Добірка наукової літератури з теми "DisA protein"

Bluetongue (BT) is a midge-borne OIE-notifiable disease of ruminants caused by the bluetongue virus (BTV). There are at least 29 BTV serotypes as determined by serum neutralization tests and genetic analyses of genome segment 2 encoding serotype immunodominant VP2 protein. Large parts of the world are endemic for multiple serotypes. The most effective control measure of BT is vaccination. Conventionally live-attenuated and inactivated BT vaccines are available but have their specific pros and cons and are not DIVA compatible. The prototype Disabled Infectious Single Animal (DISA)/DIVA vaccine based on knockout of NS3/NS3a protein of live-attenuated BTV, shortly named DISA8, fulfills all criteria for modern veterinary vaccines of sheep. Recently, DISA8 with an internal in-frame deletion of 72 amino acid codons in NS3/NS3a showed a similar ideal vaccine profile in cattle. Here, the DISA/DIVA vaccine platform was applied for other serotypes, and pentavalent DISA/DIVA vaccine for “European” serotypes 1, 2, 3, 4, 8 was studied in sheep and cattle. Protection was demonstrated for two serotypes, and neutralization Ab titers indicate protection against other included serotypes. The DISA/DIVA vaccine platform is flexible in use and generates monovalent and multivalent DISA vaccines to combat specific field situations with respect to Bluetongue.

The bluetongue virus (BTV) is transmitted by Culicoides biting midges and causes bluetongue (BT), an OIE-notifiable disease of ruminants. At least 29 BTV serotypes are described as determined by the outer shell proteins VP2 and VP5. Vaccination is the most effective control measure. Inactivated and live-attenuated vaccines (LAVs) are currently available. These vaccines have their specific pros and cons, and both are not DIVA vaccines. The BT Disabled Infectious Single Animal (DISA) vaccine platform is based on LAV without nonessential NS3/NS3a expression and is applicable for many serotypes by the exchange of outer shell proteins. The DISA vaccine is effective and completely safe. Further, transmission of the DISA vaccine by midges is blocked (DISA principle). Finally, the DISA vaccine enables DIVA because of a lack of antibodies against the immunogenic NS3/NS3a protein (DIVA principle). The deletion of 72 amino acids (72aa) in NS3/NS3a is sufficient to block virus propagation in midges. Here, we show that a prototype DISA vaccine based on LAV with the 72aa deletion enables DIVA, is completely safe and induces a long-lasting serotype-specific protection in cattle. In conclusion, the in-frame deletion of 72-aa codons in the BT DISA/DIVA vaccine platform is sufficient to fulfil all the criteria for modern veterinary vaccines.

The DNA damage checkpoint protein DisA and the branch migration translocase RecG are implicated in the preservation of genome integrity in reviving haploid Bacillus subtilis spores. DisA synthesizes the essential cyclic 3′, 5′-diadenosine monophosphate (c-di-AMP) second messenger and such synthesis is suppressed upon replication perturbation. In vitro, c-di-AMP synthesis is suppressed when DisA binds DNA structures that mimic stalled or reversed forks (gapped forks or Holliday junctions [HJ]). RecG, which does not form a stable complex with DisA, unwinds branched intermediates, and in the presence of a limiting ATP concentration and HJ DNA, it blocks DisA-mediated c-di-AMP synthesis. DisA pre-bound to a stalled or reversed fork limits RecG-mediated ATP hydrolysis and DNA unwinding, but not if RecG is pre-bound to stalled or reversed forks. We propose that RecG-mediated fork remodeling is a genuine in vivo activity, and that DisA, as a molecular switch, limits RecG-mediated fork reversal and fork restoration. DisA and RecG might provide more time to process perturbed forks, avoiding genome breakage.

Cilia-organizing basal bodies (BBs) are microtubule scaffolds that are visibly asymmetrical because they have attached auxiliary structures, such as striated fibers. In multiciliated cells, BB orientation aligns to ensure coherent ciliary beating, but the mechanisms that maintain BB orientation are unclear. For the first time in Tetrahymena thermophila, we use comparative whole-genome sequencing to identify the mutation in the BB disorientation mutant disA-1. disA-1 abolishes the localization of the novel protein DisAp to T. thermophila striated fibers (kinetodesmal fibers; KFs), which is consistent with DisAp’s similarity to the striated fiber protein SF-assemblin. We demonstrate that DisAp is required for KFs to elongate and to resist BB disorientation in response to ciliary forces. Newly formed BBs move along KFs as they approach their cortical attachment sites. However, because they contain short KFs that are rotated, BBs in disA-1 cells display aberrant spacing and disorientation. Therefore, DisAp is a novel KF component that is essential for force-dependent KF elongation and BB orientation in multiciliary arrays.

ABSTRACTAfrican horse sickness virus (AHSV) is a virus species in the genusOrbivirusof the familyReoviridae. There are nine serotypes of AHSV showing different levels of cross neutralization. AHSV is transmitted by species ofCulicoidesbiting midges and causes African horse sickness (AHS) in equids, with a mortality rate of up to 95% in naive horses. AHS has become a serious threat for countries outside Africa, since endemicCulicoidesspecies in moderate climates appear to be competent vectors for the related bluetongue virus (BTV). To control AHS, live-attenuated vaccines (LAVs) are used in Africa. We used reverse genetics to generate “synthetic” reassortants of AHSV for all nine serotypes by exchange of genome segment 2 (Seg-2). This segment encodes VP2, which is the serotype-determining protein and the dominant target for neutralizing antibodies. Single Seg-2 AHSV reassortants showed similar cytopathogenic effects in mammalian cells but displayed different growth kinetics. Reverse genetics for AHSV was also used to study Seg-10 expressing NS3/NS3a proteins. We demonstrated that NS3/NS3a proteins are not essential for AHSV replicationin vitro. NS3/NS3a of AHSV is, however, involved in the cytopathogenic effect in mammalian cells and is very important for virus release from cultured insect cells in particular. Similar to the concept of the bluetongue disabled infectious single animal (BT DISA) vaccine platform, an AHS DISA vaccine platform lacking NS3/NS3a expression was developed. Using exchange of genome segment 2 encoding VP2 protein (Seg-2[VP2]), we will be able to develop AHS DISA vaccine candidates for all current AHSV serotypes.IMPORTANCEAfrican horse sickness virus is transmitted by species ofCulicoidesbiting midges and causes African horse sickness in equids, with a mortality rate of up to 95% in naive horses. African horse sickness has become a serious threat for countries outside Africa, since endemicCulicoidesspecies in moderate climates are supposed to be competent vectors. By using reverse genetics, viruses of all nine serotypes were constructed by the exchange of Seg-2 expressing the serotype-determining VP2 protein. Furthermore, we demonstrated that the nonstructural protein NS3/NS3a is not essential for virus replicationin vitro. However, the potential spread of the virus by biting midges is supposed to be blocked, since thein vitrorelease of the virus was strongly reduced due to this deletion. VP2 exchange and NS3/NS3a deletion in African horse sickness virus were combined in the concept of a disabled infectious single animal vaccine for all nine serotypes.

Structures of Thermotoga maritima DNA-integrity-scanning protein A (DisA) in different reaction states describe the diadenylate cyclase (DAC) reaction and the possibility of its inhibition. We conclude that the mechanisms of cyclic-di-AMP (c-di-AMP) synthesis and its inhibition are conserved among different DAC enzymes and bacterial species.

Antibiotic-producing Streptomyces use the diadenylate cyclase DisA to synthesize the nucleotide second messenger c-di-AMP, but the mechanism for terminating c-di-AMP signaling and the proteins that bind the molecule to effect signal transduction are unknown. Here, we identify the AtaC protein as a c-di-AMP-specific phosphodiesterase that is also conserved in pathogens such as Streptococcus pneumoniae and Mycobacterium tuberculosis. AtaC is monomeric in solution and binds Mn2+ to specifically hydrolyze c-di-AMP to AMP via the intermediate 5′-pApA. As an effector of c-di-AMP signaling, we characterize the RCK_C domain protein CpeA. c-di-AMP promotes interaction between CpeA and the predicted cation/proton antiporter, CpeB, linking c-di-AMP signaling to ion homeostasis in Actinobacteria. Hydrolysis of c-di-AMP is critical for normal growth and differentiation in Streptomyces, connecting ionic stress to development. Thus, we present the discovery of two components of c-di-AMP signaling in bacteria and show that precise control of this second messenger is essential for ion balance and coordinated development in Streptomyces.

The development, final appearance and digestion (during germination) of seed reserves in a number of genera of the Orchidaceae (tribe Orchideae) has been studied comprehensively, using ultrastructural and histochemical techniques complemented by gas chromatographic analysis of free sugars. Mature seeds of Disa, Disperis and Huttonaea contain substantial reserves of lipid and protein in the embryo. The protodermal cells of Disperis also contain protein-carbohydrate bodies. Free sugars are present but starch occurs only in immature seeds. Glyoxysomes are absent and lipolysis does not occur in seeds incubated without an external source of sucrose, and although a little starch is formed it is apparently synthesised from endogenous sucrose reserves. In the presence of exogenous sucrose, however, proteins are hydrolysed and glyoxysomes appear. Substantial quantities of starch are formed in such seeds. From these observations it is apparent that orchid seeds are unable to utilise endogenous reserves of lipid unless simple sugars are supplied to the medium but can utilise the free sugars present in the embryo. Resultant conclusions on the role of mycorrhizae in the germination of orchid seeds are discussed.

Abstract The discoidin I protein has been studied extensively as a marker of early development in the cellular slime mold Dictyostelium discoideum. However, like most other developmentally regulated proteins in this system, no reliable information was available on the linkage of the discoidin genes to other known genes. Analysis of the linkage of the discoidin I genes by use of restriction fragment length polymorphisms revealed that all three discoidin I genes as well as a pseudogene are located on linkage group II. This evidence is consistent with the discoidin I genes forming a gene cluster that may be under the control of a single regulatory element. The discoidin I genes are linked to three genetic loci (disA, motA, daxA) that affect the expression of the discoidin I protein. Linkage of the gene family members to regulatory loci may be important in the coordinate maintenance of the gene family and regulatory loci. A duplication affecting the entire discoidin gene family is also linked to group II; this appears to be a small tandem duplication. This duplication was mapped using a DNA polymorphism generated by insertion of the Tdd-3 mobile genetic element into a Tdd-2 element flanking the gamma gene. A probe for Tdd-2 identified a restriction fragment length polymorphism in strain AX3K that was consistent with generation by a previously proposed Tdd-3 insertion event. A putative duplication or rearrangement of a second Tdd-2 element on linkage group IV of strain AX3K was also identified. This is the first linkage information available for mobile genetic elements in D. discoideum.

The fission yeast mutant dis3-54 is defective in mitosis and fails in chromosome disjunction. Its phenotype is similar to that of dis2-11, a mutant with a mutation in the type 1 protein phosphatase gene. We cloned the dis3+ gene by transformation. Nucleotide sequencing predicts a coding region of 970 amino acids interrupted by a 164-bp intron at the 65th codon. The predicted dis3+ protein shares a weak but significant similarity with the budding yeast SSD1 or SRK1 gene product, the gene for which is a suppressor for the absence of a protein phosphatase SIT4 gene or the BCY1 regulatory subunit of cyclic AMP-dependent protein kinase. Anti-dis3 antibodies recognized the 110-kDa dis3+ gene product, which is part of a 250- to 350-kDa oligomer and is enriched in the nucleus. The cellular localization of the dis3+ protein is reminiscent of that of the dis2+ protein, but these two proteins do not form a complex. A type 1 protein phosphatase activity in the dis3-54 mutant extracts is apparently not affected. The dis3+ gene is essential for growth; gene disruptant cells do not germinate and fail in cell division. Increased dis3+ gene dosage reverses the Ts+ phenotype of a cdc25 wee1 strain, as does increased type 1 protein phosphatase gene dosage. Double mutant dis3 dis2 is lethal even at the permissive temperature, suggesting that the dis2+ and dis3+ genes may be functionally overlapped. The role of the dis3+ gene product in mitosis is unknown, but this gene product may be directly or indirectly involved in the regulation of mitosis.

Understanding how proteins fold and bind is interesting since these processes are central to most biological activity. Protein folding and protein-protein interaction are by themselves very complex but using a good and robust system to study them could ease some of the hurdles. In this thesis I have tried to answer some of the fundamental questions of protein folding and binding. I chose to work with PDZ domains, which are protein domains consisting of 90-100 amino acids. They are found in more than 400 human proteins and function mostly as protein-protein interaction units. These proteins are very stable, easy to express and purify and their folding reaction is reversible under most laboratory conditions. I have characterized the interaction of PSD-95 PDZ3 domain with its putative ligand under different experimental conditions and found out that its binding kinetics is sensitive to salt and pH.  I also demonstrated that the two conserved residues R318 and H372 in PDZ3 are responsible for the salt and pH effect, respectively, on the binding reaction. Moreover, I determined that for PSD 95 PDZ3 coupling of distal residues to peptide binding was better described by a distance relationship and there was a very weak evidence of an allosteric network. Further, I showed that another PDZ domain, SAP97 PDZ2 undergoes conformational change upon ligand binding. Also, I characterized the binding mechanism of a dimeirc ligand/PDZ1-2 tandem interaction and showed that despite its apparent complexity the binding reaction is best described by a square scheme. Additionally, I determined that for the SAP 97 PDZ/HPV E6 interaction that all three PDZ domains each bind one molecule of the E6 protein and that a set of residues in the PDZ2 of SAP 97 could operate in an unexpected long-range manner during E6 interaction. Finally, I showed that perhaps all members in the PDZ family could fold via a three state folding mechanism. I characterized the folding mechanism of five different PDZ domains having similar overall fold but different primary structure and the results indicate that all five fold via an intermediate with two transition states. Transition state one is rate limiting at low denaturant concentration and vice versa for transition state two. Comparing and characterizing the structures of the transition states of two PDZ domains using phi value analysis indicated that their early transition states are less similar as compared to their late transition states.

The Z-disc of striated muscle cells is a highly specialized three-dimensional structure which delineates the boundary of the individual sarcomeres. It accomplishes a unique role by anchoring actin filaments and acts as a molecular trigger for contraction. Beyond a well-defined structural role, in recent years it is emerging the hypothesis that Z-disc may be directly involved in the perception and transmission of muscular stress signals. To achieve these complex functions, many Z-disc proteins are involved in multiple protein interactions. The importance of these interactions is indicated by the fact that mutations in several Z-disc proteins can result in muscular dystrophies and/or cardiomyopathies in human and mice. The knowledge of Z-disc interactome and its regulation would improve by far the comprehension of the Z-disc biology and the onset of muscular disorders. The main goal of my project was to understand the complex network of protein-protein interactions occurring at the Z-disc of skeletal and cardiac muscle. In particular, my work was focused on two groups of Z-disc proteins: the FATZ and myotilin protein families on one hand, and some proteins belonging to the enigma family on the other hand. This work led to the identification of a specific interaction between the PDZ domains of enigma family members and the C-terminal five amino acids of the FATZ and myotilin families. The work of this thesis is part of a wider project involving the groups of Dr. G. Faulkner at ICGEB, Trieste, and Prof. O. Carpen at University of Turku, Finland. Together with our collaborators we noted that the C-terminal five amino acids of FATZ-1 (ETEEL), FATZ-2 (ESEDL), FATZ-3 (ESEEL), myotilin (ESEEL), palladin (ESEDL) and myopalladin (ESDEL) are highly similar. Searches in protein sequence database revealed that this E-[S/T]-[D/E]-[D/E]-L motif is restricted in Vertebrates to the FATZ and myotilin families of proteins, and it is evolutionary conserved from zebrafish to humans, indicating its importance for their biological function. The ELM program (a source for predicting functional sites in eukaryotic proteins) predicted that the terminal four amino acids of the FATZ family, myotilin, palladin and myopalladin constitute a binding motif for class III PDZ domain proteins (X-[D/E]-X-[V/I/L]). The first object of my work was to determine if the proteins with this new type of class III PDZ binding motif at their C-terminal could effectively bind PDZ domains. We knew from the literature that ZASP binds to all the three members of the FATZ family by means of its N-terminal PDZ domain, and that the C-terminal region of myotilin interacts with ZASP. In addition to ZASP, other two members of the enigma family of PDZ proteins, ALP and CLP-36, were included in this study. Both the full-length and the truncated (lacking the last five amino acids) version of the FATZ and myotilin families were produced as native proteins and tested for PDZ binding using the AlphaScreen (Amplified Luminescence Proximity Homogeneous Assay) technique. Biotinylated phosphorylated and non-phosphorylated peptides corresponding to the C-terminal five amino acids of the FATZ family, myotilin, palladin and myopalladin were also used in AlphaScreen interaction experiments, as well as a control peptide having E instead of L as its last amino acid (ESEEE). The results presented in this thesis show that the final five amino acids of the FATZ and myotilin families of proteins are responsible for the binding to the PDZ domains of ZASP, ALP and CLP-36, and that the nature of the last amino acid of the motif is crucial for the interaction. We also show that phosphorylation of the ligand sequence modulates the ability of the peptides to bind to the PDZ domains of the enigma family. ?-actinin-2 was included in this study as its C-terminus (GESDL) is classified as a class I PDZ binding motif that is able to bind to ZASP and ALP PDZ domains. AlphaScreen experiments confirm the binding of both the full-length and the C-terminal phosphorylated and non-phosphorylated peptides of ?-actinin-2 to the PDZ domains of ZASP and ALP, and they also reveal an interaction with the PDZ domain of CLP-36. These interactions were verified using another in vitro binding technique, the TranSignal PDZ Domain Array. Based on the results of the PDZ arrays, RIL was found to be another member of the enigma family capable to bind to the E-[S/T]-[D/E]-[D/E]-L motif. Therefore, these final five amino acids can be considered a novel type of class III PDZ binding motif specific for the PDZ domains of enigma proteins. To better quantify the strength of the noted interactions, SPR (Surface Plasmon Resonance) experiments were performed in the laboratory of Dr. A. Baines at University of Kent, UK. The affinities of the interactions between the PDZ domain of ZASP and some of the phosphorylated and non-phosphorylated peptides of the FATZ and myotilin families result to be in the nM range. The SPR results also demonstrate a new interaction between the PDZ domain of ZASP and ANKRD2. This protein is a member of the MARP family and it is thought to be involved in muscle stress response pathways. ANKRD2 localizes both in the sarcomeric I-band and the nucleus, and it is able to bind to several transcription factors, including YB-1, PML and p53. This interaction strengthens the hypothesis that, besides a structural function, Z-disc could have a role in cell signalling. The fact that at the Z-disc many proteins can interact with the same partners, it would be helpful to define the pattern and level of expression of the individual proteins in different muscle tissues. Another aim of my work was to measure the abundance of mRNAs of some Z-disc proteins using the Real-Time PCR technique. Four different muscles from adult mice were considered: tibialis (a fast-twitch skeletal muscle), soleus (a slow-twitch skeletal muscle), gastrocnemius (a skeletal muscle with mixed fibers) and heart (cardiac muscle). The different distribution of the FATZ proteins, myotilin and the alternatively spliced variants of ZASP suggest that, at least in mouse, the interactions between these proteins could be compartmentalized in distinct fiber types.
Il disco-Z del muscolo striato è una struttura molecolare altamente specializzata a livello della quale si instaurano numerose interazioni proteina-proteina. Il disco-Z delinea il confine dei singoli sarcomeri, fornendo un punto di ancoraggio per i filamenti sottili di actina; il loro scorrimento sui filamenti spessi di miosina produce la forza meccanica responsabile della contrazione. Uno dei ruoli chiave del disco-Z, dunque, è quello di trasmettere la tensione generata dalla struttura seriale dei sarcomeri lungo le miofibrille e, di conseguenza, lungo tutto il muscolo. Al di là di un evidente significato strutturale, negli ultimi anni sta diventando sempre più consistente l’ipotesi di un suo coinvolgimento anche nella percezione e nella trasmissione di segnali. L’importanza delle interazioni tra le proteine del disco-Z è indicata dal fatto che mutazioni in molte di queste proteine possono risultare in distrofie muscolari e/o cardiomiopatie sia in uomo sia in topo. Una più ampia conoscenza delle interazioni che si articolano a livello del disco-Z e, in generale, degli eventi che le regolano, aiuterebbe a chiarire la biologia del disco-Z e l’insorgenza di eventuali patologie associate. Il mio progetto di Dottorato è stato incentrato su due gruppi di proteine sarcomeriche e sulle loro interazioni: le proteine delle famiglie FATZ e miotilina da un lato, e alcune proteine appartenenti alla famiglia enigma dall’altro. Questo lavoro ha portato all’identificazione di un’interazione specifica tra i domini PDZ delle proteine della famiglia enigma e gli ultimi cinque residui aminoacidici presenti nelle proteine delle famiglie FATZ e miotilina. Il lavoro di questa tesi fa parte di un progetto più ampio che coinvolge i gruppi coordinati dalla Dr.ssa G. Faulkner dell’ICGEB, Trieste, e il Prof. O. Carpen dell’Università di Turku, Finlandia. Grazie alla loro collaborazione, è stato possibile notare che i cinque residui C-terminali delle proteine FATZ-1 (ETEEL), FATZ-2 (ESEDL), FATZ-3 (ESEEL), miotilina (ESEEL), palladina (ESEDL) e miopalladina (ESDEL) sono molto simili. Una ricerca effettuata in database di sequenze proteiche ha rivelato che questo motivo, E-[S/T]-[D/E]-[D/E]-L, è quasi esclusivamente ristretto nei Vertebrati alle proteine delle famiglie FATZ e miotilina; inoltre, esso sembra essere conservato da zebrafish ad uomo, suggerendo la sua importanza per le proteine che lo contengono. Il programma ELM (che predice siti funzionali in proteine eucariotiche) ha predetto che gli ultimi quattro amino acidi delle proteine FATZ, miotilina, palladina e miopalladina costituiscono un motivo di legame per le proteine con domini PDZ di classe III (X-[D/E]-X-[V/I/L]). Il mio primo obiettivo è stato quello di verificare se le proteine caratterizzate da questo nuovo motivo C-terminale potessero effettivamente legare domini PDZ. E’ noto dalla letteratura che tutti e tre i componenti della famiglia FATZ legano il PDZ di ZASP, e che l’interazione tra ZASP e miotilina è mediata dalla regione C-terminale di quest’ultima. Oltre a ZASP, altri due membri della famiglia enigma, ALP e CLP-36, sono stati inclusi nello studio. Le proteine della famiglia FATZ e miotilina sono state prodotte sia in versione full-length sia priva degli ultimi cinque amino acidi per essere utilizzate in saggi di interazione AlphaScreen (Amplified Luminescence Proximity Homogeneous Assay). Peptidi biotinilati, fosforilati e non, corrispondenti ai cinque amino acidi finali delle FATZ, miotilina, palladina e miopalladina sono stati inoltre impiegati nei saggi AlphaScreen, così come un peptide di controllo avente in ultima posizione un acido glutammico (E) invece che una leucina (L). I risultati riportati in questa tesi dimostrano che gli ultimi cinque amino acidi delle proteine delle famiglie FATZ e miotilina sono responsabili del legame ai domini PDZ di ZASP, ALP e CLP-36, e che la natura dell’ultimo residuo aminoacidico è cruciale per questa interazione. Inoltre, la fosforilazione del residuo di serina o treonina del ligando C-terminale può influenzare il legame dei peptidi nei confronti dei domini PDZ della famiglia enigma. La proteina ?-actinina-2 è stata introdotta nello studio, poiché la sua sequenza C-terminale (GESDL) è classificata come motivo di legame per i domini PDZ di classe I (X-[S/T]-X-[V/I/L]). Gli esperimenti AlphaScreen hanno confermato l’interazione di ?-actinina-2 (sia della forma full-length sia dei peptidi C-terminali, fosforilati e non) con i PDZ di ZASP e ALP, e hanno fatto emergere una nuova interazione con il PDZ di CLP-36. Molte di queste interazioni sono state verificate con un altro metodo di interazione proteina-proteina in vitro, il TranSignal PDZ Domain Array. Sulla base dei risultati di PDZ array è stato possibile identificare un altro membro della famiglia di proteine enigma, RIL, in grado di legare il motivo E-[S/T]-[D/E]-[D/E]-L. Possiamo considerare questi cinque amino acidi C-terminali come un nuovo motivo di legame per le proteine con domini PDZ di classe III, specifico per i domini PDZ delle proteine enigma. Per poter meglio quantificare la forza delle interazioni studiate, alcuni esperimenti di SPR (Surface Plasmon Resonance) sono stati eseguiti nel laboratorio del Dr. A. Baines all’Università di Kent, UK. Le affinità delle interazioni tra il dominio PDZ di ZASP e alcuni dei peptidi fosforilati e non-fosforilati delle famiglie di proteine FATZ e miotilina risultano essere nell’ordine del nM. Gli esperimenti di SPR hanno portato anche all’identificazione di un’interazione tra il PDZ di ZASP e ANKRD2. Si pensa che questa proteina, membro della famiglia MARP, sia coinvolta nelle vie di risposta a stress muscolari. ANKRD2 può trovarsi sia nella banda-I del sarcomero sia nel nucleo ed è in grado di legare diversi fattori di trascrizione, come YB-1, PML e p53. La scoperta di questa interazione rafforza l’ipotesi che il disco-Z, oltre ad un ruolo specificamente strutturale, potrebbe essere coinvolto in vie di segnalazione. Dal momento che a livello del disco-Z molte proteine hanno più di un partner proteico, sarebbe utile cercare di definire il livello e il profilo di espressione delle singole proteine in tessuti muscolari con diverse caratteristiche. Un altro obiettivo del mio lavoro è stato quindi quello di valutare l’abbondanza degli mRNA di alcune delle proteine del disco-Z da me studiate con la Real-Time PCR. Allo scopo sono stati presi in considerazione quattro tessuti muscolari di topo adulto: il tibiale (un muscolo scheletrico a contrazione rapida), il soleo (un muscolo scheletrico a contrazione lenta), il gastrocnemio (un muscolo scheletrico con fibre miste) e il muscolo cardiaco. La differente distribuzione delle FATZ, miotilina e ZASP (con le sue varianti di splicing) suggerisce che, almeno in topo, le interazioni tra queste proteine potrebbero essere compartimentalizzate in distinte fibre muscolari.

Low back pain (LBP) is associated with intervertebral disc (IVD) degeneration and exerts enormous socioeconomic burdens on the society. The nucleus pulposus (NP) is the structural and functional core of the IVD, and plays vital roles in its homeostasis. Although the etiology of IVD degeneration is not fully understood, the cellular changes of the NP have been proposed to be associated with degeneration. Conventional management for IVD degeneration primarily targets to relieve LBP and other symptoms without restoring or preserving disc function. Novel therapeutic strategies have emerged with an aim to retard or even reverse disc degeneration. In particular, the use of growth factors, such as the bone morphogenetic proteins (BMP), has received considerable attention due to their anabolic effects on extracellular matrix (ECM) synthesis by NP cells. BMP-2 and BMP-7 are of great interest for their involvement in osteogenesis, chondrogenesis, and development and maintenance of the IVD. To date, the benefits of BMP-2 on disc degeneration are controversial, given the inconsistent findings from animal model studies. The effectiveness of BMP-7 in disc repair, however, has been well demonstrated both in vitro and in vivo. A better understanding of the differences between BMP-2 and BMP-7 regulatory action on NP cells may facilitate future applications of BMP in disc repair/regeneration. This study hypothesized that BMP-2 and BMP-7 act differentially on human NP cells via different signal transduction processes. The differential effect of BMP-2 and BMP-7 was first tested in bovine NP cells using a three-dimensional culture system (alginate beads). Both BMP-2 and BMP-7 enhanced ECM production and phenotypic characteristics of bovine NP cells. Notably, BMP-7 was significantly more potent than BMP-2 in this regard. The effects of BMPs were further tested on non-degenerated (ND-NP) and degenerated (D-NP) human NP cells. The DMMB assay revealed that BMP-7 exerted a superior up-regulatory action on GAG production of D-NP cells compared to BMP-2. Furthermore, the overall response of D-NP cells to BMP-2 and BMP-7 was significantly lower than ND-NP cells. Immunohistochemical staining and quantitative RT-PCR assays demonstrated that D-NP cells possess a more fibroblastic and less chondrocyte-like phenotype than ND-NP cells. At the mRNA level, the BMP receptor BMPR1A was not expressed in D-NP cells. BMP-7, but not BMP-2, induced expression of BMPR1A in D-NP cells. On the other hand, gene expression of selected TGF-β pathway components and hypoxia pathway components were significantly up-regulated by BMP-2 but down-regulated by BMP-7. These findings suggest that D-NP cells can activate differential molecular cascades in response to BMP-2 and BMP-7. In conclusion, this study showed a superior effect of BMP7 in up-regulation of classical BMP signaling components including BMP receptor BMPR1A. The reduced responsiveness of D-NP cells to BMP-2 and BMP-7 stimulation may be related to a different expression pattern of BMP receptors. This study provides insights into the differential regulatory actions of BMP-2 and BMP-7 on human NP cells and facilitates the future application of BMPs in managing disc degeneration.
published_or_final_version
Orthopaedics and Traumatology
Doctoral
Doctor of Philosophy

The main focus of my PhD studies was aimed at understanding the general mechanism of nuclear translocation and isolating novel components of the nuclear translocation pathway in neurons. Using the Drosophila visual system as an in vivo model to study nuclear motility in developing photoreceptor cells (R-cells), I have identified a novel role for the Ser/Thr kinase Misshapen (Msn) and the endocytic trafficking pathway in regulating the nuclear translocation process.
The development of R-cells in the Drosophila eye disc is an excellent model system for the study of nuclear motility owing to its monolayer organization and the stereotypical translocation of its differentiating R-cell nuclei along the apical-basal plane. Prior to my thesis work, several laboratories had identified dynein and its associating proteins in R-cell nuclear translocation, however nothing was known about the signalling pathway that controlled their function in nuclear migration. Thus, one of my thesis goals was to elucidate the signalling mechanism controlling nuclear translocation in R-cells.
Using a combination of molecular and genetic approaches, I identified Msn as a key component of a novel signalling pathway regulating R-cell nuclear translocation. Loss of msn causes a failure of R-cell nuclei to migrate apically. Msn appears to control R-cell nuclear translocation by regulating the localization of dynein and Bicaudal-D (Bic-D). My results also show that Msn enhances Bic-D phosphorylation in cultured cells, suggesting that Msn regulates R-cell nuclear migration by modulating the phosphorylation state of Bic-D. Consistently, my results show that a Bic-D-phosphorylation-defective mutation disrupted the apical localization of both Bic-D and dynein. I propose a model in which Msn induces the phosphorylation of Bic-D, which in turn modulates the activity and/or subcellular localization of dynein leading to the apical migration of R-cell nuclei.
In addition to studying Msn, I have also searched for additional players in R-cell nuclear migration. From a gain-of-function approach, I found that the misexpression of the GTPase-activating-protein (GAP) RN-Tre caused a severe defect in R-cell nuclear migration. Since mammalian RN-Tre is involved in negatively regulating Rab protein activity, I speculated that the RN-Tre misexpression phenotype reflected a role for Rab-mediated vesicular transport in regulating R-cell nuclear migration. I systematically examined the potential role of Rab family proteins in R-cell nuclear migration and found that interfering with the function of Rab5, Rab11 or Shibire caused a similar nuclear migration phenotype. I propose that an endocytic pathway involving these GTPases is required for the targeting of determinants to specific subcellular locations, which in turn drive the apical migration of R-cell nuclei during development.

The aim of the study we present in this PhD thesis is to gain a deeper insight into structure of Z-disc proteins FATZ 1 and y-filamin. Z-discs are multi-protein complexes which are the primary conduits of the force generated by striated muscle contraction. The protein composition of Z-disc is not well defined and new proteins are continuously being reported. FATZ 1 is expressed early during myofibrilogenesis and it is presumed to have an important role in Z-disc assembly and functioning. At the moment, it is the only Z-disc protein for which a direct connection with the signal transducer protein phosphatase calcineurin has been demonstrated. The protein sequence of FATZl does not account for its interaction with numerous proteins as it does not contain any known protein interaction domains. For this reason we investigated the structural characteristics of this Z-disc protein. We studied also y-filamin, a binding partner of FATZl. The role of filamins in cytoskeleton organization and signal transduction has been well documented. y-filamin is the muscle specific isoform of filamins and it has not been studied as extensively as other filamin isoforms. y-filamin can be found both at sarcolemma and at the Z-disc of striated muscle cells and for this reason is considered to be a structural and functional link between contractile apparatus and sarcolemma. y-filamin repeat 23 was reported to be necessary for the interaction of y-filamin and FATZ. We determined crystal structure of y-filamin repeat 23, and found that y-filamin repeat 23 bound nickel. Detailed analysis of y-filamin protein sequence indicated that other filamin repeats could bind nickel or similar divalent cations. Physiological importance of nickel in Eukaryotes is not well studied, but in Prokaryotes nickel is an essential component of many enzymes. Nickel binding to a protein with mainly structural role would be an exception, since nickel is bound mainly to proteins with enzymatic activities. We analyzed also structural characteristics of y-filamin repeat 20. The y-filamin repeat 20 has the unique muscle specific insertion responsible for protein localization in the Z-disc. The muscle specific insertion presents no homology with any sequence with known protein structure. Our results from structural studies of repeat 20 indicate that the insertion in repeat 20 influences the fold of this repeat and makes it structurally different from y-filamin repeat 23.

AbstractDisk, file, and database encryption are technologies that protect data confidentiality when stored. Full disk encryption encrypts all data on a disk except the part containing the code to unlock the rest of the disk, which is usually not encrypted. File-based encryption operates at the file level and can be done by the operating system or an application. Database encryption can be done using transparent database encryption, column-level encryption, or field-level encryption. Both full disk encryption and File-based encryption allow good protection against data leakage in the case of device theft or loss. However, due to their nature, they cannot protect against an attacker who gets her hands on a device turned on or any other intruders who can run programs on the computer.

AbstractPost-translational modifications (PTMs) occur dynamically, allowing cells to quickly respond to changes in the environment. Lysine residues can be targeted by several modifications including acylations (acetylation, succinylation, malonylation, glutarylation, and others), methylation, ubiquitination, and other modifications. One of the most efficient methods for the identification of post-translational modifications is utilizing immunoaffinity enrichment followed by high-resolution mass spectrometry. This workflow can be coupled with comprehensive data-independent acquisition (DIA) mass spectrometry to be a high-throughput, label-free PTM quantification approach. Below we describe a detailed protocol to process tissue by homogenization and proteolytically digest proteins, followed by immunoaffinity enrichment of lysine-acetylated peptides to identify and quantify relative changes of acetylation comparing different conditions.

AbstractIn this chapter 1 discuss how political groups use tools to protect EU values at both member state and EU level. I first survey the different tools used to protect EU values at member state level. I then focus on those available vis-à-vis the EU level itself, particularly the Electoral Act, requirements incumbent on Europarties and European political foundations to comply with EU values and the EPRoP. I conclude that political group’s track-record is disappointing and discuss what improvements could be envisioned to (better) reach the aim of protecting EU values across all political groups’ activities and across all levels of government.

The aim of this study is to perform detail experimental mapping of the lubricating film thickness of bovine serum (BS) within the contact between an artificial metal or ceramic femoral head and a glass disc and analyze effect of proteins on the film formation under rolling/sliding conditions. The film thickness was studied experimentally using an optical test rig as a function of time under variety of constant mean speeds. Chromatic interferograms were recorded with a high-speed digital camera and evaluated with thin film colorimetric interferometry. Under pure rolling conditions it was observed that the central film thicknesses increased with time for all measurements. When the disc was slower than head then the measured central film thicknesses achieved values only about some few nanometres, whereas when the tests were realized with faster disc then measured central film thicknesses achieved significantly higher values. Distribution of the film thickness within the contact zone is not homogeneous and two different film thickness regions can be found; thicker protein film and thinner base film that both show specific behaviour over time. This study showed that protein formation plays an important role in the lubrication processes of artificial joints of the human. Due to challenging of this study the more complex research work is carried out at the present time.

Introdução - O fungo Moniliophthora perniciosa (Mp), causador da vassoura-de-bruxa no cacaueiro é hemibiotrófico. Sua infecção inicia com a germinação dos esporos na superfície do tecido vegetal, seguido da penetração nos espaços intercelulares, estabelecendo a fase biotrófica da doença. Essa fase pode durar de 60-90 dias, evoluindo para uma fase saprofítica. Em ambas as fases, o fungo utiliza proteínas efetoras que manipulam a fisiologia da célula hospedeira, por meio da modulação ou supressão da resposta imune. O estudo do modo de ação dessas proteínas pode levar a compreensão dos mecanismos infecciosos utilizados pelo fungo. Objetivo - O objetivo do trabalho foi compreender o efeito de uma potencial proteína efetora de Mp em plantas mono e dicotiledôneas. Metodologia - A proteína candidata a efetora Mp4145-3305 foi expressa em bactéria e foi testada em bioensaios com plantas dicotiledôneas. Nesse contexto, foi analisado o efeito da proteína recombinante na espécie Phalaris canariensis (alpiste) que é uma monocotiledônea. Para isso, foram feitos teste de atividade de necrose e também foi avaliado o envolvimento da proteína na geração de espécies reativas de oxigénio em folhas do alpiste. Resultados - Foi analisado em revisão que os fungos de mono e dicotiledônea podem apresentar diferenças nos tamanhos dos genomas e nas especificidades dos genes e efetores. Porém, existem evidências de efetores homólogos em espécies de patógenos em monocotiledônea e dicotiledôneas distantemente relacionados. A proteína efetora MpNEP (Necrosis and ethylene induced protein) promove morte celular em dicotiledôneas, mas não afeta monocotiledôneas. Tal fato aventa hipóteses que indicam a falta de receptores nas superfícies da célula de monocotiledôneas que possam reconhecer tal proteína efetora. Os resultados mostraram alterações fisiológicas nas plantas de alpiste induzida pela proteína. Isto promoveu um melhor entendimento dos sistemas evolutivos envolvidos com a relação planta patógeno. Conclusões - Os resultados sugerem que a proteína possui potencial uso como indutora de crescimento e resistência em plantas cultivadas.

Aging and degeneration of the intervertebral discs are cell mediated processes that include biochemical, mechanical and structural changes. Although these processes are similar, disc degeneration is defined as an accelerated aging process that results in a detriment in the function of the disc. Biochemical changes include protein cross-linking, proteoglycan depletion and changes on collagen type. These compositional changes are related to changes in the mechanical properties of the disc and its tissues. For instance, it has been shown that an increase of protein cross-linking by glycation or genipin treatment causes an increase of the stiffness in disc tissues [1,2]. On the other hand, a decrease on the amount of proteoglycan has been shown to cause a decrease on tissue stiffness due to a reduction of the osmotic pressure [3,4]. However, during aging and degeneration, these two processes occur simultaneously with opposing effects on the mechanical properties of the tissue. Consequently, it is important to analyze these effects separately. Additionally, many multiphasic models for soft charged tissues, such articular cartilage and intervertebral disc, also consider the ionic phases separately from non-charged solids. Although multiphasic models for the disc have been used in the past, the mechanical properties of the non-charged extra-fibrillar matrix (EFM) have not been measured directly.

When replacing plowing with disk tillage, there is a steady downward trend in the yield of barley grain, but the differences are not always reliable. On average, the decrease was 5.7%. In arid conditions, the use of minimal tillage leads to a decrease in the protein content in the grain of barley, and in conditions of increased moisture, to an increase. With traditional tillage, there was a tendency to an increase in protein harvest with grain harvest,

Modelos matemáticos são imprescindíveis para uma atividade rural que busca precisão em sua produção. O objetivo desta pesquisa foi ajustar equações matemáticas para estimar o crescimento de tilápias do Nilo em provas de desempenho para progênies. Para modelar o crescimento em peso corporal (g) em função do tempo (dias) da criação das tilápias após a fase da reversão sexual (idade?), foram avaliados 100 animais por caixa de alvenaria com volume útil de 3,0 x 1,0 x 0,80 m, sendo duas repetições para cada lote oriundo da reprodução. Cada lote foi representado pela coleta de larvas. Foram realizadas duas coletas sequentes e dois lotes de formas jovens formados (idade?). Os animais foram alimentados quatro vezes ao dia com ração em pó com 55% de proteína bruta durante todo período experimental de cada lote e grupo. Biometrias de 100 % dos animais foram realizadas em média a cada sete dias para peso vivo em balança de precisão (0,0001 g). Os conjuntos de dados obtidos nas biometrias dos alevinos de tilápia do Nilo para peso vivo (g) em relação ao tempo em dias e o conjunto de dados de taxa de crescimento especifico (%/dia) em função do tempo em dias foram submetidos à análise de regressão exponencial, logarítmica, polinomial e de potência. Os alevinos do primeiro lote apresentaram curvas de crescimento polinomial de segundo grau, entretanto, para os alevinos do segundo lote, as curvas se adequaram a equação de potência. Curvas para taxa de crescimento especifico adequaram a equação polinomial de segundo grau para todos os lotes e suas respectivas repetições.

Biological and anatomical changes of intervertebral disc (IVD) degeneration frequently occur in the nucleus pulposus (NP) [1]. Changes in NP matrix composition coincide with the loss of a distinct notochord derived cell population [2],[3], which may have the potential to generate or maintain a functional NP-like matrix. Immature NP cells reside in an environment rich in laminin and express specific laminin-binding receptors [4],[5]. Additionally, NP cells attach in higher numbers to laminins as compared to cells isolated from other regions of the IVD [6]. Our initial work demonstrated that matrix protein and stiffness modulate NP cell-cell interactions upon surfaces [7], with results that suggest soft, laminin-functionalized hydrogels may be useful for promoting an NP-like cell phenotype.

Antifreeze proteins (AFPs) allow biological organisms, including insects, fish, and plants, to survive in freezing temperatures. While in solution, AFPs impart cryoprotection by creating a thermal hysteresis (TH), imparting ice recrystallization inhibition (IRI), and providing dynamic ice shaping (DIS). To leverage these ice-modulating effects of AFPs in other scenarios, a range of icing assays were performed with AFPs to investigate how AFPs interact with ice formation when tethered to a surface. In this work, we studied ApAFP752, an AFP from the beetle Anatolica polita, and first investigated whether removing the fusion protein attached during protein expression would result in a difference in freezing behavior. We performed optical microscopy to examine ice-crystal shape, micro-structure, and the recrystallization behavior of frozen droplets of AFP solutions. We developed a surface chemistry approach to tether these proteins to glass surfaces and conducted droplet-freezing experiments to probe the interactions of these proteins with ice formed on those surfaces. In solution, ApAFP752 did not show any DIS or TH, but it did show IRI capabilities. In surface studies, the freezing of AFP droplets on clean glass surfaces showed no dependence on concentration, and the results from freezing water droplets on AFP-decorated surfaces were inconclusive.

Potyviruses form one of the largest and most economically important groups of plant viruses. Individual potyviruses and their isolates vary in symptom expression, host range, and ability to overcome host resistance genes. Understanding factors influencing these biological characteristics is of agricultural importance for epidemiology and deployment of resistance strategies. Cucurbit crops are subject to severe losses by several potyviruses including the highly aggressive and variable zucchini yellow mosaic virus (ZYMV). In this project we sought to investigate protein domains in ZYMV that influence systemic infection and host range. Particular emphasis was on coat protein (CP), because of known functions in both cell to cell and long distance movement, and helper component-protease (HC-Pro), which has been implicated to play a role in symptom development and long distance movement. These two genes are also essential for aphid mediated transmission, and domains that influence disease development may also influence transmissibility. The objectives of the approved BARD project were to test roles of specific domains in the CP and HC-Pro by making sequence alterations or switches between different isolates and viruses, and testing for infectivity, host range, and aphid transmissibility. These objectives were largely achieved as described below. Finally, we also initiated new research to identify host factors interacting with potyviral proteins and demonstrated interaction between the ZYMV RNA dependent RNA polymerase and host poly-(A)-binding protein (Wang et al., in press). The focus of the CP studies (MSU) was to investigate the role of the highly variable amino terminus (NT) in host range determination and systemic infection. Hybrid ZYMV infectious clones were produced by substituting the CP-NT of ZYMV with either the CP-NT from watermelon mosaic virus (overlapping, but broader host range) or tobacco etch virus (TEV) (non- overlapping host range) (Grumet et al., 2000; Ullah ct al., in prep). Although both hybrid viruses initially established systemic infection, indicating that even the non-cucurbit adapted TEV CP-NT could facilitate long distance transport in cucurbits, after approximately 4-6, the plants inoculated with the TEV-CPNT hybrid exhibited a distinct recovery of reduced symptoms, virus titer, and virus specific protection against secondary infection. These results suggest that the plant recognizes the presence of the TEV CP-NT, which has not been adapted to infection of cucurbits, and initiates defense responses. The CP-NT also appears to play a role in naturally occurring resistance conferred by the zym locus in the cucumber line 'Dina-1'. Patterns of virus accumulation indicated that expression of resistance is developmentally controlled and is due to a block in virus movement. Switches between the core and NT domains of ZYMV-NAA (does not cause veinal chlorosis on 'Dina-1'), and ZYMV-Ct (causes veinal chlorosis), indicated that the resistance response likely involves interaction with the CP-NT (Ullah and Grumet, submitted). At the Volcani Center the main thrust was to identify domains in the HC-Pro that affect symptom expression or aphid transmissibility. From the data reported in the first and second year report and in the attached publications (Peng et al. 1998; Kadouri et al. 1998; Raccah et al. 2000: it was shown that: 1. The mutation from PTK to PAK resulted in milder symptoms of the virus on squash, 2. Two mutations, PAK and ATK, resulted in total loss of helper activity, 3. It was established for the first time that the PTK domain is involved in binding of the HC-Pro to the potyvirus particle, and 4. Some of these experiments required greater amount of HC-Pro, therefore a simpler and more efficient purification method was developed based on Ni2+ resin.

This paper provides a broad overview of how multilateral peace operations have responded to cholera and Ebola epidemics and the HIV/ AIDS and Covid-19 pandemics over the past 20 years. Such public health crises can be especially lethal in fragile and conflict-affected areas. Peace operations possess resources and capacities that enable them to contribute in varying ways to state and humanitarian responses. Multilateral peace operations have acted to protect the health of peacekeepers and to prevent peacekeepers from spreading infectious diseases. They have also directly provided security to health and humanitarian personnel, health services and supplies to some non-mission personnel and local communities, and communications capacities to dispel dis/misinformation and inform local populations about health measures. Another area where peace operations have given indirect support to epidemic/ pandemic response measures is by offering political engagement, coordination, training and material support to host state actors as well as supporting the rule of law and capacity building of local security and police personnel. The paper concludes by considering arguments against and in favour of more strategic involvement of peace operations in future epidemics and pandemics.

The avian influenza virus, subtype H9N2 subtype, defined as having a low pathogenicity, causes extensive economical losses in commercial flocks, probably due to management and synergism with other pathogens. AIV H9N2 was first identified in Israel in the year 2000, and since then it became endemic and widespread in Israel. Control by vaccination of commercial flocks with an inactivated vaccine has been introduced since 2007. In face of the continuous H9N2 outbreaks, and the application of the vaccination policy, we aimed in the present study to provide a method of differentiating naturally infected from vaccinated animals (DIVA). The aim of the assay would be detect only antibodies created by a de-novo infection, since the inactivated vaccine virus is not reproducing, and might provide a simple tool for mass detection of novel infections of commercial flocks. To fulfill the overall aim, the project was designed to include four operational objectives: 1. Evaluation of the genetic evolution of AIV in Israel; 2. Assessment of the diagnostic value of an NS1 ELISA; 3. NS1 ELISA as evaluation criteria for measuring the efficacy of vaccination against H9N2 AIV; 4. Development of an AIV H9 subtype specific ELISA systems. Major conclusion and implications drawn from the project were: 1. A continuous genetic change occurred in the collection of H9N2 isolates, and new introductions were identified. It was shown thatthe differences between the HA proteins of viruses used for vaccine productionand local fieldisolatesincreasedin parallelwith the durationand intensity ofvaccine use, therefore, developing a differential assay for the vaccine and the wild type viruses was the project main aim. 2. To assess the diagnostic value of an NS1 ELISA we first performed experimental infection trials using representative viruses of all introductions, and used the sera and recombinant NS1 antigens of the same viruses in homologous and heterologous NS1 ELISA combination. The NS1 ELISA was evidently reactive in all combinations, and did not discriminate significantly between different groups. 3. However, several major drawbacks of the NS1 ELISA were recognized: a) The evaluation of the vaccination effect in challenged birds, showed that the level of the NS1 antibodies dropped due to the vaccination-dependent virus level drop; b) the applicability of the NS1-ELISA was verified on sera of commercial flocks and found to be unusable due to physico-chemical composition of the sera and the recombinant antigen, c) commercial sera showed non-reactivity that might be caused by many factors, including vaccination, uncertainty regarding the infection time, and possibly low antigen avidity, d) NS1 elevated antibody levels for less than 2 months in SPF chicks. Due to the above mentioned reasons we do not recommend the application of the DIVA NS1 ELISA assay for monitoring and differentiation AIV H9N2 naturally-infected from vaccinated commercial birds.