A manipulação de materiais em escala nanométrica representa uma das fronteiras em nanociência e nanotecnologia, devido à possibilidade de controle de propriedades específicas do material. No caso de materiais biológicos, em particular, a manipulação e imobilização na forma de filmes ou camadas ultrafinas é crucial para seu emprego em dispositivos biotecnológicos. Neste trabalho, objetivou-se o estudo de detecção de diferentes hormônios tireoidianos (HTs) e análogos a partir da imobilização da região LBD do receptor de hormônio tireoidiano humano TRTRβ1 em um eletrodo interdigitado, para o desenvolvimento de um biossensor capacitivo. Este sistema consiste em um arranjo estrutural na forma de filme fino capaz de distinguir a interação específica receptor-ligante de outras interações possivelmente interferentes, visando a quantificação dos níveis de HTs. Para isto, a técnica de SAMs (Self-Assembled Monolayers) foi empregada, por permitir um alto controle da espessura e ordenamento molecular dos filmes, assim como a preservação das atividades das biomoléculas. Análises espectroscópicas e morfológicas foram realizadas para o estudo de adsorção das biomoléculas no filme. As interações específicas receptor-ligante foram avaliadas por meio de respostas elétricas (impedância) do biossensor contendo o TRβ1-LBD imobilizado em um filme orgânico ultrafino, e também por SPR (Surface Plasmon Resonance). Os resultados mostraram a capacidade dos eletrodos contendo TRTRβ1-LBD de detectar e diferenciar entre diferentes HTs em concentrações da ordem de nanomolar, compatível com níveis fisiológicos, evidenciando o grande potencial de aplicação para este sistema no diagnóstico e tratamento de disfunções tireoidianas. / Manipulation of materials at the nanoscale represents one of the frontiers in nanoscience and nanotechnology, mainly due to the possibility of specific controlling, improved properties, not observed if conventional bulk processing is applied. For biomolecules, in particular, processing via immobilization in the form of nanostructured films has allowed their use in biotechnological applications and devices. In this master dissertation, we aimed at investigating the immobilization of the LBD domain of human thyroid hormone receptor TRTRβ1 on interdigitated electrodes, to be used as capacitive biosensors for thyroid hormones (THs) and analogues detection. The nuclear receptors were immobilized via SAMs (Self-Assembled Monolayers), since this technique allows a high control of molecular order and thickness of the films, as well as the preservation of biological activities. Spectroscopic and morphological analyses were performed to investigate the adsorption of biomolecules on the nanostructured film. The interactions between receptor - ligand were also evaluated by means of electrical response (impedance) and SPR (Surface Plasmon Resonance). The bioelectrodes containing immobilized TRTRβ1 were capable of detecting and distinguishing among different HTs, including T3, T4, TRIAC and GC-1 at concentrations down to nanomolar, compatible with physiological levels. The latter results point to the possibility of applications of the bioelectrodes in the diagnosis and treatment of thyroid dysfunctions.

A manipulação de materiais em escala nanométrica representa uma das fronteiras em nanociência e nanotecnologia, devido à possibilidade de controle de propriedades específicas do material. No caso de materiais biológicos, em particular, a manipulação e imobilização na forma de filmes ou camadas ultrafinas é crucial para seu emprego em dispositivos biotecnológicos. Neste trabalho, objetivou-se o estudo de detecção de diferentes hormônios tireoidianos (HTs) e análogos a partir da imobilização da região LBD do receptor de hormônio tireoidiano humano TRTRβ1 em um eletrodo interdigitado, para o desenvolvimento de um biossensor capacitivo. Este sistema consiste em um arranjo estrutural na forma de filme fino capaz de distinguir a interação específica receptor-ligante de outras interações possivelmente interferentes, visando a quantificação dos níveis de HTs. Para isto, a técnica de SAMs (Self-Assembled Monolayers) foi empregada, por permitir um alto controle da espessura e ordenamento molecular dos filmes, assim como a preservação das atividades das biomoléculas. Análises espectroscópicas e morfológicas foram realizadas para o estudo de adsorção das biomoléculas no filme. As interações específicas receptor-ligante foram avaliadas por meio de respostas elétricas (impedância) do biossensor contendo o TRβ1-LBD imobilizado em um filme orgânico ultrafino, e também por SPR (Surface Plasmon Resonance). Os resultados mostraram a capacidade dos eletrodos contendo TRTRβ1-LBD de detectar e diferenciar entre diferentes HTs em concentrações da ordem de nanomolar, compatível com níveis fisiológicos, evidenciando o grande potencial de aplicação para este sistema no diagnóstico e tratamento de disfunções tireoidianas. / Manipulation of materials at the nanoscale represents one of the frontiers in nanoscience and nanotechnology, mainly due to the possibility of specific controlling, improved properties, not observed if conventional bulk processing is applied. For biomolecules, in particular, processing via immobilization in the form of nanostructured films has allowed their use in biotechnological applications and devices. In this master dissertation, we aimed at investigating the immobilization of the LBD domain of human thyroid hormone receptor TRTRβ1 on interdigitated electrodes, to be used as capacitive biosensors for thyroid hormones (THs) and analogues detection. The nuclear receptors were immobilized via SAMs (Self-Assembled Monolayers), since this technique allows a high control of molecular order and thickness of the films, as well as the preservation of biological activities. Spectroscopic and morphological analyses were performed to investigate the adsorption of biomolecules on the nanostructured film. The interactions between receptor - ligand were also evaluated by means of electrical response (impedance) and SPR (Surface Plasmon Resonance). The bioelectrodes containing immobilized TRTRβ1 were capable of detecting and distinguishing among different HTs, including T3, T4, TRIAC and GC-1 at concentrations down to nanomolar, compatible with physiological levels. The latter results point to the possibility of applications of the bioelectrodes in the diagnosis and treatment of thyroid dysfunctions.

Receptores ativados da proliferação de peroxissomos (PPAR) são receptores nucleares que regulam o metabolismo de gordura e glicose, adipogênese e polarização de macrófagos, e são os mediadores da ação de uma grande classe de fármacos usada no tratamento de diabetes tipo 2, as tiazolidinadionas (TZD). Enquanto as TZDs reduzem a glicose do sangue e aumentam efetivamente a sensibilidade à insulina, elas podem também apresentar efeitos colaterais como aumento do risco de complicações cardiovasculares, ganho de peso, retenção de fluido e toxicidade hepática. Por causa disso, novos fármacos que possuem respostas mais favoráveis devem ser desenvolvidos, e o mecanismo de ativação do PPAR por ligantes vem sendo intensamente examinado. Para entender a relação entre a ligação de agonistas ao PPAR e a ativação transcricional, pretendíamos primeiramente obter cristais de PPAR-LBD (domínio de ligação ao ligante) humano na forma apo. Porém, surpreendentemente, a análise do sítio de ligação ao ligante revelou a presença de três pequenas moléculas, identificadas como ácidos nonanoicos e octanoicos. Este trabalho reporta a análise da estrutura cristalográfica do PPAR LBD complexado simultaneamente com três ácidos graxos de cadeia média (AGCM), provindos de bactérias (organismo de expressão), localizados no sítio de ligação ao ligante. A análise estrutural e funcional sugere que os AGCM são agonistas parciais que estabilizam a conformação do LBD do PPAR por mecanismo independente da hélice 12. / PPARs (peroxisome proliferator activated receptors) are nuclear receptors that regulate glucose and fat metabolism, adipogenesis and macrophage polarization and mediate actions of a major class of drugs that are used to treat type 2 diabetes, the thiazolidinediones. While TZDs reduce blood glucose and improve insulin sensitivity effectively, they can also exhibit deleterious side effects such as increased cardiovascular risk, weight gain, fluid retention and liver toxicity. Because it is desirable to develop new PPAR drugs with more favorable spectrums of response, mechanisms of PPAR ligand activation have come under intense scrutiny. To understand relationships between PPAR ligand binding and transcriptional activation, we sought to obtain apo human PPAR-LBD (ligand binding domain) crystals that diffract to high resolution. More surprisingly, close analysis of the ligand binding pocket revealed the presence of three small molecules, identified as nonanoic acid and octanoic acid. Here, we report the X-ray structural analysis of the PPAR LBD complexed with three bacterial (expression organism) medium chain fatty acids (MCFAs) that simultaneously occupy the buried ligand binding pocket (LBP). Structural and functional analysis suggests that MCFAs are partial agonists that stabilize PPAR LBD conformation, through a helix 12 independent mechanism.

Orientador: Munir Salomão Skaf / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-22T05:21:38Z (GMT). No. of bitstreams: 1 Souza_PauloCesarTellesde_D.pdf: 16006519 bytes, checksum: 82d3ddc86356ae8c995467ed56947170 (MD5) Previous issue date: 2013 / Resumo: Receptores Nucleares (NRs) são proteínas que regulam a transcrição de genes, sendo alvos importantes para o desenho de fármacos. NRs são formados por quatro domínios, sendo o mais essencial deles, o Domínio de Ligação com o Ligante (LBD), responsável pelo reconhecimento seletivo de ligantes e ativação de sua função. Nesta Tese são utilizadas simulações de Dinâmica Molecular (MD) para o estudo do LBD de dois importantes NRs: Receptor do Hormônio Tireoideano (TR) e Receptor de Estrogênios (ER). Os estudos envolvendo o LBD do TR iniciaram-se pela investigação de um novo segundo sítio de ligação dos hormônios tireoideanos (T3 e T4). Foi mostrado que os hormônios se mantêm ancorados ao segundo sítio, possuindo grande mobilidade e múltiplos modos de ligação. Estimativas do DG de dissociação indicam que este novo sítio deve existir em solução aquosa, sendo T4 o hormônio com maior afinidade e candidato a ligante natural. O segundo objetivo da Tese foi a modelagem molecular da estrutura do LBD do TR sem ligantes (apo-TR) através da combinação de resultados de simulações de MD e experimentos de troca de hidrogênio/deutério. O modelo do apo-TR obtido mostra que a-hélice H12 ancora-se na H3, o que explica as mudanças de hidratação nesta região apontadas pelos experimentos. O terceiro objetivo da Tese foi elucidar os mecanismos moleculares que levam a alterações da atividade em duas mutações dos TRs: M369Ra e P452Lb. As simulações de M369Raindicam que o resíduo mutado interage com T3 no segundo sítio, o que pode explicar o aumento de sua afinidade por este ligante. As simulações de P452Lbsugeriram que esta mutação altera a posição da H12, levando a redução da cavidade de interação com co-ativadores e das interações do T3 com o primeiro sítio. O último estudo da Tese investigou uma conformação alternativa do LBD do ERb , que tem potencial para explicar como este subtipo promove repressão parcial da transcrição de genes regulada pelo ERa. Os cálculos de DG entre as conformaçõs clássica e alternativa indicam que a alternativa é estável, sendo o mínimo global de energia livre. / Abstract: Nuclear receptors (NRs) are proteins that regulate the gene transcription and thus are important targets for drug development. NRs are composed of four structural domains. The most important of them is the Ligand Binding Domain (LBD), responsible for the selective recognition of ligands and activation of NR function. In this Doctoral Thesis, Molecular Dynamics (MD) Simulation are used to study two important NRs LBD: Thyroid Hormone Receptor (TR) and Estrogen Receptor (ER). Studies involving TR began by investigating a new second binding site of thyroid hormones (T3 and T4) in the TR LBD. It has been shown that hormones remain anchored to the second site and have high mobility and multiple binding modes. Estimates of dissociation DG indicate that this new site can exist in aqueous solution. T4 has the higher affinity and may be the natural ligand of this site. The second objetive of the Thesis was the molecular modeling of the TR LBD structure without ligands (apo-TR) by combining results of MD simulations and hydrogen deuterium exchange experiments. The obtained model of apo-TR shows that H12 a-helix is anchored in H3 which explains the hydration changes in this region indicated by the experiments. The third goal was to elucidate the molecular mechanisms that lead to changes in the activity of two TR mutations: M369Ra and P452Lb. Simulations of M369Ra indicate that the mutated residue can interact directly with T3 in the second binding site, explaining the increase of its affinity. Simulations of P452Lbsuggested that this mutation changes the H12 position, leading to loss of ligand interaction with the rst binding site and reduction of coactivator cavity. The last study investigated a new alternative conformation of ERb LBD, which has the potential to explain how this subtype promotes the partial repression of ERagene transcription. Calculation of DG between classic and alternative conformations indicate that the alternative is stable and the global minimum of free energy. / Doutorado / Físico-Química / Doutor em Ciências

Os receptores ativadores da proliferação de peroxissomos (PPARs) pertencem à superfamília de receptores nucleares que funcionam como fatores transcricionais. Eles exercem um papel fundamental em processos que envolvem, principalmente, o metabolismo lipídico, em resposta à ativação por ligantes naturais e sintéticos como os ácidos graxos e os fibratos, respectivamente. A crescente descoberta de importantes funções fisiológicas, coordenadas pelos PPARs, e a necessidade de se conhecer como os agonistas, atualmente disponíveis, atuam nesses receptores, têm incitado pesquisas que vislumbram sua melhor exploração nos tratamentos de doenças metabólicas e inflamatórias, minimizando os efeitos adversos de ativações suprafisiológicas. Nesse cenário, o presente trabalho buscou compreender melhor as bases estruturais envolvidas nas funções atribuídas aos PPARs e explicar como as interações com seus ligantes ocorrem. Para isso, foram realizadas a subclonagem do domínio de ligação ao ligante do PPARα, sua expressão e purificação, seguidas de ensaios cristalográficos e biofísicos, além da abordagem de testes funcionais. Uma vez que a formação de oligômeros está relacionada à funcionalidade desses receptores, foram abordados estudos de oligomerização dos PPARs α e γ, compreendendo tanto o processo de homo- quanto o de heterodimerização. Os ensaios de cristalização do hPPARα LBD complexado a ligantes naturais e sintéticos, resultaram em estruturas cristalográficas que permitiram a identificação dos resíduos envolvidos no reconhecimento dos ligantes e a caracterização de sítios de ligação nunca antes descritos. A presença de ligantes nessas regiões afeta a conformação da proteína e, consequentemente, a modulação de sua função e o recrutamento da maquinaria transcricional. Adicionalmente, as estruturas cristalográficas da proteína complexada a ácidos graxos auxiliaram na compreensão de como essa importante classe de ligantes naturais possui efeitos farmacológicos similares aos de ligantes sintéticos. Esses resultados têm imediato impacto na procura racional de agonistas para esses receptores e se inserem em uma perspectiva de promoção do desenvolvimento científico-tecnológico na área de endocrinologia molecular. / The peroxisome proliferation-activated receptors (PPARs) belong to the nuclear receptors superfamily, acting as transcriptional factors. They play a key role in processes involving essentially lipid metabolism in response to activation by natural and synthetic ligands such as fatty acids and fibrates, respectively. The rising discovery of important physiological functions coordinated by PPARs and the necessity to know how the currently available agonists act on these receptors, have encouraged researches envisioning a better receptor exploration in the treatment of metabolic and inflammatory diseases, minimizing the adverse effects of supraphysiological activations. In this scenario, the present study aimed to better understand the structural basis involved in PPARs functions and elucidates how the interactions with their ligands takes place. For this, the ligand-binding domain of PPARα was subjected to subcloning, expression and purification steps, followed by crystallographical and biophysical assays, in addition to functional testing approaches. Since the degree of oligomerization is related to the functionality of these receptors, oligomeric studies of PPARs α and γ oligomerization were also achieved, comprising both homo- and hetero-dimerization. The co-crystallization assays of hPPARα LBD complexed with natural and synthetic ligands resulted in crystallographic structures that allowed the identification of residues involved in ligand recognition and the characterization of novel binding sites. The presence of ligands in these regions affects the conformation of the protein and thereby modulates their function and transcriptional machinery recruitment. Additionally, the crystallographic structures of the protein complexed to fatty acids were valuable for the understanding of how this important class of natural ligands has similar pharmacological effects to those of synthetic ligands. These results have direct impact on rational agonists design to these receptors and are inserted in a perspective of scientifical promotion and technological development in the field of molecular endocrinology.

Os receptores ativadores da proliferação de peroxissomos (PPARs) pertencem à superfamília de receptores nucleares que funcionam como fatores transcricionais. Eles exercem um papel fundamental em processos que envolvem, principalmente, o metabolismo lipídico, em resposta à ativação por ligantes naturais e sintéticos como os ácidos graxos e os fibratos, respectivamente. A crescente descoberta de importantes funções fisiológicas, coordenadas pelos PPARs, e a necessidade de se conhecer como os agonistas, atualmente disponíveis, atuam nesses receptores, têm incitado pesquisas que vislumbram sua melhor exploração nos tratamentos de doenças metabólicas e inflamatórias, minimizando os efeitos adversos de ativações suprafisiológicas. Nesse cenário, o presente trabalho buscou compreender melhor as bases estruturais envolvidas nas funções atribuídas aos PPARs e explicar como as interações com seus ligantes ocorrem. Para isso, foram realizadas a subclonagem do domínio de ligação ao ligante do PPARα, sua expressão e purificação, seguidas de ensaios cristalográficos e biofísicos, além da abordagem de testes funcionais. Uma vez que a formação de oligômeros está relacionada à funcionalidade desses receptores, foram abordados estudos de oligomerização dos PPARs α e γ, compreendendo tanto o processo de homo- quanto o de heterodimerização. Os ensaios de cristalização do hPPARα LBD complexado a ligantes naturais e sintéticos, resultaram em estruturas cristalográficas que permitiram a identificação dos resíduos envolvidos no reconhecimento dos ligantes e a caracterização de sítios de ligação nunca antes descritos. A presença de ligantes nessas regiões afeta a conformação da proteína e, consequentemente, a modulação de sua função e o recrutamento da maquinaria transcricional. Adicionalmente, as estruturas cristalográficas da proteína complexada a ácidos graxos auxiliaram na compreensão de como essa importante classe de ligantes naturais possui efeitos farmacológicos similares aos de ligantes sintéticos. Esses resultados têm imediato impacto na procura racional de agonistas para esses receptores e se inserem em uma perspectiva de promoção do desenvolvimento científico-tecnológico na área de endocrinologia molecular. / The peroxisome proliferation-activated receptors (PPARs) belong to the nuclear receptors superfamily, acting as transcriptional factors. They play a key role in processes involving essentially lipid metabolism in response to activation by natural and synthetic ligands such as fatty acids and fibrates, respectively. The rising discovery of important physiological functions coordinated by PPARs and the necessity to know how the currently available agonists act on these receptors, have encouraged researches envisioning a better receptor exploration in the treatment of metabolic and inflammatory diseases, minimizing the adverse effects of supraphysiological activations. In this scenario, the present study aimed to better understand the structural basis involved in PPARs functions and elucidates how the interactions with their ligands takes place. For this, the ligand-binding domain of PPARα was subjected to subcloning, expression and purification steps, followed by crystallographical and biophysical assays, in addition to functional testing approaches. Since the degree of oligomerization is related to the functionality of these receptors, oligomeric studies of PPARs α and γ oligomerization were also achieved, comprising both homo- and hetero-dimerization. The co-crystallization assays of hPPARα LBD complexed with natural and synthetic ligands resulted in crystallographic structures that allowed the identification of residues involved in ligand recognition and the characterization of novel binding sites. The presence of ligands in these regions affects the conformation of the protein and thereby modulates their function and transcriptional machinery recruitment. Additionally, the crystallographic structures of the protein complexed to fatty acids were valuable for the understanding of how this important class of natural ligands has similar pharmacological effects to those of synthetic ligands. These results have direct impact on rational agonists design to these receptors and are inserted in a perspective of scientifical promotion and technological development in the field of molecular endocrinology.

Effects of man made pollutants on an ecosystem are initiated at the cellular level where a prime determinant for survival of an organism is its ability to metabolise and excrete toxic chemicals or their metabolites, thereby preventing cellular toxicity or damage to germ cell DNA. Cytochrome P450 (CYP) enzymes are responsible (in concert with the remainder of the Ah battery enzymes) for the metabolism of numerous xenobiotics and endogenous compounds, including the metabolic activation of most environmental toxic chemicals and carcinogens. Genetic polymorphisms which affect performance of these enzymatic detoxification systems may alter tolerance to pollutants and thus survival in polluted environments. Alterations in the susceptibility of individuals and the development of resistant populations has arisen by forced selection of populations with variant genes, resulting in increased detoxification capacity. There is evidence for such scenarios of variations in activities of pollutant biotransforming enzymes of fish contributing to survival in polluted estuarine environments and several chemically resistant populations have been identified in the USA and Europe. In fish it has been demonstrated that CYP1A enzyme activity is required to activate some carcinogenic xenobiotics to a metabolic state in which they can form DNA adducts. The mechanism of reduced CYP1A expression in highly contaminated populations may therefore represent resistance to chemical stressors. European flounder (Platichthys flesus) from some waterways which have a long history of severe sedimentary contamination do not show elevated levels of CYP1A. The aim of the current study was to investigate whether any heritable differences were apparent between offspring from parents inhabiting long-term polluted and pristine areas. Flounder were obtained from a highly polluted estuary in the UK and crossed with fish from a relatively pristine environment. Offspring were raised in communal tanks in order to standardise environmental conditions, and allow investigations into the genetic variation of CYP1A. To allow identification of offspring to parental fish, polymorphic microsatellite loci were isolated and characterised for the flounder. Novel cDNA probes to transcription factors in the detoxification pathway (AhR2 and ARNT2) were cloned for flounder, and RT-PCR / Southern blot methods were developed for quantitation of gene transcript levels. A novel method of CYP1A quantification using real-time PCR was developed. PAH and PCB exposure trials were carried out on mixed batch offspring, and CYP1A gene transcript levels assessed using Northern blot and real-time PCR techniques. Offspring were genotyped to their parents using the microsatellites obtained, and CYP1A transcript levels were correlated with clean and polluted areas. CYP1A was further correlated to transcription factor expression, and data are presented. Following exposure to the commercial PCB mixture, Aroclor 1254, CYP1A transcript levels were found to be significantly lower in families whose parents originated from a polluted area. This observation indicates that there is a possible genetic component to variation in CYP1A levels, and that these fish may have acquired a heritable tolerance to polluted areas. The lack of induction, or correlation with CYP1A levels, of AhR2 and ARNT2 expression indicates a possible AhR independent pathway for the metabolism of PCBs in the flounder. © Tom Dixon 2003 http://www.tomdixon.org

Abstract Cytochrome P450 (CYP) enzymes are the major metabolizers of xenobiotics, e.g. drugs, and environmental toxins. Thus, changes in CYP expression have an important impact on drug metabolism and susceptibility to chemical toxicity. In the present study, the transcriptional mechanisms of both constitutive and inducible regulation of the Cyp2a5 gene in mouse liver were investigated. Mouse primary hepatocyte cultures were used as the main model system together with cell and molecular biology methods. The key activation regions of the Cyp2a5 5' promoter were determined using reporter gene assays. Two major transcription activation sites of the Cyp2a5 5' promoter, called the proximal and the distal, were found. Transcription factors hepatocyte nuclear factor-4 (HNF-4) and nuclear factor I were shown to bind to the proximal promoter. Aryl hydrocarbon receptor nuclear translocator (ARNT) and upstream stimulatory factor bound to a common palindromic E-box element in the distal promoter region. All three response elements were shown to be essential for constitutive expression of CYP2A5 in murine hepatocytes. ARNT appeared to control Cyp2a5 transcription without a heterodimerization partner suggesting active involvement of the ARNT homodimer in mammalian gene regulation. Aryl hydrocarbon receptor (AHR) ligands were shown to induce Cyp2a5 transcriptionally by an AHR-dependent mechanism, and established Cyp2a5 as a novel AHR-regulated gene. The AHR response element and the E-box, identified in these studies, were located near to each other and close to a separately defined nuclear factor (erythroid-derived 2)-like 2 binding site in the distal region of the Cyp2a5 promoter, suggesting cooperation between these elements. Peroxisome proliferator-activated receptor gamma coactivator-1α was shown to up-regulate Cyp2a5 transcription through coactivation of HNF-4α. This indicates that xenobiotic metabolism can be regulated by modification of co-activation. The present results show that CYP2A5 is regulated by several different cross-regulatory pathways. The regulatory mechanisms involved in the transcription of the Cyp2a5 gene may also control other CYP genes, especially the human ortholog CYP2A6, and may explain some of the individual variations in the metabolism of xenobiotics.

The aryl hydrocarbon receptor (AhR) heterodimerizes with the aryl hydrocarbon receptor nuclear translocator (Arnt) for transcriptional regulation. We generated three N-terminal deletion constructs of the human AhR of 12-24 KDa in size—namely D1 (aa 84-295), D2 (aa 84-192) and D3 (aa 191-295)—to suppress the Arnt function. We observed that all three constructs interact with the human Arnt with similar affinities. D2, which contains part of the AhR PAS-A domain and interacts with the PAS-A domain of Arnt, inhibits the formation of the AhR gel shift complex. D2 suppresses the 3-methylcholanthrene-induced, dioxin response element (DRE)-driven luciferase activity in Hep3B cells and exogenous Arnt reverses this D2 suppression. D2 suppresses the induction of CYP1A1 at both the message and protein levels in Hep3B cells; however, the CYP1B1 induction is not affected. D2 suppresses the recruitment of Arnt to the cyp1a1 promoter but not to the cyp1b1 promoter, partly because the AhR/Arnt heterodimer binds better to the cyp1b1 DRE than to the cyp1a1 DRE. Interestingly, D2 has no effect on the cobalt chloride-induced, hypoxia inducible factor-1 (HIF-1)-dependent expression of vegf, aldolase c, and ldh-a messages. Our data reveal that the flanking sequences of the DRE contribute to the binding affinity of the AhR/Arnt heterodimer to its endogenous enhancers and the function of AhR and HIF-1 can be differentially suppressed by the D2 inhibitory molecule. In chapter 2, a Pichia Pastoris expression system was constructed expressing codon optimized human full length AhR. This codon optimization is necessary for overexpression of huAhR. RT-PCR data showed that the codon optimized mRNA was more stably expressed than wild types. Overexpressed huAhR protein was degraded by proteinase when using a regular P. Pastoris strain yJC100 whereas the proteinase deficient ySMD1163 maintained a much higher level of huAhR. P. Pastoris expressed huAhR was natively purified and analyzed. Coimmunopricipitation assay shows its interaction with endogenous Arnt. A ligand-dependent gel shift was also observed. In addition, we performed an in vitro coprecipitation assay to study its binding to endogenous cyp1b1 DREs. The result shows that the DRE3, known as a critical DRE for cyp1b1 transcriptional activity, has the highest binding affinity to AhR/Arnt complex. Taking together, we constructed a novel P. Pastoris expression system to overexpress human full length AhR. Purified huAhR is a good reagent for studing its ligand and DNA binding. In chapter 3, an adeno-associated virus (AAV) expression system was constructed to express an AhR deletion contruct CΔ553 (aa1-295) for tumor injection. Western blot shows the expression of CΔ553 (aa1-295) in hela cells infected by AAV-553, but the low yield of AAV-553 limited its application on tumor treatment. Possible solutions were discussed for future work.