Using Proteomics to Discover New Connections in the Arabidopsis Circadian Clock

The plant circadian clock is an endogenous timekeeping mechanism that uses daylength and temperature cycles to synchronize internal physiology with the external environment. Much of our understanding of the clock in the model plant Arabidopsis thaliana comes from genetic approaches. In this thesis, I use affinity purification coupled with mass spectrometry (APMS) to identify protein-protein interactions for core clock components on a proteomic scale. I developed and optimized a protocol to perform APMS on a core set of circadian clock proteins: CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYL (LHY), PSEUDORESPONSE REGULATOR 5 (PRR5), PRR7, PRR9, TIMING OF CAB 1 (TOC1)/PRR1, FIONA 1 (FIO1), JUMONJI DOMAIN CONTAINING 5 (JMJD5), NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED 1 (LNK1), LNK2, and REVEILLE 8 (RVE8). The combined dataset of proteins coprecipitated with these clock factors represents a circadian clock “interactome” that is publicly available for future studies. I chose to follow up on an interaction between RVE8/LNK1/LNK2 and two proteins previously unrelated to these clock components, COLD-REGULATED PROTEIN 27 (COR27) and COR28. I found that these proteins form a complex in the early evening that serves to regulate RVE8 protein stability and to block the transcriptional activity of RVE8-LNK1/2. Together, this work demonstrates the power of proteomics to make new discoveries in the plant circadian clock and I hope that my datasets will be a useful tool for future studies

Development of molecular tools for expression and trafficking studies of the human monocarboxylate transporters

Most cancer cells rely on glycolysis to sustain their high proliferation rates with the production of lactate. For many years, lactate was seen as a metabolic waste of glycolytic metabolism in the tumor microenvironment, however, lactate has been recently associated as a key metabolic fuel and as an important signaling molecule 1,2. This substrate is responsible for extracellular acidification, which, is a feature of the tumor environment, and favors tumor invasion. The transport of lactate across the plasma membrane is mediated by a family of proton coupled monocarboxylate transporters (MCTs), which comprises 14 members 3. MCT1 and MCT4 serve as metabolic links between cancer cells via lactate exchange within tumors. This form of metabolic symbiosis illustrates how the apparent waste product from hypoxic tumor cells may be exploited by oxidative tumor cells to sustain their energy production under nutrient deprived conditions 4. MCTs are not only gatekeepers of intercellular metabolic cooperation, but also important regulators of angiogenesis and tumor migration, invasion and metastasis 5 . However, the role of MCTs in tumors is far from being well understood and their potential as therapeutic targets is poorly explored. Given the relationships between MCT1 and MCT4 in cancer cells, they offer a unique opportunity for novel treatment strategies. In this work, a set of molecular tools was generated for the expression and trafficking analyses of MCT1 and MCT4. Plasmids were designed harboring MCT1 or MCT4 with GFP or mCherry at the C- or N- terminal following the classical DNA cloning method. These molecular tools will be essential to study the expression and localization of MCT1 and MCT4 and to study the conditions and mechanisms underlying the endocytic trafficking of both transporters to further elucidate the significance of MCTs expression in tumor cells

Novel Health Information Technology to Aid Provider Recognition and Treatment of Major Depressive Disorder and Posttraumatic Stress Disorder in Primary Care.

BACKGROUND: Millions of traumatized refugees worldwide have resettled in the United States. For one of the largest, the Cambodian community, having their mental health needs met has been a continuing challenge. A multicomponent health information technology screening tool was designed to aid provider recognition and treatment of major depressive disorder and posttraumatic stress disorder (PTSD) in the primary care setting. METHODS: In a clustered randomized controlled trial, 18 primary care providers were randomized to receive access to a multicomponent health information technology mental health screening intervention, or to a minimal intervention control group; 390 Cambodian American patients empaneled to participating providers were assigned to the providers randomized group. RESULTS: Electronic screening revealed that 65% of patients screened positive for depression and 34% screened positive for PTSD. Multilevel mixed effects logistic models, accounting for clustering structure, indicated that providers in the intervention were more likely to diagnose depression [odds ratio (OR), 6.5; 95% confidence interval (CI), 1.48-28.79; P=0.013] and PTSD (OR, 23.3; 95% CI, 2.99-151.62; P=0.002) among those diagnosed during screening, relative to the control group. Providers in the intervention were more likely to provide evidence-based guideline (OR, 4.02; 95% CI, 1.01-16.06; P=0.049) and trauma-informed (OR, 15.8; 95% CI, 3.47-71.6; P<0.001) care in unadjusted models, relative to the control group. Guideline care, but not trauma-informed care, was associated with decreased depression at 12 weeks in both study groups (P=0.003), and neither was associated with PTSD outcomes at 12 weeks. CONCLUSIONS: This innovative approach offers the potential for training primary care providers to diagnose and treat traumatized patients, the majority of whom seek mental health care in primary care (ClinicalTrials.gov number, NCT03191929)

New generalized nonspherical black hole solutions

We present numerical evidence for the existence of several types of static black hole solutions with a nonspherical event horizon topology in $d\geq 6$ spacetime dimensions. These asymptotically flat configurations are found for a specific metric ansatz and can be viewed as higher dimensional counterparts of the $d=5$ static black rings, dirings and black Saturn. Similar to that case, they are supported against collapse by conical singularities. The issue of rotating generalizations of these solutions is also considered.Comment: 47 pages, 11 figures, some comments adde

COLD REGULATED GENE 27 and 28 Antagonize the Transcriptional Activity of the RVE8/LNK1/LNK2 Circadian Complex

Many molecular and physiological processes in plants occur at a specific time of day. These daily rhythms are coordinated in part by the circadian clock, a timekeeper that uses daylength and temperature to maintain rhythms of ∼24 h in various clock-regulated phenotypes. The circadian MYB-like transcription factor REVEILLE 8 (RVE8) interacts with its transcriptional coactivators NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED 1 (LNK1) and LNK2 to promote the expression of evening-phased clock genes and cold tolerance factors. While genetic approaches have commonly been used to discover connections within the clock and between clock elements and other pathways, here, we used affinity purification coupled with mass spectrometry (APMS) to identify time-of-day-specific protein interactors of the RVE8-LNK1/LNK2 complex in Arabidopsis (Arabidopsis thaliana). Among the interactors of RVE8/LNK1/LNK2 were COLD-REGULATED GENE 27 (COR27) and COR28, which coprecipitated in an evening-specific manner. In addition to COR27 and COR28, we found an enrichment of temperature-related interactors that led us to establish a previously uncharacterized role for LNK1 and LNK2 in temperature entrainment of the clock. We established that RVE8, LNK1, and either COR27 or COR28 form a tripartite complex in yeast (Saccharomyces cerevisiae) and that the effect of this interaction in planta serves to antagonize transcriptional activation of RVE8 target genes, potentially through mediating RVE8 protein degradation in the evening. Together, these results illustrate how a proteomic approach can be used to identify time-of-day-specific protein interactions. Discovery of the RVE8-LNK-COR protein complex indicates a previously unknown regulatory mechanism for circadian and temperature signaling pathways

Requirements for F-BAR Proteins TOCA-1 and TOCA-2 in Actin Dynamics and Membrane Trafficking during Caenorhabditis elegans Oocyte Growth and Embryonic Epidermal Morphogenesis

The TOCA family of F-BAR–containing proteins bind to and remodel lipid bilayers via their conserved F-BAR domains, and regulate actin dynamics via their N-Wasp binding SH3 domains. Thus, these proteins are predicted to play a pivotal role in coordinating membrane traffic with actin dynamics during cell migration and tissue morphogenesis. By combining genetic analysis in Caenorhabditis elegans with cellular biochemical experiments in mammalian cells, we showed that: i) loss of CeTOCA proteins reduced the efficiency of Clathrin-mediated endocytosis (CME) in oocytes. Genetic interference with CeTOCAs interacting proteins WSP-1 and WVE-1, and other components of the WVE-1 complex, produced a similar effect. Oocyte endocytosis defects correlated well with reduced egg production in these mutants. ii) CeTOCA proteins localize to cell–cell junctions and are required for proper embryonic morphogenesis, to position hypodermal cells and to organize junctional actin and the junction-associated protein AJM-1. iii) Double mutant analysis indicated that the toca genes act in the same pathway as the nematode homologue of N-WASP/WASP, wsp-1. Furthermore, mammalian TOCA-1 and C. elegans CeTOCAs physically associated with N-WASP and WSP-1 directly, or WAVE2 indirectly via ABI-1. Thus, we propose that TOCA proteins control tissues morphogenesis by coordinating Clathrin-dependent membrane trafficking with WAVE and N-WASP–dependent actin-dynamics

Persistent cAMP-Signals Triggered by Internalized G-Protein–Coupled Receptors

Real-time monitoring of G-protein-coupled receptor (GPCR) signaling in native cells suggests that the receptor for thyroid stimulating hormone remains active after internalization, challenging the current model for GPCR signaling