Plaquetas são fragmentos celulares anucleados, derivados de megacariócitos, que estão envolvidos em diversos processos fisiológicos e patológicos, como coagulação, inflamação, trombose, aterosclerose, e metástase e angiogênese tumorais. Para executar estas funções, plaquetas ativadas secretam uma fração solúvel de moléculas presentes em seus conteúdos granulares, que passam a interagir com outras moléculas e células adjacentes ao local da injúria, e com os próprios receptores plaquetários. No entanto, os mecanismos que regem a secreção em plaquetas ainda são pouco conhecidos. Neste sentido, o objetivo deste estudo foi analisar comparativamente a agregação de plaquetas ativadas por dois diferentes agonistas enzimáticos: a trombina, um dos mais importantes agonistas plaquetários, e a PA-BJ, uma serinoproteinase do veneno da Bothrops jararaca, que assim como a trombina, ativa plaquetas através dos receptores PAR-1 e PAR-4. Neste estudo foram utilizadas abordagens de espectrometria de massas e de bioinformática para caracterizar alterações nos proteomas do sedimento de plaquetas não ativadas e ativadas, e também, para em paralelo analisar as frações proteicas e peptídicas presentes no sobrenadante (secretoma). Nas análises do sedimento de plaquetas ativadas tanto por PA-BJ quanto por trombina, foi verificada a menor abundância das proteínas PBP, PF4, proteína S, fibronectina, fator V e alfa-1 antitripsina, entre outras, e que também foram identificadas no sobrenadante (secretadas), e o aumento de abundância das proteínas ADAM-10, tromboxano A2 sintase, integrina αlIb, miosina-9 e fosforilase b, que estão diretamente envolvidas na ativação/agregação. Por outro lado, verificamos que na secreção plaquetária induzida por trombina ocorreu o aumento de abundância de proteínas envolvidas na regulação da formação do coágulo, como a proteína S, PAI1 e antitrombina III, sugerindo que nos eventos disparados pela trombina, exista uma regulação rigorosa de sua ação no local da injúria vascular. Já na secreção induzida por PA-BJ, verificamos o aumento significativo das proteínas amiloide beta A4 e do fibrinogênio, envolvidas na ativação/agregação plaquetária, além da liberação e ativação de MMP1, indicando que esta metaloproteinase atue sinergicamente com a PA-BJ para a formação e estabilização do agregado plaquetário. Nas análises do secretoma de plaquetas não ativadas, identificamos pela primeira vez, a presença das proteínas catalase, anidrase carbônica, inibidor de elastase leucocitária e a glicoproteína rica em histidina, que estão envolvidas na inibição e regulação da ativação plaquetária. A análise da fração peptídica do sobrenadante plaquetário permitiu avaliar pela primeira vez o degradoma gerado no processo de agregação por PA-BJ e trombina. O conjunto de peptídeos resultante da ativação plaquetária pela PA-BJ é maior e mais complexo do que aquele gerado pela ação da trombina, sugerindo que as vias ativadas por ambas sejam diferenciais e sujeitas a diferentes controles de regulação da proteólise. Além disso, a degradação seletiva de algumas proteínas, e o conjunto de peptídeos gerados, poderiam ter um papel no controle da ativação e agregação plaquetárias. Em conjunto, nossos resultados demostram que, embora a PA-BJ e a trombina induzam a agregação plaquetária mediada pelos receptores PAR-1 e PAR-4, estas enzimas induzem vias diferentes, alterando a secreção plaquetária para levar à agregação. / Platelets are anucleated cell fragments derived from megakaryocytes which are involved in many physiological and pathological processes, such as coagulation, inflammation, thrombosis, atherosclerosis, and tumor angiogenesis and metastasis. To perform these functions, activated platelets secrete a soluble fraction of molecules present in their granules, which then interact with other molecules and cells adjacent to the site of injury, and with platelet receptors. However, the mechanisms governing secretion in platelets are still poorly understood. Therefore, the objective of this study was to comparatively analyze the aggregation of platelets activated by two different enzyme agonists: thrombin, one of the most important platelet agonists, and PA-BJ, a serine proteinase from Bothrops jararaca venom, which, like thrombin, causes platelet aggregation mediated by the receptors PAR-1 and PAR-4. For this purpose, approaches of mass spectrometry and bioinformatics were used to characterize changes in the proteome of non-activated and activated platelets, and also to analyze proteins and peptides present in the supernatant of aggregated platelets (secretome). In the analysis of the sediment of platelets activated by PA-BJ and thrombin, various proteins, such as PBP, PF4, protein S, fibronectin, factor V, and alpha-1 antitrypsin, were detected in lower abundance while they were also identified as secreted, in the supernatant; likewise, proteins that are directly involved in the activation/aggregation, such as ADAM-10, thromboxane A2 synthase, integrin αIIb, myosin-9 and phosphorylase b were identified in higher abundance in platelets activated by PA-BJ and thrombin. Moreover, we found that in the thrombin-induced platelet secretion there was increased abundance of proteins involved in the regulation of blood clot formation, such as protein S, and antithrombin III PAI1, suggesting that in the events triggered by thrombin, there is strict regulation of its action at the site of vascular injury. In the analysis of the secretion induced by PA-BJ, we found a significant increase in amyloid beta A4 protein and fibrinogen, which are involved in the platelet activation/aggregation, in addition to the release and activation of MMP-1, indicating that this metalloproteinase acts synergistically with PA-BJ in the formation and stabilization of the platelet thrombus. In the analysis of the non-activated platelet secretome, we identified for the first time the presence of catalase, carbonic anhydrase, leukocyte elastase inhibitor and histidine-rich glycoprotein, which are involved in the inhibition and regulation of platelet activation. The analysis of the peptide fraction of the supernatant of activated platelets enabled the characterization, for the first time, of the degradome generated in the process of aggregation by thrombin and PA-BJ. The resulting set of peptides generated upon platelet activation by PA-BJ is larger and more complex than that generated by the action of thrombin, suggesting that the pathways activated by both are differential and are subject to different controls of proteolysis. Furthermore, the selective degradation of some proteins, and the set of generated peptides could play a role in the control of platelet activation and aggregation. Taken together, our findings demonstrate that although both PA-BJ and thrombin induce platelet aggregation via PAR-1 and PAR-4, these enzymes activate different pathways to cause platelet secretion and aggregation.

Neste trabalho foram aprofundados nossos estudos para obtenção da (S)-2-terc-butilamida-4-(3-picolil)piperazina, pela abertura da (S)-2-terc-butilcarboxamida-N-p-tosilaziridina seguida de ciclização, em 78% de rendimento, com o triflato de vinildifenilsulfônio. A aziridina foi preparada por um processo de ciclização, em condições de transferência de fase, partindo-se da L-serina, um aminoácido natural de baixo custo. Esta rota sintética rendeu um material que apresenta a mesma estereoquímica S do fragmento piperazínico usado na síntese do Indinavir, podendo vir a constituir uma via alternativa para a obtenção deste fármaco. / In this work we performed a deeper study for obtaining (S)-2-tert-butylamide-4-(3-picolyl)piperazine by opening (S)-2-tert-butylcarboxamide-N-p-tosylaziridine followed by cyclization, in 78% yield, with diphenylvinylsulfonium trifluoromethanesulfonate. The aziridine were prepared by a cyclization process in phase transfer conditions, starting from L-serine, a low cost amino acid. This synthetic route yielded a material which has the same S piperazinic fragment stereochemistry used in the synthesis of Indinavir, and may constitute an alternative route for obtaining this drug.

Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) derivatives possess cancerostatic and anti-infective properties thus making them candidates of possible antibacterial agents. ²-lactam antibiotics are vital weapons for the treatment of bacterial infections, but their existence and effectiveness has been faced with resistance from ²-lactamases. Therefore, the need for new effective antimicrobial drugs is very crucial. In this work, we synthesized in high yields, PBD analogs 1−3, 5 and 7−9 in three to four synthetic steps from commercially available L-proline and isatoic anhydride. MTT Assay was employed to test the in vitro cytotoxicity of PBD analogs 1, 2, 5 and 7 on cancer cell lines including MCF-7, SKBR-3, SKMEL-2, CaCo 2 and Mia Paca. These compounds decreased the cell viability of MCF-7 by roughly 20% however, 1 and 5 had no effect on the SKMEL-2 cell lines. The inhibitory efficacy of these PBDs were also tested against TEM-1 and P99 Serine class A and C ²-lactamases.

ABSTRACT PURIFICATION AND CHARACTERIZATION OF CYTOPLASMIC AND PROTEASOME ASSOCIATED CHYMOTRYPSIN-LIKE PROTEASES FROM THERMOPLASMA VOLCANIUM &Ouml / zdemir, F.inci Ph.D., Department of Biology Supervisor: Prof. Dr. Semra Kocabiyik September, 147 pages In this study, two novel cytoplasmic serine proteases were isolated and characterized from thermophilic archaea Thermoplasma volcanium. The first protease was purified by ion exchange and affinity chromatographies and identified as a chymotrypsin-like serine protease mainly based on its substrate profile and inhibition pattern. The presence of protease activity was analyzed by gelatin zymography which was detected as a single band (35 kDa). Optimum temperature was found to be 60oC for azocasein hydrolysis and 50oC for N-Suc-Phe-pNA hydrolysis. Optimum activity was observed in the pH range of 6.0-8.0 with a maximum value at pH 7.0. The Km and Vmax values for the purified protease were calculated to be 2.2 mM and 40 &micro / moles of p-nitroanilide released min-1.ml-1, respectively, for N-Suc-Phe-PNA as substrate. Ca2+ and Mg2+ at 4 mM concentrations were the most effective divalent cations in activating the enzyme. In the second stage of this study, 20S proteasome of Tp. volcanium with substantial chymotrypsin-like activity was purified and characterized. This enzyme complex was purified with 19.1 U/mg specific activities from cell free extract by a four-step procedure. SDS-PAGE analysis revealed two strong bands with relative molecular masses of 26 kDa (&amp / #945 / -subunit) and 21.9 kDa (&amp / #946 / -subunit). Tp. volcanium 20S proteasome predominantly catalyzed cleavage of peptide bonds carboxyl to the acidic residue Glu (postglutamyl activity) and the hydrophobic residue Phe (chymotrypsin-like activity) in short chromogenic peptides. Low-level hydrolyzing activity was also detected carboxyl to basic residue Arg (trypsin-like activity). Chymotrypsin-like activity of Tp. volcanium 20S proteasome was significantly inhibited by chymotrypsin specific serine protease inhibitor chymostatin. When N-CBZ-Arg was used which is a substrate for trypsin, 20S proteasome was strongly inhibited by TLCK. The optimum temperature for Ala-Ala-Phe-pNA hydrolysis by the Tp. volcanium 20S proteasome was 55oC and the optimum pH was 7.5. The chymotryptic activity was significantly enhanced by divalent cations such as Ca+2 and Mg2+ at high concentrations, i.e. 125-250 mM. Keywords:Serine protease, 20S proteasome, archaea, thermophilic protease, Thermoplasma volcanium, chymotrypsin-like serine protease.

Phosphorylation of the alpha-subunit of the eukaryotic initiation factor 2 (eIF2alpha) on Serine 51 (Ser51) is an early event associated with downregulation of protein synthesis at the level of translation and constitutes a potent mechanism to overcome various stress conditions. In mammals, four eIF2alpha-kinases PERK, PKR, HRI and GCN2, activated following specific stresses, have been involved in this process. Our laboratory has previously demonstrated that the adaptor protein Nck, composed only of Src homology domains and classically implicated in cell signaling by activated plasma membrane receptor tyrosine kinases, modulates translation through its interaction with the beta-subunit of the eukaryotic initiation factor 2 (eIF2beta). Moreover, we reported that Nck-1 overexpression antagonizes the inhibition of translation in endoplasmic reticulum stress condition and prevents the PERK-mediated phosphorylation of the alpha-subunit of eIF2 on Ser51. In this thesis, I demonstrate that the adaptor protein Nck-1 modulates eIF2alpha-kinase-mediated eIF2alphaSer51 phosphorylation in a specific manner. More particularly, I show that Nck-1 overexpression reduces eIF2alpha phosphorylation in conditions activating PKR or HRI as described previously for PERK. In contrast, I observe that overexpression of Nck-1 in mammalian cells fails to attenuate eIF2alphaSer51 phosphorylation in response to amino acid starvation, a stress condition activating GCN2. I further confirm this observation by showing that Nck-1 fails to alter eIF2alphaSer51 phosphorylation in Saccharomyces cerevisiae, for which the sole eIF2alpha-kinase is GCN2. In addition, I report that Nck-1 reduces PKR activation in response to dsRNA. I also find that Nck-1 reduces dsRNA-induced activation of p38 MAPK, a PKR-downstream substrate, and cell death. Finally, I show that Nck-1 interacts exclusively with the inactivated form of PKR in a Src homology domain independent manner. All together these data uncover the existence of a novel mechanism regulating phosphorylation of eIF2alphaSer51 under various stress conditions and identifies Nck-1 as a modulator of the tumor suppressor and antiviral protein kinase PKR.

Spinal cord injury (SCI) results in a well-orchestrated inflammatory response which causes secondary tissue damage. Activated macrophages contribute to this cytotoxic response, which includes damage to neurons, glia and myelin, and tissue loss that worsens functional outcomes after SCI. However, activated macrophages in the spinal cord under other conditions are not cytotoxic, such as after intraspinal injection of lysophosphatidylcholine (LPC), a potent demyelinating agent. Recovery from SCI may be optimized by reducing the detrimental effects of macrophages while promoting their beneficial ones. Therefore, I compared spinal cord tissue, as well as purified macrophages, from mice after SCI (cytotoxic response) and intraspinal LPC injection (non-cytotoxic response). As a first step to carry out this work, I characterized the injury parameters for SCI contusion injury (i.e. injury force and spinal cord displacement) in mice using the Infinite Horizons impactor (Chapter 2). This lesioning model was used in other work for the thesis. The role T cells may play in mediating macrophage activation after LPC microinjection and SCI was also assessed using Nude mice (Chapter 3). Next, Affymetrix GeneChip analysis was carried out on spinal cord tissue obtained at the peak of the macrophage response after SCI and intraspinal LPC injection to identify potential candidate genes that may control the divergent inflammatory responses (Chapter 4). Several potential genes were identified. I next characterized the expression and role of one of these genes, MAPK activated protein kinase 2 (MK2), and showed that it mediates secondary tissue damage after SCI via several mechanisms (Chapter 5). The differences in gene expression profiles of macrophages purified from the spinal cord after SCI and LPC-injection were also assessed (Chapter 6). This microarray analysis of macrophages led to the identification of 10 novel candidate genes, two of which were validated at the protein level. Finally, I also examined the expression and role of secretory leukocyte protease inhibitor (SLPI) in SCI (Chapter 7). Using a combination of knockout/overexpressing transgenic mice and recombinant SLPI, I found that SLPI mediates protective anti-inflammatory effects after SCI. In conclusion, work done for this thesis has led to the identification of several novel molecules that influence the inflammatory response after injury and thus have led to the identification of potentially novel targets for the development of pharmacological approaches to treat acute SCI.

Using the known sequences for serine palmitoyltransferase (SPT) and ceramide kinase (CERK) from Arabidopsis, candidates for the corresponding genes in Loblolly pine were cloned and examined during embryogenesis. The cloned two cDNA sequences from Loblolly pine, which has similarity of 81% and 88% respectively to two subunits of SPT1 and SPT2 in Arabidopsis, were presumed as the Loblolly pine SPT1 and SPT2 (Pt-SPT1 and Pt-SPT2). A few different versions of Pt-SPT1 mRNAs (2223 nts, 756 nts, 822 nts, and 754 nts respectively), most likely the alternative splicing results, were found. Three of these mRNAs are capable of encoding proteins. The long version (2223 nts) encodes a protein with 484 amino acids (Pt-SPT1); two short versions (822 nts, 756 nts) encode a 90 a.a. protein. Another cDNA sequence of 2396 nts encodes a protein of 493 a.a. (Pt-SPT2). Both predicted Pt-SPT1 and Pt-SPT2 proteins possess highly conserved serine palmitoyltransferase functional domains (E value 5.7e-61). Their expression patterns are different between somatic and zygotic embryogenesis. Two different versions of mRNAs, with 2786 nts (long), and 2320 nts (short) respectively, of ceramide kinases in Loblolly pine (Pt-CERKs) have been cloned. The long version encodes a protein with 721 a.a.; the short version with 560 a.a. The expression patterns for these two CERK mRNAs are different during embryo development. The long version is constitutively expressed, while the short one is only expressed in some stages with much lower expression level. Overexpression Pt-CERKL, Pt-CERKS, and Pt-CERKF in E.coli and function analysis in vitro show that all Pt-CERKs appear to have the same catalytic functions as their homologs in human and Arabidopsis, but with different efficiency. The catalytic efficiency was dramatically lower in the short Pt-CERK protein compared with the long Pt-CERK protein and Pt-CERKF. The membrane system is not necessary for the catalytic reactions of these three Pt-CERKs in vitro and Pt-CERKs were less dependent on the Ca2+ ions. Thus, these studies have provided the first information about SPT- and CERK- like proteins in loblolly pine, and open new avenues of investigation for the roles of sphingolipids in embryonic development.

Mast cells are key effector cells in allergic and inflammatory diseases. However, their primary role is most likely in host defence against parasitic and bacterial infections. Mast cells are a particularly rich source of serine proteases. These proteases belong to the chymase or the tryptase family, which are encoded from the mast cell chymase and the multigene tryptase loci, respectively. To better understand the biological functions and the molecular evolution of these enzymes we have studied the organisation of these two loci in species ranging from fish to human. We show that the mast cell chymase locus has evolved from a single founder gene to a complex locus during the past 200 Myr of mammalian evolution. Forty-five fish candidate genes for hematopoietic serine proteases were also identified. However, in phylogenetic analyses none of them grouped with individual branches holding mammalian mast cell chymase locus genes, indicating an independent parallel evolution in fish. Studies of the evolution of the multigene tryptase locus showed that this locus has been highly conserved between marsupials and eutherians. However, no genes belonging to the individual subfamilies identified in eutherians could be identified in fish, amphibians or in birds, which also here indicates parallel evolution. To study the evolution of specific cleavage specificities associated with these proteases, the extended cleavage specificity of opossum α-chymase was determined and found to be nearly identical to human mast cell chymase and the major mouse mast cell chymase mMCP-4. This indicates a strong pressure to maintain this specificity during mammalian evolution. Basophils are rare blood cells with functions similar to mast cells that when mature almost completely lack mRNA. To study the proteome and to primarily characterize the granule protein content of basophils, an in vitro purification protocol was developed to obtain transcriptionally active umbilical cord blood-derived basophil precursors.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.