The role of the DNA sequence in protein-coding genes has been widely established due to the obvious functional relevance and well-defined relationships between DNA variation and protein sequence. ...However, most disease-associated variants are not in protein-coding sequences. This has led to much investigation of non-coding variation as it relates to protein-coding gene regulation. Critically, non-coding regulatory elements within DNA are intricately linked to cell type-specific protein-coding gene expression. This dissertation contributes to the goal of uncovering the underlying cis-regulatory logic of non-coding DNA sequences driving cell type-specific gene expression in homeostasis and disease. Cell type-specific non-coding cis-regulatory elements are known to drive important gene expression programs. However, it is still unclear what combinatorial DNA sequences underlie cell type-specific gene regulatory mechanisms. The objective of this dissertation is to progress our understanding of cis-regulatory logic through integration of genome-wide regulatory maps followed by modeling of combinatorial DNA motif logic in cis-regulatory modules regulating cell type-specific gene expression programs and focused identification of functional regulatory DNA motif combinations (DMCs) in homeostasis and complex disease. In Chapter 2, I generate epigenomic and transcriptomic profiles of 15 primary human cell types and perform integrated analysis to define cell type-specific open chromatin peak-long range looped-expressed target gene transcripts (PLTs). I then incorporate disease-associated single nucleotide variants (SNVs) from the NHGRI GWAS catalog and Genotype-Tissue Expression (GTEx) data to link SNVs enriched in cell type-specific PLTs to putative target genes. In Chapter 3, I model the genomic information necessary to derive cis-regulatory modules linked to cell type-specific gene expression programs and nominate transcription factor (TF) DMCs underlying cell type-specific regulatory logic. Finally, in Chapter 4, I validate DMC logic in four primary human cell types and identify cancer-specific regulatory logic in human squamous cell carcinoma and melanoma cells. The findings and approaches described in this dissertation add to the existing annotation of functional combinatorial TF motif logic and help build a framework for future studies of cis-regulatory logic.
Somatization, or physical symptoms that are inconsistent with a physiological cause that may or may not involve an identified stressor, is common in outpatient pediatrics. When these symptoms ...persist, they can impair function and progress to a somatic symptom and related disorder (SSRD), resulting in increased health care use and increased demands on primary care providers (PCPs). We performed a needs assessment among PCPs to better understand how best to support providers caring for children with SSRDs. Pediatric PCPs (n = 77) were surveyed to better understand their training, experience, perceptions, and practices of SSRD care. Findings indicate that PCPs have limited training in SSRD care but express interest in learning more. Many barriers to effective care were reported. We hope to use these findings to develop training materials and support services for pediatric PCPs managing SSRDs.
Between 1960 and 2000 the US responded to growing threats of terrorism with wide ranging measures. As discussion in the US progresses on the best course of action for dealing with conventional, ...chemical, biological, nuclear, or radiological terrorism, it is useful to take stock of where the country stands in the development of its counterterrorism strategy and to consider what factors have shaped the American response. Provides a classified review of efforts to address the threat, and argues that, while some gaps may need to be addressed, a more serious concern is the long term effect of the steady expansion of US counterterrorist measures. (Original abstract - amended)
Gain-of-function mutations in Notch receptor genes occur in 10-15% of cases of chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), and are associated with inferior clinical outcomes. ...Nearly all Notch mutations reported in B cell tumors lead to loss of the C-terminal negative regulatory PEST domain and result in stabilization of the activated form of Notch (intracellular Notch ICN), whereas mutations that lead to ligand-independent Notch activation (which are common in T cell acute lymphoblastic leukemia T-ALL) are rare. ICN can be detected in tumor cells within lymph nodes of >80% of patients with CLL, suggesting that Notch may have a broader oncogenic role than the incidence of Notch mutations would suggest. However, the downstream targets of Notch in B-cell tumors have not been identified.
We used a gamma-secretase inhibitor (GSI) washout strategy to determine the immediate, direct effects of Notch activation in three MCL cell lines with Notch gain-of-function mutations, including two cell lines with unusual Notch gene rearrangements that lead to ligand-independent Notch activation, as well as a third line with a Notch PEST domain mutation in which signaling was activated with recombinant Notch ligand. Using these models, we identified likely direct target genes and their associated genomic Notch response elements using RNA-seq and ChIP-Seq in the Notch-on and Notch-off states. Most of these response elements corresponded to long-range enhancers that showed Notch-dependent changes in H3K27 acetylation, and were bound by components of the Notch transcription complex (NTC) in both cell lines. We confirmed these associations by performing ChIP-Seq on primary CLL and MCL biopsies, and by identifying specific looping interactions with Notch target gene promoters in public genome-wide proximity ligation datasets (RNA Pol2 ChIA-PET) from a lymphoblastoid cell line expressing the EBV-encoded Notch surrogate protein EBNA2.
MYC was among the most strongly Notch-activated genes in Notch-dependent MCL cell lines and was associated with NTC binding at two B cell-specific 5' enhancers distinct from the Notch-dependent MYC enhancer previously identified in T-ALL. MCL cell line proliferation was blocked by Cas9 nuclease or epigenetic repressors targeting the 5' MYC enhancers, whereas cells were rescued from Notch inhibition by GSI via transduction with MYC. Gene set enrichment analysis of other direct Notch target genes identified in MCL models showed enrichment for regulators of B cell receptor (BCR) signaling, including the Src family kinase genes FYN, LYN, and BLK, and the signaling complex adaptor BLNK, as well as regulators of CD40 and cytokine signaling.
RNA-seq analysis of primary CLL lymph node biopsies revealed significantly higher expression of many Notch target genes in biopsies with high levels of ICN. To functionally validate Notch target genes in primary tumors, we co-cultured CLL and MCL cells obtained from peripheral blood with Notch ligand-expressing stromal cells in the presence ("notch off") or absence ("notch on") of GSI, and demonstrated increased expression of Notch target genes, including MYC, in the "notch-on" cells. Furthermore, "notch-on" CLL cells showed increased phosphorylation of the BCR signaling intermediates SYK and PLCg2 upon BCR crosslinking compared to GSI-treated cells. Finally, we validated Notch-dependent regulation of target genes in vivo in a patient-derived xenograft model of NOTCH1-mutant MCL. Notch target gene expression was significantly higher in MCL cells within the spleen versus bone marrow or blood, but was markedly reduced in animals treated for five days with GSI. Additional xenograft studies are ongoing, and will be described at the meeting.
Our data link active Notch signaling to two well-characterized oncogenic drivers in B cell lymphoma, MYC and BCR signaling, and may have important implications for the development of treatment strategies involving Notch antagonists and other targeted therapeutics, such as BCR targeting agents.
Weinstock:Novartis: Consultancy, Research Funding.
Donohue comments on Smith and colleagues' article, "The Prosecution and Punishment of International Terrorists on Federal Courts: 1980-1998," saying that Smith et al fail to offer remedies for the ...consequent bias entailed from criminal sentencing of foreign terrorists than domestic terrorists.
Although the 1922–43 Special Powers Acts (SPAs) played a
central role in prompting
the Northern Irish civil rights movement in the late 1960s, virtually no
secondary literature exists on
the ...operation of the statutes. This article examines the manner in which
the unionist government
employed the acts during the tenure of the northern parliament. It suggests
that the 1922–43 SPAs
became a central grievance of the minority community because of the manner
in which regulations
introduced under their auspices were exercised: the ministry of home affairs
initially used the emergency
statutes to return civil order to the province; however, as violence declined
the government began to
utilize regulations to prevent the expression of republican ideals. Any
attempt to garner support for a
united Ireland was perceived as an attack on the Northern Irish constitution.
Concurrent with this shift
was a change in justification for the 1922–43 SPAs: from being required
in order to establish law and
order in the face of rising violence, they were soon heralded by unionists
as necessary to maintain the
constitutional structure of the North. In preventing the public exposition
of republicanism, expressions
of nationalism were likewise limited. This impacted upon a large portion
of the minority community,
giving rise to claims that the statutes were being unfairly applied.
In the late twentieth century, the United States' federal government responded to the threat of terrorism by passing a wide range of counterterrorist laws. The vigor that accompanied these ...initiatives echoed at a state level where, virtually unnoticed, states passed similar legislation. This article examines state measures in three areas: the funding of foreign terrorist organizations, the use or threatened use of weapons of mass destruction, and definitions of terrorist activity. While these statutes, as a legal matter, may not violate any specific federal provisions or constitutional prohibitions, they raise important questions about federal supremacy in foreign affairs and the constitutional protections afforded citizens. More significantly, as a policy concern, these provisions threaten America's ability to speak in one voice, introducing divisions into the domestic realm and diminishing the ability of the federal government to negotiate with foreign states and organizations. They also mask an appropriate role for the states in fighting terrorism. Both the policy implications and legal considerations suggest that such measures may ultimately undermine America's ability to counter the terrorist threat.
This article explores three sets of legislative authorities that have dominated the anti-terrorist finance realm post-9/11: Specially Designated Global Terrorists, Foreign Terrorist Organizations, ...and financial surveillance. Drawing from examples in each area, this article suggests that the constitutional concerns are more than just growing pains. In some sense, they are an inevitable byproduct of legislative action in this area, which the courts are not particularly well-positioned to address. Equal protection claims have failed, and due process concerns relating to notice and hearing have been a persistent issue in the evolution of the statutory authorities. But this article focuses more narrowly on counterterrorist law, where these concerns play out in a particularly severe way. In light of the relatively weak position of the judiciary, it is all the more important for Congress to take due account of constitutional considerations -- particularly in considering any new initiatives -- in the anti-terrorist finance realm.
PDF
