315 research outputs found
MicroRNA-275 targets sarco/endoplasmic reticulum Ca2+ adenosine triphosphatase (SERCA) to control key functions in the mosquito gut.
No full textThe yellow fever mosquito Aedes aegypti is the major vector of arboviruses, causing numerous devastating human diseases, such as dengue and yellow fevers, Chikungunya and Zika. Female mosquitoes need vertebrate blood for egg development, and repeated cycles of blood feeding are tightly linked to pathogen transmission. The mosquito's posterior midgut (gut) is involved in blood digestion and also serves as an entry point for pathogens. Thus, the mosquito gut is an important tissue to investigate. The miRNA aae-miR-275 (miR-275) has been shown to be required for normal blood digestion in the female mosquito; however, the mechanism of its action has remained unknown. Here, we demonstrate that miR-275 directly targets and positively regulates sarco/endoplasmic reticulum Ca2+ adenosine triphosphatase, which is implicated in active transport of Ca2+ from the cytosol to the sarco/endoplasmic reticulum. We utilized a combination of the gut-specific yeast transcription activator protein Gal4/upstream activating sequence (Gal4/UAS) system and miRNA Tough Decoy technology to deplete the endogenous level of miR-275 in guts of transgenic mosquitoes. This gut-specific reduction of miR-275 post blood meal decreased SERCA mRNA and protein levels of the digestive enzyme late trypsin. It also resulted in a significant reduction of gut microbiota. Moreover, the decrease of miR-275 and SERCA correlated with defects in the Notch signaling pathway and assembly of the gut actin cytoskeleton. The adverse phenotypes caused by miR-275 silencing were rescued by injections of miR-275 mimic. Thus, we have discovered that miR-275 directly targets SERCA, and the maintenance of its level is critical for multiple gut functions in mosquitoes
Members of the miRNA-200 Family Regulate Olfactory Neurogenesis
No full textMicroRNAs (miRNAs) are highly expressed in vertebrate neural tissues, but the contribution of specific miRNAs to the development and function of different neuronal populations is still largely unknown. We report that miRNAs are required for terminal differentiation of olfactory precursors in both mouse and zebrafish but are dispensable for proper function of mature olfactory neurons. The repertoire of miRNAs expressed in olfactory tissues contains over 100 distinct miRNAs. A subset, including the miR-200 family, shows high olfactory enrichment and expression patterns consistent with a role during olfactory neurogenesis. Loss of function of the miR-200 family phenocopies the terminal differentiation defect observed in absence of all miRNA activity in olfactory progenitors. Our data support the notion that vertebrate tissue differentiation is controlled by conserved subsets of organ-specific miRNAs in both mouse and zebrafish and provide insights into control mechanisms underlying olfactory differentiation in vertebrates.National Institutes of Health. (U.S.) (grant NS049319)National Institutes of Health. (U.S.) (grant GM56211)Wellcome Trust (London, England) (grant 066790/B/02/Z
Non-self RNA sensing in virus infected cells and activation of antiviral immunity
No full textColloque organisé par Jean-Luc Imler (Université de Strasbourg) et Raul Andino (UCSF, USA) du 27 août au 1er septembre 2012 Participants Shizuo Akira, Raul Andino, Bruce Beutler, Stephen Cusack, Ding Shou-Wei, Takashi Fujita, Akira Tajima-Goto, Otto Haller, Jules Hoffmann, Veit Hornung, Jean-Luc Imler, Karim Majzoub, Carine Meignin, Eric Miska, Jan Rehwinkel, Carla Saleh, Rotem Sorek, Volker Thiel, Joao Trindade Marques, Ronald van Rij, Olivier Voinnet, Friedemann Weber -- Abstract Innate imm..
Microscopic online simulation for real-time traffic management
No full textThe thesis deals with the design and implementation of a distributed framework for real-time traffic management using microscopic online simulation.Trai
Genetic Mapping and Characterization of a piRNA Mutant Library
No full textHistorically, several studies aimed to better understand the genetic regulation by small RNAs, have used C.elegans as a model organism. From the pioneering work on the regulation of lin-14 by lin-4, to the elucidation of the role of dsRNA in the so-called RNAi pathway, the nematode C.elegans has been regarded as a powerful tool to investigate different aspects of small RNA pathways.
In this work, an in vivo assay has been used to characterize a novel small RNA pathway, the Piwi/piRNA pathway in C.elegans.
The Piwi/piRNA pathway plays crucial roles during germline development and gametogenesis of many metazoan species, from germline determination and germline stem cell maintenance, to meiosis and spermiogenesis. Its function in all these processes lies in protecting the integrity of the genome from parasite nucleic acids, namely from transposons.
However, little is known about how the final biological function of the pathway is achieved: recent evidences support the hypothesis of a mechanism which involves both a post-trascriptional regulation (partially obtained by the activation of a distinct endo-siRNA pathway) and an epigenetic/co-transcriptional regulation.
A clearer view of this pathway will significantly advance our understanding of complex gene regulation strategies.
Previously, using a forward genetic screen, a large number of mutant alleles for genes involved in this pathway have been isolated. The aim of this study was to investigate the identity of those genes with high-throughput sequencing and linkage analysis, as well as by complementation tests. Finally, the involvement of each screened allele in the pathway was assessed through specific assays (chromosome non-disjunction assays, RNAi against a germ line specific gene, qRT-PCR for Tc3 expression).
Taken together with previous findings in the lab, these data enable us to provide a preliminary model for the general structure of the Piwi/piRNA pathway in C.elegans, pointing out its overlapping with the endogenous RNAi pathway, based on the activity of 22G endo-siRNAs
Effluent filtration for more than particle removal
No full textwatermanagementCivil Engineering and Geoscience
Outlining a balance-point model of homeostasis in the small intestine of broiler chickens
No full textSince the removal of in feed antibiotics in the past few years commercial poultry production is especially sensitive to the health of the small intestine. Healthy small intestines balance nutrient absorption and defensive barrier functions to ensure the chicken is able to meet the whole-body nutritional needs and is able to help prevent internalization of pathogens or potentially toxic components. This balance can only be maintained under stable conditions. When a disturbance event occurs the intestine imbalances until a new, and less efficient, balance can be achieved. The objective of this dissertation is to propose a novel model to understanding intestinal homeostasis in the face of various disturbance events. Chapter 2 investigated the effects of Runting Stunting Syndrome on broiler chickens in four different groups of chicks displaying clinical symptoms. The major finding in this study was that in two of the four groups the expression of stem cell gene Olfactomedin 4 was absent from the crypt though other functional genes were found to still be expressed there. Chapter 3 characterized intestinal gene expression following a single challenge of Eimeria acervulina in broiler chickens. During Eimeria infection gene expression of multiple host defense peptide genes were decreased compared to uninfected chickens. Further, Eimeria infected chickens increased cell proliferation within the crypt and post-peak infection showed signs of intestinal recovery. Additionally, chapter 3 developed a novel method for visualizing Eimeria as it infects the intestine. In chapters 4 and 5 cell type population changes during the peri-hatch intestinal maturation process were evaluated. Peri-hatch intestinal maturation is critical for the successful transition from embryonic to post-hatch life. Chapter 4 profiled changes in proliferative cells and gene expression of various stem cell marker genes during the peri-hatch period: the last three days of embryogenesis and the first week post-hatch. The stem cell marker gene Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (Lgr5) decreased during the post-hatch period while Olfactomedin 4 increased post-hatch. Both stem cell genes were expressed within the intestinal crypt, though prior to hatch Lgr5 was expressed in the lamina propria and villi as well. Additionally, the marker of proliferation Ki67 gene was expressed in cells throughout the intestine prior to hatch but became restricted to the crypts and along the center of the villi. Chapter 5 assessed the effect of providing probiotics to late term embryos via in ovo feeding (IOF). The effects of IOF were primarily observed on embryonic day 20 (e20), roughly 48 hours after IOF. On e20 the embryos in ovo fed probiotics in saline had increased expression in the ileum of Peptide Transporter 1 (PepT1) a marker gene for enterocytes and Mucin-2 (Muc2) a marker gene for goblet cells compared to non-injected control embryos. Also, on e20 the embryos in ovo fed saline only had numerically increased PepT1 and Muc2 compared to non-injected control embryos. The difference in responses between the probiotic and saline fed embryos on e20 suggests different routes of stimulation. These investigations illustrate various possible scenarios and means of investigating intestinal homeostasis during disturbance events.Doctor of PhilosophyIn healthy birds, the small intestine absorbs nutrients while preventing the free passage of microbes or toxic chemicals into the body. The two functions: absorption and barrier exclusion seem contradictory, but a balance is struck to ensure both functions continue. This balance-point, homeostasis, persists until an event disturbs it. Once disturbed the balance-point is changed and the intestine is unable to maintain both functions, until a new balance is found following recovery. The objective of the dissertation is to better understand intestinal homeostasis, through four different research projects. Experiment 1 characterized the intestinal cell population changes in broiler chickens during Runting Stunting Syndrome, a viral infection. The major finding of this chapter was that a stem cell gene, that is normally robustly expressed was not expressed in some groups of infected chicks but not all. Experiment 2 investigated the intestinal response of broiler chickens to Eimeria acervulina, an intestinal parasitic infection. Eimeria, which infects intestinal enterocytes, caused a decrease in defensive genes during the peak of infection. Then after the peak the intestine began to recover, as indicated by increased cell proliferation. Experiment 3 profiled changes in the expression patterns of stem cell and proliferation genes in the small intestine during the last days before hatch and the first week post-hatch. Pre-hatch stem and proliferative gene expression occurred in the crypt and villus, but became restricted to the crypt early during the post-hatch period. Experiment 4 assessed the effect of feeding probiotics to embryos before hatching on intestinal gene expression. Embryos fed probiotics had increased Mucin-2 and Peptide Transporter 1 gene expression in the last segment of the intestine, the ileum compared to non-fed embryos 48 hours after feeding. Additionally, treatments fed saline also showed increased gene expression, though to a lesser extent. Together these projects illustrate various disturbances to intestinal homeostasis and how intestinal cells change and respond during the disturbance and recovery periods
Piwi und piRNAs reprimieren Tc3 Transposon Mobilität in der Caenorhabditis elegans Keimbahn durch Regulation endogener siRNAs
No full textIn den letzten Jahren wurden verschiedene Klassen
kleiner nicht-kodierender RNA Moleküle identifiziert,
zu denen u.a. die sogenannten microRNAs (miRNAs), die
repeat-associated small interfering RNAs (rasiRNAs) und
die small-interfering RNAs (siRNAs) zählen. Diese RNAs
werden als Einzelstrang in funktionell verschiedene
Protein-RNA Effektorkomplexe integriert, welche als
zentralen Bestandteil ein Mitglied der Argonaute (AGO)
Proteinfamilie enthalten. Argonaute-Proteinkomplexe mit
ihren assoziierten RNAs spielen eine Rolle in
transkriptioneller Genrepression,
Translationsrepression und mRNA-Abbau. Piwi Proteine
bilden eine Untergruppe innerhalb der Argonaute Familie
und weisen konservierte Funktionen in der
Keimbahnentwicklung und der Erhaltung von
Keim(stamm)zellen auf. Im Nematoden Caenorhabditis
elegans existieren zwei Piwi Proteine, PRG-1 und PRG-2.
Um die Funktion von PRG-1 und PRG-2 während der
Keimbahnentwicklung von C. elegans zu untersuchen,
wurden prg-1 und prg-2 Deletionen generiert. Die
Mutation beider Gene (prg-1; prg-2) führt zu stark
ausgeprägten Keimbahndefekten. In dieser Arbeit wird
gezeigt, dass eine Klasse von 21 Nukleotiden langen,
zuvor als 21U-RNA bezeichneten RNAs die
Piwi-interagierenden RNAs (piRNAs) in C. elegans sind.
Piwi und piRNAs sind spezifisch in der Keimbahn
exprimiert. Die Biogenese der piRNAs ist unabhängig von
vielen Proteinen, die eine Funktion in anderen kleinen
RNA Signalwegen haben, wie zum Beispiel Dicer (DCR-1).
Im Gegensatz zu anderen Organismen gibt es keinen
Hinweis auf den sogenannten Ping-Pong Mechanismus zur
Amplifikation von piRNAs. Piwi Proteine reprimieren die
Mobilität von Tc3 DNA Transposons in der Keimbahn. Die
Exzisionsrate in piwi Doppelmutanten ist hundertfach
höher als in Wildtyp Würmern. Zudem kann gezeigt
werden, dass ein Piwi-piRNA Komplex einen endogenen
siRNA Signalweg reguliert, der für die Tc3 Suppression
in der Keimbahn notwendig ist, was auf eine
funktionelle Verbindung zwischen piRNAs und siRNAs
hindeutet.Several hundred endogenous small RNAs, namely
microRNAs (miRNAs), repeat- associated small
interfering RNAs (rasiRNAs), small interfering RNAs
(siRNAs) have been discovered in diverse organisms.
These small RNAs are derived from different sources and
they cause transcriptional gene silencing,
translational repression and mRNA cleavage through
effector complexes. Several functionally distinct RNA
silencing effector complexes have already been isolated
from different organisms. Effector complexes are
composed of members of the Argonaute (AGO) protein
family and single-stranded small RNAs, along with other
associated proteins. Members of the Piwi subfamily of
Argonaute proteins have conserved roles in germline
development and stem cell maintenance. In C. elegans,
PRG-1 and PRG-2 are the two members of Piwi sub-family
of Argonaute proteins. In order to study the role of
PRG-1 and PRG-2 in C. elegans germline development, we
generated prg-1 and prg-2 single mutants as well as
prg-1; prg-2 double mutant worms, which show severe
germline defects. I identify a class of 21 nucleotide
RNAs, previously named 21U-RNAs, as the
Piwi-interacting RNAs in C. elegans. Piwi and piRNA
expression is restricted to the male and female
germline and piRNA biogenesis is independent of many
other proteins involved in small RNA pathways including
DCR-1. I show that Piwi is specifically required for
suppression of Tc3 transposons. The excision rate of
Tc3 is 100-fold higher in piwi mutants compared to
wild-type. There is no evidence for a ping-pong
mechanism in C. elegans for amplification of piRNAs.
Finally, I demonstrate that a Piwi-piRNA complex acts
upstream of the endogenous siRNA pathway for Tc3
silencing and that might suggest a link between the
function of endogenous siRNAs and piRNAs
Cellular Events During Coccidial Infection in Chickens
No full textAvian coccidiosis is caused by the intestinal protozoa Eimeria. The parasite's site of infection in the intestine is site specific. Eimeria acervulina mainly affects the duodenum, E. maxima the jejunum, and E. tenella the ceca. Lesions in the intestinal mucosa cause reduced feed efficiency and body weight gain in Eimeria-challenged chickens. My previous studies showed that the growth reduction may be due to changes in expression of digestive enzymes and nutrient transporters in the intestine. This can also lead to diminished intracellular pools of nutrients and inhibit pathogen replication. In this dissertation, further analysis of cellular events was performed. Expression of host defense peptides (HDPs), apoptosis and autophagy related genes were examined in Eimeria challenged broilers. The results showed that upon Eimeria infection, LEAP2 was consistently downregulated in the target tissues, while the avian beta-defensins (AvBDs) showed many variations in expression patterns. Downregulation of LEAP2 may be a mechanism for Eimeria to combat the host defense system, and to promote its survival inside the host cell. The in situ hybridization results showed that LEAP2 was expressed only along the villus in the small intestine and not in the crypt. This is the first time LEAP2 has been localized to epithelial cells of the chicken intestine. Eimeria infection can also induce an anti-apoptotic and anti-autophagy state in the host cells. This condition can be both favorable and unfavorable to parasite survival and replication inside the host cell.
A comparison of gene expression between Ross and Eimeria resistant Fayoumi (line M5.1 and M15.2) chickens challenged with Eimeria maxima was conducted. The comparison among different lines of chickens showed differential gene expression patterns in lines with different resistance to Eimeria. The similar body weight reduction indicated that there may not be a significant Eimeria resistant line among the Ross, Fayoumi M5.1 and M15.2 birds. The interaction between Eimeria and the host cell is very complex. Studying the mechanisms behind the changes of gene expression during Eimeria infection may give rise to potential therapeutic targets of coccidiosis.Ph. D
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