Genetic noise in the cyanobacterial circadian oscillator

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Physics, June 2005.Includes bibliographical references (p. 155-161).Clocks are generally considered as quintessential examples of accurate and precise devices. Biological clocks however, are continually subjected to intracellular and extracellular fluctuations that might reduce the fidelity of this periodic timer. One fundamental limitation that might set an upper bound on the fidelity is the stochastic nature of gene expression creating a noisy intracellular environment. Circadian rhythms, driven by oscillators which provide cells with an internal clock that controls the gene expression program, have been observed in a wide range of organisms from cyanobacteria to mammals. To explore the impact of stochastic expression fluctuations on the performance of the circadian clock, it is necessary to monitor single cells, since population experiments will average out these fluctuations. The cyanobacterium Synechococcus elongatus PCC7942 is an excellent candidate for this study since its core circadian oscillator is well explored. We therefore measured, in single cells, the expression fluctuations of a fluorescent reporter driven by the cyanobacterial circadian oscillator. Repeated microscopic imaging of individual cells and their progeny revealed a robust circadian rhythm, and experiments with cells lacking the proposed central clock proteins confirm the crucial role they play in Synechococcus. Experiments conducted by microscopy and flow cytometry establish that the majority of genetic noise in Synechococcus arises from fluctuations uncorrelated between multiple genes (and therefore does not originate with a global clock noise).(Cont.) We observed via flow cytometry measurements a significant variability in cell-to-cell gene expression which is most pronounced during periods of net protein synthesis. An analytic model reveals that this nonequilibrium effect arises when protein lifetimes are comparable to the circadian period and the mean number of proteins translated per mRNA transcript is large. Stochastic Monte Carlo simulations verify this interpretation by providing a window into genetic behavior on an individual cell level. Estimates of the genetic noise supplied by intrinsic (uncorrelated), extrinsic (correlated within individual cells), and "clock" (unexplained sources with a 24 hour period) noise are developed. A quantitative description of noisy oscillators will be necessary for ultimately understanding the fidelity of circadian timekeeping and might inspire the design of robust synthetic oscillators.by Jeffrey R. Chabot.Ph.D

A Sea Turtle Population Assessment for Florida\u27s Big Bend, Northeastern Gulf of Mexico

Coastal waters of Florida’s Big Bend, Gulf of Mexico (GOM) once supported one of the largest sea turtle fisheries in the United States. To fill an information gap in this region on abundance and distribution of sea turtles, we used vessel—based distance sampling and active capture methods to characterize current foraging aggregations near the St. Martins Marsh Aquatic Preserve. Over 10 sampling periods between 2012—2018, we completed 513 km of transects and recorded 819 turtles among 4 species—green turtle (Chelonia mydas, n = 624), Kemp’s ridley (Lepidochelys kempii, n = 147), loggerhead (Caretta caretta, n = 47), and a single hawksbill (Eretmochelys imbricata). Turtle densities in 4 study plots within the 200 km2 study site ranged from 57—221 immature green turtles/km2, 16—56 immature Kemp’s ridleys/km2, and 1—14 juvenile—to—adult loggerheads/km2. Of 200 green turtles captured, 67.5% showed skin tumors consistent with fibropapillomatosis, a frequency similar to that from urbanized estuaries of Florida’s east coast. The largest green turtles (\u3e 60 cm straight standard carapace length), abundant in the southern portion of our study area, are of note because this size class is uncommonly recorded within US territorial waters. Analyses of green turtle mtDNA haplotypes found contributions from rookeries in the western GOM, Mexican Caribbean, and Costa Rica. Although Big Bend protected areas were principally designed to conserve marine and coastal habitats, these regulatory zones have also effectively encompassed a hotspot for foraging sea turtles

Two additive mechanisms impair the differentiation of 'substrate-selective' p38 inhibitors from classical p38 inhibitors in vitro

<p>Abstract</p> <p>Background</p> <p>The success of anti-TNF biologics for the treatment of rheumatoid arthritis has highlighted the importance of understanding the intracellular pathways that regulate TNF production in the quest for an orally-available small molecule inhibitor. p38 is known to strongly regulate TNF production via MK2. The failure of several p38 inhibitors in the clinic suggests the importance of other downstream pathways in normal cell function. Recent work has described a 'substrate-selective' p38 inhibitor that is able to preferentially block the activity of p38 against one substrate (MK2) versus another (ATF2). Using a combined experimental and computational approach, we have examined this mechanism in greater detail for two p38 substrates, MK2 and ATF2.</p> <p>Results</p> <p>We found that in a dual (MK2 and ATF2) substrate assay, MK2-p38 interaction reduced the activity of p38 against ATF2. We further constructed a detailed kinetic mechanistic model of p38 phosphorylation in the presence of multiple substrates and successfully predicted the performance of classical and so-called 'substrate-selective' p38 inhibitors in the dual substrate assay. Importantly, it was found that excess MK2 results in a stoichiometric effect in which the formation of p38-MK2-inhibitor complex prevents the phosphorylation of ATF2, despite the preference of the compound for the p38-MK2 complex over the p38-ATF2 complex. MK2 and p38 protein expression levels were quantified in U937, Thp-1 and PBMCs and found that [MK2] > [p38].</p> <p>Conclusion</p> <p>Our integrated mechanistic modeling and experimental validation provides an example of how systems biology approaches can be applied to drug discovery and provide a basis for decision-making with limited chemical matter. We find that, given our current understanding, it is unlikely that 'substrate-selective' inhibitors of p38 will work as originally intended when placed in the context of more complex cellular environments, largely due to a stoichiometric excess of MK2 relative to p38.</p

Pan-Cancer Analysis of lncRNA Regulation Supports Their Targeting of Cancer Genes in Each Tumor Context

Long noncoding RNAs (lncRNAs) are commonly dys-regulated in tumors, but only a handful are known toplay pathophysiological roles in cancer. We inferredlncRNAs that dysregulate cancer pathways, onco-genes, and tumor suppressors (cancer genes) bymodeling their effects on the activity of transcriptionfactors, RNA-binding proteins, and microRNAs in5,185 TCGA tumors and 1,019 ENCODE assays.Our predictions included hundreds of candidateonco- and tumor-suppressor lncRNAs (cancerlncRNAs) whose somatic alterations account for thedysregulation of dozens of cancer genes and path-ways in each of 14 tumor contexts. To demonstrateproof of concept, we showed that perturbations tar-geting OIP5-AS1 (an inferred tumor suppressor) andTUG1 and WT1-AS (inferred onco-lncRNAs) dysre-gulated cancer genes and altered proliferation ofbreast and gynecologic cancer cells. Our analysis in-dicates that, although most lncRNAs are dysregu-lated in a tumor-specific manner, some, includingOIP5-AS1, TUG1, NEAT1, MEG3, and TSIX, synergis-tically dysregulate cancer pathways in multiple tumorcontexts

Genomic, Pathway Network, and Immunologic Features Distinguishing Squamous Carcinomas

This integrated, multiplatform PanCancer Atlas study co-mapped and identified distinguishing molecular features of squamous cell carcinomas (SCCs) from five sites associated with smokin

Spatial Organization and Molecular Correlation of Tumor-Infiltrating Lymphocytes Using Deep Learning on Pathology Images

Beyond sample curation and basic pathologic characterization, the digitized H&E-stained images of TCGA samples remain underutilized. To highlight this resource, we present mappings of tumorinfiltrating lymphocytes (TILs) based on H&E images from 13 TCGA tumor types. These TIL maps are derived through computational staining using a convolutional neural network trained to classify patches of images. Affinity propagation revealed local spatial structure in TIL patterns and correlation with overall survival. TIL map structural patterns were grouped using standard histopathological parameters. These patterns are enriched in particular T cell subpopulations derived from molecular measures. TIL densities and spatial structure were differentially enriched among tumor types, immune subtypes, and tumor molecular subtypes, implying that spatial infiltrate state could reflect particular tumor cell aberration states. Obtaining spatial lymphocytic patterns linked to the rich genomic characterization of TCGA samples demonstrates one use for the TCGA image archives with insights into the tumor-immune microenvironment

Pan-cancer Alterations of the MYC Oncogene and Its Proximal Network across the Cancer Genome Atlas

Although theMYConcogene has been implicated incancer, a systematic assessment of alterations ofMYC, related transcription factors, and co-regulatoryproteins, forming the proximal MYC network (PMN),across human cancers is lacking. Using computa-tional approaches, we define genomic and proteo-mic features associated with MYC and the PMNacross the 33 cancers of The Cancer Genome Atlas.Pan-cancer, 28% of all samples had at least one ofthe MYC paralogs amplified. In contrast, the MYCantagonists MGA and MNT were the most frequentlymutated or deleted members, proposing a roleas tumor suppressors.MYCalterations were mutu-ally exclusive withPIK3CA,PTEN,APC,orBRAFalterations, suggesting that MYC is a distinct onco-genic driver. Expression analysis revealed MYC-associated pathways in tumor subtypes, such asimmune response and growth factor signaling; chro-matin, translation, and DNA replication/repair wereconserved pan-cancer. This analysis reveals insightsinto MYC biology and is a reference for biomarkersand therapeutics for cancers with alterations ofMYC or the PMN

Copy Number Variation of CCL3-like Genes Affects Rate of Progression to Simian-AIDS in Rhesus Macaques (Macaca mulatta)

Variation in genes underlying host immunity can lead to marked differences in susceptibility to HIV infection among humans. Despite heavy reliance on non-human primates as models for HIV/AIDS, little is known about which host factors are shared and which are unique to a given primate lineage. Here, we investigate whether copy number variation (CNV) at CCL3-like genes (CCL3L), a key genetic host factor for HIV/AIDS susceptibility and cell-mediated immune response in humans, is also a determinant of time until onset of simian-AIDS in rhesus macaques. Using a retrospective study of 57 rhesus macaques experimentally infected with SIVmac, we find that CCL3L CNV explains approximately 18% of the variance in time to simian-AIDS (p<0.001) with lower CCL3L copy number associating with more rapid disease course. We also find that CCL3L copy number varies significantly (p<10−6) among rhesus subpopulations, with Indian-origin macaques having, on average, half as many CCL3L gene copies as Chinese-origin macaques. Lastly, we confirm that CCL3L shows variable copy number in humans and chimpanzees and report on CCL3L CNV within and among three additional primate species. On the basis of our findings we suggest that (1) the difference in population level copy number may explain previously reported observations of longer post-infection survivorship of Chinese-origin rhesus macaques, (2) stratification by CCL3L copy number in rhesus SIV vaccine trials will increase power and reduce noise due to non-vaccine-related differences in survival, and (3) CCL3L CNV is an ancestral component of the primate immune response and, therefore, copy number variation has not been driven by HIV or SIV per se

Integrated Genomic Analysis of the Ubiquitin Pathway across Cancer Types

Protein ubiquitination is a dynamic and reversibleprocess of adding single ubiquitin molecules orvarious ubiquitin chains to target proteins. Here,using multidimensional omic data of 9,125 tumorsamples across 33 cancer types from The CancerGenome Atlas, we perform comprehensive molecu-lar characterization of 929 ubiquitin-related genesand 95 deubiquitinase genes. Among them, we sys-tematically identify top somatic driver candidates,including mutatedFBXW7with cancer-type-specificpatterns and amplifiedMDM2showing a mutuallyexclusive pattern withBRAFmutations. Ubiquitinpathway genes tend to be upregulated in cancermediated by diverse mechanisms. By integratingpan-cancer multiomic data, we identify a group oftumor samples that exhibit worse prognosis. Thesesamples are consistently associated with the upre-gulation of cell-cycle and DNA repair pathways, char-acterized by mutatedTP53,MYC/TERTamplifica-tion, andAPC/PTENdeletion. Our analysishighlights the importance of the ubiquitin pathwayin cancer development and lays a foundation fordeveloping relevant therapeutic strategies