Molecular Biology Commons^™

Hd2d Is A Regulator Of Abscisic Acid Responses In Arabidopsis, Joshua A. Farhi

Electronic Thesis and Dissertation Repository

Histone deacetylases have important roles in development and stress response in plants. To further investigate their function, the HD2D gene, of the plant specific HD2 family, was studied. An hd2d-1 mutant and two HD2D overexpression lines were used in this study. Germination was delayed in hd2d-1 and HD2D overexpression seeds only in the presence of ABA. HD2D was found to positively regulate the expression of members of the ABA-response pathway (ABI1, ABI5, and RD29A) leading to increased resistance to drought and salinity treatments. Furthermore, HD2D expression delayed flowering by positively regulating FLC expression. Using bimolecular fluorescence complementation, the HD2D protein …

Preventing Thymus Involution In K5.Cyclin D1 Transgenic Mice Sustains The Naïve T Cell Compartment With Age, Michelle L. Bolner

Dissertations & Theses (Open Access)

The thymus maintains T cell receptor (TCR) repertoire diversity through perpetual release of self-MHC restricted naive T cells. However, thymus involution during the aging process reduces naïve T cell output, leading to defective immune responsiveness to newly encountered antigens. We have found that early thymus involution precipitates the age-associated shift favoring memory T cell dominancy in young control mice. Furthermore, we have shown that age-related thymus involution is prevented in mice expressing a keratin 5 promoter-driven Cyclin D1 (K5.D1) transgene in thymic epithelial cells (TECs). Thymopoiesis occurs normally in K5.D1 transgenic thymi and sustains T cell output to prevent the …

Reactive Oxygen Species-Mediated Neurodegeneration Is Independent Of The Ryanodine Receptor In Caenorhabditis Elegans, Lyndsay E.A. Young, Daniel C. Williams

Journal of the South Carolina Academy of Science

Despite the significant impacts on human health caused by neurodegeneration, our understanding of the degeneration process is incomplete. The nematode Caenorhabditis elegans is emerging as a genetic model organism well suited for identification of conserved cellular mechanisms and molecular pathways of neurodegeneration. Studies in the worm have identified factors that contribute to neurodegeneration, including excitotoxicity and stress due to reactive oxygen species (ROS). Disruption of the gene unc-68, which encodes the ryanodine receptor, abolishes excitotoxic cell death, indicating a role for calcium (Ca²⁺) signaling in neurodegeneration. We tested the requirement for unc-68 in ROS-mediated neurodegeneration using the …

Biodistribution And Function Of Extracellular Mirna-155 In Mice, Shashi Bala, Timea Csak, Fatemeh Momen-Heravi, Dora Lippai, Karen Kodys, Donna Catalano, Abhishek Satishchandran, Victor R. Ambros, Gyongyi Szabo

Gyongyi Szabo

Circulating miRNAs can be found in extracellular vesicles (EV) and could be involved in intercellular communication. Here, we report the biodistribution of EV associated miR-155 using miR-155 KO mouse model. Administration of exosomes loaded with synthetic miR-155 mimic into miR-155 KO mice resulted in a rapid accumulation and clearance of miR-155 in the plasma with subsequent distribution in the liver, adipose tissue, lung, muscle and kidney (highest to lowest, respectively). miR-155 expression was detected in isolated hepatocytes and liver mononuclear cells of recipient KO mice suggesting its cellular uptake. In vitro, exosome-mediated restoration of miR-155 in Kupffer cells from miR-155 …

Caenorhabditis Elegans Alg-1 Antimorphic Mutations Uncover Functions For Argonaute In Microrna Guide Strand Selection And Passenger Strand Disposal, Anna Y. Zinovyeva, Isana Veksler-Lublinsky, Ajay A. Vashisht, James A. Wohlschlegel, Victor R. Ambros

Victor R. Ambros

MicroRNAs are regulators of gene expression whose functions are critical for normal development and physiology. We have previously characterized mutations in a Caenorhabditis elegans microRNA-specific Argonaute ALG-1 (Argonaute-like gene) that are antimorphic [alg-1(anti)]. alg-1(anti) mutants have dramatically stronger microRNA-related phenotypes than animals with a complete loss of ALG-1. ALG-1(anti) miRISC (microRNA induced silencing complex) fails to undergo a functional transition from microRNA processing to target repression. To better understand this transition, we characterized the small RNA and protein populations associated with ALG-1(anti) complexes in vivo. We extensively characterized proteins associated with wild-type and mutant ALG-1 and found that the mutant …

Biodistribution And Function Of Extracellular Mirna-155 In Mice, Shashi Bala, Timea Csak, Fatemeh Momen-Heravi, Dora Lippai, Karen Kodys, Donna Catalano, Abhishek Satishchandran, Victor R. Ambros, Gyongyi Szabo

Victor R. Ambros

Circulating miRNAs can be found in extracellular vesicles (EV) and could be involved in intercellular communication. Here, we report the biodistribution of EV associated miR-155 using miR-155 KO mouse model. Administration of exosomes loaded with synthetic miR-155 mimic into miR-155 KO mice resulted in a rapid accumulation and clearance of miR-155 in the plasma with subsequent distribution in the liver, adipose tissue, lung, muscle and kidney (highest to lowest, respectively). miR-155 expression was detected in isolated hepatocytes and liver mononuclear cells of recipient KO mice suggesting its cellular uptake. In vitro, exosome-mediated restoration of miR-155 in Kupffer cells from miR-155 …

Control Of Stem Cell Self-Renewal And Differentiation By The Heterochronic Genes And The Cellular Asymmetry Machinery In Caenorhabditis Elegans, Omid F. Harandi, Victor Ambros

Victor R. Ambros

Transitions between asymmetric (self-renewing) and symmetric (proliferative) cell divisions are robustly regulated in the context of normal development and tissue homeostasis. To genetically assess the regulation of these transitions, we used the postembryonic epithelial stem (seam) cell lineages of Caenorhabditis elegans. In these lineages, the timing of these transitions is regulated by the evolutionarily conserved heterochronic pathway, whereas cell division asymmetry is conferred by a pathway consisting of Wnt (Wingless) pathway components, including posterior pharynx defect (POP-1)/TCF, APC related/adenomatosis polyposis coli (APR-1)/APC, and LIT-1/NLK (loss of intestine/Nemo-like kinase). Here we explore the genetic regulatory mechanisms underlying stage-specific transitions between self-renewing …

The Decapping Scavenger Enzyme Dcs-1 Controls Microrna Levels In Caenorhabditis Elegans, Gabriel Bosse, Stefan Ruegger, Maria Ow, Alejandro Vasquez-Rifo, Evelyne Rondeau, Victor Ambros, Helge Grosshans, Martin Simard

Victor R. Ambros

In metazoans, microRNAs play a critical role in the posttranscriptional regulation of genes required for cell proliferation and differentiation. MicroRNAs themselves are regulated by a multitude of mechanisms influencing their transcription and posttranscriptional maturation. However, there is only sparse knowledge on pathways regulating the mature, functional form of microRNA. Here, we uncover the implication of the decapping scavenger protein DCS-1 in the control of microRNA turnover. In Caenorhabditis elegans, mutations in dcs-1 increase the levels of functional microRNAs. We demonstrate that DCS-1 interacts with the exonuclease XRN-1 to promote microRNA degradation in an independent manner from its known decapping scavenger …

Developmental Decline In Neuronal Regeneration By The Progressive Change Of Two Intrinsic Timers, Yan Zou, Hui Chiu, Anna Zinovyeva, Victor Ambros, Chiou-Fen Chuang, Chieh Chang

Victor R. Ambros

Like mammalian neurons, Caenorhabditis elegans neurons lose axon regeneration ability as they age, but it is not known why. Here, we report that let-7 contributes to a developmental decline in anterior ventral microtubule (AVM) axon regeneration. In older AVM axons, let-7 inhibits regeneration by down-regulating LIN-41, an important AVM axon regeneration-promoting factor. Whereas let-7 inhibits lin-41 expression in older neurons through the lin-41 3' untranslated region, lin-41 inhibits let-7 expression in younger neurons through Argonaute ALG-1. This reciprocal inhibition ensures that axon regeneration is inhibited only in older neurons. These findings show that a let-7-lin-41 regulatory circuit, which was previously …

The Evolution Of Our Thinking About Micrornas, Victor Ambros

Victor R. Ambros

Our appreciation of the significance of microRNAs to biology at large continues to be an evolving process.

Victor Ambros: The Broad Scope Of Micrornas. Interview By Caitlin Sedwick, Victor R. Ambros

Victor R. Ambros

Interview with Victor Ambros, who studies how microRNAs impact development.

Mutations In Conserved Residues Of The C. Elegans Microrna Argonaute Alg-1 Identify Separable Functions In Alg-1 Mirisc Loading And Target Repression, Anna Y. Zinovyeva, Samir Bouasker, Martin J. Simard, Christopher M. Hammell, Victor R. Ambros

Victor R. Ambros

microRNAs function in diverse developmental and physiological processes by regulating target gene expression at the post-transcriptional level. ALG-1 is one of two Caenorhabditis elegans Argonautes (ALG-1 and ALG-2) that together are essential for microRNA biogenesis and function. Here, we report the identification of novel antimorphic (anti) alleles of ALG-1 as suppressors of lin-28(lf) precocious developmental phenotypes. The alg-1(anti) mutations broadly impair the function of many microRNAs and cause dosage-dependent phenotypes that are more severe than the complete loss of ALG-1. ALG-1(anti) mutant proteins are competent for promoting Dicer cleavage of microRNA precursors and for associating with and stabilizing microRNAs. However, …

The Developmental Timing Regulator Hbl-1 Modulates The Dauer Formation Decision In Caenorhabditis Elegans, Xantha Karp, Victor Ambros

Victor R. Ambros

Animals developing in the wild encounter a range of environmental conditions, and so developmental mechanisms have evolved that can accommodate different environmental contingencies. Harsh environmental conditions cause Caenorhabditis elegans larvae to arrest as stress-resistant "dauer" larvae after the second larval stage (L2), thereby indefinitely postponing L3 cell fates. HBL-1 is a key transcriptional regulator of L2 vs. L3 cell fate. Through the analysis of genetic interactions between mutations of hbl-1 and of genes encoding regulators of dauer larva formation, we find that hbl-1 can also modulate the dauer formation decision in a complex manner. We propose that dynamic interactions between …

Mir-14 Regulates Autophagy During Developmental Cell Death By Targeting Ip3-Kinase 2, Charles Nelson, Victor Ambros, Eric Baehrecke

Victor R. Ambros

Macroautophagy (autophagy) is a lysosome-dependent degradation process that has been implicated in age-associated diseases. Autophagy is involved in both cell survival and cell death, but little is known about the mechanisms that distinguish its use during these distinct cell fates. Here, we identify the microRNA miR-14 as being both necessary and sufficient for autophagy during developmentally regulated cell death in Drosophila. Loss of miR-14 prevented induction of autophagy during salivary gland cell death, but had no effect on starvation-induced autophagy in the fat body. Moreover, misexpression of miR-14 was sufficient to prematurely induce autophagy in salivary glands, but not in …

Circulating Micrornas In Cardiovascular Disease, David Mcmanus, Victor Ambros

Victor R. Ambros

Comment on: Transcoronary concentration gradients of circulating microRNAs. [Circulation. 2011]

Micrornas And Developmental Timing, Victor Ambros

Victor R. Ambros

MicroRNAs regulate temporal transitions in gene expression associated with cell fate progression and differentiation throughout animal development. Genetic analysis of developmental timing in the nematode Caenorhabditis elegans identified two evolutionarily conserved microRNAs, lin-4/mir-125 and let-7, that regulate cell fate progression and differentiation in C. elegans cell lineages. MicroRNAs perform analogous developmental timing functions in other animals, including mammals. By regulating cell fate choices and transitions between pluripotency and differentiation, microRNAs help to orchestrate developmental events throughout the developing animal, and to play tissue homeostasis roles important for disease, including cancer.

Dauer Larva Quiescence Alters The Circuitry Of Microrna Pathways Regulating Cell Fate Progression In C. Elegans, Xantha Karp, Victor Ambros

Victor R. Ambros

In C. elegans larvae, the execution of stage-specific developmental events is controlled by heterochronic genes, which include those encoding a set of transcription factors and the microRNAs that regulate the timing of their expression. Under adverse environmental conditions, developing larvae enter a stress-resistant, quiescent stage called 'dauer'. Dauer larvae are characterized by the arrest of all progenitor cell lineages at a stage equivalent to the end of the second larval stage (L2). If dauer larvae encounter conditions favorable for resumption of reproductive growth, they recover and complete development normally, indicating that post-dauer larvae possess mechanisms to accommodate an indefinite period …

Mirwip: Microrna Target Prediction Based On Microrna-Containing Ribonucleoprotein-Enriched Transcripts, Molly Hammell, Dang Long, Liang Zhang, Andrew Lee, C. Steven Carmack, Min Han, Ye Ding, Victor Ambros

Victor R. Ambros

Target prediction for animal microRNAs (miRNAs) has been hindered by the small number of verified targets available to evaluate the accuracy of predicted miRNA-target interactions. Recently, a dataset of 3,404 miRNA-associated mRNA transcripts was identified by immunoprecipitation of the RNA-induced silencing complex components AIN-1 and AIN-2. Our analysis of this AIN-IP dataset revealed enrichment for defining characteristics of functional miRNA-target interactions, including structural accessibility of target sequences, total free energy of miRNA-target hybridization and topology of base-pairing to the 5' seed region of the miRNA. We used these enriched characteristics as the basis for a quantitative miRNA target prediction method, …

Circulating Cell And Plasma Microrna Profiles Differ Between Non-St-Segment And St-Segment-Elevation Myocardial Infarction, Jeanine Ward, Nada Esa, Rahul Pidikiti, Jane E. Freedman, John F. Keaney, Kahraman Tanriverdi, Olga Vitseva, Victor R. Ambros, Rosalind Lee, David D. Mcmanus

Victor R. Ambros

BACKGROUND: Differences in plasma and whole blood expression microRNAs (miRNAs) in patients with an acute coronary syndrome (ACS) have been determined in both in vitro and in vivo studies. Although most circulating miRNAs are located in the cellular components of whole blood, little is known about the miRNA profiles of whole blood subcomponents, including plasma, platelets and leukocytes in patients with myocardial ischemia. METHODS: Thirteen patients with a ST-segment-elevation (STEMI) or non-ST-segment elevation (NSTEMI) myocardial infarction were identified in the University of Massachusetts Medical Center Emergency Department (ED) or cardiac catheterization laboratory between February and June of 2012. Whole blood …

The Embryonic Mir-35 Family Of Micrornas Promotes Multiple Aspects Of Fecundity In Caenorhabditis Elegans, Katherine Mcjunkin, Victor R. Ambros

Victor R. Ambros

MicroRNAs guide many aspects of development in all metazoan species. Frequently, microRNAs are expressed during a specific developmental stage to perform a temporally defined function. The C. elegans mir-35-42 microRNAs are expressed abundantly in oocytes and early embryos and are essential for embryonic development. Here, we show that these embryonic microRNAs surprisingly also function to control the number of progeny produced by adult hermaphrodites. Using a temperature-sensitive mir-35-42 family mutant (a deletion of the mir-35-41 cluster), we demonstrate three distinct defects in hermaphrodite fecundity. At permissive temperatures, a mild sperm defect partially reduces hermaphrodite fecundity. At restrictive temperatures, somatic gonad …

An Efficient And Sensitive Method For Preparing Cdna Libraries From Scarce Biological Samples, Catherine H. Sterling, Isana Veksler-Lublinsky, Victor R. Ambros

Victor R. Ambros

The preparation and high-throughput sequencing of cDNA libraries from samples of small RNA is a powerful tool to quantify known small RNAs (such as microRNAs) and to discover novel RNA species. Interest in identifying the small RNA repertoire present in tissues and in biofluids has grown substantially with the findings that small RNAs can serve as indicators of biological conditions and disease states. Here we describe a novel and straightforward method to clone cDNA libraries from small quantities of input RNA. This method permits the generation of cDNA libraries from sub-picogram quantities of RNA robustly, efficiently and reproducibly. We demonstrate …

Immunopurification Of Ago1 Mirnps Selects For A Distinct Class Of Microrna Targets, Xin Hong, Molly Hammell, Victor Ambros, Stephen Cohen

Victor R. Ambros

microRNAs comprise a few percent of animal genes and have been recognized as important regulators of a diverse range of biological processes. Understanding the biological functions of miRNAs requires effective means to identify their targets. Combined efforts from computational prediction, miRNA over-expression or depletion, and biochemical purification have identified thousands of potential miRNA-target pairs in cells and organisms. Complementarity to the miRNA seed sequence appears to be a common principle in target recognition. Other features, including miRNA-target duplex stability, binding site accessibility, and local UTR structure might affect target recognition. Yet computational approaches using such contextual features have yielded largely …

Drosophila Let-7 Microrna Is Required For Remodeling Of The Neuromusculature During Metamorphosis, Nicholas S. Sokol, Peizhang Xu, Yuh-Nung Jan, Victor R. Ambros

Victor R. Ambros

The Drosophila let-7-Complex (let-7-C) is a polycistronic locus encoding three ancient microRNAs: let-7, miR-100, and fly lin-4 (miR-125). We find that the let-7-C locus is principally expressed in the pupal and adult neuromusculature. let-7-C knockout flies appear normal externally but display defects in adult behaviors (e.g., flight, motility, and fertility) as well as clear juvenile features in their neuromusculature. We find that the function of let-7-C to ensure the appropriate remodeling of the abdominal neuromusculature during the larval-to-adult transition is carried out predominantly by let-7 alone. This heterochronic role of let-7 is likely just one of the ways in which …

Effect Of Life History On Microrna Expression During C. Elegans Development, Xantha Karp, Molly Hammell, Maria C. Ow, Victor R. Ambros

Victor R. Ambros

Animals have evolved mechanisms to ensure the robustness of developmental outcomes to changing environments. MicroRNA expression may contribute to developmental robustness because microRNAs are key post-transcriptional regulators of developmental gene expression and can affect the expression of multiple target genes. Caenorhabditis elegans provides an excellent model to study developmental responses to environmental conditions. In favorable environments, C. elegans larvae develop rapidly and continuously through four larval stages. In contrast, in unfavorable conditions, larval development may be interrupted at either of two diapause stages: The L1 diapause occurs when embryos hatch in the absence of food, and the dauer diapause occurs …

Micrornas: Genetically Sensitized Worms Reveal New Secrets, Victor Ambros

Victor R. Ambros

Why do many microRNA gene mutants display no evident phenotype? Multiply mutant worms that are selectively impaired in genetic regulatory network activities have been used to uncover previously unknown functions for numerous Caenorhabditis elegans microRNAs.

Prb/Cki Pathways At The Interface Of Cell Cycle And Development, Victor Ambros

Victor R. Ambros

Comment on: The cyclin-dependent kinase inhibitors, cki-1 and cki-2, act in overlapping but distinct pathways to control cell-cycle quiescence during C. elegans development. Buck SH, et al. Cell Cycle 2009; 8:2613-20.

Mutations Of Adjacent Amino Acid Pairs Are Not Always Independent, Jyotsna Ramanan, Peter Revesz

CSE Conference and Workshop Papers

Evolutionary studies usually assume that the genetic mutations are independent of each other. This paper tests the independence hypothesis for genetic mutations with regard to protein coding regions. According to the new experimental results the independence assumption generally holds, but there are certain exceptions. In particular, the coding regions that represent two adjacent amino acids seem to change in ways that sometimes deviate significantly from the expected theoretical probability under the independence assumption.

A Unified Framework For The Prioritization Of Variants Of Uncertain Significance In Hereditary Breast And Ovarian Cancer Patients, Natasha G. Caminsky

Electronic Thesis and Dissertation Repository

A significant proportion of hereditary breast and ovarian cancer (HBOC) patients receive uninformative genetic testing results, an issue exacerbated by the overwhelming quantity of variants of uncertain significance identified. This thesis describes a framework where, aside from protein coding changes, information theory (IT)-based sequence analysis identifies and prioritizes pathogenic variants occurring within sequence elements predicted to be recognized by proteins involved in mRNA splicing, transcription, and untranslated region binding and structure. To support the utilization of IT analysis, we established IT-based variant interpretation accuracy by performing a comprehensive review of mutations altering mRNA splicing in rare and common diseases.

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An Exploration Of The Phylogenetic Placement Of Recently Discovered Ultrasmall Archaeal Lineages, Jeffrey M. O'Brien

Honors Scholar Theses

In recent years, several new clades within the domain Achaea have been discovered. This is due in part to microbiological sampling of novel environments, and the increasing ability to detect and sequence uncultivable organisms through metagenomic analysis. These organisms share certain features, such as small cell size and streamlined genomes. Reduction in genome size can present difficulties to phylogenetic reconstruction programs. Since there is less genetic data to work with, these organisms often have missing genes in concatenated multiple sequence alignments. Evolutionary Biologists have not reached a consensus on the placement of these lineages in the archaeal evolutionary tree. There …

Three-Dimensional Confocal Microscopy Indentation Method For Hydrogel Elasticity Measurement, Donghee Lee, Md Mahmudur Rahman, You Zhou, Sangjin Ryu

Md Mahmudur Rahman

No abstract provided.