FAPESP n: 01/02460-1

Principal Investigator: Ana Maria de Lauro Castrucci

Institution: Inst. Biociências/USP

Area of Knowledge: Physiology

Title: Melanopsin: Comparative gene physiology and light signal transduction in fish, amphibian and mammalian pigment cells.

Abstract:

The knowledge of a novel class of opsins, the melanopsins, raised new questions concerning its mechanisms of light signal transduction, and its gene regulation by light and hormones. These questions will be addressed with a comparative approach in teleost fish, amphibians and mammals, to test the following hypotheses:

1. the expression of melanopsins in cutaneous pigment cells appeared early and was conserved along evolution;

2. the response to light (pigment dispersionor aggregation) in GEM-81 fish erythrophoroma cell line, Xenopus melanophore cell line and mouse S-91 and Melan-A cell lines depends on melanopsin expression;

3. melanopsin expression may be regulated by light and hormones.

The following experimental strategies will be pursued:

1. the quantitation of melanopsin expression, proliferative rate and tyrosine activity in pigment cells in response to visible light;

2. the expression of melanotropic hormones' receptors, aiming to select those to be analyzed regarding their ability to modulate melanopsin expression;

3. the quantitation of melanopsin expression in response to hormones;

4. the determination of the signaling pathways evoked by light-melanopsin interaction;

5. the transfection with melanopsin gene of non-expressing melanopsin cells to rescue their ability to respond to light.

FAPESP n: 98/12139-1

Principal Investigator: Antonio Carlos Boschero

Institution: Inst. Biologia UNICAMP

Area of Knowledge: Physiology

Title: Mechanism of Insulin Secretion and Action In Different Animal Models: Neonatal, Malnourished, Hyperlipidemic, and Insulin Resistant.

Abstract:

Diabetes mellitus (DM) is defined as a sindrome of persistent hyperglycemia resulting from the lack of insulin production generally associated to increased resistance to the hormone. DM is classified in at least four categories: 1) Insulin dependent diabetes mellitus (IDDM) or type 1 diabetes; 2) Non insulin dependent diabetes mellitus (NIDDM) or type 2 diabetes; 3) Diabetes mellitus associated to other illness, or secondary diabetes mellitus, and 4) Gestational diabetes mellitus. The DM type 1, usually manifested during childhood and adolescence, has autoimmune origin. Some variation of this modality has been proposed recently. Among these, the latent autoimmune diabetes (LADA), that represents an expressive percentage of the adult diabetic patients with the onset of the illness around age 20. Almost 90% of diabetic patients belong to the type 2. This is an endocrine disorder characterized by multiple defects in insulin action and insulin secretion.

I. Insulin action in target celis involves the tyrosine phosphorylation of the insulin receptor (IR) and other proteins named insulin receptor substrates (IRSs).These substrates couple the IR to different intracellular proteins like PI-3-kinase,Grb2/S0S, SHP2, NCK, CRK, etc. We have recently demonstrated that IR, IRS-1 and IRS-2 are present in isolated islets of Langerhans. These proteins had tyrosine phosphorylation increased by high concentrations of glucose (1 1 or 22 mM) and by insulin (10-7M) indicating a regulatory role (feed back) of insulin on its own secretion. It was also demonstrated that IRS-2 knockout mouse (but not IRS-I)displayed features similar to that observed for type 2 diabetes; i.e.increased insulin resistance followed by reduction of insulin secretion. Interestingly, control of transcription of pro-insulin gene by insulin involves a cascade of reactions including IRS-2, PI-3-kinase, and p70 s6K showing that IRSS, especially the IRS-2,participate in the mechanism of insulin synthesis and secretion. In a first project we aim at studying the possible alterations in the cascade of events responsible for the insulin signal in the insulin secreting cells in: neonatal rats (where the insulin secretion mechanism is not yet fully developed), in malnourished rats (where the insulin secretion is reduced), and in different animal models of insulin resistance(that could provoke diabetes).

II. Hypertension and insulin resistance is generally associated to hyperlipidemia mainly hypertriglyceridemia. In a second project we will analyze the glucose homeostasis in an experimental model of primary hyperglyceridemia, the apolipoproteic CIII transgenic mice. The main goal is to determine if thisanomaly is related to dysfunction of insulin sensitivity and/or secretion.

III. Reactive oxygen species (ROS) have been implicated in the destruction of pancreatic B cells during the onset of type 1 diabetes induced by autoimmune process as well as by drugs such as alloxan and streptozootocin. It is suggestive that B cells possess reduced amount of enzymes such as; superoxid dismutase (SOD),catalase (CAT) and glutathione peroxidase (Gpx) responsible for the transformation of free radicals. Recently, a new enzyme, a thiol specific antioxidant (TSA), as cavenger of H202, was isolated firom yeast. The presence of this enzyme was also detected in nervous tissues and muscle of rodents,1 but its presence in the islets of Langerhans was not been investigated. Thus, in a third project we will study the possible presence and role of TSA in isolated islets and in an insulinoma cell line(HIT cells).

FAPESP n: 03/01577-8

Institution: Inst. Biociências/USP

Area of Knowledge: Physiology

Title: Causes and correlations of physiological variation: role of environmental and behavioral gradients at different levels of organization.

Abstract:

Evolutionary physiology integrates comparative physiology, ecology, evolution and systematics, aiming to understand how the evolution of physiological characters occur. Given its integrative nature, evolutionary physiology has a leading role in the discussion about the nature of animal adaptation, and has influenced significantly recent research tendencies in comparative physiology. However, given that evolutionary physiology is both conceptually and methodologically young, many relevant questions remain to be answered. The nature of variation of physiological traits figures among the least understood essential issues, for comparative physiology has traditionally stressed central tendency while neglecting the discussion about variance. It is frequent, for example, that studies in comparative physiology consider individual variation, a key issue in the neo-Darwinian concept of evolution by natural selection, as part of the unexplained variance. However, to understand how physiology evolves, it is fundamental to understand the scope, origin, and implications of physiological variance, the relationship between this variance and animal ecology, and the role of different sources of variation at different levels of organization. This proposal focuses on the nature of physiological variance at different levels of organization, and its relationships with ecology, evolution and animal distribution. The project focuses on five general questions that are conceptually related, and that will be studied in the context of ecological and behavioral gradients. The emphasis on these gradients serves to limit the study to problems in which a solid interaction between physiology, ecology and evolution is evident. The main general questions to be addressed are:
1. What is the role of the interaction between physiology and behavior to extend the geographical ranges of ectotherm vertebrates?;
2. How does simultaneous physiological adaptation occur as an evolutionary response to more than one eco-behavioral variables?;
3. 15 physiological variance additive?
4. What are the ecological correIates of physiological variance in ectotherm vertebrates at different levels of organization?;
5. What is the scope of physiological plasticity and the factors responsible for morpho-physiological differentiation within genera and species in ectotherm vertebrates?

We propose specific sub-projects that address, in specific contexts, the issues raised by each one of the main questions. Most, but not all sub-projects use ectotherm tetrapods as research models. Model selection was based mainly on adequacy to answer the question raised, but other factors such as previous experience of the main team and infrastructure already available were considered also. The proposal involves field and lab research, as well as theoretical considerations. When necessary, formal comparative analyses will be applied.

FAPESP n: 01/00009-0

Principal Investigator: Eduardo Moacyr Krieger

Institution: Inst. Coração/SSSP

Area of Knowledge: Physiology

Title: An integrated approach for the dissection of primary hipertension: molecular and functional characterization of the cardiovascular system

Abstract:
Cardiovascular diseases and in particular hypertension represent a major burden in terms of Public health worldwide. Hypertension is a complex disease where multiple genetic alterations influenced by a variety of environmental factors are essential for the manifestation of the phenotype. It is believed that hypertension results from an imbalance between the interplay of complex vasopressor/growth stimulating versus vasodepressor/growth inhibiting systems. Thus, one of the major challenges in the area is to dissect each component of these systems, its genes and their functional variants, their regulation, and their relative importance within this complex interplay which determines the levels of blood pressure within normal or pathological ranges throughout the life span. In this project we want to use a multidisciplinary approach with the participation of a team of researches with a wide range of expertise to address the topics listed below. All sections attempt to share the same approach aimed to integrate knowledge from DNA to bedside with more or less emphasis in cell, animal, or clinical work.

FAPESP n: 00/08982-7

Principal Investigator: Esper Abrão Cavalheiro

Institution: Esc. Paulista de Medicina UNIFESP

Area of Knowledge: Physiology

Title: Epileptogenesis in human epileptic tissue. Structural and electrophysiological characteristics of the human epileptogenic tissue obtained from surgeries for epilepsy treatment. Clinical, electrographical and pathological correlations.

Abstract:

Epilepsy is a frequent neurological disturbance which affects around 1 % of the population. Thirty to fourty per cent of patients present epileptic seizures which are considered refractory to antiepileptic drugs. The understanding of the epileptogenicity in this population would allow the development of new therapeutic modalities and adequate medical approach of this population.

Mesial temporal sclerosis, cortical developmental malformations and indolent tumors of the nervous system are among the pathological findings more commonly seen in this group of epileptic patients. The objective of this research aims to elucidate the mechanisms of epileptogenicity in these lesions whose electrophysiological and morphological basis will be approached in three distinct steps. These permitted the differenciation of three subprojects:

Subproject 1. Electroclinical characterization of patients with refractory partial epilepsy due to lesions whose mechanisms of epileptogenicity are not yet well known as double pathology of the temporal lobe (mesial temporal sclerosis associated with other lesions) and the cortical developmental malformations and indolent tumors of the nervous system.

Subproject 2. In vitro electrophysiological studies of the human epileptogenic tissue aiming at the investigation of the microphysiological basis involved in the epileptogenicity of mesial temporal sclerosis, cortical developmental malformations and indolent tumors of the nervous system.

Subproject 3. Anatomopathological characterization and molecular biology studies of the human epileptic tissue removed from these three types of lesions.

The data obtained from these three subprojects will be correlated according to Graphic 1 and the electroclinical studies and the epileptic tissue analysis will be developed at the Setor de Epilepsia and at the Laboratório de Neurologia Experimental of the Escola Paulista de Medicina, Universidade Federal de São Paulo.

Graphic 1. Epileptogenesis study in the human tissue.

FAPESP n: 00/12154-2

Principal Investigator: Francisco Rafael Martins Laurindo

Institution: Inst. Coração/SSSP

Area of Knowledge: Physiology

Title: Molecular physiology of redox signaling in the vascular system and cultured cell models.

Abstract:
Vascular endothelial dysfunction is primarily a dysfunction of redox signaling. These processes involve the effect of free radicals, redox-active metals and reducing equivalents on specific proteins that govern cell function. The enzymatic pathways underlying superoxide radical and other reactive oxygen species (ROS) production are yet unclear. A number of evidences, including work from our laboratory, suggest that NAD(P)H oxidase activity is a major source of vascular superoxide. Our data suggest that vascular NAD(P)H oxidase-mediated superoxide production has a signaling role in the early program of the vascular injury repair reaction. However, the enzymatic pathways underlying such NAD(P)H oxidase activity remain controversial. In particular, the extent to which the vascular enzyme resembles that of professional phagocytes is unclear. Other points are also unclear, namely: the physiological role of this(ese) enzyme(s) in vasomotricity and apoptosis, the factors that modulate enzyme activity and the role of reactive nitrogen species, e.g. peroxynitrite in enzyme function. Recently, our laboratory showed that reactive thiol groups have a potential permissive or inhibitory role in enzyme function, in a way unrelated to the intracellular glutathione redox status. It is possible, therefore, that the crucial thiol groups are susceptible to modulation by thiol oxidoreductases, e.g., protein disulfide isomerase, which could, therefore, have an important role in the regulation of the oxidase and consequently of overall redox signaling. Other important question is the possible existence of more than one oxidase, which could interact with NADPH oxidase in such a way to constitute a complex ROS generating system working at the same time as second messengers and as electron transport systems in membranes. These ROS sources interact with specific cellular targets involved in the control of apoptosis, differentiation and proliferation.

The overall aim of the project is to further our understanding of the enzymatic pathways and physiological processes related to the production of ROS, particularly superoxide, by the vascular system, as well as its interactions with nitric oxide. The major focus is the characterization of structural and physiological aspects of NAD(P)H oxidase(s) in vascular cells, in particular the role of thiol regulatory proteins and effects of the enzyme(s) in apoptosis, vasomotricity and cell differentiation. Those questions are investigated under the perspective of a role for ROS in the vascular response to injury.

Subproject 1 - Methodological aspects related to the detection of vascular NAD(P)H oxidase through cehmiluminescence and EPR techniques.

The goal is to assess the influence of lucigenin concentrations in the detection of vascular NAD(P)H oxidase activity, to validate EPR techniques for this purpose and to critically assess the composition of vascular homogenates with respect of possible contamination by mitochondrial material. Clarification of these aspects is a fundamental prerequisite to the execution of this thematic project.

Subproject 2 - Similarities between vascular and phaqocyte oxidase: the role of gp91phox subunit in the
production of superoxide and vascular reactivit

The objectives are: a) to assess superoxide production via NAD(P)H oxidase through EPR and chemiluminescence techniques in rings and homogenates of mice aortas, both from wild type specimens (C57/BL-6 background) and mice knockout for gp91phox subunit of the phagocyte oxidase; b) To assess superoxide production after ex vivo vascular injury in this model; c) To develop a model of vasoreactivity in response to exogenous NADH/NADPH.

Subproject 3 - Role of an endothelial NADPH oxidase bearing the Phagocyte-simile subunit gp91 phox in superoxide production and shear stress-dependent vascular remodeling.

The goal is to assess the role of this subunit, a putative constituent of the endothelial NADPH oxidase, in both the remodeling process and superoxide radical production in response to flow changes.

Subproject 4 - Influence of peroxynitrite anion in vascular NAD(P)H oxidase activity.

The objective is to study the effect of several concentrations of authentic exogenous peroxynitrite on vascular NAD(P)H oxidase activity. In addition, possible physiological consequences of this interaction will be studied, as well as possible mechanisms of peroxynitrite effects, with emphasis on reactive thiol groups and nitration of tyrosine residues. Eventually, the finding of an important peroxynitrite effect in the oxidase may be a relevant component of a redox cell signaling paradigm.

Subproject 5 - Role of the thiol oxidoreductase protein disulfide isomerase in the regulation of vascular NADPH oxidase and superoxide production.

The aim is to characterize the expression and activity of protein disulfide isomerase in vascular cells and to assess its functional correlation of NAD(P)H oxidsae activity and to the apoptosis process in cultured cells.

Subproject 6 - Redox signaling pathways in cell differentiation: role of intracellular thiol/disulfide equilibrium and thiol oxidoreductases.

The aim is to assess the effects of changes in intracellular glutathione or thioredoxin levels in cell differentiation, using a model of human monocytes. Redox-dependent modulation of several protein kinases will also be investigated.

Subproject 7 - The search for pharmacological evidences for a role of plasma membrane NADH oxidase(s) ("PMOR" - plasma membrane oxidoreductase system) in ROS reduction in vascular cells.

The goal is to collect pharmacological evidence for the existence of a functionally relevant PMOR system in ROS production in vascular smooth muscle cells, as well as its correlation with the phagocyte-simile NADPH oxidase and protein disulfide isomerase activity.

Subproject 8 - Additional characterization of the vascular redox. status throughout the vascular repair reaction to a mechanical injury.

Objectives: a) To study the time course, some underlying mechanisms and consequences of the depletion of superoxide dismutase activity in the vascular wall; b) To characterize the expression of protein disulfide isomerase as well as phagocyte-simile NADPH oxidase subunits in the vascular wall at different time points of the vascular response to injury and to correlate these variables to the occurrence of apoptosis; c) To study NADPH-stimluated superoxide-dependent vasomotricity after arterial injury.

Subproiect 9 - Characterization of NADPH/lucigenin reductase activity secrete vascular wall after arterial injurv.

In studies investigating the mechanism of NADPH oxidase attenuation to repeated injuries, we verified after ex vivo vascular injury the appearance in the incubation medium of a strong NADPH oxidase activity. The objectives of this study are: a) To characterize the kinetics and pharmacological profile of secreted NADPH oxidase activity; b) To tentatively reproduce this phenomenon in cultured vascular smooth muscle cells; c) To asses the role of protein disulfide isomerase in oxidase shedding; d) To purify enzymatic activity and try to identify some known components of the phagocyte-simile oxidase; e) Based on the results of the above projects, to identify an eventual correlation of this shedding phenomenon in the apoptosis process.

Subproject 10 - The role of thioredoxin in nitric oxide-dependent apoptosis

The aim is to develop a model of apoptosis triggered by NO in cultured cells and to assess, through manipulation of gene transcription, the effects of thioredoxin, a major intracellular thiol oxidoreductase, in that process. Superexpression or transfection with dominant negative gene will be performed through the TET-system.

Subproject 11 - C242T polymorphism of the NADPH subunit p22phox and superoxide production in human vessels.

The objective is to assess, in patients submitted to myocardial revascularization, the production of ROS in segments of mammary or radial artery, the production of ROS in peripheral blood leukocytes and to correlate each variable to the presence of homo or heterozygosis for the 242T allele. The purpose is to understand function all consequences of this mutation, as well as the role of p22phox-centered oxidase in free radical production in man.

The experiments proposed in this project are designed to allow the elucidation of some mechanisms governing enzymatic systems underlying redox signal transduction in vascular cells. The functional characterization of these interactions may constitute a basis for the future development of cell-free models of these enzymes, an advance that shall provide the detailed knowledge necessary to design rational pharmacological or genetic therapeutic interventions. Another potential advance is the identification of genotypic markers of increased cardiovascular disease risk. Finally, this project may contribute to the study of novel mechanisms or possibly novel markers of apoptosis, differentiation and proliferation of vascular cells.

FAPESP n: 99/03523-5

Principal Investigator: Janete Aparecida Anselmo Franci

Institution: Fac. Odontologia de Ribeirão Preto/USP

Area of Knowledge: Physiology

Title: Female Reproductive System: Neuroendocrine control and effects of stress.

Abstract:

Relevance- Understanding regulation of the ovarian cycle is important for control of fertility in animals, to elucidate and treat cases of infertility in women, to adapt anticonception strategies to personal needs, to treat behavioral alterations, such as premenstrual syndrome, and to promote adequate therapies for different female pathologies which are subjected to interference by variations in hormone titers during the reproductive cycles or ovarianquiescence. The hormonal variations that normally occur during the estrous cycle make the research on female animals more difficult, and consequently, most of the published investigations were performed on males. In Brazil there is only a small number of researchers in this field, including the ones which have joined forces in this project to study mechanisms of control in the release of reproductive hormones and on the effects of stress during there productive phase of life.

Objetives: The aims of this work are to investigate- 1) the central control of the reproductive process in cycling and in ovariectomized rats with or without hormone replacement. We will focus the research on the function of several central neuromediators (noradrenaline, angiotensin II, neuropeptide Y, nitric oxide) in the secretion of hormones related to reproduction, as well as on the funetion of ovarian steroids on the release of these neuromediators. 2) how sexual hormones can alter stress responses, and 3) how stress can alter reproductive functions.

Methods- The following techniques will be used in this project- vaginal smears to study estrous cycles, ovariectomv followed or not by hormonal replacement (estrogen and progesterone) to study the role of ovarian steroids on the responses observed and to compare them to those observed in cycling rats, electrolytic lesions to study the role of cerebral nuclei in the control of hypothalamus-pituitary-ovary function, juqular cannulation to collect serial samples for investigation of hormonal responses to different physiological challenges, radioimmunoassay to measure several hormones, catecholamines measurements by HPLC in dyalisate as well as cerebral tissue, ",punch " and microdialysis of cerebral nuclei to measure content and secretion of hormones and neurotransmitters in cerebral nuclei, activity measurements of nitric oxide synthase to analyze nitric oxide action, liquid or in situ hibridization to evaluate expression of genes coding for NPY and All receptors, neonatal stimulation to study its effects on the reproductive performance during adulthood, estimation of the number of brain cells to study stress-evoked morphologic alterations in cerebral nuclei, and behavioral computerized recording to evaluate the effects of stress and sexual hormones on behavior.

FAPESP n: 98/15846-0

Principal Investigator: José Antunes Rodrigues

Institution: Fac. Medicina Ribeirão Preto USP

Area of Knowledge: Physiology

Title: Neuroendocrine Control of Fluid and Electrolyte Metabolism.

Abstract:

FAPESP n 97/09904-5

Principal Investigator: José Cipolla Neto

Institution: Inst. Ciências Biomédicas USP

Area of Knowledge: Physiology

Title: Study the Relationship between the central nervous system and the pineal gland and its hormone-melatonin.

Abstract:

The aim of this project is to study the relationship between the central nervous system and the pineal gland and its hormone-melatonin. In the part of the study dedicated to the neural control of the pineal metabolism two phenomena will be approached: 1) the daily profile of the main pineal indolic compounds (looking at they amplitude, mesor, and acrophase) and 2) the pineal gland metabolic reactivity to nocturnal retinal short term photic stimulation.

These studies will be done in control intact animals as well as in ibotenic acid or electrolytic lesioned animals. In isolated cells from the central nervous system of intact or pinealectomized animals, we intend to study the action meltaonin on the cell metabolism and the ionic membrane currents (probed by patch clamp metodology). In addition, using the immunohistochemical c- fos detection we intend to study the neural reactivity to sistemic injection of melatonin in intact or pinealectomized rats.

FAPESP n: 97/12749-1

Principal Investigator: Luiz Roberto Giorgetti de Britto

Institution: Inst. Ciências Biomédicas USP

Area of Knowledge: Physiology

Title:

Abstract:

The nervous system has been the object of intensive investigation, which has generated a great deal of knowledge that is relevant not only for a better understanding of the neural basis of behavior and of neurological diseases, but also for the advancement of Science in general. Our laboratory has long been involved with the study of the functional organization of the visual system. We initially used in these studies an electrophysiological approach, and later started to use anatomical techniques. Most recently, and following a general trend in the Neurosciences, we have begun to investigate neural processes also from a cellular and/or molecular point of view, which permits a more detailed analysis of neural operations. The general objective of the present project is to chemically characterize neurons of the visual system of vertebrates, by means of the identification of their content of neuromediators, receptors and neuronal markers, such as the glutamate receptors, acetylcholine receptors, calcium-binding proteins and the Fos protein. Some aspects of the regulation of those proteins will also be addressed. Two main techniques will be employed to accomplish that objective, which are immunohistochemical methods and in situ hybridization, that will be often combined with heural lesions and tracer and toxin injections. We antecipate that this project will generate new and relevant information about the functional organization of the visual system and also about the neurochemical organization of the nervous system as a whole.

FAPESP n: 98/06231-2

Principal Investigator: Oswaldo Úbriaco Lopes

Institution: Esc. Paulista de Medicina UNIFESP

Area of Knowledge: Physiology

Title: Neural Cardiovascular Control: Different levels, Distinct Integrations.

Abstract:

The present project comprise several sub-projects which main objective, through a integrative approach, is to contribute to the understanding how the Central Nervous System (CNS) contributes to the maintenance of the cardiovascular function in the awake and the anesthetized animal. Two levels were initially selected: the medullary and the hypothalamic level.

FAPESP n: 98/11714-2

Principal Investigator: Rui Curi

Institution: Inst. Ciências Biomédicas USP

Area of Knowledge: Physiology

Title:

Abstract: