Molecular characterization of animal microRNAs : sequence, expression, and stability

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2004.Includes bibliographical references.(cont.) miRNAs. These cells lines may also be useful for other functional studies, such as validation of putative mRNA target genes.Multicellular organisms possess natural gene-regulatory pathways that employ small RNAs to negatively regulate gene expression. In nematodes, the small temporal RNAs (stRNAs), lin-4 and let-7, negatively regulate genes important in specifying developmental timing. A gene-silencing pathway present in plants, fungi and animals called RNA interference, involves the conversion of long double-stranded RNA into short interfering RNAs, which can serve to negatively regulate endogenous genes or suppress the replication of viruses and transposons. To investigate how wide a role small RNAs play in regulating gene expression in animals, we developed a RNA cloning procedure and first applied it to the cloning of small RNAs from the nematode, Caenorhabditis elegans. In addition to cloning lin-4 and let-7 sequences, our study revealed a large number of conserved and highly expressed small RNAs with features reminiscent of stRNAs. Because not all of these small RNAs were expressed in temporal fashion, we and others have referred to this novel class of tiny RNAs as microRNAs. We completed an extensive census of microRNA (miRNA) genes in C.elegans by cloning and bioinformatics searches to lay the groundwork for future functional studies. Our census marked the detection of nearly 90 C.elegans miRNAs, estimated an upper-bound of about 120 miRNA genes in C.elegans, and detailed the conservation and clustering of miRNA sequences. We also determined the high molecular abundance of several miRNAs in C.elegans and Hela cells. In an effort to understand the reason for the high molecular abundance of miRNAs, we constructed an inducible miRNA-expressing cell line to measure the stability of animal miRNAs. Time course measurements suggested a long (>24 hours) half-life for twoby Nelson C. Lau.Ph.D

The \u3cem\u3elet-7\u3c/em\u3e MicroRNA Family Members \u3cem\u3emir\u3c/em\u3e-48, \u3cem\u3emir\u3c/em\u3e-84, and mir-241 Function Together to Regulate Developmental Timing in \u3cem\u3eCaenorhabditis elegans\u3c/em\u3e

The microRNA let-7 is a critical regulator of developmental timing events at the larval-to-adult transition in C. elegans. Recently, microRNAs with sequence similarity to let-7 have been identified. We find that doubly mutant animals lacking the let-7 family microRNA genes mir-48 and mir-84 exhibit retarded molting behavior and retarded adult gene expression in the hypodermis. Triply mutant animals lacking mir-48, mir-84, and mir-241 exhibit repetition of L2-stage events in addition to retarded adult-stage events. mir-48, mir-84, and mir-241 function together to control the L2-to-L3 transition, likely by base pairing to complementary sites in the hbl-1 3′ UTR and downregulating hbl-1 activity. Genetic analysis indicates that mir-48, mir-84, and mir-241 specify the timing of the L2-to-L3 transition in parallel to the heterochronic genes lin-28 and lin-46. These results indicate that let-7 family microRNAs function in combination to affect both early and late developmental timing decisions

New variables, the gravitational action, and boosted quasilocal stress-energy-momentum

This paper presents a complete set of quasilocal densities which describe the stress-energy-momentum content of the gravitational field and which are built with Ashtekar variables. The densities are defined on a two-surface $B$ which bounds a generic spacelike hypersurface $\Sigma$ of spacetime. The method used to derive the set of quasilocal densities is a Hamilton-Jacobi analysis of a suitable covariant action principle for the Ashtekar variables. As such, the theory presented here is an Ashtekar-variable reformulation of the metric theory of quasilocal stress-energy-momentum originally due to Brown and York. This work also investigates how the quasilocal densities behave under generalized boosts, i. e. switches of the $\Sigma$ slice spanning $B$. It is shown that under such boosts the densities behave in a manner which is similar to the simple boost law for energy-momentum four-vectors in special relativity. The developed formalism is used to obtain a collection of two-surface or boost invariants. With these invariants, one may ``build" several different mass definitions in general relativity, such as the Hawking expression. Also discussed in detail in this paper is the canonical action principle as applied to bounded spacetime regions with ``sharp corners."Comment: Revtex, 41 Pages, 4 figures added. Final version has been revised and improved quite a bit. To appear in Classical and Quantum Gravit

Most \u3cem\u3eCaenorhabditis elegans\u3c/em\u3e MicroRNAs are Individually Not Essential for Development or Viability

MicroRNAs (miRNAs), a large class of short noncoding RNAs found in many plants and animals, often act to post-transcriptionally inhibit gene expression. We report the generation of deletion mutations in 87 miRNA genes in Caenorhabditis elegans, expanding the number of mutated miRNA genes to 95, or 83% of known C. elegans miRNAs. We find that the majority of miRNAs are not essential for the viability or development of C. elegans, and mutations in most miRNA genes do not result in grossly abnormal phenotypes. These observations are consistent with the hypothesis that there is significant functional redundancy among miRNAs or among gene pathways regulated by miRNAs. This study represents the first comprehensive genetic analysis of miRNA function in any organism and provides a unique, permanent resource for the systematic study of miRNAs

Label-free Raman spectroscopic imaging to extract morphological and chemical information from a formalin-fixed, paraffin-embedded rat colon tissue section.

Animal models and archived human biobank tissues are useful resources for research in disease development, diagnostics and therapeutics. For the preservation of microscopic anatomical features and to facilitate long-term storage, a majority of tissue samples are denatured by the chemical treatments required for fixation, paraffin embedding and subsequent deparaffinization. These aggressive chemical processes are thought to modify the biochemical composition of the sample and potentially compromise reliable spectroscopic examination useful for the diagnosis or biomarking. As a result, spectroscopy is often conducted on fresh/frozen samples. In this study, we provide an extensive characterization of the biochemical signals remaining in processed samples (formalin fixation and paraffin embedding, FFPE) and especially those originating from the anatomical layers of a healthy rat colon. The application of chemometric analytical methods (unsupervised and supervised) was shown to eliminate the need for tissue staining and easily revealed microscopic features consistent with goblet cells and the dense populations of cells within the mucosa, principally via strong nucleic acid signals. We were also able to identify the collagenous submucosa- and serosa- as well as the muscle-associated signals from the muscular regions and blood vessels. Applying linear regression analysis to the data, we were able to corroborate this initial assignment of cell and tissue types by confirming the biological origin of each layer by reference to a subset of authentic biomolecular standards. Our results demonstrate the potential of using label-free Raman microspectroscopy to obtain superior imaging contrast in FFPE sections when compared directly to conventional haematoxylin and eosin (H&E) staining

The role of microtubule movement in bidirectional organelle transport

We study the role of microtubule movement in bidirectional organelle transport in Drosophila S2 cells and show that EGFP-tagged peroxisomes in cells serve as sensitive probes of motor induced, noisy cytoskeletal motions. Multiple peroxisomes move in unison over large time windows and show correlations with microtubule tip positions, indicating rapid microtubule fluctuations in the longitudinal direction. We report the first high-resolution measurement of longitudinal microtubule fluctuations performed by tracing such pairs of co-moving peroxisomes. The resulting picture shows that motor-dependent longitudinal microtubule oscillations contribute significantly to cargo movement along microtubules. Thus, contrary to the conventional view, organelle transport cannot be described solely in terms of cargo movement along stationary microtubule tracks, but instead includes a strong contribution from the movement of the tracks.Comment: 24 pages, 5 figure

Acceptability of A/H1N1 vaccination during pandemic phase of influenza A/H1N1 in Hong Kong: population based cross sectional survey

Objective To investigate the intention of the Hong Kong general population to take up vaccination against influenza A/H1N1

Adjusting bone mass for differences in projected bone area and other confounding variables: an allometric perspective.

The traditional method of assessing bone mineral density (BMD; given by bone mineral content [BMC] divided by projected bone area [Ap], BMD = BMC/Ap) has come under strong criticism by various authors. Their criticism being that the projected bone "area" (Ap) will systematically underestimate the skeletal bone "volume" of taller subjects. To reduce the confounding effects of bone size, an alternative ratio has been proposed called bone mineral apparent density [BMAD = BMC/(Ap)3/2]. However, bone size is not the only confounding variable associated with BMC. Others include age, sex, body size, and maturation. To assess the dimensional relationship between BMC and projected bone area, independent of other confounding variables, we proposed and fitted a proportional allometric model to the BMC data of the L2-L4 vertebrae from a previously published study. The projected bone area exponents were greater than unity for both boys (1.43) and girls (1.02), but only the boy's fitted exponent was not different from that predicted by geometric similarity (1.5). Based on these exponents, it is not clear whether bone mass acquisition increases in proportion to the projected bone area (Ap) or an estimate of projected bone volume (Ap)3/2. However, by adopting the proposed methods, the analysis will automatically adjust BMC for differences in projected bone size and other confounding variables for the particular population being studied. Hence, the necessity to speculate as to the theoretical value of the exponent of Ap, although interesting, becomes redundant