Alisha K Holloway

University of California, Davis, Davis, CA, United States

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Publications (34)281.22 Total impact

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    ABSTRACT: Next-generation sequencing technology enables the identification of thousands of gene regulatory sequences in many cell types and organisms. We consider the problem of testing if two such sequences differ in their number of binding site motifs for a given transcription factor (TF) protein. Binding site motifs impart regulatory function by providing TFs the opportunity to bind to genomic elements and thereby affect the expression of nearby genes. Evolutionary changes to such functional DNA are hypothesized to be major contributors to phenotypic diversity within and between species; but despite the importance of TF motifs for gene expression, no method exists to test for motif loss or gain. Assuming that motif counts are Binomially distributed, and allowing for dependencies between motif instances in evolutionarily related sequences, we derive the probability mass function of the difference in motif counts between two nucleotide sequences. We provide a method to numerically estimate this distribution from genomic data and show through simulations that our estimator is accurate. Finally, we introduce the R package {\tt motifDiverge} that implements our methodology and illustrate its application to gene regulatory enhancers identified by a mouse developmental time course experiment. While this study was motivated by analysis of regulatory motifs, our results can be applied to any problem involving two correlated Bernoulli trials.
    01/2014;
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    ABSTRACT: The chicken has long served as an important model organism in many fields, and continues to aid our understanding of animal development. Functional genomics studies aimed at probing the mechanisms that regulate development require high-quality genomes and transcript annotations. The quality of these resources has improved dramatically over the last several years, but many isoforms and genes have yet to be identified. We hope to contribute to the process of improving these resources with the data presented here: a set of long cDNA sequencing reads, and a curated set of new genes and transcript isoforms not currently represented in the most up-to-date genome annotation currently available to the community of researchers who rely on the chicken genome.
    PLoS ONE 01/2014; 9(4):e94650. · 3.53 Impact Factor
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    ABSTRACT: RNA-seq is widely used to determine differential expression of genes or transcripts as well as identify novel transcripts, identify allele-specific expression, and precisely measure translation of transcripts. Thoughtful experimental design and choice of analysis tools are critical to ensure high-quality data and interpretable results. Important considerations for experimental design include number of replicates, whether to collect paired-end or single-end reads, sequence length, and sequencing depth. Common analysis steps in all RNA-seq experiments include quality control, read alignment, assigning reads to genes or transcripts, and estimating gene or transcript abundance. Our aims are two-fold: to make recommendations for common components of experimental design and assess tool capabilities for each of these steps. We also test tools designed to detect differential expression, since this is the most widespread application of RNA-seq. We hope that these analyses will help guide those who are new to RNA-seq and will generate discussion about remaining needs for tool improvement and development. Curr. Protoc. Hum. Genet. 83:11.13.1-11.13.20. © 2014 by John Wiley & Sons, Inc.
    Current protocols in human genetics / editorial board, Jonathan L. Haines ... [et al.] 01/2014; 83:11.13.1-11.13.20.
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    ABSTRACT: Large-scale annotation efforts have improved our ability to coarsely predict regulatory elements throughout vertebrate genomes. However, it is unclear how complex spatiotemporal patterns of gene expression driven by these elements emerge from the activity of short, transcription factor binding sequences. We describe a comprehensive promoter extension assay in which the regulatory potential of all 6 base-pair (bp) sequences was tested in the context of a minimal promoter. To enable this large-scale screen, we developed algorithms that use a reverse-complement aware decomposition of the de Bruijn graph to design a library of DNA oligomers incorporating every 6-bp sequence exactly once. Our library multiplexes all 4,096 unique 6-mers into 184 double-stranded 15-bp oligomers, which is sufficiently compact for in vivo testing. We injected each multiplexed construct into zebrafish embryos and scored GFP expression in 15 tissues at two developmental time points. Twenty-seven constructs produced consistent expression patterns, with the majority doing so in only one tissue. Functional sequences are enriched near biologically relevant genes, match motifs for developmental transcription factors, and are required for enhancer activity. By concatenating tissue-specific functional sequences, we generated completely synthetic enhancers for the notochord, epidermis, spinal cord, forebrain and otic lateral line, and show that short regulatory sequences do not always function modularly. This work introduces a unique in vivo catalog of short, functional regulatory sequences and demonstrates several important principles of regulatory element organization. Furthermore, we provide resources for designing compact, reverse-complement aware k-mer libraries.
    Genome biology 07/2013; 14(7):R72. · 10.30 Impact Factor
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    ABSTRACT: BACKGROUND: We describe the genome of the western painted turtle, Chrysemys picta bellii, one of the most widespread, abundant and well-studied turtles. We place the genome into a comparative evolutionary context, and focus on genomic features associated with tooth loss, immune function, longevity, sex differentiation and determination, and the species¿ physiological capacities to withstand extreme anoxia and tissue freezing. RESULTS: Our phylogenetic analyses confirm that turtles are the sister group to living archosaurs, and demonstrate an extraordinarily slow rate of sequence evolution in the painted turtle. The ability of the painted turtle to withstand complete anoxia and partial freezing appears to be associated with common vertebrate gene networks, and we identify candidate genes for future functional analyses. Tooth loss shares a common pattern of pseudogenization and degradation of tooth-specific genes with birds, although the rate of accumulation of mutations is much slower in the painted turtle. Genes associated with sex differentiation generally reflect phylogeny rather than convergence in sex determination functionality. Among gene families that demonstrate exceptional expansions or show signatures of strong natural selection, immune function and musculoskeletal patterning genes are consistently overrepresented. CONCLUSIONS: Our comparative genomic analyses indicate that common vertebrate regulatory networks, some of which have analogs in human diseases, are often involved in the western painted turtle¿s extraordinary physiological capacities. As these regulatory pathways are analyzed at the functional level, the painted turtle may offer important insights into the management of a number of human health disorders.
    Genome biology 03/2013; 14(3):R28. · 10.30 Impact Factor
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    ABSTRACT: Heart development is exquisitely sensitive to the precise temporal regulation of thousands of genes that govern developmental decisions during differentiation. However, we currently lack a detailed understanding of how chromatin and gene expression patterns are coordinated during developmental transitions in the cardiac lineage. Here, we interrogated the transcriptome and several histone modifications across the genome during defined stages of cardiac differentiation. We find distinct chromatin patterns that are coordinated with stage-specific expression of functionally related genes, including many human disease-associated genes. Moreover, we discover a novel preactivation chromatin pattern at the promoters of genes associated with heart development and cardiac function. We further identify stage-specific distal enhancer elements and find enriched DNA binding motifs within these regions that predict sets of transcription factors that orchestrate cardiac differentiation. Together, these findings form a basis for understanding developmentally regulated chromatin transitions during lineage commitment and the molecular etiology of congenital heart disease.
    Cell 09/2012; 151(1):206-20. · 31.96 Impact Factor
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    ABSTRACT: This report of independent genome sequences of two natural populations of Drosophila melanogaster (37 from North America and 6 from Africa) provides unique insight into forces shaping genomic polymorphism and divergence. Evidence of interactions between natural selection and genetic linkage is abundant not only in centromere- and telomere-proximal regions, but also throughout the euchromatic arms. Linkage disequilibrium, which decays within 1 kbp, exhibits a strong bias toward coupling of the more frequent alleles and provides a high-resolution map of recombination rate. The juxtaposition of population genetics statistics in small genomic windows with gene structures and chromatin states yields a rich, high-resolution annotation, including the following: (1) 5'- and 3'-UTRs are enriched for regions of reduced polymorphism relative to lineage-specific divergence; (2) exons overlap with windows of excess relative polymorphism; (3) epigenetic marks associated with active transcription initiation sites overlap with regions of reduced relative polymorphism and relatively reduced estimates of the rate of recombination; (4) the rate of adaptive nonsynonymous fixation increases with the rate of crossing over per base pair; and (5) both duplications and deletions are enriched near origins of replication and their density correlates negatively with the rate of crossing over. Available demographic models of X and autosome descent cannot account for the increased divergence on the X and loss of diversity associated with the out-of-Africa migration. Comparison of the variation among these genomes to variation among genomes from D. simulans suggests that many targets of directional selection are shared between these species.
    Genetics 06/2012; 192(2):533-98. · 4.39 Impact Factor
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    ABSTRACT: Currently, the diagnosis of malignant pheochromocytoma can only be made when there is clinical evidence of metastasis or extensive local invasion. Thus, there is a need for new diagnostic marker(s) to identify tumors with malignant potential. The purpose of this study was to identify microRNAs (miRNAs) that are differentially expressed between benign and malignant pheochromocytomas and assess their diagnostic accuracy. Toward this aim, we analyzed miRNA expression in benign and malignant pheochromocytoma tumor samples using whole genome microarray profiling. Microarray analysis identified eight miRNAs that were significantly differentially expressed between benign and malignant pheochromocytomas. We measured a subset of these miRNAs directly by RT-PCR and found that miR-483-5p, miR-183, and miR-101 had significantly higher expression in malignant tumors as compared to their benign counterparts. Area under the receiver operating curve (AUC) analysis indicated that miR-483-5p, miR-101, and miR-183 could be useful diagnostic markers for distinguishing malignant from benign pheochromocytomas. In addition, these miRNAs could be detected in pheochromocytoma patient serum. Overall our data suggest that misexpression of miR-483-5p, miR-101, and miR-183 is associated with malignant pheochromocytoma.
    Endocrine Related Cancer 01/2012; 19(2):157-66. · 5.26 Impact Factor
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    ABSTRACT: The comparison of related genomes has emerged as a powerful lens for genome interpretation. Here we report the sequencing and comparative analysis of 29 eutherian genomes. We confirm that at least 5.5% of the human genome has undergone purifying selection, and locate constrained elements covering ∼4.2% of the genome. We use evolutionary signatures and comparisons with experimental data sets to suggest candidate functions for ∼60% of constrained bases. These elements reveal a small number of new coding exons, candidate stop codon readthrough events and over 10,000 regions of overlapping synonymous constraint within protein-coding exons. We find 220 candidate RNA structural families, and nearly a million elements overlapping potential promoter, enhancer and insulator regions. We report specific amino acid residues that have undergone positive selection, 280,000 non-coding elements exapted from mobile elements and more than 1,000 primate- and human-accelerated elements. Overlap with disease-associated variants indicates that our findings will be relevant for studies of human biology, health and disease.
    Nature 10/2011; 478(7370):476-82. · 38.60 Impact Factor
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    ABSTRACT: Assignment of alleles to haplotypes for nearly all the variants on all chromosomes can be performed by genetic analysis of a nuclear family with three or more children. Whole-genome sequence data enable deterministic phasing of nearly all sequenced alleles by permitting assignment of recombinations to precise chromosomal positions and specific meioses. We demonstrate this process of genetic phasing on two families each with four children. We generate haplotypes for all of the children and their parents; these haplotypes span all genotyped positions, including rare variants. Misassignments of phase between variants (switch errors) are nearly absent. Our algorithm can also produce multimegabase haplotypes for nuclear families with just two children and can handle families with missing individuals. We implement our algorithm in a suite of software scripts (Haploscribe). Haplotypes and family genome sequences will become increasingly important for personalized medicine and for fundamental biology.
    The American Journal of Human Genetics 08/2011; 89(3):382-97. · 11.20 Impact Factor
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    ABSTRACT: The authors are interested in identifying molecular markers that can aid in the diagnosis of adrenocortical carcinoma (ACC). The aim of this study was to identify microRNAs (miRNAs or miRs) that are differentially expressed in malignant adrenocortical tumors as compared with benign tumors and assess their potential as diagnostic predictors. Differentially expressed miRNAs were identified using microarray profiling of adrenocortical tumors and validated by quantitative real-time RT-PCR. Microarray profiling in benign and primary malignant adrenocortical tumors revealed several significant differences between these histological groups. By using directed quantitative RT-PCR analysis on a subset of these differentially expressed miRNAs, the authors determined that miRs -100, -125b, and -195 were significantly down-regulated, whereas miR-483-5p was significantly up-regulated in malignant as compared with benign tumors. Furthermore, the current study shows that miR-483-5p expression can accurately categorize tumors as benign or malignant. The authors identified 4 miRNAs that are dysregulated in adrenocortical carcinoma. The high expression of one of these, miR-483-5p, appears to be a defining characteristic of adrenocortical malignancies, and can thus be used to accurately distinguish between benign and malignant adrenocortical tumors.
    Cancer 04/2011; 117(8):1630-9. · 5.20 Impact Factor
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    ABSTRACT: Transcription factor binding site(s) (TFBS) gain and loss (i.e., turnover) is a well-documented feature of cis-regulatory module (CRM) evolution, yet little attention has been paid to the evolutionary force(s) driving this turnover process. The predominant view, motivated by its widespread occurrence, emphasizes the importance of compensatory mutation and genetic drift. Positive selection, in contrast, although it has been invoked in specific instances of adaptive gene expression evolution, has not been considered as a general alternative to neutral compensatory evolution. In this study we evaluate the two hypotheses by analyzing patterns of single nucleotide polymorphism in the TFBS of well-characterized CRM in two closely related Drosophila species, Drosophila melanogaster and Drosophila simulans. An important feature of the analysis is classification of TFBS mutations according to the direction of their predicted effect on binding affinity, which allows gains and losses to be evaluated independently along the two phylogenetic lineages. The observed patterns of polymorphism and divergence are not compatible with neutral evolution for either class of mutations. Instead, multiple lines of evidence are consistent with contributions of positive selection to TFBS gain and loss as well as purifying selection in its maintenance. In discussion, we propose a model to reconcile the finding of selection driving TFBS turnover with constrained CRM function over long evolutionary time.
    PLoS Genetics 04/2011; 7(4):e1002053. · 8.52 Impact Factor
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    ABSTRACT: Dominant mutations in cardiac transcription factor genes cause human inherited congenital heart defects (CHDs); however, their molecular basis is not understood. Interactions between transcription factors and the Brg1/Brm-associated factor (BAF) chromatin remodelling complex suggest potential mechanisms; however, the role of BAF complexes in cardiogenesis is not known. In this study, we show that dosage of Brg1 is critical for mouse and zebrafish cardiogenesis. Disrupting the balance between Brg1 and disease-causing cardiac transcription factors, including Tbx5, Tbx20 and Nkx2-5, causes severe cardiac anomalies, revealing an essential allelic balance between Brg1 and these cardiac transcription factor genes. This suggests that the relative levels of transcription factors and BAF complexes are important for heart development, which is supported by reduced occupancy of Brg1 at cardiac gene promoters in Tbx5 haploinsufficient hearts. Our results reveal complex dosage-sensitive interdependence between transcription factors and BAF complexes, providing a potential mechanism underlying transcription factor haploinsufficiency, with implications for multigenic inheritance of CHDs.
    Nature Communications 02/2011; 2:187. · 10.74 Impact Factor
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    ABSTRACT: The developmental mechanisms through which the cerebral cortex increased in size and complexity during primate evolution are essentially unknown. To uncover genetic networks active in the developing cerebral cortex, we combined three-dimensional reconstruction of human fetal brains at midgestation and whole genome expression profiling. This novel approach enabled transcriptional characterization of neurons from accurately defined cortical regions containing presumptive Broca and Wernicke language areas, as well as surrounding associative areas. We identified hundreds of genes displaying differential expression between the two regions, but no significant difference in gene expression between left and right hemispheres. Validation by qRTPCR and in situ hybridization confirmed the robustness of our approach and revealed novel patterns of area- and layer-specific expression throughout the developing cortex. Genes differentially expressed between cortical areas were significantly associated with fast-evolving non-coding sequences harboring human-specific substitutions that could lead to divergence in their repertoires of transcription factor binding sites. Strikingly, while some of these sequences were accelerated in the human lineage only, many others were accelerated in chimpanzee and/or mouse lineages, indicating that genes important for cortical development may be particularly prone to changes in transcriptional regulation across mammals. Genes differentially expressed between cortical regions were also enriched for transcriptional targets of FoxP2, a key gene for the acquisition of language abilities in humans. Our findings point to a subset of genes with a unique combination of cortical areal expression and evolutionary patterns, suggesting that they play important roles in the transcriptional network underlying human-specific neural traits.
    PLoS ONE 01/2011; 6(3):e17753. · 3.53 Impact Factor
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    ABSTRACT: The molecular basis and characteristics of familial non-medullary thyroid cancer are poorly understood. In this study, we performed microRNA (miRNA) profiling of familial and sporadic papillary thyroid cancer tumor samples. Genome wide miRNA profiling of sporadic and familial papillary thyroid cancer was performed. Differentially expressed miRNAs were validated by quantitative RT-PCR. Ectopic expression of miR-886-3p in thyroid cancer lines was performed to identify pathways targeted by the miRNA, as well as, to determine its effect on tumor cell biology. We found four differentially expressed miRNAs between familial and sporadic papillary thyroid cancer tumor samples. MiR-886-3p and miR-20a were validated to be differentially expressed by 3- and 4-fold, respectively. Pathway analysis of genome-wide expression data on cells overexpressing miR-886-3p and target prediction analysis showed genes involved in DNA replication and focal adhesion pathways were regulated by miR-886-3p. Overexpression of miR-886-3p in thyroid cancer cell lines significantly inhibited cellular proliferation, the number and size of spheroids and cellular migration. Additionally, overexpression of miR-886-3p increased the number of cells in S phase. Our findings for the first time suggest that miR-886-3p plays an important role in thyroid cancer tumor cell biology and regulates genes involved in DNA replication and focal adhesion. Thus, miR-886-3p may play a role in the initiation and or progression of papillary thyroid cancer.
    PLoS ONE 01/2011; 6(10):e24717. · 3.53 Impact Factor
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    ABSTRACT: The molecular factors that control parathyroid tumorigenesis are poorly understood. In the absence of local invasion or metastasis, distinguishing benign from malignant parathyroid neoplasm is difficult on histologic examination. We studied the microRNA (miRNA) profile in normal, hyperplastic, and benign and malignant parathyroid tumors to better understand the molecular factors that may play a role in parathyroid tumorigenesis and that may serve as diagnostic markers for parathyroid carcinoma. miRNA arrays containing 825 human microRNAs with four duplicate probes per miRNA were used to profile parathyroid tumor (12 adenomas, 9 carcinomas, and 15 hyperplastic) samples normalized to four reference normal parathyroid glands. Differentially expressed miRNA were validated by real-time quantitative TaqMan polymerase chain reaction (PCR). One hundred fifty-six miRNAs in parathyroid hyperplasia, 277 microRNAs in parathyroid adenoma, and 167 microRNAs in parathyroid carcinomas were significantly dysregulated as compared with normal parathyroid glands [false discovery rate (FDR) < 0.05]. By supervised clustering analysis, all parathyroid carcinomas clustered together. Three miRNAs (miR-26b, miR-30b, and miR-126*) were significantly dysregulated between parathyroid carcinoma and parathyroid adenoma. Receiver-operating characteristic curve analysis showed mir-126* was the best diagnostic marker, with area under the curve of 0.776. Most miRNAs are downregulated in parathyroid carcinoma, while in parathyroid hyperplasia most miRNAs are upregulated. miRNA profiling shows distinct differentially expressed miRNAs by tumor type which may serve as helpful adjunct to distinguish parathyroid adenoma from carcinoma.
    Annals of Surgical Oncology 11/2010; 18(4):1158-65. · 4.12 Impact Factor
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    ABSTRACT: Determining the genetic basis of environmental adaptation is a central problem of evolutionary biology. This issue has been fruitfully addressed by examining genetic differentiation between populations that are recently separated and/or experience high rates of gene flow. A good example of this approach is the decades-long investigation of selection acting along latitudinal clines in Drosophila melanogaster. Here we use next-generation genome sequencing to reexamine the well-studied Australian D. melanogaster cline. We find evidence for extensive differentiation between temperate and tropical populations, with regulatory regions and unannotated regions showing particularly high levels of differentiation. Although the physical genomic scale of geographic differentiation is small--on the order of gene sized--we observed several larger highly differentiated regions. The region spanned by the cosmopolitan inversion polymorphism In(3R)P shows higher levels of differentiation, consistent with the major difference in allele frequencies of Standard and In(3R)P karyotypes in temperate vs. tropical Australian populations. Our analysis reveals evidence for spatially varying selection on a number of key biological processes, suggesting fundamental biological differences between flies from these two geographic regions.
    Genetics 11/2010; 187(1):245-60. · 4.39 Impact Factor
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    ABSTRACT: The Afrotropical mosquito Anopheles gambiae sensu stricto, a major vector of malaria, is currently undergoing speciation into the M and S molecular forms. These forms have diverged in larval ecology and reproductive behavior through unknown genetic mechanisms, despite considerable levels of hybridization. Previous genome-wide scans using gene-based microarrays uncovered divergence between M and S that was largely confined to gene-poor pericentromeric regions, prompting a speciation-with-ongoing-gene-flow model that implicated only about 3% of the genome near centromeres in the speciation process. Here, based on the complete M and S genome sequences, we report widespread and heterogeneous genomic divergence inconsistent with appreciable levels of interform gene flow, suggesting a more advanced speciation process and greater challenges to identify genes critical to initiating that process.
    Science 10/2010; 330(6003):512-4. · 31.20 Impact Factor
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    ABSTRACT: Understanding how DNA sequence polymorphism relates to variation in gene expression is essential to connecting genotypic differences with phenotypic differences among individuals. Addressing this question requires linking population genomic data with gene expression variation. Using whole genome expression data and recent light shotgun genome sequencing of six Drosophila simulans genotypes, we assessed the relationship between expression variation in males and females and nucleotide polymorphism across thousands of loci. By examining sequence polymorphism in gene features, such as untranslated regions and introns, we find that genes showing greater variation in gene expression between genotypes also have higher levels of sequence polymorphism in many gene features. Accordingly, X-linked genes, which have lower sequence polymorphism levels than autosomal genes, also show less expression variation than autosomal genes. We also find that sex-specifically expressed genes show higher local levels of polymorphism and divergence than both sex-biased and unbiased genes, and that they appear to have simpler regulatory regions. The gene-feature-based analyses and the X-to-autosome comparisons suggest that sequence polymorphism in cis-acting elements is an important determinant of expression variation. However, this relationship varies among the different categories of sex-biased expression, and trans factors might contribute more to male-specific gene expression than cis effects. Our analysis of sex-specific gene expression also shows that female-specific genes have been overlooked in analyses that only point to male-biased genes as having unusual patterns of evolution and that studies of sexually dimorphic traits need to recognize that the relationship between genetic and expression variation at these traits is different from the genome as a whole.
    Genome biology 09/2008; 9(8):R125. · 10.30 Impact Factor

Publication Stats

1k Citations
281.22 Total Impact Points

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Institutions

  • 2004–2012
    • University of California, Davis
      • • Department of Evolution & Ecology
      • • Center for Population Biology
      Davis, CA, United States
  • 2011
    • National Institutes of Health
      • Section on Endocrine Physiology
      Bethesda, MD, United States
  • 2010–2011
    • National Cancer Institute (USA)
      Maryland, United States
    • University of California, San Francisco
      • • Gladstone Institute
      • • Department of Surgery
      San Francisco, CA, United States
    • Dartmouth College
      • Department of Biological Sciences
      Hanover, NH, United States
    • J. David Gladstone Institutes
      San Francisco, California, United States
  • 2006
    • University of Texas at Austin
      • Department of Integrative Biology
      Texas City, TX, United States