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DEGREES

1964           Medical Doctor (M.D.), Facultad de Medicina, Universidad de Buenos Aires, Argentina. Honor Award.

1969           Doctor of Philosophy (Ph.D.) in Biophysics, University College, University of London.

ACADEMIC POSITIONS

2005-present Associate Director, Cardiovascular Research Laboratories,            University of California, Los Angeles, CA, USA.

2002-present Director of the Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine at UCLA, University of California, Los Angeles, CA, USA.

1999-present Professor Above Scale, School of Medicine, University of California, Los Angeles, CA, USA.

1996-present Professor, Department of Physiology,  David Geffen School of Medicine at UCLA, University of    California, Los Angeles, CA, USA.

1994-present Vice Chairman of Research, Department of Anesthesiology, David Geffen School of Medicine at UCLA, University of California, Los Angeles, CA, USA.

1994-present Professor, Department of Anesthesiology, David Geffen School of Medicine at UCLA, University of    California, Los Angeles, CA, USA.

RESEARCH STATEMENT

    In the post-genomic era we know the structures of most of the genes and proteins. The next step, to understand cell functions we need to know how the various molecular cell components made of genes and their products (RNA and proteins) are statically and dynamically localized at the molecular level in different cellular microdomains. To this end, my laboratory is focused to determine in mammalian cells: (1) the identity of the molecular constituents of macromolecular complexes, (2) the degree of proximity between these macromolecules, (3) the subcellular distribution of these complexes, and (4) dynamic changes of these complexes during resting conditions and cell stimulation. To achieve these goals our group is are using a proteomic approach and we are devoting significant effort to the development and construction of high resolution optical tools such as a multicolor nano-microscope based on laser stimulated emission depletion with a 3D spatial resolution down to the scale of 10 nm while maintaining the microscopic scale over a 10-100 µm range. We have also constructed a high resolution fast laser scanning confocal that can acquire 16-128 images per second with a resolution in the diffraction limit (100-200 nm).

We are using as model systems heart at different stages of pregnancy and during ischemia, and myometrium.  We are investigating: 1. The molecular signature in heart functional hypertrophy that occurs in pregnancy and the heart estrogen receptor alpha molecular identity and pathways involved, 2. Dynamic changes of protein signaling complexes associated with mitochondria during cardio-protection and ischemic preconditioning, and 3. The molecular mechanisms of ion channel regulation by sex hormones and their functional consequences in myometrium. To perform these investigations, we are using a multidisciplinary approach involving molecular biology, biochemical, immuno-cytochemical and functional techniques.  We are using real time PCR for accurate quantification of transcript levels, proteomics to discover molecular partners, high resolution confocal microscopy with 3D reconstruction for colocalization of various proteins in their native cellular compartments and small animal imaging techniques to follow “in vivo” changes of heart function.  We have developed new statistical methods to quantify the degree of protein-protein proximity by evaluating the intensity landscape correlation among sets of proteins and we are constructing novel microcopies to achieve beyond the diffraction limit. These studies are performed in conjunction with microarray analysis to investigate changes in global gene expression and as a discovery tool for novel genes involved in ion channel remodeling. 

 1. Molecular mechanisms and metabolic pathways of K⁺ channel remodeling in heart functional hypertrophy during pregnancy.  During pregnancy, there is an increased risk of arrhythmias and the heart develops functional hypertrophy.  We are investigating K⁺ channel molecular remodeling and metabolic pathways during heart functional hypertrophy that may explain the cardiac risks during pregnancy and their reversibility after delivery.  These studies are focused on the molecular constituents of heart transient outward currents (I_to, Kv1.4, Kv4.2 and Kv4.3 genes and regulatory subunits).  We recently discovered that transcripts and protein levels of some of the molecular constituents of heart I_to were reduced in late pregnancy.  This downregulation can also be mimicked by estrogen treatment in whole animals and in cultured cardiomyocytes.  We also discovered that heart myocytes possess membrane estrogen receptors that can be visualized with immunocytochemical methods that may underlie acute actions of estrogen on K⁺ channel activity.  Furthermore, c-Src tyrosine kinase activity, which is activated by estrogen, reduced expression of Kv4.3 resulting in action potential prolongation and I_to current reduction.  Thus, we are proposing that estrogen dependent c-Src activity is one of the molecular pathways involved in K⁺ channel remodeling during functional heart hypertrophy. In addition, molecular genetic studies are performed to investigate the promoter region as a mechanism of direct K channel gene regulation by estrogen (NIH RO1 HL071824).

2. Dynamic changes of protein signaling complexes associated with mitochondria during cardioprotection and ischemic preconditioning.  One of the goals is to investigate the signaling kinases/phosphatases cascades and other partner proteins associated with heart mouse mitochondria in control conditions and during ischemia/reperfusion injury.  In addition to determining at the biochemical level the activity of a set of signaling molecules, we are investigating their subcellular localization and potential association with mitochondria and changes under ischemia/reperfusion injury.  We are using a proteomic approach together with high resolution confocal microscopy to identify dynamic changes of protein signaling complexes associated with mitochondria.  High resolution images of heart cells reflect a snapshot of the cardiomyocyte protein organization at the moment of the animal death and heart dissection.  Both methods provide independent information on the molecular partners associated to mitochondria function. We have achieved enough spatial resolution to visualize and define the degree of proximity and association between mitochondria and signaling molecules by image restoration in conjunction with the use of analytical algorithms to evaluate the degree of protein-protein proximity. (NIH PPG P01 HL 080111; Project 3 and Mitochondria Biology Core).

 3. Mechanisms of hormonal control of ion channels in myometrium.  We are using myometrium from animals at different stages of pregnancy and under various hormonal treatments to investigate molecular remodeling of ion channels under the influence of hormones.  These studies are focused on Ca²⁺-activated K⁺ channels (MaxiK) and  fast transient K⁺ channels (Kv4.3).  One of the goals is to unravel the molecular mechanisms involved in the hormonal regulation of ion channels.  To this end, we are investigating the role of sex hormones on channel cell biology such as transcriptional regulation, intracellular trafficking and targeting to the cell membrane. We are also using microarray technology in conjunction with functional genomics to identify gene regulation mechanism(s) by hormones. We recently discovered that estrogen downregulates Kv4.3 channel protein levels by transcriptional regulation and that a novel mechanism of hormonal regulation is changes in intracellular trafficking and targeting to the plasma membrane.  We also obtained the myometrium transcriptomes before and during parturition with microarray analysis.  This new information is analyzed to define protein changes and discover new genes involved in myometrium functional and pathological changes during pregnancy.  (RO1 HD046510).

4. Development of high resolution fluorescence microscopy beyond the diffraction limit. To better understand cell function in health and disease, we need to visualize the localization of protein complexes and dynamic changes in different cellular compartments in response to normal stimuli or insult. To this end, we are developing “Nanomicroscopes” for fluorescence imaging to measure structures and their dynamics inside a cell with a 3D spatial resolution down to the scale of 20-40 nm while maintaining the microscopic whole cell scale over a 20-100 µm range. We are developing such “Nanomicroscopes” for cardiovascular research, specifically in a pressure-overload model of heart failure.  The overall hypothesis states that, stress-induced structural rearrangements -in the subcellular location and interactions- of key signaling protein complexes in the heart and blood vessels differentially contribute to the onset and progression of heart failure. We have exciting advances in the design of a novel Reflexion Nanomicroscope that achieves a full-width-half-maximum (FWHM) of ~100 nm lateral resolution. We are improving the novel reflexion confocal Nanomicroscope by constructing a fast acquisition multicolor Reflexion Confocal with FRET for living cells. Furthermore, we plan to combine STED with 4Pi microscopy to achieve 10-20 nm 3D resolution and expand to two fluorescene wavelengths for protein colocalization imaging.  Nano-imaging will be complemented by state-of-the-art molecular manipulations, biochemical and proteomic approaches. These studies will be the basis to unravel -at the nanoscale level- the structural map of protein complexes at the subcellular level, their localization and dynamic interactions in cardiovascular disease.  (R01HL088640-01 pending).

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