensitization. In contrast, our results indicate that PMA-mediated hD5R desensitization occurs through a hierarchical phosphorylation of Ser260, Ser261, Ser271 and Ser274. Notably, these hD5R mutants exhibited a PMA-induced sensitization, reminiscent of the PMA-induced hD1R sensitization. Additionally, using short hairpin RNAs (shRNAs), we showed that PKCε is the potentiating PKC while the desensitizing isoform is δ. Overall, our work suggests the presence or absence of specific Ser residues on IL3 of hD1-like receptors dictate if phosphorylation-dependent desensitization (through PKCδ) or phosphorylation-independent potentiation (via PKCε) will occur.

Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2002.
On t.p. "A̳" is subsript. Includes bibliographical references (leaves 160-177). Also available in electronic version. Access restricted to campus users.

Erythropietin (Epo), acting through its receptor (EpoR), is an essential hemotopietic growth factor that regulates the proliferation, differentiation, and survival of erythroid progenitor cells. Perturbations of Epo/EpoR function cause myeloproliferative disease, such as erythrocytosis, or myelodeficient disease, such as anemia. Therefore, defining the mechanisms by which Epo/EpoR control proliferation and differentiation of erythroid cell lineages attracts interest. Ubiquitin-dependent internalization and degradation is a common regulatory mechanisms affecting signaling from a variety of receptors. Although EpoR has been found to be ubiquitinated, the function of EpoR ubiquitination in the regulation of Epo signaling remains unclear. Therefore, the primary goal of this study was to define the role of EpoR ubiquitination in regulating Epo signaling activities and erythroid cell growth. We showed that EpoR was ubiquitinated in response to ligand stimulation, and that loss of EpoR ubiquitination reduced signaling activity and biological responses to low dosages of Epo. We also identified two EpoR lysines that were the primary targets for ubiquitination, and showed that either ubiquitination site supported the enhanced activities of wild-type-EpoR. Ubiquitination of EpoR was also associated with a change in the endocytic pathway mediating internalization of EpoR. Specifically, constitutive internalization of non-ubiquitinated EpoR was found to depend on dynamin activity, while internalization of ubiquitinated EpoR was dynamin-independent but could be inhibited by disrupting lipid raft microdomains in the plasma membrane. Interestingly, inhibiting internalization of ubiquitinated EpoR (by disrupting lipid rafts) specifically reduced signaling from ubiquitinated receptors without affecting signaling from non-ubiquitinated receptors. Conversely, reducing internalization of non-ubiquitinated EpoR (by inhibiting dynamin) reduced its signaling activity without affecting signaling from ubiquitinated receptors. This strong correlation between EpoR internalization and signaling activity suggests a novel regulatory mechanism in which internalization of EpoR facilitates its signaling activity. In this regard, Epo-induced ubiquitination of EpoR promotes more efficient internalization of ligand-activated receptor and may contribute to enhanced responsiveness to low concentrations of Epo.

Una de las principales cuestiones en farmacología molecular de los GPCR es entender los mecanismos estructurales de las siete hélices transmembrana (TM) que se producen para estabilizar ya sea Rg o los diferentes estados R*. Para entender el mecanismo que cambia el equilibrio del conjunto a un estado activo R* se construyeron tres de los receptores de la serotonina (5-HT4, 5-HT6, y 5 HT7) sobre la base de su información más reciente de cristalografía de rayos X. Dando lugar a dos modelos de cada receptor: una inactiva y otra activa. Los modelos, mejorados y evaluados con la ayuda de datos farmacológicos y químicos se utilizaron principalmente para comprender la interacción entre un ligando y su receptor y su mecanismo de acción. Estos hallazgos estructurales pueden a su vez resultar útiles para el diseño de nuevos fármacos más eficaces y selectivos.
One of the main questions in G protein coupled receptors (GPCRs) molecular pharmacology is to understand the structural arrangements of the seven transmembrane (TM) helices that occur to stabilize either the ground state (Rg) or different active states (R*) of the receptors. In order to understand the mechanism that shift the equilibrium of the ensemble to an active R* state models of the inactive and the active state of three serotonin receptors (5-HT4, 5-HT6, and 5-HT7) were built based on the latest information from X-ray crystallography. The resulting models were mainly used to understand the interaction between a ligand and its receptor and the mechanism of action. With the help of pharmacological and chemical data these models and complexes were improved and evaluated. These findings may prove valuable for structural based drug discovery efforts and facilitate the design of more effective and selective pharmaceuticals.

Orientadores: Luis Otávio Zanatta Sarian, Sophie Françoise Mauricette Derchain
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: Introdução: o câncer de ovário é comumente diagnosticado em estágios avançados, sendo atualmente a neoplasia ginecológica de maior letalidade. As pesquisas têm direcionado esforços na tentativa de descobrir novos fatores prognósticos e métodos terapêuticos. Muitos trabalhos estudam a expressão dos receptores de esteróides (dentre eles, estrógeno (RE), progesterona (RP) e andrógeno (RA)) e HER2 (receptor estimulador de crescimento epitelial - subtipo 2) como fatores prognósticos e associados à resposta terapêutica, apresentando; entretanto, resultados conflitantes. Até onde conhecemos, não há estudos desta natureza no Brasil. Objetivo: Correlacionar à expressão dos RE, RP, RA e HER2 aos fatores clínico patológicos, ao intervalo livre de doença e à sobrevida nos cânceres epiteliais de ovário. Material e métodos: Este é um estudo observacional de coorte retrospectiva. Foram incluídas 152 mulheres com tumores epiteliais malignos, selecionados através dos prontuários no período de 1993 a 2008, no Centro de Atenção Integral à Saúde da Mulher (CAISM) da Universidade Estadual de Campinas - UNICAMP, São Paulo, Brasil. A avaliação da expressão dos RE [subtipos alfa (RE?) e beta (RE?)], RP, RA e HER2 foram realizadas por imunoistoquímica através da construção de microarranjos de tecidos (TMA). Inicialmente, foi realizada análise uni variada da expressão dos receptores acima quanto à idade, estado menopausal, índice de massa corpórea (IMC), tipo histológico, grau histológico, estadiamento inicial de acordo com a classificação proposta pela FIGO e presença de doença residual pós-tratamento cirúrgico. Em seguida, as pacientes foram divididas em dois grupos: ausência da expressão de RE?, RP e HER2 (triplo negativo) e positividade para pelo menos um desses receptores (não triplo negativo); e, avaliadas em relação aos critérios clínicos e epidemiológicos acima. Foram, então, realizadas análises multivariadas dos padrões de expressão de RE ? e ?, RP, RA, HER2 e triplo negativo em relação a estes mesmos critérios. Por fim, análise de sobrevida multivariada foi realizada utilizando-se todos os padrões de expressão e os fatores clínicos epidemiológicos estudados. Resultados: Nas análises multivariadas, mostraram-se significativas as seguintes associações: do receptor de estrógeno alfa (ER?) com tumores de grau histológico menos diferenciado (p=0.01); do receptor de progesterona (RP) à obesidade (p= 0.01); do receptor de andrógeno (RA) com tumores do subtipo seroso (p= 0.01); do receptor de HER2 com tumores dos graus histológicos II-III (p=0.02); do subgrupo triplo negativo com tumores de grau histológico menos diferenciado (II-III) (p<0.01). Não houve associação do RE? com nenhum dos fatores estudados. Quanto à análise multivariada de sobrevida livre de doença e sobrevida global; dentre os padrões de expressão de receptores, apenas o RE? esteve associado com melhor sobrevida livre de doença (RR=0.39; 95%CI 0.17 -0.90). Conclusões: A expressão do RE? esteve mais significativamente associada a fatores clínicos de pior prognóstico. O RP esteve associado à obesidade. O RA esteve significativamente mais presente nos tumores serosos. A expressão do HER2 e a presença de tumores triplo negativo foram maiores em tumores menos diferenciados. Paradoxalmente; o RE? foi o único receptor a apresentar associação com maior sobrevida livre de doença apesar de sua relação significativa com fatores reconhecidos de pior evolução clínica. Não houve diferença estatística significativa na análise multivariada de sobrevida total e sobrevida livre de doença quanto ao grupo de tumores triplo negativo
Abstract: Introduction: Ovarian cancer is commonly diagnosed in advanced stages and currently is the most lethal gynecological malignancy. Surveys have focused efforts in an attempt to discover new prognostic and therapeutic methods. A plenty of studies investigates the expression of steroid receptors (among them, estrogen (ER), progesterone (PR) and androgen AR)) and HER2 (epidermal growth receptor stimulator - subtype 2) as prognostic factors and associated to therapeutic response, presenting, however, conflicting results. As far as we know, there are no studies of this nature in Brazil. Objective: The aim of this study was to correlate the expression of ER (subtypes ? (ER ?) and ? (ER ?), PR, AR and HER2 to clinical pathological factors, the disease-free survival and overall survival of epithelial ovarian cancers. Methods: This is a retrospective observational cohort study. The study included 152 women with malignant epithelial tumors, selected through the records from 1993 to 2008, in the Centro de Atenção Integral à Saúde da Mulher (CAISM) at the State University of Campinas - UNICAMP, São Paulo, Brazil. The expression of ER (? and ?), PR, AR and HER2 was evaluated by immuno histochemistry through tissue microarray (TMA) technique. Initially, univariate analyses were performed, evaluating the expression of each receptor mentioned above to age, menopausal status, body mass index (BMI), histological type, histological grade, initial staging as preconized by FIGO staging of ovarian tumors and presence of residual disease after surgical treatment. Then, the patients were divided into two groups: absence of the expression of ER?, PR and HER2 (triple negative) and positive for at least one of these receptors (not triple negative), and evaluated in relation to the clinical and epidemiological criteria mentioned above. Multivariate analyzes were performed with ER ?, ER?, PR, AR, HER2 and triple negative towards these same criteria. At last, multivariate survival analyses were conducted using all the patterns of receptors' expression, epidemiological and clinical factors studied. Results: In multivariate analyzes, there were the following significant associations: of the estrogen receptor alpha (ER?) with less differentiated histological grade tumors (p = 0.01); of the progesterone receptor (PR) to obesity (p = 0.01); of the androgen receptor (AR) with the serous tumors (p = 0.01); of the HER2 receptor with tumors of histological grades II-III ( p = 0.02); of the triple negative subgroup with less differentiated histological grade tumors (II-III) (p <0.01). There was no association of the ER? with any of the factors studied. In the multivariate analysis of disease-free survival and overall survival; considering the patterns of receptors' expression, only the ER? expression was associated with better disease-free survival (RR= 0.39, 95% CI 0.17 to 0.90). Conclusions: The expression of ER? was more significantly associated with clinical factors of poor prognosis. The PR was associated with obesity. The AR was significantly more prevalent in serous tumors. The HER2 expression and the presence of triple negative epithelial ovarian cancer tumors were higher in less differentiated tumors. Paradoxically, the ER? was the only receptor which showed association with better disease-free survival despite its significant relationship with recognized factors of worse clinical outcome. There was no statistically significant difference in multivariate analysis of overall survival and disease-free survival regarding to the triple negative group
Mestrado
Oncologia Ginecológica e Mamária
Mestra em Ciências da Saúde

The chemokine receptor CXCR4 contributes to tumour cell migration and invasion during the progression of prostate cancer. In particular, this pathway is central to the metastasis of prostate cancer to the bone marrow. Limited therapeutic options exist for prostate cancer patients who have progressed to advanced metastatic disease, and pharmacological interference of the chemokine network may serve to control tumour cell dissemination and the establishment of metastasis. A more detailed knowledge of the mechanisms regulating chemokine receptors is required, in order to further characterise and explore the capacity and effectiveness of targeting these pathways for therapeutic intervention in prostate cancer. Here, the regulation of CXCR4 protein expression and function was investigated in relation to androgens and the extracellular matrix. Accumulating evidence of CXCR4 regulation by androgens and the androgen receptor have indicated that androgens not only promote the growth and development of prostate cancer, but may actively contribute to the metastatic progression of prostate through modulation of the chemokine network. In the current study, the endogenous protein expression and functionality of the androgen receptor were firstly characterised in the androgen-insensitive prostate cancer cell lines DU145 and PC3, using the androgen-sensitive LNCaP cells as a basis for comparison. Investigations were performed using two-dimensional culture in conjunction with the more physiologically relevant three-dimensional in vitro culture model.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Eskitis Institute for Cell and Molecular Therapies
Science, Environment, Engineering and Technology
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Chemokines triggered by Toll-like receptors (TLRs) are small chemoattractant proteins, which mainly regulate leukocyte trafficking in inflammatory reactions via interaction with G protein-coupled receptors. Forty-two chemokines and 19 cognate receptors have been found in the human genome. Prior to this study, only 11 chicken chemokines and 7 receptors had been reported. The objectives of this study were to identify systematically chicken chemokines and their cognate receptor genes in the chicken genome and to annotate these genes and ligand-receptor binding by a comparative genomics approach. Twenty-three chemokine and 14 chemokine receptor genes were identified in the chicken genome. The number of coding exons in these genes and the syntenies are highly conserved between human, mouse, and chicken although the amino acid sequence homologies are generally low between mammalian and chicken chemokines. Chicken genes were named with the systematic nomenclature used in humans and mice based on phylogeny, synteny, and sequence homology. The independent nomenclature of chicken chemokines and chemokine receptors suggests that the chicken may have ligand-receptor pairings similar to mammals. The TLR family represents evolutionarily conserved components of the patternrecognizing receptors (PRRs) of the innate immune system that recognize specific pathogen-associated molecular patterns (PAMPs) through their ectodomains (ECDs). TLR's ECDs contain 19 to 25 tandem copies of leucine-rich repeat (LRR) motifs. TLRs play important roles in the activation of pro-inflammatory cytokines, chemokines and modulation of antigen-specific adaptive immune responses. To date, nine TLRs have been reported in chicken, along with a non-functional TLR8. Two non-mammalian TLRs, TLR21 and TLR22, have been identified in pufferfish and zebrafish. The objectives of this study were to determine if there is the existence of chicken genes homologous to fish-specific TLRs, and if possible ligands of these receptors exist. After searching the chicken genome sequence and EST database, a novel chicken TLR homologous to fish TLR21 was identified. Phylogenetic analysis indicated that the identified chicken TLR is the orthologue of TLR21 in fish. Bioinformatic analysis of potential PAMP binding sites within LRR insertions showed that CpG DNA is the putative ligand of this receptor.

El receptor Neuroquinin-1 (NK1R) és un GPCR que es troba en el sistema nerviós central i perifèric dels vertebrats i és responsable dels processos fisiològics com la transmissió de dolor, secreció endocrina i exocrina, vasodilatació, modulació de la proliferació cel·lular i molts altres. Els antagonistes del NK1R poden ser potencials analgèsics i antidepressius i també poden ser utilitzats per al tractament del trastorn bipolar, l'alcoholisme, càncer, malalties del sistema immune i algunes infeccions. Les tècniques espectroscòpiques i les estructurals d'alta resolució com NMR i la cristal·lografia requereixen quantitats de l'ordre de mil·ligrams del receptor actiu purificat. Una de les estratègies que permet produir els GPCRs recombinants per estudis estructurals és el sistema d'expressió en E.coli. No obstant, molts GPCRs degut al seu efecte tòxic per a la cèl·lula bacteriana s'expressen en forma dels cossos d'inclusió i han de ser sotmesos al procés de renaturalització. La renaturalització dels GPCRs és una tasca complexa que implica complexos ajustaments dels tampons. La primera part d'aquest treball s'ha centrat en la renaturalització de las formes truncades hNK1R-366 i hNK1R-311 del receptor expressades en cossos d'inclusió d'E.coli. Per a l'obtenció del receptor ben plegat hem establert un original protocol de la renaturalització en columna. Hem utilitzat diferents tècniques espectroscòpiques per a estudiar el receptor renaturalitzat. Els resultats de CD han demostrat que el hNK1R-366 i el hNK1R-311 renaturalitzats en DDM presenten un percentatge d'hèlix-α similar al de la rodopsina extreta de retines bovines i solubilitzada en DDM. En els estudis de fluorescència intrínseca de triptòfans a baixes concentracions del GuHCl hem pogut observar el desplaçament cap el blau en l'espectre d'emissió, típic de triptòfans que es troben en un entorn hidrofòbic. A més a més, els espectres d'emissió del hNK1R-366 expressat en cèl·lules COS-1 i solubilitzat en DDM presenten el màxim d'emissió a 335 nm, molt similar al del receptor renaturalitzat a partir dels cossos d'inclusió, indicant la seva correcte renaturalització. El hNK1R-366, renaturalitzat en tampó fisiològic presenta agregació en 24 hores. No obstant, la presència de 0.05% DDM és capaç d'estabilitzar el receptor. Els assajos de radioligand binding de saturació del hNK1R-366 renaturalitzat indiquen que el receptor actiu constitueix l’1% de la proteïna total de la mostra. No obstant, la unió de la SP al receptor en el rang de nanomols és significatiu i és un resultat important, donat que, per primera vegada s’ha obtingut NK1 funcional a partir de E.coli. Per altre costat, no hem pogut obtenir una corba de saturació del hNK1R-311, degut possiblement al plegament defectuós del receptor per falta dels darrers 96 residus. La segona part d'aquest estudi està centrada en l'expressió, purificació i caracterització estructural del C-terminal del receptor hNK1. El domini C-terminal és important per l'acoblament de la proteïna G i la β-arrestina, i també és essencial per a la dessensibilització, internalització i reciclatge del receptor. No obstant, el paper d'aquest domini ha estat infravalorat pels investigadors durant molt temps i existeix poca informació sobre la seva estructura. Els estudis espectroscòpics de UV i de fluorescència posen de manifest anomalies en l'absorbància a 292 nm i en l'emissió intrínseca de les tirosines a 345 nm, atribuïdes a formes ionitzades de l’aminoàcid, degut a la seva proximitat a grups carboxil de residus glutàmics o aspàrtics. A partir de la predicció de l'estructura secundària i terciària i dels resultats dels estudis espectroscòpics hem proposat un model tridimensional pel C-terminal del hNK1 que conté: 25% d’hèlix-α, 27% d’estructura desordenada i 48% de fulles-β i girs-β. En conjunt, els resultat obtinguts, indiquen que el C-terminal del hNK1R no és un domini desordenat, sinó que té una estructura secundària i terciària clarament definides que poden relacionar amb les seves funcions.
Neurokinin-1 receptor (NK1R) is a GPCR found in the central and peripheral nervous system of vertebrates, responsible for such physiological processes as pain transmission, exocrine and endocrine secretion, vasodilatation, modulation of cell proliferation and many others. NK1R antagonists could be potential analgesics and anti-depressants and may also be used for treatment of bipolar disorder, alcoholism, cancer, immune system diseases and selected infections. Spectroscopic studies and high resolution structural techniques, as NMR and crystallography, require milligram amounts of active purified receptor. One of the strategies to produce recombinant GPCRs for structural studies is an E.coli expression system. However, many GPCRs due to their toxic effect for bacterial host cell are expressed in form of inclusion bodies and require refolding. The refolding of GPCRs is a complicated task that requires screening and adjustment of buffer conditions. The first part of this work was centered on the refolding of hNK1R-366 and hNK1R-311 truncated forms expressed in E.coli inclusion bodies. To obtain properly folded receptor, we established an original on-column refolding protocol. Different spectroscopic techniques were applied to study the refolded receptor. The results obtained from CD measurements showed that hNK1R-366 refolded in DDM presents similar α-helical content as rhodopsin extracted from bovine retinas and solubilized in non-denaturing DDM micelles. In the intrinsic tryptophan fluorescence studies, at low concentrations of GuHCl we observed a blue-shift in the emission spectrum peak, typical for tryptophan in hydrophobic environment. Furthermore, the emission spectra of hNK1R-366 expressed in COS-1 cells and solubilized in DDM micelles show very similar emission maximum around 335 nm to that of the receptor refolded from the inclusion bodies, which may be indicative of proper protein refolding. The refolded in physiological buffer hNK1R-366 was prone to aggregate in about 24 hours, however, the presence of 0.05% DDM was found to stabilize the receptor. Saturation radioligand-binding assays for the refolded hNK1R-366 showed that the amount of the active receptor is about 1% of the total protein the sample. However, the binding of SP to the refolded hNK1R-366 in nanomolar range is significant and can be considered as a promising result, since until now the intents to produce any detectable amounts of functional NK1 receptor in E. coli were unsuccessful. On the other hand, we were unable to get any saturation binding curve for hNK1R-311 truncated form of the receptor, which could be explained by incorrect folding caused by the lack of 96 residues of the C-terminus of the receptor. The second part of the present study is centered on hNK1R C-terminus expression, purification and characterization to elucidate its structural properties. The C-terminus domain seems to be essential for the coupling to corresponding G protein and β-arrestin, and is essential for receptor desensitization, internalization and recycling. However, the role of this domain was underestimated by researchers for a long time and as a result very little information is known about its structure. UV and fluorescence spectroscopic studies revealed abnormal tyrosine red-shifted absorbance band at 292 nm and intrinsic tyrosine emission at 345 nm which could be attributed to ionized form of tyrosine and possibly arises from the proximity of one or more tyrosines to carboxyl groups of glutamic o aspartic residues. Based on secondary and tertiary structure prediction as well as on the results of spectroscopic studies we propose a 3D-model for hNK1R C-terminus. Th following assignment of the secondary structure was made: 25% α-helix, 27% unordered structure, 48% β-sheets and β-turns. The obtained ressults give evidence that hNK1R C-terminus is not an unordered region but has clearly defined secondary and tertiary structures which certainly are tightly related to its multiple functions.

GABAA receptors mediate the majority of fast synaptic inhibition in the adult mammalian brain, and play a critical role in controlling neuronal excitability during development. The functional properties and stability of cell-surface GABAA receptors are key determinants of the efficacy of GABAergic neurotransmission. I therefore employed a broad spectrum of biochemical, and cell and molecular biological techniques to identify molecular interactions that may be involved in regulating the activity and number of GABAA receptors at the neuronal surface. In this thesis, I identified a novel interaction between the GABAA receptor and the calcium/calmodulin-dependent protein kinase II (CaMKII). Affinity-purification assays revealed that CaMKII forms a native complex with the GABAA receptor in brain, and that the kinase binds non-selectively to the major intracellular domain (ICD) of various GABAA receptor subunits. The interaction between CaMKII and the receptor β subunits was found to be dependent upon phosphorylation of the kinase at T286, but appeared to be independent of subunit phosphorylation. Furthermore, a CaMKII binding site was identified in the ICD of the receptor β subunits, between residues 304 and 323. Further work demonstrated that CaMKII selectively phosphorylates the ICDs of the receptor β and γ subunits, and identified protein phosphatases that dephosphorylate the receptor β3 subunit at the CaMKII sites. Depolarisation of cultured immature cortical neurons was found to increase the level of enzymatically-active CaMKII that associates with the ICD of the receptor β3 subunit, and to trigger CaMKII-dependent phosphorylation of the receptor β3 subunit at both the S408 and S409 residues. Work presented in this thesis also identified multivalent interactions between GABAA receptor y subunits and the AP2 adaptor complex, a major component of the endocytic machinery. The ICD of each receptor y subunit was found to interact with the native AP2 complex from brain, and to bind selectively and directly to the u2- adaptin of AP2. Two distinct binding sites for jx2-adaptin were identified in the ICD of the y2S subunit. The N-terminal half of the ICD was found to interact with sub- domain B of u2-adaptin via a putative basic-rich motif. In contrast, the C-terminal half of the ICD was found to bind sub-domain A of u2-adaptin, likely via a tyrosine- based motif. Both u2-adaptin binding motifs were found to be common to all the y subunit isoforms.

GPCRs are rapidly phosphorylated in response to stimulation and this regulates receptor function and signalling. This process is widely considered to be mediated by the GRK family of protein kinases. In the current thesis we extend this paradigm by examining the role of the casein kinases in the phosphorylation of the M3-muscarinic receptor. Investigation of a phosphorylation deficient mutant of the M 3-receptor (mutant 6), where 17 putative serine phospho-acceptor sites had been substituted, demonstrated that receptor internalisation and efficient coupling to the phospholipase C pathway are dependent on receptor phosphorylation. However, this receptor mutant coupled normally to the ERK and JNK kinase pathway and mediated calcium entry similar to the wild type receptor. The importance of the specific sites of phosphorylation in driving defined receptor processes was illustrated by the use of another phosphorylation deficient mutant of the M3-receptor where amino acids between Lys 370-Ser425 inclusive were removed. Specific reduction of CK1alpha and CK2 protein expression levels with siRNA resulted in decreased agonist-induced receptor phosphorylation and had no effect on receptor internalisation, indicating that phosphorylation by kinase/s other than the casein kinases (probably the GRKs) are required for internalisation. Following inhibition of CK2-mediated receptor phosphorylation, receptor stimulated Ins(1,4,5)P3 production and ERK activation occurred as normal. In contrast, siRNAs against CK1alpha reduced the ability of the receptor to stimulate Ins(1,4,5)P3 production whereas coupling to the ERK pathway was unaffected. The data presented here support the hypothesis that phosphorylation at specific sites is mediated by a range of receptor kinases that include CK1alpha, CK2 and the GRKs. It appears that site specific phosphorylation by particular kinases regulates defined receptor processes.

The chemokine receptor CCR5 (CCR5) plays an integral role within the inflammatory network of cells. Importantly, CCR5 is a mediator in several disease states and can be targeted using small molecule antagonists. Within this work, CCR5’s role in prostate cancer and HIV/AIDS has been exploited in order to develop potential therapeutics and probes. First, a series of novel compounds was designed by using pharmacophore-based drug design based upon known CCR5 antagonists and molecular modeling studies of the CCR5 receptor’s three-dimensional conformation. Once synthesized, these compounds were tested for their CCR5 antagonism and their anti-proliferative effects in several prostate cancer cell lines. The data from both the calcium mobilization studies and the anti-proliferation studies suggests that the compounds synthesized have activity as CCR5 antagonists and as anti-proliferative agents in certain prostate cancer cell lines. In addition, a bivalent ligand containing both a mu opioid receptor (MOR) and a CCR5 antagonist pharmacophore was designed and synthesized in order to study the pharmacological profile of the putative CCR5-MOR heterodimer and its relation with NeuroAIDS. The structural-activity relationship between the bivalent ligand and the heterodimer was studied with radio-ligand binding assays, functional assays, HIV-1 fusion assays, cell fusion assays, and in silico molecular dynamics. The subsequent bivalent ligand was proven to be a potent inhibitor in both an artificial cell fusion assay mimicking HIV invasion and a native HIV-1 invasion assay using live virus. In all, two novel sets of compounds were synthesized that targeted either CCR5 or the CCR5-MOR heterodimer. For the CCR5 antagonists, as leads for prostate cancer therapeutics, further work needs to be done to ascertain and develop their structure-activity-relationship. This library of novel compounds was shown as promising leads as CCR5 and anti-prostate cancer agents. The bivalent ligand targeting the CCR5-MOR heterodimer proved to be a potent and tissue-specific inhibitor for neuroAIDS where the known treatment, maraviroc, is less efficacious and fails to inhibit virus entry in the presence of morphine. Both projects illustrate the roles that CCR5 plays in these two unique diseases.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação Oswaldo Ramos
Alguns estudos identificaram uma associação entre nefrolitíase e os polimorfismos do Receptor de Vitamina D (VDR) ou do Receptor Sensor de Cálcio (CaSR). O objetivo deste estudo foi avaliar uma possível associação destes polimorfismos com a excreção de cálcio urinário (CaU) em pacientes litiásicos. O polimorfismo do VDR, detectado através da digestão com a enzima de restrição BsmI, e três polimorfismos do CaSR (G/T no codon 986, G/A no codon 990 e C/G no codon 1011), detectados através de sequenciamento direto, foram avaliados em pacientes litiásicos, sendo 100 hipercalciúricos (HCa) e 101 normocalciúricos (NCa). A frequência alélica do polimorfismo do VDR na amostra total foi: 16% BB, 49% Bb e 35% bb. A prevalência do genótipo bb foi significantemente maior nos pacientes HCa quando comparados ao grupo NCa (43 versus 27%). Em relação aos polimorfismos do CaSR, as três variantes alélicas (986S, 990G e 1011E) foram detectadas respectivamente em 5, 4 e 3% da amostra total e cinco haplótipos para os polimorfismos do CaSR foram identificados: 94% ARQ (selvagem), 3% SRQ, 1,5% AGQ, 1,0% ARE e 0,5% AGE. Nenhuma diferença estatística foi observada entre os pacientes NCa e HCa em relação à distribuição destes haplótipos do CaSR. Estes achados sugerem uma associação entre a excreção urinária de cálcio com o polimorfismo BsmI do VDR mas não com os três polimorfismos do CaSR em pacientes litiásicos.
TEDE
BV UNIFESP: Teses e dissertações

Breast cancer is a great source of morbidity and mortality in the developed world, being the most common cause of cancer death in Australian women and affecting roughly 1 in 10 women. The development and progression of cancers is a multi-stage process, involving numerous perturbations to normal cellular functions, especially to those genes which control cellular growth, cellular differentiation and DNA repair. Over time, these alterations combine to change normal cells into cancerous ones that typically no longer respond to normal control stimuli and grow with great rapidity. The nuclear receptors are a family of proteins which accept incoming signals from various molecules, and then alter gene expression or affect cell behavior directly. The steroid nuclear receptors receive transducer signals from hormones such as estrogen and testosterone and are intimately involved in affecting cellular growth and differentiation. Stimulation of the steroid receptors have been used as successful treatment avenues, so how these genes behave in cancer is of great interest. Accordingly, this study has examined the expression of various nuclear receptors and some related genes in a number of tissue samples derived from breast tumours and from surrounding areas, and examined how various pathological parameters affect their expression. Tissue samples were first microdissected to separate tumour tissue from the surrounding stroma and then subjected to RNA extraction procedures to allow gene expression to be quantitated. RNA was then converted into cDNA by reverse transcription and the genes of interest amplified and quantitated using semiquantitative polymerase chain reaction. Expression data was then normalized by expressing it as a ratio of the gene of interest to the 18S ribosomal gene and differences in expression analysed by analysis of variance (ANOVA) testing. The first portion of the study dealt with the progesterone and glucocorticoid receptors in tumour tissue. Progesterone is an anti-mitogenic signal for breast tissue and the stimulation of the receptor is a useful part of combined hormonal therapy for breast cancer. The glucocorticoid receptor plays a similar role to the progesterone receptor, slowing breast tissue growth and promoting apoptosis. The complete tissue population of 25 samples from control, grade 1, grade 2 and grade 3 tumours underwent PCR for the glucocorticoid and progesterone receptor genes and was normalized using 18S. Analysis of the expression data by ANOVA showed that the progesterone and glucocorticoid receptors were more highly expressed in grade 3 tumour tissue (p=0.023 and p=O.00033, for the progesterone and glucocorticoid receptors, respectively). Most advanced tumours cease being responsive to growth repressing hormones, so these results indicated that the control of the expression of these receptors in tumour tissue was more complicated than might have been expected. The increase in expression observed may be the result of intact growth preventing feedback mechanisms which have malfunctioned in some manner. There are several mechanisms the tumours may be using to escape an increase in progesterone or glucocorticoid sensitivity. The mRNA detected may simply not be being translated into completed proteins or be being spliced into isoforms of the receptors that favour tissue growth. The second portion of the study dealt with the estrogen receptors alpha and beta in tumour tissue. The estrogen receptors control estrogen signaling and are highly important in breast cancer, since estrogen is the primary growth inducing hormone for breast tissue. For the estrogen receptors alpha and beta, the complete tissue population of 25 samples from control, grade 1, grade 2 and grade 3 tumours underwent PCR and were normalized using 18S. ANOVA analysis found that the expression of the estrogen receptors did not change significantly in any grade of cancer (p= 0.057 and p=O.7'38, for ESRα and ESRβ, respectively) nor in tissues that are negative for the estrogen receptor alpha protein, a poor prognostic factor in breast cancer (p= 0.794 and p=O.7l6, for ESRα and ESRβ, respectively). These results indicated that the loss of estrogen receptor in advanced cancers is not controlled at the level of mRNA. The mRNA observed in this study may be being spliced into alternate and possibly inactive isoforms, or may be being degraded post transcriptionally, preventing estrogen stimulation in these tumours. This could prove an excellent area for further study, since if the mechanism that prevents or distorts ESR mRNA translation can be discovered, it would allow manipulation of one of the most important treatment avenues for breast cancer. The third portion of the study dealt with the expression of the androgen receptor in tumour tissue. Androgens are a strong anti-growth signal in breast tissue and despite the side effects that androgens have on women, they have been used to treat breast cancer with some success. The complete tissue population of 25 samples from control, grade 1, grade 2 and grade 3 tumours underwent PCR for the androgen receptor gene and normalized using 18S. The results obtained for androgen receptor expression in tumour tissue showed that androgen receptor expression was significantly elevated in grade 2 and grade 3 tumour tissue, as well as in ESRα negative tumours (p= 0.014 and p=O.O25, respectively). An increase in expression in late stage tumours would seem to be unusual for an anti-mitogenic receptor, however many advanced breast tumours have been found to be receptive to androgen stimulation, even when they no longer respond to other hormones. The increased expression of AR may be a normal response to cellular over-growth, or it may be a mechanism by which the tumour prevents stimulation by other growth retarding hormones, by sequestering all available receptor co-enzymes with a receptor that is unlikely to be stimulated. The fourth portion of the study examined the expression of the estrogen alpha, estrogen beta, progesterone, glucocorticoid and androgen nuclear receptors in stromal tissue derived from the tumours studied in previous chapters. Tumours have been observed in other studies to manipulate the activities of the cells that surround them through the release of cofactors and vice versa. These cofactors include the steroid hormones, among others, and hence the study of how the tumour and stroma interacts is a valuable extension to the results obtained in the previous sections. PCR was performed for all nuclear receptors, except for the estrogen receptor alpha, in the complete tissue population of 25 samples of tissue derived from the stroma of the grade 1, grade 2 and grade 3 tumours used in the previous studies as well as the control tissues. Due to difficulties in PCR optimization for estrogen receptor alpha, only three stromal samples from each grade and four controls were able to produce results, for a total population of 13 samples. Of all the receptors tested, only the progesterone and glucocorticoid receptors displayed significant changes in expression in stromal tissue, with PgR having significantly lower expression in all stromal samples compared to control, while GR was more highly expressed in stroma derived from high grade tumours (p= 5.908x107 and p=2.761x105, for PgR and GR, respectively). UR expression was also increased in stroma derived from ESRα negative tumours (p=5.85x105). These alterations reflect the kind of stimulation a tumour is likely to apply to the surrounding stroma, using progesterone to stimulate the cells into differentiating to provide a more suitable environment, hence the loss in PgR expression. The increase in GR expression may be the result of the high level of growth stimulating factors that tumours produce, priming the local cells to be more sensitive to growth suppressors, a situation that is also mirrored in results previously obtained for the tumour tissue. The fifth part of the study concerned the expression of the nuclear receptor coactivator 1 and nuclear receptor co-activator 3 genes. These proteins are required for the activation and function of the nuclear receptors and both have been implicated in cancer development, being found to be over-expressed in several tissues and cell lines. As integral parts of the nuclear receptor pathway, their level of expression is important for determining how effective any nuclear receptors present will be when stimulated. The complete tissue population of 25 samples from control, grade 1, grade 2 and grade 3 tumours underwent PCR for both nuclear receptor co-activator genes and normalized using 18S. The result of ANOVA analysis on the NCoA data showed that NCoA3 expression remained unaltered in all grades of cancer and stroma and in both ESRα positive and negative tissue. NCoA1 however, was significantly upregulated in grade 3 tumours as compared to grade 1 tumours and also in ESRα negative tumours. This increase in expression would seem to indicate that these tissues would be more capable of acting on any received hormonal stimulation. That this increase in expression occurs in more advanced cancers could be evidence that the nuclear receptor expression observed in prior sections is resulting in NR splice variants that favour, rather than repress, growth, as advanced cancers usually do not respond normally to hormonal stimulation. The final part of the study investigated the possibility of correlations between the expression of the nuclear receptors, between the nuclear receptor co-activators and between all of the tested genes and other pathological parameters, including tumour size, metastasis, site of tumour, carcinoma in situ invasiveness, age of patient and the presence of calcification. The data generated in the prior studies was analyzed using ANOVA for categorical data and correlation analysis for numerical data. The ANOVA and correlation analysis revealed a number of interactions between these factors, which provide additional information on the relationships between the tested genes. Expression of the progesterone receptor was found to be correlated with the expression of GR, AR and NCoA1 (p= 0.022, p=O.OO3, and p=O.0i9, respectively). Likewise the expression of GR and AR were found to be correlated (p=O.O29). Additionally, AR was found to be associated with tumour size (p=O.O36) while GR was found to be associated with both tumour size and metastasis (p= 0.006 and p=7.6x106, respectively). ESRα and ESRβ expression were found to be negatively correlated (p=O.O44M), as were patient age and the amount of ductal carcinoma in situ invasion. Given the results of previous analyses, it is not surprising that PgR, GR, AR and NCoA1 expression are related, and the negative correlations between ESRα and ESRβ expression, as well as between age and ductal carcinoma in situ invasion have been documented in other studies. Hence, these results provide reinforcement for previous observations, as well as providing new information, particularly on AR and GR.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Full Text

NMDA receptors are of particular importance in the control of synaptic strength and integration of synaptic activity. Dopamine receptor modulation of NMDA receptors in the striatum may influence the efficacy of synaptic transmission in the cortico-striatal pathway (Calabresi et al., 2000c Centonze et al., 2003) and if so, this modulation will be lost in Parkinson's disease. This change may be an important factor in the changes in the basal ganglia neural network that occur in Parkinson's Disease. In this thesis I have studied dopamine D1 and D2 receptor modulation of NMDA receptors in medium spiny neurons of 7-21 day old rat striatum. The dopamine D1 receptor agonist, SKF-82958, significantly decreased rat striatal NMDA receptor currents in patch-clamp whole-cell recordings from 7 day old rats. This inhibition was not abolished by application of a G protein inhibitor (GDP-p-S) or irreversible activator (GTP-y-S) suggesting a G protein-independent mechanism. In addition, intracellular application of protein tyrosine kinase inhibitors (lavendustin A or PP2) abolished D1 inhibition of NMDA currents. Functional NR2A receptors were absent in 7 day old rat striatum according to my experiments. Single-channel recordings showed that direct D1 receptor inhibition of NMDA receptors can not be observed in isolated membrane patches, which may indicate that D1 inhibition in whole-cell recordings is mediated by a change in NMDA receptor trafficking. Consistent with this hypothesis, intracellular application of a dynamin inhibitory peptide (QVPSRPNRAP) abolished D1 inhibition of NMDA receptor currents. I therefore conclude that a tyrosine kinase-dependent alteration of NMDA receptor trafficking underlies D1 dopamine receptor-mediated down-regulation of NMDA receptor currents in the striatum. The D2 class dopamine receptor agonist, quinpirole, significantly inhibited the NMDAR responses at 1 uM, but at a lower concentration (40 nM) there was no significant effect in 7 day old rat striatum. Replacement of GTP with GDP-P-S in the pipette solution abolished the inhibition induced by 1 uM quinpirole suggesting a G protein-dependent mechanism underlies the D2 family dopamine receptor modulation of NMDA receptors in the striatum.

Nuclear receptors are ligand-activated transcription factors that play significant roles in various biological processes within the body, such as cell development, hormone metabolism, reproduction, and cardiac function. As transcription factors, nuclear receptors are involved in many diseases, such as diabetes, cancer, and arthritis, resulting in approximately 10-15% of the pharmaceutical drugs presently on the market being targeted toward nuclear receptors. Structurally, nuclear receptors consist of a DNA-binding domain (DBD), responsible for binding specific sequences of DNA called response elements, fused to a ligand-binding domain (LBD) through a hinge region. The LBD binds a small molecule ligand. Upon ligand binding, the LBD changes to an active conformation leading to the recruitment of coactivator (CoAC) proteins and initiation of transcription. As a result of their involvement in disease, there is an emphasis on engineering nuclear receptors for applications in gene therapy, drug discovery and metabolic engineering.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on February 26, 2008) Vita. Includes bibliographical references.