Ribosoomide lagundamine bakterites

Väitekirja elektrooniline versioon ei sisalda publikatsiooneRibosoomid on makromolekulaarsed kompleksid, mis koosnevad kahest suurest ja ühest väikesest RNAst ja paljudest erinevatest valkudest. Ribosoomides sünteesitakse kõik valgud, mida organismis leida võib, ning aktiivsete ribsoomide konsentratsioon (ja seega sünteesi kiirus) limiteerib rakkude kasvu kiirust. Ehk teisisõnu, mida kiiremini sünteesitakse uusi ribosoome, seda kiiremini kasvab ja jaguneb ka rakk. Kuna ribosomaalse RNA süntees hõlmab ca 80% raku RNA sünteesi aktiivsusest ja ribosoomi valgud moodustavad kuni veerandi raku valgumassist on selge, et mitte ainult ribosoomide funktsioon valgusünteesil vaid ka nende metabolism on rakulises majapidamises määrava tähtsusega. Tõepoolest, juba mõnda aega on teada, et aeglaselt kasvavates bakterirakkudes tegeleb enamus raku RNA lagundamise võimekusest värskelt sünteesitud ribosomaalse RNA lagundamisega. Sellegipoolest on viimase 50 aasta vältel üldiselt usutud, et kord juba valmis tehtud ja kokku pakitud ribosoomid on äärmiselt stabiilsed ning, et neid lagundatakse vaid tugeva stressi tingimustes. Samuti on meie teadmised ribosoomide lagundamise molekulaarsetest mehhanismidest bakteris üsnagi piiratud. Käesoleva doktoritöö eesmärk on kirjeldada ribosoomide lagundamist kasvavates soolekepikese (Escherichia coli) rakkudes ja heita valgust ribsoomide lagundamise mehhanismidele, molekulaarsetele radadele ning ensüümidele, mis selles protsessis osalevad. Me avastasime üllatusega, et kuigi ribosoome tõepoolest lagundatakse kasvavates bakterirakkudes, toimub see protsess vaid rakukultuuri kasvu aeglustumise perioodil, mis eelneb statsionaarse kasvufaasi saabumisele. Meil ei õnnestunud tuvastada küpsete ribosoomide lagundamist ei ühtlase kiirusega kasvavates ega ka null-kiirusega kasvavates rakkudes. Võimalik, et ribosoomide lagundamine aitab rakke neid ette valmistades eluks statsionaarses faasis, mil ei vajata suurt valgusünteesi võimekust, küll aga vabu komponente, millest elutingimuste paranedes kiiresti uusi makromolekule tootma hakata. Lisaks leidsime, et osad (kuid mitte kõik) ribosoomi RNAd inaktiveerivad mutatsioonid viivad samuti ribsoomide lagundamisele, kuid miskipärast lagundatakse siis nii mutantseid ning inaktiivseid kui metsiktüüpi ning aktiivseid ribosoome. Jällegi viitab see, et ribsoomide lagundamise eesmärk võiks olla üldise ribosoomide konsentratsiooni alandamine rakus. Kui me lisasime ribsoomide lagundamise katsesüsteemi valgusünteesi pärssivat antibiootikumi kloramfenikool, päästsime me sellega ribosoomid lagundamisest. Seda tulemust võib tõlgendada viisil, et de novo valgusüntees on vajalik ribosoomide lagundamisprogrammi käivitamiseks rakus. Testides ribosoomide lagundamise võime osas bakteritüvesid, kus puuduvad erinevad RNAd lagundavad ensüümid, leidsime kaks ensüümi, mille puudumise korral ribosoome ei lagundatud. Neist esimene, RNaas R, lõhub RNAsid alates nende tagumisest ehk 3’ otsast ning tunneb erilist lõbu kõrge sekundaarstruktuuriga RNA-de hävitamisest. RNaas R on ka eelnevalt näidatud osalevat ribosoomide lagundamisel. Teine ensüüm on seevastu suhteliselt vähetuntud endoribonukleeas nimega YbeY, mis lõikab RNAd katki keskelt, mitte ei lagunda seda otstest. See huvitav valk on arvatud osalevat ribsoomide kokkupakkimise kvaliteedikontrollil, kus ta on vajalik kõige viimases etapis, mil tuntakse ära valgusünteesil ebaõnnestuvad ribosoomid ja suunatakse need lagundamisse. Meie katsed viitavad, et seesama valk võib valla päästa ka töökorras olevate ribosoomide lagundamise, tehes ribosoomi RNAsse esimese lõike ning tekitades sellega kaitsetu 3’ otsa, mida tunneb ära RNaas R, mis omakorda suudab ribosoomi RNA täielikult lagundadaRibosomes are macromolecular complexes that consist of two large and one small RNA and of many different small proteins. The ribosome synthesizes all cellular proteins and the concentration of active ribosomes is rate limiting for cell growth. As synthesis or ribosomal RNA encompasses 80% of cellular RNA synthesis activity and the ribosomal proteins can make up half of the cellular protein mass, it is clear that ribosomal metabolism, including ribosomal degradation, makes a worthy object of study. Nevertheless, during the past half century it has been widely believed that mature ribosomes are quite stable in the cells. The major goal of this dissertation is to describe the degradation of mature ribosomes in growing E. coli cells and to shed light on the molecular mechanism of degradation. We discovered that while mature ribosomes are indeed degraded in cells growing in batch cultures, this process is limited to the slowing of growth phase, which precedes entry into the stationary phase. We were unable to detect degradation during constant-rate growth and during early stationary phase. In addition, we found that some, but not all, ribosome-inactivating mutations in 23S rRNA and 16S rRNA led to degradation of both mutant and wild-type ribosomal RNAs. Thus, unlike in yeast, the ribosome degradation in E. coli is a general process that, once initiated, does not discriminate between active and inactive ribosomes. As ribosome degradation is inhibited by the protein synthesis inhibitor chloramphenicol, we further suggest that de novo protein synthesis might be needed for triggering the degradation program. To pinpoint the enzymes responsible for degradation we tested several strains defective for different RNases. We found two RNases, RNaseR and YbeY, whose deletion saved ribosomes from degradation. RNaseR is a well studied 3’ to 5’ exonuclease whose role in degrading heavily structured RNAs, including the rRNAs, is well established. In contrast YbeY is a potential endonuclease recently implicated in a late step ribosomal quality control, which could well be the initiating endonuclease, whose cut(s) in rRNA would present substrates for RNaseR to further scavenge into mononucleotides

RUSSIAN CAPABILITIES IN CONVENTIONAL HIGH INTENSITY WARFARE. LESSONS FROM THE 2022 INVASION OF UKRAINE

The Russian Federation is today one of the world’s major military powers. The most important component of the Russian military machine is the nuclear Strategic Forces, but conventional forces are also key actors in Russia’s defense and strategic policy. The present article will briefly describe the post-Soviet evolution of Russian conventional forces and their proficiency and effectiveness in the light of the 2022 Russo-Ukrainian War. The armed forces inherited from the Soviet Union in the 1990s faced a struggle to maintain even modest capabilities. After Boris Yeltsin handed power to Vladimir Putin in 1999, sustained attempts were made to increase their capabilities. Extensive reforms were made to improve personnel conditions and proficiency, organization and equipment. The Russian military involvement in the Syrian Civil War and in the 2014–2022 Donbas conflict seemed to indicate that substantial progress had been achieved. When Russia launched its invasion of Ukraine in February 2022, military experts expected that the reformed Russian military machine would overwhelm their outmanned and outgunned foes. Contrary to expectations, the Russians suffered decisive setbacks, and despite partial successes in Southern and Eastern Ukraine they are presently embroiled in slow, costly attritional warfare. The operations in the current conflict have evidenced serious shortcomings in organization and in combined arms tactics, suggesting that the reforms have only been partially successful. They also suggest the persistence of problems that the modern Russian armed forces inherited from their predecessors, and this may be rooted in their historical military heritage and in aspects of Russian society that go back a long way in the past. The present conflict offers an opportunity for the military organizations of the Western world to learn important lessons and readdress their defense policies

TANKS IN THE RUSSO-UKRAINIAN WAR

The Russo Ukrainian war, after the initial Russian advance following the invasion in February 2022, has turned into a stalemate characterised by grinding trench and urban warfare which in some instances has been compared to WWI. Despite the relative absence of fluid operations by massed mechanised formations, armoured vehicles and tanks in particular have been intensely employed by both opponents, resulting in heavy losses. When properly used in combination with other arms, tanks have been a key factor on the battlefield. During the first year of hostilities the Russian army often suffered from a tactical mishandling of their armoured units, leading to disproportionate losses of tanks and other AFVs compared to their opponent. The great majority of the tanks employed in Ukraine so far are upgraded late Cold War models, even if deliveries by NATO countries have introduced modern Western models into the Ukrainian arsenal. The aim of this article is to give a summary of the key characteristics of the main MBTs employed in Ukraine and to provide a brief analysis of their role and impact

CO adsorption on metal surfaces: a hybrid functional study with plane wave basis set

We present a detailed study of the adsorption of CO on Cu, Rh, and Pt (111) surfaces in top and hollow sites. The study has been performed using the local density approximation, the gradient corrected functional PBE, and the hybrid Hartree-Fock density functionals PBE0 and HSE03 within the framework of generalized Kohn-Sham density functional theory using a plane-wave basis set. As expected, the LDA and GGA functionals show a tendency to favor the hollow sites, at variance with experimental findings that give the top site as the most stable adsorption site. The PBE0 and HSE03 functionals reduce this tendency. In fact, they predict the correct adsorption site for Cu and Rh but fail for Pt. But even in this case, the hybrid functional destabilizes the hollow site by 50 meV compared to the PBE functional. The results of the total energy calculations are presented along with an analysis of the projected density of states.Comment: 32 pages, 6 tables, 3 figures. (Re)Submitted to Phys. Rev. B; LDA results added in the tables; minor changes in the tex

Assembling Coherent Network Topologies Using Round-Trip Graphs (short paper)

Discovering the network topology in computer networks is challenging due to limited communication and incomplete information about non-immediately connected nodes. In this paper we address the problem of assembling partial views obtained by discovery tools into a coherent representation, using round-trip graphs: labelled bipartite directed graphs representing the communications between hosts, interfaces, and networks. A merge operation is introduced, facilitating compositional and incremental assembly of partial views. This research provides a practical solution for incrementally constructing a comprehensive network topology

A Calculus for Subjective Communication

In this paper we introduce Subjective Communication, a new interaction model for CAS and generalizing the attribute-based communication introduced in the AbC calculus. In this model, a message is broadcasted to every process, but each process can view the very same message in different ways, depending on its attributes. To formalize this model, we introduce SCC, the Subjective Communication Calculus, for which we propose two semantics: Direct SCC, particularly useful when dealing with an edge computing communication paradigm, and Indirect SCC, more suited to a cloud-centric model. We then introduce a stateless bisimilarity for our semantics, which we prove to be a congruence

DBCChecker: A Bigraph-Based Tool for Checking Security Properties of Container Compositions

Despite their widespread application in modern systems, container composition is often complex and error-prone. In this work, we present DBCChecker, a tool aiming to verify security properties of systems obtained by composition of containers. From the configuration of a container-based system and an abstract description of the interface behaviour of each container, the tool builds a formal model of the overall system, which can be verified in ProVerif (an automatic symbolic protocol verifier), to check that the overall system satisfies the required properties. The system can be described in a specification language capable to express at once the interfaces and connections of containers and the relevant behavioural aspects of their interfaces, called JSON Bigraph Format (JBF), and inspired by previous formal models, based on bigraphs, for containerized architectures

Curli Fimbria: An Escherichia Coli Adhesin Associated With Human Cystitis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Escherichia coli is the major causative agent of human cystitis. In this study, a preliminary molecular analysis carried out by PCR (polymerise chain reaction) demonstrated that 100% of 31 E. coli strains isolated from patients with recurrent UTIs (urinary tract infections) showed the presence of the curli fimbria gene (csgA). Curli fimbria is known to be associated with bacterial biofilm formation but not with the adhesion of human cystitis-associated E. coli. Therefore, this work aimed to study how curli fimbria is associated with uropathogenic E. coli (UPEC) as an adhesion factor. For this purpose, the csgA gene was deleted from strain UPEC-4, which carries three adhesion factor genes (csgA, fimH and ompA). The wild-type UPEC-4 strain and its mutant (Delta csgA) were analyzed for their adhesion ability over HTB-9 (human bladder carcinoma), Vero (kidney cells of African green monkey) and HUVEC (human umbilical vein) cells in the presence of alpha-D-mannose. All the wild-type UPEC strains tested (100%) were able to adhere to all three cell types, while the UPEC-4 Delta csgA mutant lost its adherence to HTB-9 but continued to adhere to the HUVEC and Vero cells. The results suggest that curli fimbria has an important role in the adhesion processes associated with human UPEC-induced cystitis. (C) 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda.472414416CNPq (Conselho Nacional de Desenvolvimento e Tecnologico)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq