RECENTLY TENURED

Montserrat Garcia-Closas

Montserrat Garcia-Closas received an M.D. degree from the University of Barcelona in Spain in 1990 and an M.P.H. in quantitative methods and a Dr.P.H. in epidemiology from the Harvard School of Public Health in 1993 and 1996, respectively. She joined the Hormonal and Reproductive Epidemiology Branch, NCI, in 1996 as a postdoctoral fellow and is now a senior investigator in that branch.

I am interested in the application of biomarkers in molecular epidemiologic studies of breast, ovarian, endometrial, and bladder cancers, with special emphasis on the study of genetic susceptibility and etiologic heterogeneity of breast cancer.

I have also conducted a series of methodological investigations to address relevant scientific questions derived from molecular epidemiologic studies such as assessment of false-positive findings, collection and use of buccal cell DNA, and creation of pilot studies for biomarker development.

The discovery of susceptibility genes for cancer holds great promise for improving risk assessment and developing targeted preventive strategies. It also provides an opportunity to dissect the complex etiology of each cancer.

The importance of susceptibility genes in risk assessment is being evaluated in the Polish Breast, Ovarian, and Endometrial Cancer studies, a set of parallel, molecular epidemiologic studies targeting these three tumors in women and including about 2,500 breast, 500 endometrial, and 300 ovarian cancer cases and more than 2,500 control subjects. This complex study combines detailed exposure assessment and comprehensive collection of biological specimens.

In exploring genetic susceptibility of breast cancer, I have evaluated variation in genes in several candidate pathways, including DNA repair, apoptosis, and regulation of telomeres.

With the Breast Cancer Association Consortium, a consortium of studies including more than 20,000 breast cancer cases, we were able to uncover convincing evidence for modest associations between breast cancer risk and two common genetic variants, caspase 8 (CASP8 D302H) and transforming growth factor–b1 (TGFb1 L10P).

In addition, I am collaborating with two efforts to identify genetic susceptibility markers through genome-wide association studies (GWAS) at the University of Cambridge, U.K., and the Cancer Genetic Markers of Susceptibility project. Recent publications from these studies have identified at least five novel genetic susceptibility markers for breast cancer. Numerous other variants are likely to be identified in the coming years using this approach.

Ongoing work after the initial identification of markers includes:

Fine mapping and functional studies to identify causative markers

Evaluation of how lifestyle factors affect the risk from genetic variants

Learning how different genetic variants interact with each other in affecting risk

Learning whether genetic variants are differentially associated with different types of breast cancer with different clinical and pathologic features at presentation

Learning how variants affect survival

My work related to etiologic heterogeneity of breast cancer in the Polish breast cancer study has demonstrated substantial heterogeneity in breast cancer risk factors by histopathological tumor characteristics of clinical relevance, particularly tumor grade and size.

In this study, we are using tissue microarray blocks to obtain standardized, rapid, and cost-effective immunohistochemical characterization of many tumors. This process allows us to evaluate relationships between tumor markers with known breast cancer risk factors and newly discovered genetic markers.

I have also worked on the evaluation of genetic susceptibility factors for bladder cancer and the interaction of those factors with environmental and occupational exposures.

Because of the central role of tobacco smoking in bladder carcinogenesis, I initially focused on polymorphisms in genes involved in carcinogen metabolism and DNA repair. Data from this study provided compelling evidence for the associations of N-acetyltransferase 2 (NAT2) slow acetylator and glutathione S-transferase null genotype with increased risk of bladder cancer.

The data also supported an interaction between smoking and NAT2 genotype, which is one of the few consistent gene-environment interactions described to date. In addition, I showed that common variants in the nucleotide excision repair pathway are likely to alter bladder cancer risk.

More recently, we used a highly multiplexed genotyping platform to explore associations between variants in cancer-related genes and bladder cancer, which resulted in the identification of novel genes that may be involved in bladder cancer etiology.

I plan to follow up on findings from our candidate gene investigations in the recently formed Internatioinal Consortium of Bladder Cancer Study. In addition, we are planning to carry out a GWAS of bladder cancer in collaboration with other intramural and extramural studies.

Kent Hunter

Kent Hunter received his Ph.D. in biology from the Massachusetts Institute of Technology in Cambridge in 1991. He was an associate member at the Fox Chase Cancer Center in Philadelphia before joining the Laboratory of Population Genetics, NCI, in 1999. He is currently a senior investigator in the Laboratory of Cancer Biology and Genetics, NCI.

The focus of my laboratory is the study of inherited metastasis susceptibility in breast cancer. Previously, metastasis was thought to be the result of sequential random somatic alterations that occurred in tumor cells as tumors evolved. Thus, metastatic disease was was viewed as a random process, whose appearance could not be predicted before the development of the primary tumor.

While somatic mutation is clearly a part of metastatic progression, work in my laboratory has demonstrated that there is also a significant inherited predisposition to developing secondary tumors. Using a highly metastatic mouse mammary tumor model and a simple breeding scheme, we demonstrated that the genetic background upon which the primary tumor arose had a significant impact on the number of subsequent lung metastases.

These results suggest that in addition to the somatic mutations, activating or inactivating specific genes that have been previously studied in metastatic disease, polymorphisms that subtly effect gene function also play an important role in breast cancer progression.

Using an integrated genomic and complex trait genetic mapping strategy, my lab has begun to identify some of the genes that may contribute to metastasis susceptibility. The first gene we identified is the signal transduction regulator signal-induced proliferation-associated gene Sipa1.

We identified an amino-acid polymorphism in this protein in mice that reduces its enzymatic function. Modeling the effect of this polymorphism by halving the endogenous levels of Sipa1 mRNA significantly reduced the metastatic capacity of a highly malignant mammary tumor cell line, suggesting that subtle variations in this gene’s activity may play a role in human breast cancer progression.

In addition to the mouse modeling experiments, we have also performed pilot epidemiology experiments to directly investigate SIPA1 in humans. In collaboration with Hoda Anton-Culver at the University of California at Irvine, we demonstrated that polymorphisms in SIPA1 were associated with markers of poor outcome in humans, consistent with our hypothesis that this gene may be one of the factors that establish metastatic susceptibility in humans.

There are several important implications of our research. First, our work suggests that modulating gene function by relatively minor amounts may have significant impacts on cancer progression.

This possibility leads to the further hypothesis that one may subtly change the molecular state of the cell, rather than use a cytotoxic agent, to prevent relapse in breast cancer patients.

Moreover, the existence of polymorphisms in the germline DNA that establish susceptibility to metastatic progression should enable the development of a blood-based prognostic test to identify those patients at risk for disseminated disease. We hope that this test will ultimately result in better tailored treatment for patients, reducing the number of relapses and cancer-related deaths.

Aravind (Iyer)

L. Aravind (aka Aravind Iyer) received his Ph.D. in 1999 from Texas A & M University, College Station. He worked as a staff scientist at NCBI from December 1999 to December 2002 and is currently senior investigator in the Computational Biology Branch, NCBI.

I am an evolutionary biologist, and I use computational methods to decipher biological functions from genome sequences.

The past decade has been an exciting and definitive period in modern biology. Thanks to the advances in sequencing technology, we are in possession of the genome sequences of not just humans, but organisms spanning the entire tree of life. With the whole script of an organism’s biology spread before us, we are ready to obtain unprecedented glimpses of life’s workings. However, to apprehend these views, we need to be effective readers and interpreters of this script.

My group has primarily worked towards this objective, developing and using computational methods for comparing protein sequences and structures and analyzing biological networks.

To obtain a reasonably complete approximation of biological function, my group investigates different evolutionary problems at multiple organizational levels. At the "microscopic" level, we study protein domains in order to identify their structural and functional determinants. At the "mesoscopic" level, we study the various interactions between different proteins or proteins and nucleic acids in the context of entire biological functional pathways. Finally, at the "macroscopic" level we attempt to reconstruct salient aspects of organismal biology from whole genome sequences.

Our studies at the microscopic level have strongly focused on the discovery of new protein domains and the generation of testable hypotheses regarding their functions.

Some of our major findings include the computational discovery of new peptidases involved in deubiquitination; proteins involved in viral and cellular DNA packaging and segregation; and novel enzymes and factors involved in chromatin dynamics, RNA modification, and post-transcriptional gene silencing.

Overall, we are moving toward generating a complete natural classification of the protein universe.

Our studies on the next level of biological organization have followed the evolutionary trajectories of entire functional systems involved in RNA and DNA metabolism, apoptosis, ubiquitin signaling, transcriptional control, and chromatin-level regulation.

More recently, we have investigated how whole biological networks, especially those involved in transcription, evolve as their component nodes undergo diversification.

Especially striking is our finding that despite conservation of target genes, the major specific transcription factors are subject to massive lineage-specific displacement via new innovations. Different lineages may therefore differ dramatically in their transcription factors despite retaining a similar complement of target genes, suggesting that the evolutionary turnover in transcription factors is a major player in phenotypic diversity.

Our genome-scale analysis has led to the understanding of several aspects of biology at the organismal level, such as the parasitic adaptations of apicomplexans and large DNA viruses, the origins of eukaryotic cellular complexity, and sensory and signaling strategies of organizationally complex bacteria.

In particular, we have made several efforts toward understanding the biology of apicomplexans parasites, which are causative agents of malaria, toxoplasmosis, a fatal lymphoma in cattle, and cryptosporidiosis. We computationally predicted novel O-linked protein glycosylation pathways that are likely to modify surface molecules and play a critical role in the way these parasites interact with their host.

Our discovery of apicomplexan-specific transcription factors has also provided a means to unravel the hitherto unknown mechanisms of gene regulation in these organisms.

In conclusion, we hope to continue to exploit computational methods and evolutionary principles to arrive at a definitive understanding of the major biological systems. This in turn would provide vital insights regarding normal as well as disease states in humans and other organisms.

Zu-Hang Sheng

Zu-Hang Sheng received his Ph.D. from the University of Pennsylvania in Philadelphia in 1993. He completed his postdoctoral training in neuroscience at the University of Washington, Seattle, before he came to NINDS as a tenure-track investigator in 1997. He is currently a senior investigator and chief of the Synaptic Function Section, NINDS.

Our research is focused on molecular and cellular mechanisms underlying (1) the axonal transport of synaptic components and organelles essential for the assembly of synapses, and (2) the regulation of synaptic vesicle (SV) priming for fusion. Such mechanisms are crucial for the initial establishment of presynaptic terminals and for the modulation of synaptic function.

We initially identified Snapin as a SNAP-25–binding protein. The physiological role of Snapin in SV exocytosis was examined and further confirmed by microinjection or overexpression of Snapin in presynaptic neurons in culture and by overexpression of Snapin in hippocampal neurons.

Our studies using snapin-knockout mice in combination with genetic rescue experiments provide evidence that Snapin modulates neurosecretion in chromaffin cells by stabilizing the structural coupling of the calcium sensor synaptotagmin-I to the vesicle fusion machinery SNARE complex, a critical step for priming docked vesicles for fusion. The deletion of Snapin leads to a marked reduction in the amount of synaptotagmin-I–SNARE complex and defective exocytosis in the snapin (-/-) chromaffin cells.

Individual SNAREs are distributed to specific membrane compartments along secretory and endocytic pathways and contribute to the specificity of membrane trafficking. In addition to its association with SVs, our ongoing study suggests that Snapin is also involved in an endosome-lysosome trafficking pathway, possibly through interactions with endosomal SNAREs.

Syntaphilin (SNPH) is a neuron-specific protein initially identified as a candidate inhibitor of presynaptic function. We recently generated mouse mutants with a homozygous deletion for the snph gene, leading to the discovery of a novel role for SNPH as a docking receptor of axonal mitochondria.

Our findings indicate that SNPH is targeted to and required for maintaining a large portion of axonal mitochondria in stationary state through an interaction with the microtubule-based cytoskeleton. The deletion of the snph gene in mice dramatically increases mitochondrial motility, reduces their density in axons, and consequently influences short-term facilitation during prolonged high-frequency stimulation, probably by affecting calcium dynamics at presynaptic boutons.

Our studies elucidate a mechanism underlying the docking of axonal mitochondria and provide evidence that the increased motility and/or reduced density of axonal mitochondria have a significant impact on presynaptic function.

We initially identified syntabulin as a syntaxin-binding and KIF5B motor-adaptor protein that mediates anterograde transport of syntaxin-1 to neuronal processes. Recently, we showed that the syntabulin-KIF5B transport complex plays a critical role in axonal delivery of the active zone (AZ) components essential for presynaptic assembly.

Using time-lapse imaging in live hippocampal neurons, we demonstrate that syntabulin co-migrates with AZ precursor vesicles along axonal processes. The knockdown of syntabulin or interference of its interactions with either KIF5B or syntaxin-1 in developing neurons results in the deficient trafficking of the AZ components to nerve terminals, reduces the density of release sites, impairs synaptic transmission, and inhibits the activity-induced formation of new presynaptic boutons.

Our findings suggest a mechanism through which long-term presynaptic plasticity is regulated by the syntabulin-KIF5B–mediated axonal transport of the AZ components, thus contributing to the activity-induced presynaptic assembly.

I believe that our continued application of these multidisciplinary systems analysis of genetically engineered mice will contribute to an understanding of the molecular mechanisms that are crucial for regulation of activity-dependent presynaptic plasticity by (1) the anterograde axonal transport of presynaptic components, (2) the axonal mitochondrial trafficking and docking, and (3) the priming and regulation of SV exocytosis.

Given that defects in axonal transport of presynaptic cargos and mitochondria in neurons have been implicated in the pathogenesis of neurodegeneration, our studies will yield fundamental information that may have an impact on the understanding of human neurodegenerative disorders.

YihongYang

Yihong Yang received his Ph.D. degree in biophysics from the University of Illinois at Urbana-Champaign in 1995, under the supervision of Nobelist Paul Lauterbur, who invented magnetic resonance imaging (MRI). He did postdoctoral work at NIH in the Laboratory of Diagnostic Radiology Research from 1995 to 1998 and then became an assistant professor in the Functional Neuroimaging Laboratory at the Weill Medical College of Cornell University in New York before joining the Neuroimaging Research Branch, NIDA, in 2002 as an investigator. He is currently a senior investigator and chief of the MRI Physics Section in that branch.

MRI and spectroscopy are noninvasive, versatile techniques that are ideal for providing system-level information on humans and animals. My research at NIDA has focused on the development of advanced magnetic resonance techniques to study brain structure, function, and metabolism, particularly as they are related to the effects of substance abuse on the brain.

My group has developed novel functional MRI (fMRI) methods with enhanced detection and quantification power to observe brain activation elicited by cognitive tasks or administration of drugs.

These fMRI techniques are able to measure multiple functional signals simultaneously (for example, cerebral blood flow, blood volume, and blood oxygenation) and to quantify local oxygen consumption in the brain. Neuronal activity accompanied by metabolism can then be separated by potential vascular artifacts in neuropharmacological studies using the comprehensive information provided by these techniques.

We have conducted research on functional connectivity of the brain using "resting-state" fMRI, in which intrinsic interactions between brain regions are reflected by synchronized fluctuations of the fMRI signals.

Compared with healthy control subjects, cocaine users showed significant reduction of functional connectivity in the "reward circuitry" of the brain, which was heavily involved in addiction processes.

This study revealed for the first time the relationship between functional brain connectivity and chronic drug abuse, and pointed to a powerful new tool to study drug-induced neuronal dysfunction or dysregulation.

Magnetic resonance spectroscopy (MRS) can be used to measure metabolite and neurotransmitter concentrations in vivo. My group has developed a new MRS method that provides well-resolved glutamate, glutamine, and GABA signals. We performed MRS experiments on cocaine users and healthy control subjects that revealed significantly lower glutamate levels in the anterior cingulate cortex of the cocaine addicts, a finding potentially associated with chronic drug addiction.

We have also developed several new diffusion-based MRI methods for better delineating complex brain white matter structures. These methods can be used to improve fiber-tracking techniques by identifying multiple fibers coexisting in an image voxel—not possible with traditional diffusion tensor imaging. Using a group-level analysis on diffusion-based imaging data, we assessed structural integrity of neural circuits relevant to drug addiction.

Recently, we developed an animal model to investigate the underlying mechanisms of resting-state fMRI by integrating electrophysiological and fMRI signals in the resting rat brain.

Our results demonstrated that, unlike the evoked fMRI response that correlated with power changes in high-frequency bands, power coherence in low-frequency bands (particularly the delta band) correlated with the resting-state fMRI signal in a region-specific and anesthesia dose–dependent fashion.

These results provided new insights into the linkage between neuronal activity and hemodynamic-based fMRI signal at rest.

Most existing fMRI techniques measure neuronal activity indirectly through hemodynamic responses coupled to neuronal and metabolic changes. To avoid potential vascular effects of drugs, we used Mn2+-enhanced MRI to detect neuronal activity reflected by an influx of Ca2+ ions due to action potentials.

In experiments on rats given cocaine, we showed that this noninvasive method detected brain activations as consistently as nonhemodynamic invasive methods and thus appears to be a very promising tool for mapping drug-induced neuronal activity.

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