Expression of eph-family receptor tyrosine kinases and ephrins in the tadpole of the frog Xenopus laevis, and possible roles in the development of retinotectal topography

Assembling a nervous system requires exquisite specificity in the construction of neuronal connectivity. One method by which such specificity is implemented is the presence of chemical cues within the tissues, differentiating one region from another, and the presence of receptors for those cues on the surface of neurons and their axons that are navigating within this cellular environment.

Connections from one part of the nervous system to another often take the form of a topographic mapping. One widely studied model system that involves such a mapping is the vertebrate retinotectal projection-the set of connections between the eye and the optic tectum of the midbrain, which is the primary visual center in non-mammals and is homologous to the superior colliculus in mammals. In this projection the two-dimensional surface of the retina is mapped smoothly onto the two-dimensional surface of the tectum, such that light from neighboring points in visual space excites neighboring cells in the brain. This mapping is implemented at least in part via differential chemical cues in different regions of the tectum.

The Eph family of receptor tyrosine kinases and their cell-surface ligands, the ephrins, have been implicated in a wide variety of processes, generally involving cellular movement in response to extracellular cues. In particular, they possess expression patterns-i.e., complementary gradients of receptor in retina and ligand in tectum- and in vitro and in vivo activities and phenotypes-i.e., repulsive guidance of axons and defective mapping in mutants, respectively-consistent with the long-sought retinotectal chemical mapping cues.

The tadpole of Xenopus laevis, the South African clawed frog, is advantageous for in vivo retinotectal studies because of its transparency and manipulability. However, neither the expression patterns nor the retinotectal roles of these proteins have been well characterized in this system. We report here comprehensive descriptions in swimming stage tadpoles of the messenger RNA expression patterns of eleven known Xenopus Eph and ephrin genes, including xephrin-A3, which is novel, and xEphB2, whose expression pattern has not previously been published in detail. We also report the results of in vivo protein injection perturbation studies on Xenopus retinotectal topography, which were negative, and of in vitro axonal guidance assays, which suggest a previously unrecognized attractive activity of ephrins at low concentrations on retinal ganglion cell axons. This raises the possibility that these axons find their correct targets in part by seeking out a preferred concentration of ligands appropriate to their individual receptor expression levels, rather than by being repelled to greater or lesser degrees by the ephrins but attracted by some as-yet-unknown cue(s).

Molecular mechanism of sulfated carbohydrate recognition: structural and biochemical studies of the cysteine-rich domain of mannose receptor

Mannose receptor (MR) is widely expressed on macrophages, immature dendritic cells, and a variety of epithelial and endothelial cells. It is a 180 kD type I transmembrane receptor whose extracellular region consists of three parts: the amino-terminal cysteine-rich domain (Cys-MR); a fibronectin type II-like domain; and a series of eight tandem C-type lectin carbohydrate recognition domains (CRDs). Two portions of MR have distinct carbohydrate recognition properties: Cys-MR recognizes sulfated carbohydrates and the tandem CRD region binds terminal mannose, fucose, and N-acetyl-glucosamine (GlcNAc). The dual carbohydrate binding specificity allows MR to interact with sulfated and nonsulfated polysaccharide chains, and thereby facilitating the involvement of MR in immunological and physiological processes. The immunological functions of MR include antigen capturing (through binding non-sulfated carbohydrates) and antigen targeting (through binding sulfated carbohydrates), and the physiological roles include rapid clearance of circulatory luteinizing hormone (LH), which bears polysaccharide chains terminating with sulfated and non-sulfated carbohydrates.

We have crystallized and determined the X-ray structures of unliganded Cys-MR (2.0 Å) and Cys-MR complexed with different ligands, including Hepes (1.7 Å), 4SO_4-N-Acetylgalactosamine (4SO_4-GalNAc; 2.2 Å), 3SO_4-Lewis^x (2.2 Å), 3S04-Lewis^a (1.9 Å), and 6SO_4-GalNAc (2.5 Å). The overall structure of Cys-MR consists of 12 anti-parallel β-strands arranged in three lobes with approximate three fold internal symmetry. The structure contains three disulfide bonds, formed by the six cysteines in the Cys-MR sequence. The ligand-binding site is located in a neutral pocket within the third lobe, in which the sulfate group of ligand is buried. Our results show that optimal binding is achieved by a carbohydrate ligand with a sulfate group that anchors the ligand by forming numerous hydrogen bonds and a sugar ring that makes ring-stacking interactions with Trpll7 of CysMR. Using a fluorescence-based assay, we characterized the binding affinities between CysMR and its ligands, and rationalized the derived affinities based upon the crystal structures. These studies reveal the mechanism of sulfated carbohydrate recognition by Cys-MR and facilitate our understanding of the role of Cys-MR in MR recognition of its ligands.

Studies of the serotonin type 3A receptor and the chemical preparation of tRNA

This thesis describes studies surrounding a ligand-gated ion channel (LGIC): the serotonin type 3A receptor (5-HT₃AR). Structure-function experiments using unnatural amino acid mutagenesis are described, as well as experiments on the methodology of unnatural amino acid mutagenesis. Chapter 1 introduces LGICs, experimental methods, and an overview of the unnatural amino acid mutagenesis.

In Chapter 2, the binding orientation of the clinically available drugs ondansetron and granisetron within 5-HT₃A is determined through a combination of unnatural amino acid mutagenesis and an inhibition based assay. A cation-π interaction is found for both ondansetron and granisetron with a specific tryptophan residue (Trp183, TrpB) of the mouse 5-HT₃AR, which establishes a binding orientation for these drugs.

In Chapter 3, further studies were performed with ondansetron and granisetron with 5-HT₃A. The primary determinant of binding for these drugs was determined to not include interactions with a specific tyrosine residue (Tyr234, TyrC2). In completing these studies, evidence supporting a cation-π interaction of a synthetic agonist, meta-chlorophenylbiguanide, was found with TyrC2.

In Chapter 4, a direct chemical acylation strategy was implemented to prepare full-length suppressor tRNA mediated by lanthanum(III) and amino acid phosphate esters. The derived aminoacyl-tRNA is shown to be translationally competent in Xenopus oocytes.

Appendix A.1 gives details of a pharmacological method for determining the equilibrium dissociation constant, K_B, of a competitive antagonist with a receptor, known as Schild analysis. Appendix A.2 describes an examination of the inhibitory activity of new chemical analogs of the 5-HT₃A antagonist ondansetron. Appendix A.3 reports an organic synthesis of an intermediate for a new unnatural amino acid. Appendix A.4 covers an additional methodological examination for the preparation of amino-acyl tRNA.

The logic of receptor-ligand interactions in the Notch signaling pathway

The Notch signaling pathway enables neighboring cells to coordinate developmental fates in diverse processes such as angiogenesis, neuronal differentiation, and immune system development. Although key components and interactions in the Notch pathway are known, it remains unclear how they work together to determine a cell's signaling state, defined as its quantitative ability to send and receive signals using particular Notch receptors and ligands. Recent work suggests that several aspects of the system can lead to complex signaling behaviors: First, receptors and ligands interact in two distinct ways, inhibiting each other in the same cell (in cis) while productively interacting between cells (in trans) to signal. The ability of a cell to send or receive signals depends strongly on both types of interactions. Second, mammals have multiple types of receptors and ligands, which interact with different strengths, and are frequently co-expressed in natural systems. Third, the three mammalian Fringe proteins can modify receptor-ligand interaction strengths in distinct and ligand-specific ways. Consequently, cells can exhibit non-intuitive signaling states even with relatively few components.

In order to understand what signaling states occur in natural processes, and what types of signaling behaviors they enable, this thesis puts forward a quantitative and predictive model of how the Notch signaling state is determined by the expression levels of receptors, ligands, and Fringe proteins. To specify the parameters of the model, we constructed a set of cell lines that allow control of ligand and Fringe expression level, and readout of the resulting Notch activity. We subjected these cell lines to an assay to quantitatively assess the levels of Notch ligands and receptors on the surface of individual cells. We further analyzed the dependence of these interactions on the level and type of Fringe expression. We developed a mathematical modeling framework that uses these data to predict the signaling states of individual cells from component expression levels. These methods allow us to reconstitute and analyze a diverse set of Notch signaling configurations from the bottom up, and provide a comprehensive view of the signaling repertoire of this major signaling pathway.

Fluorescence microscopy of nicotinic acetylcholine receptors

Neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric ligand gated ion channels abundantly expressed in the central nervous system. Changes in the assembly and trafficking of nAChRs are pertinent to disease states including nicotine dependence, autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), and Parkinson’s disease (PD). Here we investigate the application of high resolution fluorescence techniques for the study of nAChR assembly and trafficking. We also describe the construction and validation of a fluorescent α5 subunit and subsequent experiments to elucidate the cellular mechanisms through which α5 subunits are expressed, assembled into mature receptors, and trafficked to the cell surface. The effects of a known single nucleotide polymorphism (D398N) in the intracellular loop of α5 are also examined.

Additionally, this report describes the development of a combined total internal reflection fluorescence (TIRF) and lifetime imaging (FLIM) technique and the first application of this methodology for elucidation of stochiometric composition of nAChRs. Many distinct subunit combinations can form functional receptors. Receptor composition and stoichiometry confers unique biophysical and pharmacological properties to each receptor sub-type. Understanding the nature of assembly and expression of each receptor subtype yields important information about the molecular processes that may underlie the mechanisms through which nAChR contribute to disease and addiction states.

Binding Site Structure and Stoichiometry in Serotonin Type 3 Receptors

This dissertation primarily describes studies of serotonin type 3 (5-HT₃) receptors of the Cys-loop super-family of ligand gated ion channels. The first chapter provides a general introduction to these important proteins and the methods used to interrogate their structure and function. The second chapter details the delineation of a structural unit of the ligand binding site of homomeric 5-HT₃A receptors on which the ligands serotonin (5-HT) and m-chlorophenyl biguanide (mCPBG) are reliant for effective receptor activation. Unnatural amino acid mutagenesis results show that the details of each ligand’s interaction with this organizing feature of the binding site differ, providing insights into general principles of receptor activation.

The third chapter describes a study in which florescent protein fusions of the A and B subunits of the heteromeric 5-HT₃AB receptor are employed to determine the subunit stoichiometry and order within functional receptors. Strong evidence is found for an A₃B₂ stoichiometry with A-A-B-A-B order. The fourth chapter investigates the potential for ligand binding across heteromeric binding sites in the 5-HT₃AB receptor. Unlike serotonin, mCPBG is found to bind the receptor at heteromeric binding sites. Further mCPBG is capable of allosterically modulating the response of serotonin on the 5-HT₃AB receptor from these heteromeric sites.

Finally, the fifth chapter describes progress towards the application of unnatural amino acid mutagenesis to an important new class of proteins, transcription factors. Experiments optimizing novel methods for the detection of function are described, using RARα of the nuclear receptor family of transcription factors.

Chemical studies of viral entry mechanisms: I. Hydrophobic protein-lipid interactions during sendai virus membrane fusion. II. Kinetics of bacteriophage λ DNA injection.

Viruses possess very specific methods of targeting and entering cells. These methods would be extremely useful if they could also be applied to drug delivery, but little is known about the molecular mechanisms of the viral entry process. In order to gain further insight into mechanisms of viral entry, chemical and spectroscopic studies in two systems were conducted, examining hydrophobic protein-lipid interactions during Sendai virus membrane fusion, and the kinetics of bacteriophage λ DNA injection.

Sendai virus glycoprotein interactions with target membranes during the early stages of fusion were examined using time-resolved hydrophobic photoaffinity labeling with the lipid-soluble carbene generator3-(trifluoromethyl)-3-(m-^(125 )I] iodophenyl)diazirine (TID). The probe was incorporated in target membranes prior to virus addition and photolysis. During Sendai virus fusion with liposomes composed of cardiolipin (CL) or phosphatidylserine (PS), the viral fusion (F) protein is preferentially labeled at early time points, supporting the hypothesis that hydrophobic interaction of the fusion peptide at the N-terminus of the F_1 subunit with the target membrane is an initiating event in fusion. Correlation of the hydrophobic interactions with independently monitored fusion kinetics further supports this conclusion. Separation of proteins after labeling shows that the F_1 subunit, containing the putative hydrophobic fusion sequence, is exclusively labeled, and that the F_2 subunit does not participate in fusion. Labeling shows temperature and pH dependence consistent with a need for protein conformational mobility and fusion at neutral pH. Higher amounts of labeling during fusion with CL vesicles than during virus-PS vesicle fusion reflects membrane packing regulation of peptide insertion into target membranes. Labeling of the viral hemagglutinin/neuraminidase (HN) at low pH indicates that HN-mediated fusion is triggered by hydrophobic interactions, after titration of acidic amino acids. HN labeling under nonfusogenic conditions reveals that viral binding may involve hydrophobic as well as electrostatic interactions. Controls for diffusional labeling exclude a major contribution from this source. Labeling during reconstituted Sendai virus envelope-liposome fusion shows that functional reconstitution involves protein retention of the ability to undergo hydrophobic interactions.

Examination of Sendai virus fusion with erythrocyte membranes indicates that hydrophobic interactions also trigger fusion between biological membranes, and that HN binding may involve hydrophobic interactions as well. Labeling of the erythrocyte membranes revealed close membrane association of spectrin, which may play a role in regulating membrane fusion. The data show that hydrophobic fusion protein interaction with both artificial and biological membranes is a triggering event in fusion. Correlation of these results with earlier studies of membrane hydration and fusion kinetics provides a more detailed view of the mechanism of fusion.

The kinetics of DNA injection by bacteriophage λ. into liposomes bearing reconstituted receptors were measured using fluorescence spectroscopy. LamB, the bacteriophage receptor, was extracted from bacteria and reconstituted into liposomes by detergent removal dialysis. The DNA binding fluorophore ethidium bromide was encapsulated in the liposomes during dialysis. Enhanced fluorescence of ethidium bromide upon binding to injected DNA was monitored, and showed that injection is a rapid, one-step process. The bimolecular rate law, determined by the method of initial rates, revealed that injection occurs several times faster than indicated by earlier studies employing indirect assays.

It is hoped that these studies will increase the understanding of the mechanisms of virus entry into cells, and to facilitate the development of virus-mimetic drug delivery strategies.

Computational Predictions of G Protein-Coupled Receptor Structures and Binding Sites

G protein-coupled receptors (GPCRs) are the largest family of proteins within the human genome. They consist of seven transmembrane (TM) helices, with a N-terminal region of varying length and structure on the extracellular side, and a C-terminus on the intracellular side. GPCRs are involved in transmitting extracellular signals to cells, and as such are crucial drug targets. Designing pharmaceuticals to target GPCRs is greatly aided by full-atom structural information of the proteins. In particular, the TM region of GPCRs is where small molecule ligands (much more bioavailable than peptide ligands) typically bind to the receptors. In recent years nearly thirty distinct GPCR TM regions have been crystallized. However, there are more than 1,000 GPCRs, leaving the vast majority of GPCRs with limited structural information. Additionally, GPCRs are known to exist in a myriad of conformational states in the body, rendering the static x-ray crystal structures an incomplete reflection of GPCR structures. In order to obtain an ensemble of GPCR structures, we have developed the GEnSeMBLE procedure to rapidly sample a large number of variations of GPCR helix rotations and tilts. The lowest energy GEnSeMBLE structures are then docked to small molecule ligands and optimized. The GPCR family consists of five subfamilies with little to no sequence homology between them: class A, B1, B2, C, and Frizzled/Taste2. Almost all of the GPCR crystal structures have been of class A GPCRs, and much is known about their conserved interactions and binding sites. In this work we particularly focus on class B1 GPCRs, and aim to understand that family’s interactions and binding sites both to small molecules and their native peptide ligands. Specifically, we predict the full atom structure and peptide binding site of the glucagon-like peptide receptor and the TM region and small molecule binding sites for eight other class B1 GPCRs: CALRL, CRFR1, GIPR, GLR, PACR, PTH1R, VIPR1, and VIPR2. Our class B1 work reveals multiple conserved interactions across the B1 subfamily as well as a consistent small molecule binding site centrally located in the TM bundle. Both the interactions and the binding sites are distinct from those seen in the more well-characterized class A GPCRs, and as such our work provides a strong starting point for drug design targeting class B1 proteins. We also predict the full structure of CXCR4 bound to a small molecule, a class A GPCR that was not closely related to any of the class A GPCRs at the time of the work.

Binding Studies of Neuronal Nicotinic Acetylcholine Receptors Expressing Unnatural Amino Acids

Nicotinic acetylcholine receptors are pentameric ligand-gated ion channels mediating fast synaptic transmission throughout the peripheral and central nervous systems. They have been implicated in various processes related to cognitive functions, learning and memory, arousal, reward, motor control and analgesia. Therefore, these receptors present alluring potential therapeutic targets for the treatment of pain, epilepsy, Alzheimer’s disease, Parkinson’s disease, Tourette’s syndrome, schizophrenia, anxiety, depression and nicotine addiction. The work detailed in this thesis focuses on binding studies of neuronal nicotinic receptors and aims to further our knowledge of subtype specific functional and structural information.

Chapter 1 is an introductory chapter describing the structure and function of nicotinic acetylcholine receptors as well as the methodologies used for the dissertation work described herein. There are several different subtypes of nicotinic acetylcholine receptors known to date and the subtle variations in their structure and function present a challenging area of study. The work presented in this thesis deals specifically with the α4β2 subtype of nicotinic acetylcholine receptor. This subtype assembles into 2 closely related stoichiometries, termed throughout this thesis as A3B2 and A2B3 after their respective subunit composition. Chapter 2 describes binding studies of select nicotinic agonists on A3B2 and A2B3 receptors determined by whole-cell recording. Three key binding interactions, a cation-π and two hydrogen bonds, were probed for four nicotinic agonists, acetylcholine, nicotine, smoking cessation drug varenicline (Chantix®) and the related natural product cytisine.

Results from the binding studies presented in Chapter 2 show that the major difference in binding of these four agonists to A3B2 and A2B3 receptors lies in one of the two hydrogen bond interactions where the agonist acts as the hydrogen bond acceptor and the backbone NH of a conserved leucine residue in the receptor acts as the hydrogen bond donor. Chapter 3 focuses on studying the effect of modulating the hydrogen bond acceptor ability of nicotine and epibatidine on A3B2 receptor function determined by whole-cell recording. Finally, Chapter 4 describes single-channel recording studies of varenicline binding to A2B3 and A3B2 receptors.

Investigation of competitive antagonist binding to the nicotinic acetylcholine receptor using voltage-jump and light-flash techniques

1. The effect of 2,2’-bis-[α-(trimethylammonium)methyl]azobenzene (2BQ), a photoisomerizable competitive antagonist, was studied at the nicotinic acetycholine receptor of Electrophorus electroplaques using voltage-jump and light-flash techniques.

2. 2BQ, at concentrations below 3 μΜ, reduced the amplitude of voltage-jump relaxations but had little effect on the voltage-jump relaxation time constants under all experimental conditions. At higher concentrations and voltages more negative than -150 mV, 2BQ caused significant open channel blockade.

3. Dose-ratio studies showed that the cis and trans isomers of 2BQ have equilibrium binding constants (K _ᵢ) of .33 and 1.0 μΜ, respectively. The binding constants determined for both isomers are independent of temperature, voltage, agonist concentration, and the nature of the agonist.

4. In a solution of predominantly cis-2BQ, visible-light flashes led to a net cis→trans isomerization and caused an increase in the agonist-induced current. This increase had at least two exponential components; the larger amplitude component had the same time constant as a subsequent voltage-jump relaxation; the smaller amplitude component was investigated using ultraviolet light flashes.

5. In a solution of predominantly trans-2BQ, UV-light flashes led to a net trans→cis isomerization and caused a net decrease in the agonist-induced current. This effect had at least two exponential components. The smaller and faster component was an increase in agonist-induced current and had a similar time constant to the voltage-jump relaxation. The larger component was a slow decrease in the agonist-induced current with rate constant approximately an order of magnitude less than that of the voltage-jump relaxation. This slow component provided a measure of the rate constant for dissociation of cis-2BQ (k_ = 60/s at 20°C). Simple modelling of the slope of the dose-rate curves yields an association rate constant of 1.6 x 10⁸/M/s. This agrees with the association rate constant of 1.8 x 10⁸/M/s estimated from the binding constant (K_i). The Q₁₀ of the dissociation rate constant of cis-2BQ was 3.3 between 6° and 20°C. The rate constants for association and dissociation of cis-28Q at receptors are independent of voltage, agonist concentration, and the nature of the agonist.

6. We have measured the molecular rate constants of a competitive antagonist which has roughly the same K _ᵢ as d-tubocurarine but interacts more slowly with the receptor. This leads to the conclusion that curare itself has an association rate constant of 4 x 10⁹/M/s or roughly as fast as possible for an encounter-limited reaction.

Structure-Function Studies of Nicotinic Acetylcholine Receptors Using Selective Agonists and Positive Allosteric Modulators

This dissertation primarily describes chemical-scale studies of nicotinic acetylcholine receptors (nAChRs) in order to better understand ligand-receptor selectivity and allosteric modulation influences during receptor activation. Electrophysiology coupled with canonical and non-canonical amino acids mutagenesis is used to probe subtle changes in receptor function.

The first half of this dissertation focuses on differential agonist selectivity of α4β2-containing nAChRs. The α4β2 nAChR can assemble in alternative stoichiometries as well as assemble with other accessory subunits. Chapter 2 identifies key structural residues that dictate binding and activation of three stoichiometry-dependent α4β2 receptor ligands: sazetidine-A, cytisine, and NS9283. These do not follow previously suggested hydrogen-bonding patterns of selectivity. Instead, three residues on the complementary subunit strongly influence binding ability of a ligand and receptor activation. Chapter 3 involves isolation of a α5α4β2 receptor-enriched population to test for a potential alternative agonist binding location at the α5 α4 interface. Results strongly suggest that agonist occupation of this site is not necessary for receptor activation and that the α5 subunit only incorporates at the accessory subunit location.

The second half of this dissertation seeks to identify residue interactions with positive allosteric modulators (PAMs) of the α7 nAChR. Chapter 4 focuses on methods development to study loss of potentiation of Type I PAMs, which indicate residues vital to propagation of PAM effects and/or binding. Chapter 5 investigates α7 receptor modulation by a Type II PAM (PNU 120596). These results show that PNU 120596 does not alter the agonist binding site, thus is relegated to influencing only the gating component of activation. From this, we were able to map a potential network of residues from the agonist binding site to the proposed PNU 120596 binding site that are essential for receptor potentiation.

Agonistas PPAR (Rosiglitazona, Bezafibrato e Fenofibrato) e alterações bioquímicas e estruturais em órgãos-alvo de camundongos C57BL/6 alimentados com dieta hiperlipídica rica em sacarose

Este trabalho teve o objetivo de estudar o efeito de medicamentos com diferentes ações agonista PPAR (rosiglitazona, fenofibrato e bezafibrato) sobre o perfil lipídico, glicídico e alterações na massa corporal e morfologia do tecido adiposo e pancreático em modelo de diabetes e sobrepeso induzido por dieta. Camundongos C57BL/6 (2 meses de idade) foram alimentados com dieta padrão (SC, n=10) ou dieta hiperlipídica rica em sacarose (HFHS, n=40) por 6 semanas. Logo após, os animais HFHS foram subdividos em: HFHS não tratado e HFHS tratado com rosiglitazona (HFHS-Ro), fenofibrato (HFHS-Fe) ou bezafibrato (HFHS-Bz) (5 semanas). Os camundongos alimentados com dieta HFHS apresentaram maior glicemia e insulina de jejum (+33% e +138%, respectivamente), intolerância à glicose, resistência à insulina, aumento da massa corporal (MC) (+20%) e adiposidade, hipertrofia de adipócitos e redução da imunocoloração para adiponectina no tecido adiposo. No pâncreas houve aumento da massa (+28%), acúmulo de gordura (+700%), hipertrofia da ilhota (+38%) e redução da imunocoloração para GLUT-2 (-60%). A rosiglitazona diminuiu a glicemia e insulina de jejum, porém induziu o ganho de MC e hipertrofia cardíaca. O fenofibrato estabilizou a MC, enquanto o bezafibrato levou a perda de MC. Apenas o bezafibrato impediu a hipertrofia da ilhota. A imunocoloração para GLUT-2 foi aumentada por todos os medicamentos, e não houve alterações na imunocoloração para o PPARα. Sinais morfológicos de pancreatite foram vistos no grupo HFHS-Fe, apesar dos níveis normais de amilase e lipase séricos. A rosiglitazona exacerbou a infiltração intrapancreática de gordura (+75% vs. HFHS), e o bezafibrato aumento a imunocoloração para o PPARβ/δ nas ilhotas pancreáticas. Em conclusão, o bezafibrato apresentou um efeito mais amplo sobre as alterações metabólicas, morfológicas e biométricas decorrentes da dieta HFHS, sugerindo que a inibição das três isoformas do PPAR seria melhor do que a inibição de apenas uma isoforma. A rosiglitazona exacerbou o ganho de MC, a infiltração de gordura no pâncreas e induziu hipertrofia cardíaca, assim, é necessário cautela ao prescrever este medicamento a um paciente obeso.

Cambios en las sinapsis del hipocampo de rata inducidos por el receptor de neurokinina 3 (NK3-R)

Mediante Western blot y cultivos celulares, en el presente trabajo se han observado las variaciones en el número de espinas dendríticas y sinapsis en neuronas del hipocampo de ratas macho de tres meses de edad, cuando se activa o se inhibe el receptor de neurokinina 3 (NK3-R) mediante drogas agonistas o antagonistas. Se ha visto que el gen de este receptor tiene una menor expresión en ratas muy ansiosas en comparación con las poco ansiosas, por lo que se pretende estudiar las implicaciones que pueda tener esta proteína en el trastorno de ansiedad.

Detección e inmunolocalización del receptor Mas en los espermatozoides humanos

En los último años el concepto clásico del Sistema Renina-Angiotensina (SRA)como un regulador de la función renal y cardiovascular ha cambiado. La identificación de nuevos componentes con diversas funciones ha ampliado el marco de actuación de este sistema. Estudios han demostrado que el SRA está involucrado en diversos procesos de reproducción masculina. En esta investigación se ha descubierto la presencia del receptor Mas de la angiotensina-(1-7) y de su mRNA en espermatozoides humanos mediante técnicas de inmunofluorescencia y RT-PCR.

Estudio del receptor 5HT2A y de la vía Akt/GSK3 como mecanismos moleculares de la psicosis inducida por el consumo crónico de cannabis

La esquizofrenia es una enfermedad mental de carácter crónico que afecta aproximadamente al 1% de la población , principalmente con edades comprendidas entre los 15 y 4 5 años . Se trata de una enfermedad de inicio en la adolescencia o comienzo de la madurez y cuyo potencial discapacitante persiste y se agrava a lo largo de la vida. La etiología de la esquizofrenia es multifactorial con evidencias de factores genéticos y ambientales. El carácter hereditario de la esquizofr e nia se ha estimado en un 73 - 90% y los factores genéticos que predisponen a la enfermedad son múltiples y heterogéneos. Los estudios epidemiológicos parecen sugerir que el mayor riesgo de desarrollar esquizofrenia es prod ucto de interacciones genético/ ambi entales (predisposición genética a agresiones ambientales) y del fenómeno de epistasis (fenotipo dependiente de la interacción entre diversos genes). Las manifestaciones clínicas de la esquizofrenia se dividen en síntomas positivos, síntomas negativos y dé ficits cognitivos. A causa de los déficits asociados y de su carácter crónico, la esquizofrenia se encuentra entre las diez causas principales de discapacidad por enfermedad del mundo y según la World Health Organization (2001), se estima que es la quinta enfermedad más costosa para la s ociedad en términos de atención requ erida y pérdida de productividad, con un coste anual en la Unión Europea que supera los 35 mil millones de €, según Andlin - Sobocki y Rössler (2005) . A nivel nacional, en el año 2009 los an tipsicóticos atípicos más empleados (risperidona y olanzapina) se encontraron entre los 8 primeros fármacos que más gasto generaron, con un coste económico superior a 350 millones de euros (Sistema Nacional de Salud (2010)) . Además, según el estudio de Pal mer et al. (2005), el riesgo de muerte prematura en la población esquizofrénica es aproximadamente dos veces mayor en la población general, siendo el suicidio la principal causa de este exceso de mortalidad, con una prevalencia estimada entre 5 - 10%. En est e sentido, se ha estimado que entre el 25% y 50% de los pacientes con esquizofrenia cometen por lo menos un intento de suicidio en su vida , según el estudio llevado a cabo por Meltzer (2002) . Dada su prevalencia, la tendencia a la cronicidad y el riesgo su icida de pacientes con esquizofrenia así como los elevados costes socio - sanitarios, la investigación de esta enfermedad es un objetivo prioritario de los sistemas de salud mundiales