DITS

CNC – The Center for Neuroscience and Cell Biology

Introduction

The Center for Neuroscience and Cell Biology (CNC) was founded in 1990 at the University of Coimbra. The principal founding members of CNC were the University of Coimbra, JNICT, recently integrated in the Ministry of Science and Technology, and the University Hospital.

Members of the staff of three faculties - Faculty of Science and Technology, Faculty of Medicine and Faculty of Pharmacy - form the scientific core of the CNC. This unique partternship was formalized in 1990 in agreement with each of the three faculties which permitted CNC attracting substantial financing for equipment, installations and for research activities. This agreement envisages the use of common existing installations, and, more important, brings together a faculty of graduate studies with research interests and scientific affinities that span a large spectrum of Fundamental Biology and Biomedicine.

There is a wide variety of training opportunities in Fundamental Biology and in Biomedicine available to graduate students at CNC. Faculty members from the various schools - Medicine, Science and Technology, Pharmacy - and staff members linked to the Hospital interested in various fields of Biology or Biomedical Sciences are encouraged to develop research programmes adapted for their objectives. Each research programme is headed by the proposing faculty member linked to any of the faculties, or to the Hospital, and by a research member at CNC.Biology and Medicine are going through an unprecedented revolution which is unravelling the molecular mechanisms governing cellular processes, such as replication, intracellular signalling, neuronal communication, embryonic development. From these insights have evolved new strategies and technologies for addressing the diagnosis and therapy of disease. Most of our programmes offer research opportunities which span from Fundamental Biology to basic and clinical research. We hope that in the future our programmes will link "the bench to bed side", and will open doors for rewarding carreers in many fields including academia, health and environmental services, industry, and government affairs.

Main Intervention Areas

Departments
The CNC is structurally subdivided in ten departments, each headed by a director, and the Laboratory of NMR. The departments join in developing departmental and interdepartmental research programmes, such as:
 Cell Biology;
 Biophysics;
 Biochemistry;
 Neurochemistry;
 Neuropharmacology;
 Molecular Biotechnology;
 Neurobiology;
 Microbiology;
 Vectors and Gene Therapy;
 Graduate Studies;
 Advanced Strategic Studies.

Strategic Research Programmes
The various departments collaborate in implementing the following strategic research programmes linked to the strategic areas of Health and Environment:
 Neuroscience and Disease;
 Molecular Biotechnology and Health;
 Cell and Molecular Toxicology;
 Microbiology;
 Biophysics and Biomedical NMR;
 Cell and Development Biology.

Neuroscience and Disease

Introduction

The area of Neurosciences and disease at CNC maintains its previous general organization with two parallel research areas: 1) basic mechanisms of modulation of neurotransmission, and 2) mechanisms involved in neurogeneration and neuroprotection.

This year, a major effort was started to unite and potentiate the research effort in the areas of neuromodulation and neuroprotection around some particular candidate systems, namely adenosine, neurotrophins and NPY. Thus, there was a detailed exploitation of these modulatory systems both in terms of their physio-pharmacological characterization as well as to their role as neuroprotective agents in different models of neurodegeneration.

In parallel, different groups continued to characterise different models of neurodegeneration, namely: beta-amyloid-induced toxicity (model of Alzheimer’s disease), prion-induced toxicity, amygdala-kindling (model of epilepsy), STZ and GK rats (as a model of diabetes), 3-NP and cells transfected with expanded polyglutamine-cointaining proteins (polyglutamine-associated diseases) and drugs of abuse.

These groups were particularly active in exploring the role of demises of cell death (apoptotic and necrotic pathways), in particular of caspases and calpains, of NO, of immediate early genes and also of the modification of the signalling by ionotropic glutamate receptors in terms of pre- and post-synaptic roles, of their different subunit assembly, of the impact of phosphorylation on their actions and trafficking and on the identification of new interacting partners.
A major trend has been initiated trying to enhance the scope of the available disease models by preferentially using both in vitro as well as vivo models in parallel.

The groups in these areas interact strongly with other areas, namely: Molecular Biotechnology and Health, Cell and Molecular Toxicology and Biophysics and Biomedical NMR within CNC.

In addition to this collaboration all the groups have established strong collaboration with national and international laboratories as can be evaluated by the authorship of published papers and organisation of Advanced Courses (see Graduate Programme section).

Neuromodulation

Adenosine is a neuromodulator that mainly acts via two types of G protein-coupled receptors: the more abundant A1 receptor (A1R) and facilitatory A2A receptors (A2AR). Adenosine is considered a neuroprotective agent because it is released in high amounts on stressful situations and because A1R activation delays the onset of noxious brain dysfunction and neuronal damage based on its ability to hyperpolarise neurons and to depress glutamatergic transmission. Recent studies have also reported that the blockade of A2AR also confers a robust neuroprotection by mechanisms that remain unresolved.

The major aims are: 1) To explore how the balanced activation of these two receptors with opposite actions control excitatory transmission and how this balanced A1R / A2AR modulation is modified in chronic stressful situations that characterise neurodegenerative disease. 2) To test in animal models of chronic neurodegenerative diseases the potential therapeutic interest of pharmacological manipulation of adenosine neuromodulation.

Research Highlights
In hippocampal synapses we observed that A1R are 10 times more abundant than A2AR, both receptors being enriched in nerve terminals. Stressful situations, both physiological (aging) and pathological (epilepsy), decrease in A1R and increase in A2AR density and efficiency. Thus, A2AR blockade may be a prominent target to control neurodegeneration . In fact, we first demonstrated a full prevention of b -amyloid neurotoxicity (a peptide thought to cause neurotoxicity in Alzheimer’s disease) by A2AR blockade. Likewise A2AR blockade prevented the bacterial toxin (LPS)-induced neuroinflammation (which occurs in several neurodegenerative diseases) and consequent neuronal dysfunction.

Ongoing and Future Work
To extend the proof of concept for the usefulness of A2AR antagonists in in vivo models of diseases like epilepsy and Alzheimer’s disease.

Molecular Biology of Glutamate Receptors

Glutamate receptors mediate the majority of excitatory neurotransmission in the central nervous system, and changes in the characteristics and cellular localization of these receptors are thought to underlie the mechanisms for synaptic plasticity.
Our group focuses on investigating the mechanisms that regulate glutamate receptor diversity, activity and cellular traffic.

Research Highlights
We are studying the regulation of the GluR4 subunit of AMPA receptors by phosphorylation, and find that GluR4 is phosphorylated, and its phosphorylation regulates receptor activity (Carvalho et al, 1998 J. Neurochem., 1999 J. Neurosci., 2002 Eur. J. Neurosci.). When GluR4 is phosphorylated by PKA, it is targeted to the cell surface and to the synapse. Moreover, in the retina, metabotropic receptors of glutamate and dopamine co-regulate GluR4-containing AMPA receptors via PKA (Gomes et al, 2004, J. Neurochem.; see figure below).
We are also interested in the mechanisms that generate AMPA receptor diversity. Alternative splicing originates the flip and flop isoforms of AMPA receptor subunits. We found that truncated isoforms are also generated, and that these proteins have a dominant negative effect when they are integrated in the receptor complexes. We are currently investigating the functional significance of the truncated versions of AMPA receptor subunits.

Another major interest of the group concerns proteins that interact with AMPA receptor subunits and that have a role in the receptor traffic. We have particular interest for interacting partners of GluR4, since this AMPA receptor subunit is the first to be targeted to functional synapses in the hippocampus during development, and its synaptic targeting relies on synaptic activity. So far, we have found that GluR4 interacts with PKCγ (Correia et al, 2003, J. Biol. Chem.), and that this interaction is necessary for efficient GluR4 phosphorylation by PKC. Ongoing work focuses on the identification of other GluR4 binding partners.

Ongoing and future work
We focus on understanding the mechanisms that regulate AMPA receptor subunits traffic by looking for AMPA receptor interacting partners, using proteomics approaches. Electrophysiology and imaging studies are used to test the functional relevance of the interactions.

Another project addresses the effect of external stimuli on the expression of glutamate receptor subunits in cultured hippocampal neurons.

Neurotoxicity and Epilepsy

Hyperexcitability of the hippocampal neuronal network can cause hyperactivation of glutamate receptors and neuronal death. The search for new antiepileptogenic and neuroprotective compounds is of critical relevance for the treatment of the pharmaco-resistant forms of temporal lobe epilepsy. Neuropeptide Y (NPY) and NPY receptors are highly up-regulated in temporal lobe epilepsy and its plausible to think that NPYergic modulation of glutamate release may limit hyperexcitability and support neuroprotection. The major aims of this group are:

1) To determine a role for NOS and calpain activity in excitotoxicity, 2) To identify the NPY receptor modulating glutamate release in the rat hippocampus, 3) To identify a neuroprotective role of NPY receptor activation against excitotoxic insult.

 Research Highlights
We have determined that NO is involved in the neurotoxicity caused by the activation of non-desensitizing AMPA receptors although to a limited extent, since AMPA receptor activation triggers mechanisms that lead to nNOS proteolysis by calpains, preventing further contribution of NO to the neurotoxic process (key publication: Araújo et al., 2003, J. Neurochem.).

NPY Y2 receptor play a major role in inhibiting the release of glutamate from rat hippocampal synaptosomes. We have described for the first time strong evidences for the functional interaction between Y2 and Y1 and Y2 and Y5 receptors in the CA3 and DG. Interestingly in the CA1 subregion only functional Y2 receptors able to inhibit the release of glutamate were identified (Y2/Y1 and Y2/Y5 oligomerization?) (key publication: Silva et al, 2003, Neuropharmacol.).

Using organotypic cultures of rat hippocampal slices we firstly identified a neuroprotective role for NPY through Y1, Y2 and Y5 receptors against excitotoxicity. Accordingly with our findings on the modulation of glutamate release in the CA1 subregion only Y2 receptors play a neuroprotective role, whereas in the CA3 and dentate gyrus the activation of Y1, Y2 or Y5 receptors protected the cultured slices from neuronal death caused by exposure to AMPA or kainate (key publication: Silva et al, 2003, FASEB J.)

 Ongoing and Future Work:
We are searching differences between pre- and postsynaptic kainite receptors subunit composition, pharmacological and electrophysiological properties, aiming at identify key elements in the physiological basis of kainate-induced epilepsy and excitotoxicity. Moreover, we are also correlating in vivo activation of calpains and excitotoxicity in the kainate model of temporal lobe epilepsy. The neuroprotective role of NPY receptors and its intracellular signalling pathway is now one of our key research projects. The neuroprotective/neurotoxic role of inflammatory cytokines and microglia activation in temporal lobe epilepsy is also a key issue for the year 2004.

Neuroprotection

In addition to promoting the survival and differentiation of developing neurons, neurotrophic factors protect neurons against toxic injury. We are studying two aspects of neurotrophic factors biology: i) control of the endogenous expression upon neuronal injury; ii) signaling mechanisms responsible for protection against excitotoxic conditions.

Research Highlights:
Glial cell line-derived neurotrophic factor (GDNF) has been shown to protect against injury and to rescue damaged dopaminergic neurons in animal models of Parkinson’s disease (PD). We are looking at GDNF expression in substantia nigra postnatal cell cultures, upon selective damage to dopaminergic neurons due to increased oxidative stress or L-DOPA, the main symptomatic treatment for PD. We found that mild, but not strong toxic conditions upregulated the expression of GDNF , both at the mRNA and the protein levels, in mixed neuron-glia cultures but not in astrocyte cultures . GDNF upregulation was mediated by soluble modulators, likely released by damaged neurons, since conditioned media from injured mixed neuron-glia cultures increased GDNF expression in astrocyte cultures.

In the same conditions, we found a transient increase in the expression of heme-oxygenase 1 (HO-1, also known as heat shock protein-32) in substantia nigra mixed cell cultures. We showed that the secondary downregulation of HO-1 was associated to the increased expression of GDNF. Ongoing experiments aim at identifying the diffusible signals involved in neuron-glia crosstalk upon dopaminergic cell damage.

In vivo and in vitro studies showed that endogenous BDNF protects a subpopulation of hippocampal neurons after ischemia/reperfusion, and pre-incubation with BDNF also protected cultured hippocampal neurons from glutamate-induced cell death. Since activation of the TrkB receptors by BDNF sets in motion parallel signaling pathways, with distinct functions in the cell, we determined the relative role of the Ras/MAPK and of the PI3-K (phosphatidylinositol 3-kinase) pathways in the protection of hippocampal neurons by BDNF under excitotoxic conditions. Using pharmacological inhibitors and by transfecting neurons with dominant negative constructs we showed that both signaling pathways are involved in the protection of hippocampal neurons by BDNF (see figure). Protection of hippocampal neurons occured upstream of caspase-3 activation and was mediated by de novo protein synthesis.

Ongoing and Future Work:
Ongoing studies focus on the identification of changes in the proteome that may account for the observed neuroprotective effects of BDNF. We found that BDNF upregulates Bcl-2 protein levels, a well know anti-apoptotic protein, but does not down-regulate glutamate receptor subunits of the NR (NMDA receptor subunits) and GluR (AMPA receptor subunits) families. Proteomics studies are also being carried out, in order to screen for effects of BDNF on the proteome of cultured hippocampal neurons. Future studies will show which of the changes in the proteome are relevant for neuroprotection by BDNF.

Mitochondrial Dysfunction and Cell Death

Neuronal cell death has been associated with changes in mitochondrial function, deregulation of calcium homeostasis and oxidative stress, which are on the basis of excitotoxic processes. Mitochondria play a central role in apoptotic pathways. Subsequent disruption of the electron transport chain results in increased superoxide production, originating other deleterious reactive oxygen species.
The main aim of our research is to examine the intricate mechanisms underlying selective neurodegeneration in Alzheimer’s and Huntington’s diseases, with the perspective of bringing about new hypothesis for therapeutic targets against these neurodegenerative disorders.

Research Highlights:
We have made important contributions to elucidate the downstream pathways upon selective activation of non-desensitizing AMPA receptors in cultured rat hippocampal neurons. AMPA-mediated excitotoxic neuronal death (apoptosis and necrosis) was linked with a moderate generation of mitochondrial superoxide and an increase in Mn-superoxide dismutase activity (Rego et al, J. Neurochem, 2003). The importance of NMDA-R subunits, 2A and 2B, on A b toxicity was investigated using HEK293 cells transfected with NR1/NR2A or NR1/NR2B subunits. We determined an increased toxicity in cells expressing NR1/NR2A upon exposure to A b 1-40.

We showed that endogenous reactive oxygen species and a rise in intracellular calcium are important inter-players in staurosporine-triggered apoptosis in cultured retinal neurons. Thus, antioxidants and inhibitors of endogenous sources of radical production prevented retinal cell death (Gil et al, Free Radic. Biol. Med., 2003). We also demonstrated that insulin protected against loss of cell viability and oxidation induced by ascorbate/Fe 2+ in cultured cortical neurons. Insulin neuroprotection was due to an increase in uric acid and glutathione antioxidant defences. Insulin also stimulated neuronal glucose metabolism, by restoring cellular energy charge (Duarte et al, submitted for publication).

By using striatal cells expressing mutant (Q111) or normal (Q7) huntingtin, derived from knock-in mice, we are studying the mitochondrial potential and the changes in intracellular Ca 2+ homeostasis by fluorescence microscopy in single cells. The analysis of neuronal cell death induced by proteins with polyglutamine expansions will be extended to the study of mutant ataxin-3. We will further examine insulin receptor-mediated n europrotection upon oxidative stress and beta-amyloid toxicity in neuronal cultures. Furthermore, we will explore the molecular determinants of NMDA receptor subunits on the toxicity of beta-amyloid peptide.

Molecular Mechanisms of Disease

The scrapie isoform of prion protein (PrPSc) and the amyloid-beta protein (Aβ), resulting from the abnormal proteolytic cleavage of amyloid precursor protein, are involved in the pathogenesis of prion-related encephalopathies (PRE) and in Alzheimer’s disease (AD), respectively. These are protease-resistant proteins that have a structure enriched in β-sheet conformation and aggregate extracellulary in brain as amyloid fibrils.

The main objectives of the research performed during 2003 were to investigate: i) the effect of Aβ and PrP peptides on synaptic function focusing in particular on the role of endoplasmic reticulum stress; ii) the alterations in signalling phosphorylation pathways induced by the Aβ and PrP peptides; iii) the role of mitochondria in Aβ- and PrP-induced cell death. For this propose, we used cultured cortical and hippocampal rat neurons, synaptosomes, isolated brain mitochondria, mtDNA-depleted NT2 cells (γ0 cells) and cybrids from AD patients treated with synthetic peptides of Aβ and PrP.

Research Highlights:
The Aβ and PrP peptides induce neuritic dystrophy and loss of synaptic function by a mechanism involving perturbation of endoplasmic reticulum (ER) Ca 2+ homeostasis due to the release of Ca 2+ through Ca 2+ channels associated with the 1,4,5-inositol triphosphate receptor (IP 3R) and ryanodine receptor (RyR). These alterations were early events of neuronal injury ( J. Neurosci. Res., in press) . The Ca 2+ overload caused by Aβ and PrP peptides induced calcineurin upregulation. This Ca2+/calmodulin phosphatase, by mediating BAD dephosphorylation, triggers a mitochondrial-dependent neuronal apoptotic pathway ( Eur. J. Neurosci. 2003, 17: 1189-96).

Using mtDNA-depleted NT2 cells (γ0 cells) it was observed that A b induces oxidative stress and apoptosis only when a functioning mitochondrial electron transport chain is present ( Free Rad. Res. 2003, 37: 967-73) . In AD cybrids it was found an increase in ROS production and reduced cytochrome oxidase activity and ATP levels. In the presence of A b , events associated with programmed cell death are activated in AD NT2 cybrids to a greater extent than they are in control cybrids or the native NT2 cell line, suggesting a role for mtDNA-derived mitochondrial dysfunction in AD degeneration (J. Neurochem., in press).

The type-2 diabetes related mitochondrial dysfunction is exacerbated by aging and/or by A b , supporting the idea that type-2 diabetes and aging are risk factors for the neurodegeneration induced by A b peptides (Diabetes 2003, 52: 1449-56) . Insulin seems to prevent the decline of mitochondrial oxidative phosphorylation efficiency and the oxidative stress induced by Aβ peptides.

Ongoing and future work:
Neuroinflamation in Alzheimer’s disease and Prion-related encephalopathies. The involvement of microglia in neurodegeneration caused by amyloid-beta and prion peptides.

Synaptotoxicity induced by the AD-associated amyloid-beta peptides. Role of peptide oligomerization state.

Molecular Biotechnology and Health

Introduction
The general objectives of this area are: 1) The study of the structure-function relationship of proteins, in particular of proteins involved in disease, aiming at the design of modulators and inhibitors, 2) The development of carriers for drug and oligonucleotide delivery target to specific cell and tissues.
The investigators of this area are organized in four research groups, each having specific scientific objectives, but collaborating with each other, for the general objectives of the area:
The Molecular Biotechnology group has focused its attention on the structure-function relationship of proteases and developed a particular Know-how on protein purification, protein chemistry and production of recombinant proteins.

The Protein Stability and Folding group focused its research on conformation plasticity and amyloide formation. Recently the group initiated the implementation of methodologies to carry out Molecular Dynamic Simulations of proteins in parallel computer environment.

The Protein Crystallography group defined as prime line of research the structural characterization of proteins associated with human diseases, this led to a strong interaction with several groups of CNC.

The Vectors and Gene Therapy group have develloped successfully nanosystemswith adequate features for in vivo use, capable of targeting specific cell or tissues. More recently the group acquired expertise in the devellopment and application of lentiviral vectors for gene delivery. This group interacts strongly with the Molecular Biotechnology group, which provides recombinant peptides, as well as, advice and support in cloning processes and design of oligonucleotides. Another crucial interaction of the group, within CNC, is with the Molecular Mechanisms of Disease Group which provides advise on the development of animal disease models.

In addition to the collaborations mentioned above, all the groups have established strong collaborations, with national and international laboratories, these collaborations can be evaluated on the bases of: authorship of published papers, co-supervision of theses and organization of advanced courses.

Molecular Biotechnology

In recent years proteases have become or have been exploited as potential targets for drug-based therapy. Examples of this trend are: HIV protease in AIDS, ACE in blood – pressure regulation and BACE in Alzheimer’s disease. A detailed analysis of protease structure and function is therefore crucial not only to fully understand biochemistry and biology of these enzymes, but also to assess whether they can be good therapeutic targets.

Protein Stability and Folding

The issues of protein stability and folding have recently become issues of large impact with the public and the press, specially due to the implications for animal and human health. However, several human and animal pathologies have been related with problems of protein folding and stability. These diseases range from Cystic Fibrosis to all the know amyloid diseases, such as BSE, Alzheimer´s, Creutzfeld-Jacob and many others.

Research Highlights
In the past few years the group has devoted his research efforts to problems of protein folding, stability, conformational plasticity and amyloid formation. The group has contributed with significant advances for the understanding of the molecular mechanisms of amyloid formation by Transthyretin (TTR), the protein responsible for the development of FAP (Familial Amyloid Disease), a genetic disease of very poor outcome in most patients. Culminating these efforts, it was published in 2003 an invited review paper on the molecular mechanism of amyloid formation and their relationship to TTR conformational stability.

Additionally, the group continued the work on the mechanisms of conformational plasticity of membrane peptides, in particular delta-toxin. The group is interested in studying the aggregation states and peptide structural changes produced by different chemical environments and membrane mimetic environments, in order to understand the mechanisms of peptide insertion into biomembranes.

During 2003, the group also initiated a significant effort in the implementation of methodologies to carry on Molecular Dynamics simulations of proteins in a parallel computer environment using the Linux cluster of the Centre of Computational Physics of the "Universidade de Coimbra". This effort is already producing very interesting outcomes with results on computational simulations of TTR unfolding, which we believe is the basis for protein aggregation and amyloid formation in TTR. These results, will allow the publication of several papers in the near future, one of them already in press. Additionally, invitations were received for two oral communications, one in France and another in the USA, early in 2004.

Vectors and Gene Therapy

The CNC laboratory of vectors and gene therapy is devoted to the design and development of carriers for gene, antisense oligonucleotide, siRNA and drug delivery as well as to the evaluation of their potential for in vitro and in vivo applications. The vectors are used for different purposes including molecular functional studies, establishment of novel models for disease and development of new molecular therapeutic strategies (antisense and gene therapies).

Research Highlights:
Regarding the development of non-viral carriers our specific aims are to generate nanosystems with adequate features for in vivo use, while allowing targeting to specific cells or tissues and enhanced intracellular delivery. The potential of karyophilic cell permeable peptides to enhance nuclear transfer of genetic material has also been explored. Cancer has been the main target disease in which approaches dealing with the addition of therapeutic genes or antisense oligonucleotides have been evaluated. Different animal models of human and murine cancer have been generated, namely of oral cancer, lung cancer and acute lymphoblastic leukaemia. The group has also expertise in the development and application of lentiviral vectors for gene delivery. Studies on viral vectors production, optimization of the doses, duration of gene expression and neuronal tropism have been addressed, both in vitro and in vivo. During 2003 , the group achieved a specific inhibition of DHFR mRNA upon treatment of leukaemia cells with antisense oligonucleotides encapsulated into targeted sterically stabilized pH-sensitive liposomes , which resulted in a significant reduction in cell proliferation. A significant antitumor effect (as evaluated by the reduction in tumor size) was observed in a mammary carcinoma murine model developed in our group, upon treatment of the animals with lipoplexes containing genes encoding either IL-12 or HSV-TK (followed by ganciclovir administration) or using a combination of both strategies. Regarding the potential of the developed gene therapy strategiesfor CNS, in vivo efficient transfection of the cortex upon stereotactic administration of transferrin-lipoplexes into the mouse brain was also demonstrated. In addition a comparative study of GDNF delivery systems for the CNS involving polymer rods, encapsulated cells, and lentiviral vectors has shown that all the systems were able to mediate efficient brain delivery.
The group also maintains a direct research interest in understanding the mechanisms of nucleo-cytoplasmic trafficking of African Swine Fever virus (ASFV) genome aiming at the development of novel and efficient therapeutic anti-viral strategies. This work is a continuation of our longstanding research interest in the molecular mechanisms involved in the initial steps of enveloped virus-cell interaction, particularly in the process of membrane fusion.During 2003, the group has identified two ASFV proteins (p10 and p14.5) that represent potential candidates to mediate nuclear import of the viral DNA. Two other viral structural proteins (p37 and p14) were shown to exhibit nucleo-cytoplasmic transport activities. Critical residues (NLS and NES) to the functionality of these various proteins have also been identified. The role that these proteins play in the replication cycle of ASFV is currently being investigated.
Our current efforts are aimed at achieving the goals mentioned to above, namely to further improve the nucleic acid delivery systems for applications in gene therapy and gene silencing. In addition, significant efforts will be devoted to evaluate the potential of the developed carriers to mediate the delivery of therapeutic genes aiming at the treatment of neurodegenerative and vascular diseases.

Cell and Molecular Toxicology

Introduction
This area includes three main research groups working on partially overlapping areas:
Mitochondrial Toxicity Group particularly interested in drug- and disease-induced mitochondrial dysfunction and cytotoxicity, correlating mitochondrial toxicity and tissue damage. In connection with these studies, and interfacing with the Area of Biomedical NMR, a complementary research area is emerging on metabolic changes (NMR) underlying pathophysiological events of different tissues;
Free radicals and Antioxidants Group , involved in studies of free radicals and oxidants, either as messenger molecules, relevant to physiological functions, or as promoters of toxic pathways in the pathogenesis of some disease processes. In addition, it is engaged in research on the molecular mechanisms underlying the protective role of dietary antioxidants against damage due to free radicals and oxidants, particularly in the context of atherosclerosis prevention. Studies on free radicals mechanisms critically inherent to neuromodulation and aging are in connection with the Area of Neuroscience and Disease;
Membrane Toxicity Group (biophysical approach), focused on the interaction of xenobiotic molecules, including pesticides, environmental pollutants and pharmaceutical drugs, with membranes in order to elucidate the molecular mechanisms underlying their toxicological effects upon biological systems.

Mitochondrial toxicity

Mitochondria play a pivotal role in cellular metabolism and in energy production. Research over the years has demonstrated that mitochondria, besides being the powerhouses of the cells, have also important roles on cell calcium homeostasis and on several pathways for cell death.

Numerous examples of mitochondria-mediated cell injury can be found in the literature; not only chemicals can negatively affect mitocondrial function but also the origin and progression of several pathologies is closely related with disruption of mitochondrial homeostasis. The main and general objective of the Mitochondrial Toxicology Group is to provide an insight into the role of mitochondria as a primary intracellular target in the initiation of drug- and disease-induced cell dysfunction.

Research Highlights:
1) Cholestasis, steatosis and hepatic ischemia: bile acids therapy. Cellular mechanisms with relevance to mitochondrial dysfunction: Our present understanding of the pathogenesis of cholestasis and fatty liver disease (FLD) is based on studies performed with experimental animal models. Both pathologies result in more susceptible organs to hepatic ischemia/reperfusion injury and in severe mitochondrial damage. We have performed studies showing mitochondrial alterations present in the livers of animal models and the toxicity/protection of bile acids at the hepatocytes level.
2) Drug-Induced Mitochondrial Dysfunction and Cytotoxicity: Mechanisms of drug-induced mitochondrial dysfunction are studied. Tamoxifen, an anti-neoplastic agent, and 4-hydroxytamoxifen were shown to protect brain mitochondria from oxidative stress. In a different approach, we observed that all deleterious effects induced by tamoxifen were highly exacerbated in the presence of 17b-estradiol. We investigated the mechanisms by which doxorubicin (DOX), another potent anti-neoplastic agent, is toxic to the heart, with a special focus on the role of mitochondria. In vivo studies showed that DOX alters the regulation of the mitochondrial permeability transition through thiol oxidation and alteration of protein expression. Endurance exercise but not vitamin E was demonstrated to prevent DOX-induced cardiac mitochondrial dysfunction. On the other hand, we showed that DOX and tert-butylhydroperoxide cause mitochondrial dysfunction and apoptosis on H9c2 myoblasts. The group has also investigated the effects of other compounds such as ecstasy or cerebrocast on mitochondrial function.
3) Diabetes-induced mitochondrial dysfunction: The group has demonstrated how diabetes affects the mitochondrial function of brain, kidney, heart and liver mitochondria. A special highlight is the study of the interrelation between diabetes, mitochondrial dysfunction and Alzheimer’s disease, specially the protective role of insulin or coenzyme Q10.

Ongoing and future work:
Newly synthesized compounds are being tested in different cancer lines to identify powerful and novel anti-cancer agents. In this context, phenolic acids and polyamines complexed with platinum or palladium are being used to distinguish mitochondrial vs. nuclear effects. We are now investigating the role of mitochondrial dependent and independent mechanisms of cell death induced by DOX on H9c2 myoblasts. The regulation of the expression of FXR (a bile acid receptor) targets is also being subject of research. A possible link with regulation of mitochondrial biogenesis is also focus of attention. Another project addresses the effects of bile acids therapy on mitochondrial function and fatty acid metabolism of steatotic animals, looking for early molecular alterations that will be responsible for a progressive alteration of mitochondrial and cell function. We will be complementing studies performed on isolated mitochondria with studies performed with intact cell systems.

Free Radicals and Antioxidants

Free radicals and oxidants are messenger molecules that modulate physiological functions but inappropriate or delocalized production may trigger toxic pathways. The molecular mechanisms that determine how the balance between physiology and pathology tilts remain largely unknown. In particular, nitric oxide performs a variety of functions in the central nervous system, ranging from neurotransmission to neurodegeneration. On the other hand, a network of antioxidants prevent and inhibit damage caused by free radicals and oxidants. For instance, the oxidation of low density lipoproteins and the oxidative/nitrosative stress of vascular endothelial cells are considered to be critical events in the development of atherosclerotic lesion. Long term objectives of this group involve: 1) the study of molecular mechanisms inherent to neuromodulation, and aging that critically involve free radicals and oxidants, particularly nitric oxide; 2) the establishment of the molecular mechanisms underlying the health-promoting role of plant-derived dietary phenolic antioxidants, particularly those present in wine and olive oil, in the context of atherosclerosis prevention.

Research Highlights
Real-time measurement of nitric oxide in hippocampal brain slices: implications for neuronal degeneration

We established the dynamics of NO production and removal/decay in the subregions of hippocampus evoked by activation of NMDA sub type glutamate receptors. For that we have implemented an electrochemical strategy for the selective and real-time measurement of NO in hippocampal tissue that involves the use of microsensors that can be placed into brain with minimal damage to the tissue. Specifically, a gradient of NO was found along the trisynaptic loop of hippocampus (highest transient concentration found in CA1 region) which is likely to determine differential responses of the sub regions to signaling pathways mediated by NO.

Dynamic interactions of phenolic compounds from wine and olive oil in plasma and lipoproteins and protective effects against human LDL oxidation: relevance to atherosclerosis prevention.

We have implemented a micelar model using Electron Paramagnetic Resonance, to assess the crtitical chemical features of procyanidins from wine and olive oil that support the efficient reduction of tocopherol radical at water: lipid interfaces. Moreover, it was demonstrated the ability of caffeic acid and other phenolic compounds to reduce inorganic nitrite to nitric oxide in a pH-dependent fashion, a mechanism with potential biological implications in situations of ischemia/reperfusion.

On the other hand, we have compared the antioxidant potential of some structurally related compounds present in red wines, anthocyanidins and anthocyanins (the glycosidic forms), toward human LDL oxidation induced by several oxidants and the ability to recycle alfa-tocopherol, observing that their antioxidant activities are highly modulated by the substitution pattern in the B ring and glycosylation at C3 position. Also, we characterized the anthocyanin profiles of some varietal Portuguese red wines and correlated them with the radical scavenging activities of anthocyanin fractions against LDL induced oxidation. Anthocyanins, as highly potent inhibitors of LDL oxidation, could be a key component of red wine in the light of its protection to cardiovascular diseases.

Ongoing and future work:
To establish a functional connection between the gradients of NO in the different subregions of hippocampus and its biological activity. In particular, to verify the hypothesis that tissue redox status is a key determinant of NO responses in the different subregions. To focus on the functional properties of hydroxycinnamates and procyanidins in connection with its ability to interact dynamically with major primary physiological antioxidants, vitamins E and C in the protection of LDL against oxidation. To establish the molecular mechanisms underlying the beneficial effects of red wine phenolic compounds in preventing cardiovascular diseases, beyond the protection of LDL from oxidation, particularly related with direct vascular effects.

Membrane Toxicity

The molecular mechanisms involved in the pharmacological/toxicological effects of xenobiotics upon membrane interaction are a fundamental question in drug research. Lipid bilayers are structured fluids with dynamic heterogeneity, both in space and time. A growing body of evidence has shown that lipid-bilayer structure and dynamics play a key role in membrane functionality. In a pharmacological/ toxicological context, the cellular effects of a variety of molecular compounds interacting with membranes may be understood in terms of their ability to affect and modulate lipid-membrane physical properties. Therefore, soluble (e.g., charged) and amphiphilic drugs, interacting and binding to lipid membrane-water interface, may disrupt the hydration shell and interfere with the normal H-bonding pattern, affecting lipid-protein interactions; lipophilic drugs, partitioning in membranes, may induce alterations of the lateral pressure profile of the lipid membrane, affecting the conformational flexibility and function of membrane-spanning proteins; additionally, alterations of the dynamic structural heterogeneity of the membrane with softening of the bending rigidity and fluctuations of the local membrane curvature may have important consequences in passive and active membrane functions.

Research Highlights
Identification of membrane lipid-mediated cellular effects of compounds of biological/pharmacological interest.
Different model systems have been used in modelling studies of the mechanisms underlying the action of membrane active compounds, such as pharmaceutical drugs (e.g., the anticancer tamoxifen and its metabolite hydroxytamoxifen, the antiarrythmic amiodarone) and pollutants (e.g., the pesticides DDT and its metabolite DDE, endosulfan, isomers α and α , tributhyltin, methoprene). Studies were designed to investigate the effects of xenobiotics on cell viability and functionality and the physical effects on lipid bilayers reconstituted with the respective lipid extracts. A remarkable parallelism has been established between the results obtained with prokaryotic models and artificial or eukaryotic model systems, namely the differential toxic effects of xenobiotics as compared with their metabolites.

Ongoing and Future Work
Further identification of membrane biophysical alterations putatively involved in drug action on target cells and/or in non-selective effects.

Study of the molecular mechanisms of bacterial adaptation to identify strategies that can improve bacterial resistance to adverse effects of toxic pollutants, in the perspective of using microorganisms for bioremediation.

Microbiology

 Introduction
 In the last years the research in this area has focused the work in extremophiles studying in particular:

1-microbial ecology in extremophilic enviroments and the classification of organisms found there;
2-The molecular biology , enzimology and biotechnological application of compatible solutes form extremophiles. In this respect the genes involved in the synthesis of mannosylglicerate from Thermus thermophilus as well as a bifunctional gene of the mesophile Dehalococcoides ethanogenes were cloned and overexpresed.

In addition to the interest in termophiles this group of researchers had develloped a considerable amount of work on the screening and characterization of Legionella species found in Portugal, particularly in the ground water used as water sources in different thermal spas.

Recently a second group joint the Microbiology area.
The main interest of this group is on yeast. Two projects were iniciated, one that aims at the establishment of Pathogenic Yeast Methods, the other is focused on the study of the alteration of the stress resistant patterns in Sacharomyces cerevisiae.

Microbiology of Extreme Enviroments

During the last year we obtained new and exciting results on the biosynthetic pathways leading to the synthesis of the compatible solute mannosylglycerate. This compatible solute had been found only in (hyper)thermophilic bacteria and archaea, but we were able to find a fused gene in the mesophilic Dehalococcoides ethenogenes that had two activities that converted UDP-mannose and 3-phosphoglycerate via a phosphorylated intermediate into mannosylglycerate. This bifunctional enzyme had a much lower temperature optimum for activity that the (hyper)thermophilic monofunctional counterparts from Pyrococcus spp., Thermus thermophilus and Rhodothermus marinus. It is noteworthy that the the characterization of the pathways leading to the synthesis of mannosylglycerate were also published this past year.

In a separate study we also found, by genetic delection of trehalose-phosphate-synthase and trehalose-phosphate phosphatase that the primary compatible solute against salt stress in Thermus thermophilus is trehalose. The importance of this compatible solute was proven, since absence of accumulation of trehalose in the double knock-out mutant did not allow the organism to grow in media containing over 2% NaCl, while the wildtype of the mutant frown in the presence of exogenous trehalose restored growth in medium containing 6% NaCl. The only compatible solute accumulated by the mutant in trehalose-deprived medium was mannosylglycerate. This compatible solute never accumulates to levels that would allow the organism to grow at salinities higher than 2%. We concluded that mannosylglycerate is important for low level osmotic adaptation while trehalose is necessary for osmotic adjustment in media containing higher levels of salt.

These results were confirmed by screening a large number of Thermus strains for the presence of the genes that lead to the synthesis of trehalose and mannosylglycerate. The results correlated perfectly: the strains with the genes for the synthesis of mannosylglycerate and trehalose lead to growth in media containing 6-7% NaCl. Those that only had the genes for the synthesis of mannosylglycerate could only gorw in medium containing salt as high as 2%, while the strains without any of the genes or those that contained only trehalose but not the genes for mannosylglycerate synthesis could not grow in media containing 1% salt.

We were also able to clone the Dehalococcoides bifunctional mpgs/mpgp gene into Saccharomyces cerevisiae and found that the yeast accumulated mannosylglycerate. This observation as well as others led to a patent apllication.
During the last year we have continued to search for novel species of bacteria and described Porphyrobacter cryptus, Thermomonas hydrothermalis, Tepidimonas aquatica, Aquicella lusitana and Aquicella siphonis. The two species of Aquicella are closely related to Ricketziella and Legionella, and are intracellular parasites of amoebae.

We evaluated and demonstrated the persistence of Legionella spp. in groundwater, used as water source in different thermal spas. Legionella strains were isolated from 176 water samples, collected from four boreholes, in two distinct geographical areas. FAME profiles analysis were used to identify Legionella species detected and the isolates were typed by Random Amplifed Polymorphic DNA; PCR technique - RAPD. Three major species were detected and identified as L. pneumophila, L. oakridgensis and L. sainthelensi. The 127 isolates from one of the areas, identified as L. pneumophila, had 6 distinct RAPD patterns, indicating the presence of different genetic groups. Two of these six clones were persistent in the groundwater for at least tem years. In the other area analysed, L. oakridgensis constituted the major Legionella specie isolated form the groundwater, during a three years sampling period. These isolates had only one RAPD type. The same was observed for the L. sainthelensi isolates.

Despite all attempts to eliminate these strains from the environment, their presence and persistence was established, demonstrating that once groundwaters are colonized by legionellae, those organisms are very difficult, if not impossible, to eradicate.

Heterotrophic populations were isolated and characterized from a rare natural alkaline groundwater environment. The alkalinity in this particular environment probably generated by active serpentinization results in an Ca(OH) 2 enriched, extremely diluted groundwater with pH 11.4.

One hundred and eighty five strains were isolated, in different media, at different pH values, during two sampling periods. To achieve the degree of diversity present in the environment and to select representative strains for further characterization of the populations, the isolates were screened using RAPD-PCR profiles, and grouped based on similarities determined by FAME analysis. Phenotypic characterization, determination of G+C content, phylogenetic analysis by direct sequencing of 16S rRNA genes and determination of pH tolerance were performed in the selected isolates.

Although 38 different populations were identified and characterized, the vast majority of the isolates was Gram positive with high G+C content and could be affiliated to three distinct groups. One comprising 32% of the isolates includes strains closely related with the species Dietzia natrolimnae; another accounts for 29% of the isolates and was related with Frigoribacterium, Clavibacter lineages. The third major group represents 20% of the isolates and was closely related with the type strain of Microbacteriumkitamiense. Other isolates were phylogenetic related with strains of the genera Agrococcus, Leifsonia, Kytococcus, Janibacter, Kocuria, Rothia, Nesterenkonia, Citrococcus, Micrococcus, Actinomyces, Rhodococcus, Bacillus and Staphylococcus.
 Only five isolates were Gram negative: one related with Sphingobacteria lineage, while the other four were related with the α -proteobacteria lineage.

Despite the pH of the environment, the vast majority of the populations were alkali-tolerant and only two strains were able to grow at pH 11.

Yeast Research

The group has been developing two projects:
1-Pathogenic Yeast Methods
The main objectives of this research project are:
1-Characterisation of pathogenic yeast flora and/or infection, in our country, in patients with no immune underlying disease and in immunodepressed patients.
2-Establishment of a data bank, including a collection of potentially emergent pathogenic yeast (EPY) strains together with the corresponding clinical data. Other partners will use this bank of EPY’s to test new specific growth/identification media.

Research Highlights
During these first 6 months 500 diferent yeast strains were isolated and identified. During this period, an adequation f the classical protocols of identification were revised. Due to the low accuracy of such a protocols, the decision was made to use molecular biology techniques for the correct identification of the isolated strains. The identification is now performed using RFLP of two ITS regions and of the 5.8S rDNA gene.
2-Expression of the oncoprotein E6 in Saccharomyces cerevisiae: alteration of the stress resistance patterns

The main objectives of this project are:

1-Identification of cellular signalling pathways with which the oncoprotein E6 can functionally interact, independently of the tumor supressor protein p53. Identification of the expression/activation of specific elements of those pathways.
2- Construction of a yeast two-hybrid system for the studying the interaction of E6 with novel targets.
3-Possibility of construction of a yeast model as a tool for the screening of new antiviral drugs.

Research Highlights
1.The expression of HPV16E6 in the yeast Saccharomyces cerevisiae results in a stress resistance phenotype upon the exposure to specific insultants like pheromone and caffeine (“HPV16 E6 oncoprotein expressed in Saccharomyces cerevisiae increases caffeine and pheromone stress resistance” – sent for publication). These results indicate that, besides the increased resistance, the presence of E6 in yeast, results in morphological changes that makes us hypothesise that the HPV16 E6 protein can specifically interact with intermediates of the protein cascades activated by pheromone, enhancing (activating) the pathway responsible for polarized growth and inhibiting the pathway that induces the cell cycle arrest. The caffeine resistance pattern together with a unchanged response to high temperatures suggests that this oncoprotein interacts with the yeast cell integrity pathway.

2. The construction of a yeast two-hybrid system, using the entire protein sequence, with no activation domain and several fragments of the sequence resulted in autoactivation. Nevertheless, while failing to provide information about specific molecular targets, this technique showed that E6 functions as a strong transcriptional activator of the yeast genome.
3. Deletion mutants of the genes PKC1, BCK1 and MPK1 (cell integrity pathway), are already transformed with the plasmid pYES2.E6, in order to study the phenotypic complementarity of E6, in the presence of caffeine and pheromone. A strain was transformed with the pYES2 plasmid containing the E6 fused to the GFP gene. The fusion was obtained using the flanking homology PCR cassette technology.

Cell and Development Biology

 Introduction
The Cell Biology Area of CNC is dedicated to the study of inter-and intracellular signaling mechanisms that are involved in the coordinated activity of cells in the nervous and immune and reproductive systems, allowing cells to communicate and respond to external cues.
These events are frequently segregated within specific subcellular compartments, contributing to their specificity, and research at CNC is contributing to elucidate the trafficking of intracellular proteins until they reach their functionally relevant location.

Traffic of receptors to the plasma membrane affects the response of the cells to extracellular signals and the translocation of proteins to the nucleus regulates gene expression. In the case of terminally differentiated cells, such as gametes, missorting of proteins may lead to impaired function, in this case with implications in terms of fertility. This is another area of interest at the CNC.

Cellular and Molecular Immunology

 This group share interests in the study of cell function under normal conditions, and how changes in the homeostatic mechanisms lead to disease. We have been studying the signalling pathways that mediate the expression of key proteins, relevant in inflammatory and allergic processes, in response to cytokines, steroid hormones, allergens and endotoxin. As models, we have been using cellular cultures of articular chondrocytes, a human T lymphocyte cell line and a mouse skin dendritic cell line.

Research Highlights
Nitric oxide synthase type II (iNOS) plays an important role in many pathological processes, including chronic inflammation and allergy. Thus, one of the key objectives of our group has been the elucidation of the signaling mechanisms that lead to its expression in articular chondrocytes and dendritic cells, in response to Interleukin-1 β (IL- 1) and to GM-CSF and the contact sensitizers, DNFB and NiSO4, respectively.

The promotor region of the iNOS gene has been reported to contain binding sites for several transcription factors, including nuclear factor kappaB (NF- κ B) and Activator Protein- 1 (AP- 1). In articular chondrocytes and dendritic cells, respectively, we found that NF- κ B is essential for iNOS expression to occur in response to the pro-inflammatory cytokine, IL- 1, and to the epidermal cytokine, GM-CSF, and the contact sensitizers DNFB and NiSO4. On the other hand, AP- 1, which is also potently activated by IL- 1, is not required for IL- 1-induced iNOS expression in articular chondrocytes. We also found that tyrosine kinases, probably including the Janus kinase 2, are required for NF- κ B activation and iNOS expression, both in articular chondrocytes and in dendritic cells. In both these cell types, the p38MAPK is also required for iNOS expression.
Furthermore, in articular chondrocytes, the superoxide anion radical and hydrogen peroxide are both produced in response to IL- 1, but only the superoxide anion radical is required to mediate NF- κ B activation and the subsequent expression of iNOS. On the other hand, NO inhibits IL- 1-induced NF- κ B activation and iNOS expression, which, together with our observation that IL- 1-induces the degradation of the constitutive NOS isoform, NOS I, indicates that NO is involved in a negative feed-back mechanism that regulates NF- κ B activation and NF- κ B-dependent gene transcription. On the other hand, NO and reactive oxygen species, namely hydrogen peroxide, mediate IL- 1-induced AP- 1 activation. Thus, inhibitors of ROS production and antioxidants can simultaneously prevent NF- κ B and AP- 1 activation, whereas inhibitors of NO production can inhibit AP- 1 activation, but may favor NF- κ B activation.

In dendritic cells, GM-CSF and the contact sensitizers DNFB and NiSO4 increased the expression of the dendritic cell activation markers, CD40 and IL- 12R. When tested together with the contact sensitizers, GM-CSF had an additive effect. We are now investigating the differential effect of allergens (DNFB and NiSO4) on the activity of the three MAPKs signaling pathways (ERK, p38 and JNK) and how they modulate the synthesis and secretion of IL-1β and CD40.

We have also been interested in finding compounds that can counteract inflammation and allergy. Thus, we found that, in articular chondrocytes, the anthracene compounds Diacerhein and Rhein prevent IL- 1-induced NF- κ B activation and iNOS expression through inhibition of I κ B- α degradation. In dendritic cells, the synthetic corticosteroid Dexamethasone (Dex) inhibits GM-CSF-induced NF- κ B activation and iNOS expression by blocking NF- κ B migration to the nucleus. Moreover, in the lymphoblastic cell line, CCRF-CEM, Dex prevented IL- 1-induced NF- κ B activation through upregulation of I κ B- α synthesis. Furthermore, Dex and estradiol (E 2 β ) induced the secretion and de novo synthesis of the anti-inflammatory protein annexin 1, which seems to require the activation of the transcription factor CREB, by a mechanism involving cAMP and the p38 MAPK.

These studies are providing a better understanding of the molecular mechanisms that regulate cell activation in inflammatory and allergic conditions and may contribute to the identification of new approaches to modify these processes.

Endothelial Dysfunction and Diabetes

The endothelium plays an important role in maintaining vascular tone and function, in part by the synthesis and release of vasoactive substances such as nitric oxide. Endothelial dysfunction is a hallmark of multiple vascular diseases including diabetes mellitus.

The mechanism of endothelial dysfunction in diabetes mellitus is not known, but one possibility is increased inactivation of endothelium-derived nitric oxide by oxygen-derived free radicals. Thus, we will focus our attention in the study of vascular endothelium dysfunction in Goto-Kakizaki (GK) rats, a model of type 2, and we will try to evaluate the importance of the balancing of basal NO production as a fundamental process in the vascular microenvironment.

Research Highlights
Free radical generation is undoubtedly raised in diabetes but the evidence for decreased antioxidant status is debatable. Our research work aimed to examine the action of established antioxidants that prevent lipid peroxidation and oxidative stress (egα-tocopherol and coenzyme Q 10), as well as antioxidant-rich dietary supplements (eg. soybean oil), on metabolic control and pancreatic mitochondrial function of GK rats. Combining bioavailability studies with biochemical assessment and metabolic screening, it was observed that α-tocopherol and coenzyme Q 10 treatment did not prevent the pancreatic lesions in diabetic GK rats.

Biology of Reproduction and Human Fertility

The group has consolidated its line of research at the CNC, related to gametogenesis and fertilization. Special attention is being paid to sperm function and clinical implications in terms of human fertility, as well as to the development of relevant animal model systems.

The collaborations with both the Reproduction Laboratory, University Hospital of Coimbra, as well as with the Medical School, specifically to work in animal models (rat) are both in full swing. A collaboration with the Agricultural School to evaluate stallion fertility and develop novel tools for horse sperm analysis is also underway, and will focus especially on the Lusitano horse.

Research Highlights
We have continued to address issues related to male infertility, namely searching for markers for the mature human acrosome that may be used as markers for human sperm with relevance towards the diagnosis of male infertility
We have used some of these molecular markers to analyse horse sperm, and were able to both describe novel proteins on the acrosome of stallion sperm and correlate their presence with fertility, thus creating novel tools for the improvement of horse breeding techniques. Using the rat model we have initiated studies on the influence of diabetes (types I and II) on male gametogenesis.

On the other had we have concluded our characterization of the ubiquitin-based mechanism responsible for the labeling and removal of defective sperm, with the characterization of a mitochondrial protein, prohibitin, that seems to be involved in this process, and this analysis is being currently expanded to the human infertility field. We have also continued the studies on cellular organization of the female gamete and early mammalian embryo, with possible interest to human infertility Finally, we have taken advantage of collaborations and initiated novel work on stem cell biology and differentiation, focusing on some of the aspects ( such as membrane trafficking and mitochondrial DNA) that we are already analysing in mammalian gametes.(In this way we intend to become involved in this exciting field.)

We have shown that molecules identified on the mammalian acrosome can be used to detect abnormal sperm in infertile patients and stallions, and we are still expanding this study on a larger scale.

Furthermore, after learning the basic technology of sperm mtDNA analysis we are expanding this area (in collaboration with the University of Birmingham, UK, where four of the team members are currently working ) and determining if specific mutations in mtDNA may be correlated with mitochondrial energy production and male infertility. We plan to transfer this technology to Coimbra very son, and carry out a study with a large ammount of samples, but this requires optimization of all the necessary techniques and tools.

Biophysics and Biomedical NMR

Introduction
The research activity in this area is organized in three sub-areas:

 Inorganic Biochemistry , whose goal is the study of inorganic drugs for medical diagnostics and therapy, in particular, new Gd(III)-based chelates as targeted contrast agents for MRI, targeted agents for gamma scintigraphy in Nuclear Medicine, the mechanism of action of lithium salts in bipolar disease and development of new vanadium complexes as oral insulin-mimetic agents; the study of environmental and toxicological effects of inorganic species such as chromium(VI) compounds is also undertaken;
 Biomedical NMR , involving studies of intermediate metabolism using stable isotope tracers and NMR, from the cellular level to perfused organs and humans with clinical applications;
 Cell Biophysics whose research is centred on fundamental mechanisms of cellular excitability with emphasis in electrophysiological pathways and signal transduction. Special attention is devoted to the study of mechanisms underlying stimulus-secretion coupling in endocrine and neuroendrocrine models of hormone secretion (pancreatic β-cells and adrenal medulla chromaffin cells)

Inorganic Biochemistry

The research activity of this group comprises the following projects:
1- Inorganic Agents for Molecular Imaging
The general objective of the work is to develop new targeted metal chelates useful in Molecular Imaging with the aim of Medical Diagnostics (Collaborations: COST D18 Action and EMIL NoE of the EU). With the objective of obtaining new contrast agents for Magnetic Resonance Imaging (MRI) endowed with optimised properties, such as higher relaxivities and specific tissue targeting capacities, several Gd 3+ complexes with different types of ligands were prepared and their solution structure was studied using paramagnetic NMR techniques, nuclear magnetic relaxation dispersion (NMRD), EPR and solution equilibrium techniques. The physico-chemical characterization studies were followed up in some cases by in vivo studies by MRI of rabbits, as well as by by gamma scintigraphy in Wistar rats injected with chelates labelled with gamma emitters, 111In and 157Sm, using as ligands DTPA derivatives containing chemical groups (“syntons”) which endow them with tissue and cell specificity, such as dendrimeric galactose glyconconjugates targeting the asialoglycoprotein receptor in liver and the tetraazaphosphonate DOTP targeting bone tissue.

Research Highlights
The synthesis and characterization of a variety of Ln(III) complexes and systems were studied, such as chelates of a bibrachial lariat ether (cryptand-like), chelates of mixed carboxylate/phosphonate DTPA derivatives, Ln(III)DO3A dendrimeric glycoconjugates, and the tetra-phosphonate Gd(DOTP) chelate. This later one was studied very thoroughly, including its X-ray crystal structure, and its imaging study in vitro and in vivo as a bone targeted contrast agent in rabbits using MRI (article published in Invest. Radiology) .
X-Ray crystal structure of GdDOTP Gamma image of a Wistar rat 24 h after injection of 153 Sm-DOTP

 2- Molecular and Cellular Mechanisms of the Therapeutic Action of Lithium Ion
 ( Collaboration: Loyola University of Chicago, USA). Research in order to clarify the molecular and cellular mechanisms underlying Li + therapeutic action in the treatment of bipolar disease has been pursued. Studies were performed with neuronal cell models, either primary cultures, such as bovine chromaffin cells and rat cortical neurons, or cell lines like the human neuroblastoma cell line (SH-SY 5Y) and the human lymphoblastoma cell line (CCRF-CEM), using multinuclear ( 7Li, 31P) NMR Spectroscopy, Fluorescence Techniques (spectroscopy and microscopy) and Atomic Absorption Spectrophotometry. Li + influx and intracellular immobilization were studied, as well as the effects of Li + binding on Mg 2+ metabolism in those cells, providing support to the ionic Li +/Mg 2+ competition model. The effects of Li + on the metabolism of SH-SY 5Y neuroblastoma cells were studied using 1H and 13C NMR spectroscopy with U- 13C-glucose as substrate.

Metabolic studies in rat neurons and astrocytes primary cultures by NMR Spectroscopy using [U- 13C]glucose, [1- 13C]glucose and [2- 13C]-acetate were also undertaken in order to investigate the effects of Li + in the metabolism of these cells. Similar studies were carried out in the extracts of rat brain after previous injection of Li + solution and labeled substrates in the living rats. Finally, the effect of Li + on intracellular cAMP levels and the influence of this second messenger in the uptake of Li + by SH-SY5Y neuroblastoma cells and rat cortical and hippocampal neurons were also studied using Atomic Absorption Spectrophotometry and radioactive biochemical assays to determine intracellular Li + and cAMP concentrations, respectively. Similar studies were also started in vivo in rat models using NMR and microdialysis.

Research Highlights
[ Mg 2+] f (in mmol/L) in human lymphoblastoma cells, human neuroblastoma cells and human RBCs under Li +-free conditions (white bars), when the cells were loaded for 30 min in 50 mmol/L LiCl PBS (black bars), and when the intracellular [Li +] was approximately 15 mmol/L (striped bars).(Biochem. Pharmacol.)
3-Development and study of new Insulino-Mimetic Vanadium Compounds:
(Collaborations: Instituto Superior Técnico, Lisboa, Instituto Abel Salazar, Porto).
The main objective of this work is to obtain more effective, less toxic, vanadium complexes as oral insulin mimetic agents for type II diabetes. The synthesis and chemical characterization in the solid state and in solution of a series of new complexes of Vanadium (IV, V) was undertaken, such as the chemical studies in aqueous solution, of the complexes of V(IV) and V(V) with the ligand N,N‚-ethylene bis(pyrido-xylideneiminato) (Rpyr2en),and the study of the interaction of V(V) with the ligand 1,2-dimethyl-3-hydroxi-4-piridinone (Hdmpp), in a methanol solution, originating a binuclear complex. Biological studies of these complexes were started. A new method was developed to q uantitate oxidative stress in human erythrocytes, using 1H NMR Spectroscopy together with 13-C 2-glucose, to evaluate the metabolic fluxes through glycolysis versus pentose phosphate pathway. This was applied to study the oxidative toxicity of sodium vanadate and two potential insulin-mimetic vanadium(IV) compounds –V IVO(dmpp) 2 and V IVO(Rpyr 2en) complexes - in human erythrocytes by 1H NMR. The uptake, toxicity and glucose consumption of vanadate, V IVO(dmpp) 2 and V IVO(Rpyr 2en) in human erythrocytes was studied using 1H and 51V NMR and EPR Spectroscopy, and biochemical assays.

Research Highlights:
4-Toxicity of Metal Ions
“Cr(VI)-induced genotoxic stress: The signalling mechanisms that link cell cycle and cell fate”.
One of the aims of this study was to establish whether the non-lethal Cr(VI) concentration that we tested, which are potential relevant for environmental and occupational exposures, could have any effect on the cell cycle and thus on the cell proliferation. Therefore, time- and concentration-dependent Cr(VI) effects on cell viability, cell proliferation and percentage of dead cells in PC12 cells cultures were evaluated.

Research Highlights
The results obtained, which were accepted for publication in Metal Ions in Biology and Medicine, indicate that Cr(VI)-induced cell death on PC12 cells is closely associated with a perturbation on the cellular redox status and the energy charge of these cells, implicating oxidative stress and mitochondria in the basal mechanisms leading to cell death by apoptosis. The contribution of necrosis to cell death increased with the intensity of the Cr(VI) insult. A decrease in cell proliferation could be observed for some time after the removal of a small, temporary and non-cytotoxic Cr(VI) insult, thus suggesting that small sub-lethal concentrations of Cr(VI), which are potentially relevant to environmental and occupational exposures, may delay DNA replication and mitosis.
“On the Mechanisms of Cr(VI)-Induced Lung Carcinogenesis and Cell Death”.
The aim of the first part of this research project was to create an in vitro model for Cr(VI)-induced carcinogenesis. We have thus investigated which Cr(VI) exposures were likely to induce carcinogenesis in cultured cells. The results obtained so far are:

Depending on the concentration and duration of the insult, Cr(VI) may induce either an increase or a decrease in cell viability, as assessed by the MTT assay. For some concentrations, it was observed that, as the duration of the insult increased (while the concentration was kept constant), the effect of the Cr(VI) insult was partially or totally reversed. We are currently investigating the mechanisms behind the results obtained.
Bioremediation of Cr(VI) contaminated soils and ground waters is another important strategy to prevent Cr(VI) carcinogenic effects. The success of microbial based metal remediation strategies requires the understanding of the response of individual bacteria isolated from a chromium contaminated activated sludge to metal stress. In order to evaluate the potential of strain 5bvl-1 as chromate reducer, it is important to allocate and characterize strain 5bvl-1 Cr(VI)-reductive ability. Therefore, the chromate reductase activity was studied on both resting cells suspensions and cell extracts of Ochrobactrum tritici strain 5bvl-1, and it was found that chromate reductase activity is chiefly associated with the cellular soluble extract and dependent on the availability of energy sources and thus on the cells metabolic state. The results obtained were accepted for publication in Metal Ions in Biology and Medicine

Intermediate Metabolism
Carbon fluxes through the central biochemical pathways of glucose and fat metabolism can be studied using stable isotope tracers such as [6,6- 2H 2]glucose and [U- 13C]fatty acids coupled with NMR analysis of their distribution in metabolic intermediates or end-products. These intermediary metabolic fluxes are disrupted in the setting of tissue injury, or from an inherited mutation of a component enzyme, or through nutritional imbalance. While these characteristics feature in many key diseases such as heart failure and diabetes, the ensuing alterations in metabolic fluxes remain poorly defined. Nevertheless, the intent of many therapeutic interventions is to correct a putative metabolic flux defect (for example excessive hepatic glucose production in Type 2 diabetes). Metabolic measurements in principle allow us to identify abnormal metabolic fluxes and to determine if and how they are modified by therapy. To this end, our aims are to create realistic models that relate the principal fluxes of glucose and fat metabolism to the highly informative metabolite 13C or 2H labelling distributions obtained by NMR measurements. We can group our research interests in two main areas: i) development of methodologies using stable tracers and NMR for measuring glucose and lipid fluxes in animal models and ii), adapting such methodologies for non-invasive human metabolic flux measurements.

Research Highlights

 Glycogen metabolism in the heart and its cardioprotective role during ischemia and reperfusion;
 Relationship between metabolic alterations and pathophysiological conditions;
 TCA cycle and transaminase exchange fluxes as indicators of tissue physiology;
 Quantification of endogenous glucose production from hepatic glycogen and gluconeogenesis in healthy and cirrhotic subjects;
 Hepatic glycogen synthesis and utilization in Type 1 diabetics;
 Non-invasive quantification of hepatic glucose and Krebs cycle fluxes in children with Glycogen Storage Disease.

Cell Biophysics

The main aims of the work developed by this group are: a) To determine whether glucose-induced electrical activity is modulated by K ATP channels, the prevailing K + conductance in non-stimulated β-cells; b) To determine the role played by protein kinase C (PKC) isoforms in cholinergic potentiation of PIR; c) To investigate the involvement of different calcium mechanisms in long term potentiation (LTP).

1- Mechanisms of Insulin Release
Pulsatile insulin release (PIR) from pancreatic islets is determined by oscillatory electrical activity and cytosolic calcium. Islets are richly innervated by sympathetic nerve endings.

Research Highlights
Combining electrophysiological monitoring of membrane potential and 5-HT/insulin release with biochemical assessment of PKC isoforms and cytosolic calcium measurements by microfluorometry, it was found that a novel ionic conductance distinct from sulphonylurea-sensitive K ATP channels contributes to glucose-induced electrical activity. Moreover, PKC activation plays an important role in cholinergic stimulation of PIR while playing a minor role, if any, on glucose-induced release. Cholinergic stimulation is specifically mediated by the PKC-α isoform, via enhancement of the Ca 2+ sensitivity of the exocytotic machinery.

2-Calcium Involvement in LTP
The main aim of the work was to investigate the involvement of different calcium mechanisms in long term potentiation (LTP) at the Schaffer collaterals - CA1 pyramidal cell synapses. Combining the measurement of fast intracellular postsynaptic calcium changes, using the fluorescent calcium indicator Fura-2, with field potential recordings, it was found that part of the calcium accumulated enters through NMDA and L-type voltage dependent calcium channels (VDCCs) during the induction of LTP and through L-type VDCCs during the expression of LTP. Thus, a substantial fraction of calcium entry may occur through other types of VDCCs or be due to calcium release from intracellular stores, this process being essential for the formation of CA1 LTP. Presynaptic calcium and zinc changes associated with mossy fiber LTP, from CA3 hippocampal area, were also investigated using fluorescent indicators.

Research Highlights
 The findings indicate that at the mossy fiber synapses presynaptic calcium and zinc changes are maintained during mossy fiber LTP and that the calcium changes are inhibited by endogenous zinc.