Les associations mutualistes, en permettant l'acquisition de nouvelles fonctions, ont joué un rôle majeur dans l'évolution des espèces. Pour comprendre en quoi ces associations sont impliquées dans l'adaptation des espèces nous avons étudié un cas unique de mutualisme associant un virus de type polydnavirus et une guêpe parasitoïde. Dans cette association, le virus est injecté dans l'hôte lépidoptère lors de l'oviposition et joue un rôle majeur dans le succès parasitaire en induisant une altération des fonctions physiologiques de l'hôte. En regard du nombre d'espèces de guêpes caractérisées par cette association, le virus doit constituer une innovation adaptative majeure et jouer un rôle déterminant dans l'évolution et la diversification des espèces de guêpes. En quoi cette association joue t-elle un rôle déterminant dans l'évolution et l'adaptation des guêpes ? Quelles sont les fonctions physiologiques ciblées chez l'hôte ? Pour répondre à ces questions nous avons étudié l'évolution de deux familles de gènes codant pour des facteurs de virulence potentiels et nous avons exploré les fonctions physiologiques d'une protéine potentiellement ciblée lors du parasitisme. Nous avons mis en évidence le rôle important de la sélection naturelle dans l'évolution des familles de gènes viraux. Par modélisation de la structure tridimensionnelle d'un facteur de virulence codant pour des cystatines, nous avons montré que cette sélection agissait préférentiellement au niveau des sites d'interaction avec les protéines cibles. De plus, cette étude souligne le caractère dynamique de l'évolution des facteurs de virulence incluant de multiples évènements de duplication, caractérisés par des processus de perte et d'acquisition au cours de l'évolution de l'association. Le caractère adaptatif et dynamique de l'évolution des gènes viraux a aussi été étudié en regard de l'évolution des espèces de guêpes et de leur spectre d'hôte. Par une approche fonctionnelle, nous avons étudié le rôle physiologique de protéases à cystéine qui constituent des cibles potentielles des cystatines virales. Nous avons montré que ces protéases sont régulées spécifiquement au cours du parasitisme au niveau protéique et transcriptionnel. Nous avons également montré que l'activité de ces protéases est modifiée après parasitisme. L'évolution adaptative et dynamique des facteurs de virulence reflètent leur rôle important dans le parasitisme. Il reste maintenant à montrer comment ces facteurs interagissent sur la physiologie de l'hôte lépidoptère. Des protéases à cystéine sont spécifiquement ciblées par le parasitisme, en étudiant les mécanismes d'interaction de ces protéases avec les cystatines virales et les processus coévolutifs mis en jeu, nous

Proteins are a highly diversified group of molecules, andfor their study, advanced analytical tools are required. Inparticular, a need for high-throughput techniques has emergedin order to enable the characterization of large sets ofproteins. In this thesis, improved techniques for proteinseparations as well as new tools for the mass spectrometricanalysis of proteins are described. In the work, presented in the first part of the thesis, arefined extract containing proteases from Antarctic krill (Euphausia superba) was separated and characterized bymeans of capillary electrophoresis (CE) and mass spectrometry(MS). Tailored CE separations of the krill extract revealed thepresence of approximately 50 components. In addition, adetailed CE and MS analysis of fractions, containing individualkrill proteases has been carried out. Trypsin-like proteasesfrom krill exhibited a 12-fold and a 60-fold higher digestionefficiency at 37 °C and 2 °C respectively compared todigests performed with bovine trypsin. Furthermore, thecleavage specificity of the trypsin-like proteases wasstudied. In the last part of the thesis, novel concepts forchip-based nanoelectrospray (nanoESI) and matrix-assisted laserdesorption/ionization (MALDI) mass spectrometry are described.First, a micromachined silicon chip with a two-dimensionalmatrix of out-ofplane nanoESI needles for high-throughputanalysis was fabricated. A two-fold improvement insignal-to-noise reproducibility was obtained. Second, achip-based target for MALDI was developed, which featured pairsof elevated 50×50 µm anchors in close proximity. Theanchors were individually addressable with sample solution. Theminiaturized sample preparations at close distance to eachother allowed a simultaneous ionization of a physicallyseparated sample and standard by one single laser pulse. Thisresulted in a twofold reduction of relative mass errors.Moreover, ion suppression of the analyte was significantlyreduced. The effective utilization of the sample resulted in adetection limit of ca 200 zeptomole of angiotensin I. Key words:Proteins, peptides, proteases, Antarctickrill,Euphausia superba, capillary electrophoresis,fluorosurfactants, mass spectrometry, nanoelectrospray, ESI,MALDI, chip, high-throughput, reproducibility, sensitivity andmass accuracy.

Molecular interactions lie at the heart of myriad biological processes. Knowledge of molecular recognition processes and the ability to model and predict interactions of any biological molecule to any chemical compound are the key for better understanding of cell functions and discovery of more efficacious medicines. This thesis presents contributions to the development of a novel chemo-bioinformatics approach called proteochemometrics; a general method for interaction space analysis of biological macromolecules and their ligands. In this work we explore proteochemometrics-based interaction models over broad groups of protein families, evaluate their validity and scope, and compare proteochemometrics to traditional modeling approaches. Through the proteochemometric analysis of large interaction data sets of multiple retroviral proteases from various viral species we investigate complex mechanisms of drug resistance in HIV-1 and discover general physicochemical determinants of substrate cleavage efficiency and binding in retroviral proteases. We further demonstrate how global proteochemometric models can be used for design of protease inhibitors with broad activity on drug-resistant viral mutants, for monitoring drug resistance mechanisms in the physicochemical sense and prediction of potential HIV-1 evolution trajectories. We provide novel insights into the complexity of HIV-1 protease specificity by constructing a generalized IF-THEN rule model based on bioinformatics analysis of the largest set of HIV-1 protease substrates and non-substrates. We discuss how proteochemometrics can be used to map recognition sites of entire protein families in great detail and demonstrate how it can incorporate target variability into drug discovery process. Finally, we assess the utility of the proteochemometric approach in evaluation of ADMET properties of drug candidates with a special focus on inhibition of cytochrome P450 enzymes and investigate application of the approach in the pharmacogenomics field.

<p>Proteins are a highly diversified group of molecules, andfor their study, advanced analytical tools are required. Inparticular, a need for high-throughput techniques has emergedin order to enable the characterization of large sets ofproteins. In this thesis, improved techniques for proteinseparations as well as new tools for the mass spectrometricanalysis of proteins are described.</p><p>In the work, presented in the first part of the thesis, arefined extract containing proteases from Antarctic krill (<i>Euphausia superba</i>) was separated and characterized bymeans of capillary electrophoresis (CE) and mass spectrometry(MS). Tailored CE separations of the krill extract revealed thepresence of approximately 50 components. In addition, adetailed CE and MS analysis of fractions, containing individualkrill proteases has been carried out. Trypsin-like proteasesfrom krill exhibited a 12-fold and a 60-fold higher digestionefficiency at 37 °C and 2 °C respectively compared todigests performed with bovine trypsin. Furthermore, thecleavage specificity of the trypsin-like proteases wasstudied.</p><p>In the last part of the thesis, novel concepts forchip-based nanoelectrospray (nanoESI) and matrix-assisted laserdesorption/ionization (MALDI) mass spectrometry are described.First, a micromachined silicon chip with a two-dimensionalmatrix of out-ofplane nanoESI needles for high-throughputanalysis was fabricated. A two-fold improvement insignal-to-noise reproducibility was obtained. Second, achip-based target for MALDI was developed, which featured pairsof elevated 50×50 µm anchors in close proximity. Theanchors were individually addressable with sample solution. Theminiaturized sample preparations at close distance to eachother allowed a simultaneous ionization of a physicallyseparated sample and standard by one single laser pulse. Thisresulted in a twofold reduction of relative mass errors.Moreover, ion suppression of the analyte was significantlyreduced. The effective utilization of the sample resulted in adetection limit of ca 200 zeptomole of angiotensin I.</p><p><b>Key words:</b>Proteins, peptides, proteases, Antarctickrill,<i>Euphausia superba</i>, capillary electrophoresis,fluorosurfactants, mass spectrometry, nanoelectrospray, ESI,MALDI, chip, high-throughput, reproducibility, sensitivity andmass accuracy.</p>

Over the years, the view on proteases as relatively non-specific protein degradation enzymes, mainly involved in food digestion and intracellular protein turnover, has shifted and they are now recognized as key regulators of many biological processes that determine the fate of a cell. Besides their biological role, proteases have emerged as important tools in various biotechnical, industrial and medical applications. At present, there are worldwide efforts made that aim at deciphering the biological role of proteases and understanding their mechanism of action in greater detail. In addition, with the growing demand of novel protease variants adapted to specific applications, protease engineering is attracting a lot of attention. With the vision of contributing to the field of protein science, we have developed a platform for the identification of site-specific proteolysis, consisting of two intracellular genetic assays; one fluorescence-based (Paper I) and one antibiotic resistance-based (Paper IV). More specifically, the assays take advantage of genetically encoded short-lived reporter substrates that upon cleavage by a coexpressed protease confer either increased whole-cell fluorescence or antibiotic resistance to the cells in proportion to the efficiency with which the substrates are processed. Thus, the fluorescence-based assay is highly suitable for high-throughput analysis of substrate processing efficiency by flow cytometry analysis and cell sorting, while the antibiotic resistance assay can be used to monitor and identify proteolysis through (competitive) growth in selective media. By using the highly sequence specific tobacco etch virus protease (TEVp) as a model in our systems, we could show that both allowed for (i) discrimination among closely related substrate peptides (Paper I &amp; IV) and (ii) enrichment and identification of the best performing substrate-protease combination from a background of suboptimal variants (Paper I &amp; IV). In addition, the fluorescence-based assay was used successfully to determine the substrate specificity of TEVp by flow cytometric screening of large combinatorial substrate libraries (Paper II), and in a separate study also used as one of several methods for the characterization of different TEVp mutants engineered for improved solubility (Paper III). We believe that our assays present a new and promising path forward for high-throughput substrate profiling of proteases, directed evolution of proteases and identification of protease inhibitors, which all are areas of great biological, biotechnical and medical interest. / QC 20110516

Thesis by publication. / "August 2006" / Thesis (PhD)--Macquarie University, Division of Environmental & Life Sciences, Dept. of Biological Sciences & Dept. of Chemistry & Biomolecular Sciences), 2007. / Bibliography: leaves 157-183. / 1. Introduction -- 1.1. Proteomics and two-dimensional electrophoresis -- 1.2. A proteomic approach to study the filamentous fungus Trichoderma -- 1.3. Aims of the thesis -- 2. Materials and methods -- 3. Results and discussion -- 3.1. Method development for the display and identification of fungal proteins by 2DE and mass spectrometry -- 3.2. Discovery of novel determinants in the biological control of phytopathogens by Trichoderma atroviride -- 3.3. Summary and concluding remarks. / Trichoderma harzianum and T. atroviride are filamentous fungi commonly found in soil. Both display biocontrol capabilities against a range of phytopathogenic fungi including Rhizoctonia solani and Botrytis cinerea which are known pests of hundreds of commercially important crops including tomatoes, potatoes, beans, cucumber, strawberries, cotton and grapes. These Trichoderma species secrete a combination of enzymes degrading cell walls and antibiotics to overgrow and kill fungal phytopathogens. They are seen as an environmentally friendly alternative to chemical fungicides currengly used on crops. / A proteomic approach was taken to separate and identify proteins from a strain of T. harzianum with well established biocontrol properties. Several methods were developed in this thesis to display the whole proteome content and several subcellular proteome fractions from T. harzianum. Proteins were separated by two-dimensional electrophoresis and identified by mass spectrometric methods. The resulting proteomic maps represent the first extensive array of cellular and sub-cellular proteomes for T. harzianum. / Cellular protein patterns of T. atroviride (T. harzianum P1) grown on media containing either glucose or R. solani cell walls were compared by differential gel electrophoresis to identify a suite of new proteins involved in the biological control response. Twenty four T. atroviride protein spots up-regulated in the presence of the R. solani cell walls were identified by mass spectrometry and N-terminal sequencing. Proteins identified from this study included previously implicated enzymes degrading cell walls and three novel proteases, vacuolar serine protease, vacuolar protease A and trypsin-like protease. The genes encoding two of these proteases, vacuolar protease A and vacuolar serine protease have been cloned by degenerate primer PCR and genomic walking PCR and sequenced. The gene sequences and protein sequences derived from these genes have been partially characterised. / Mode of access: World Wide Web. / 194 leaves ill

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2007. / Härtill 4 uppsatser.

Made available in DSpace on 2015-03-26T13:30:51Z (GMT). No. of bitstreams: 1 texto completo.pdf: 841800 bytes, checksum: 783321f8508db8b49dbbe62fa1a549c8 (MD5) Previous issue date: 2014-02-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The spider mite Tetranychus evansi interferes in the lipoxygenases pathway of plant defenses respond where are produced jasmonic acid that activates expressing genes of proteinase inhibitors which affect the capability of digest proteins in herbivores. The not PI induction ability of T. evansi promotes his performance and reproduction but other species too like the spider mite T. urticae. Until now, there has not been study how digestive enzyme activity of these and other herbivores could be affected by T. evansi interference in tomato defense respond neither which digestive enzymes are present in this particular plant- herbivore relation what became in our research objective. Tomato plants were infested with T. evansi, T. urticae or moth larvae Tuta absoluta, while some plants were infested with a combination of T.evansi with each one of the other species simultaneously. When T. evansi was in the same plant with T. urticae the defense respond by lipoxygenases pathway were induced and the proteinase inhibitors were equal that plants attacked by only T.urticae, and plants attacked by T. evansi and T. absoluta simultaneously. T. evansi oviposition rate were only affected in plants previously attacked by T. urticae. In plants previously attacked by the combination of herbivores or just T. absoluta the oviposition rate doesn t differs of rates in clean plants, despite of high PI concentration. This suggests that T. evansi performance is affected by PI but for T. urticae plant injuries and traces and T. evansi has to overcome more factors involved in food competition. Digestive enzymes profile suggest a higher serine than cysteine proteinase activity been mainly tripsyn-like proteinases. In spider mites, tripsyn-like proteinases increase when PI were higher, possible to overcome PI toxic effects plus a higher aminoacids demand in T. evansi when competes with T. urticae for food resources. The quimotripsyn-like proteinases seems to be sensible to PI and decrease with high tripsyn-like ativity. / O ácaro Tetranychus evansi interfere na resposta de defesa pela via das lipoxigenases do tomateiro, na qual ocorre a produção de ácido jasmônico que ativa os genes que expressam inibidores de proteases os quais afetam a capacidade dos herbívoros para digerir proteínas. Ao não induzir inibidores de proteases, T. evansi favorece o seu desenvolvimento e reprodução, mas também de outros herbívoros como T. urticae. Ate agora não tem sido demostrado quais enzimas digestivas e como a suas atividades em este e outros herbívoros podem ser afetados por essa interferência na reposta de defensa do tomateiro, o que consistiu em nosso objetivo de trabalho. Plantas de tomate foram infestadas com ácaros das espécies T. evansi, T. urticae e com a lagarta Tuta absoluta, outras foram infestadas com uma combinação de T. evansi com as outras duas espécies. Quando T. evansi esteve na mesma planta com T. urticae a reposta de defesa pela via das lipoxigenases foi induzida e a concentração de inibidores de proteases igual que a indução gerada por T. urticae, o mesmo quando T. evansi esteve junto com T. absoluta na planta. A oviposição de T. evansi foi afetada em plantas que foram infestadas unicamente com T. urticae, mas em plantas atacadas pela combinação de herbívoros ou unicamente por T. absoluta a oviposição não diferiu com aquela em plantas limpas, apesar das altas concentrações de IP. Isto sugere que a oviposição de T. evansi não é afetada só pelos IP, mas possivelmente pelos danos e rastros deixados por T. urticae nas folhas. O perfil das enzimas digestivas indica uma maior atividade de serino proteases que de cisteíno proteases, sendo as Tripsinas-like as de maior atividade. Nos ácaros as tripsinas incrementam com a concentração de IP, possivelmente pela necessidade de superar o efeito deletério destes e no caso de T. evansi de digerir mais aminoácidos quando compete com T. urticae. As quimotripsinas-like parecem ser mais sensíveis aos IP e diminuir com o incremento da atividade das tripsinas.

As aminoacil tRNA sintetases (AaRSs) são enzimas essenciais na síntese de proteínas assegurando a correta relação entre os aminoácidos e seus tRNA cognatos. O genoma mitocondrial dos tripanossomatídeos perdeu os genes codificantes dos tRNAs, assim os tRNA mitocondriais são codificados no núcleo e importados do citoplasma. O código genético do kinetoplasto desvia do código genético pela utilização do códon de terminação UGA para a decodificação do códon do triptofano. Um único gene codificando o tRNATrp(CCA) observado no genoma de Leismania é responsável pela incorporação do aminoácido triptofano durante a síntese proteíca na mitocôndria. Para decodificar os dois códons do Trp (UGA e UGG) a base na posição 34 do tRNATrp(CCA) passa por um evento de editoração, convertendo o ribunuclotídeo C34 em U34, produzindo o tRNATrp(UCA) capaz de decodificar o códon UGA. Nesse trabalho foram caracterizadas duas triptofanil tRNA sintetases de Leishmania major. De acordo com experimentos de ?western blotting? e análises ?in silico? das seqüências de aminoácidos, uma enzima tem localização citoplasmática (LmTrpRS1) enquanto a outra mitocondrial (LmTrpRS2). Os mRNAs dos dois genes foram definidos por experimentos de 5? e 3? RT-PCR. As duas enzimas foram clonadas em diversos vetores de expressão procariotos e eucariotos. A LmTrpRS1 foi obtida somente na fração insolúvel, já a LmTrpRS2 foi obtida na fração solúvel quando clonada no vetor de expressão pET28a. Esta porém mostrou-se instável precipitando rapidamente após sua purificação. Os ensaios enzimáticos realizados com a mesma mostraram que ela é capaz de reconhecer os tRNAsTrp editado e não editado. Modelagem molecular por homologia com as duas proteínas foi realizada usando a proteína citoplasmática humana como molde, para estudar a interação entre a proteína e o tRNATrp. Xylella fastidiosa é um bactéria gram negativa limitada ao xilema, responsável por um grande número de doenças economicamente importantes, como a doença de Pierces em videiras, Clorose variegata do Citrus (CVC) e a doença da requeima das folhas em outras plantas incluindo, amendoeira, ameixeira, louro, amoreira e café. Em todos os casos a X. fastidiosa afeta o xylema da planta causando redução na produção de frutos. Nesse trabalho nós mostramos a estrutura da Xylellaína, uma cisteíno protease desse patógeno. A estrutura foi resolvida por dispersão anômala a um único comprimento de onda, utilizando cristais de xylellaína selenometionina substituídos. A estrutura da Xylellaína foi refinada até 1,65 Å de resolução, mostrando enovelamento similar às proteínas da família da papaína, porém algumas características interessantes como uma região N-terminal composta por 38 aminoácidos cobrindo o sulco ativo da enzima, um intrigante ribonucleotídeo encontrado fora do sítio ativo da enzima e um ?loop? semelhante ao ?loop? de oclusão presente na catepsina B. / The aminoacyl tRNA synthetases (aaRSs) are essential enzymes in protein synthesis that ensure the correct match between amino acids and their cognate tRNAs. The mitochondrial (kinetoplast) genome of trypanossomatids lacks tRNA genes, and therefore nucleus-encoded tRNAs are imported from the cytoplasm, the kinetoplast genetic code deviates from the universal code in that UGA instead of UGG encodes for tryptophan. A single nucleus-encoded tRNATrp(CCA) is responsible for Trp insertion during organellar protein synthesis. To decode both Trp codons (UGA and UGG), tRNATrp(CCA) undergoes a single C to U editing event at position 34 of the anticodon yielding to versions of the tRNA in the mitochondria with anticodon CCA and UCA, permitting UGA decoding. This work have characterized two Leishmania major tryptophanyl-tRNA synthetase, acording western blotting experiments and ?in silico? sequence analisis one of cytoplasmatic localization (LmTrpRS1) and another from mitochondria localization (LmTrpRS2). The mature mRNA transcripts for both genes were defined by 5? and 3? RT-PCR. Both enzymes were cloned into several expressions vectors. LmTrpRs1 was obtained as an insoluble protein and LmTrpRs2 expressed into the soluble fraction in pET28a expression system. LmTrpRS2 protein, however, is unstable precipitating shortly after purification. The enzymatic assay showed that this enzyme is able to recognize both tRNATrp. Molecular modeling for LmTrpRS1 and LmTrpRS2 were constructed using the cytoplasmatic human tryptophanyl tRNA synthetase as a model, to study the interaction between proteins and tRNATrp. Xylella fastidiosa is a xylem-limited, gram-negative bacteria responsible for a large number of economically important plant diseases, such as Pierces disease in grapevines, citrus variegated chlorosis (CVC) in sweet oranges and leaf scorch diseases in other plants, including almond, plum, oleander, mulberry and coffee. In all cases, X. fastidiosa infects the plant xylem and impairs fruit production. Here, we report the crystal structure of xylellain, a cystein protease from X. fastidiosa. The structure was solved by single-wavelength anomalous dispersion (SAD) using seleno-methionine containing xylellain crystals. The final structure of Xylellaína was refined against the best native data set (1.65 Å) showing R/Rfree= 17/21. Xylellain shares fold similar to Papain like Family, but contains some interesting features, like a 38 N-terminal tail covering the active site cleft; one intriguing ribonucleotide found outside the active site and one loop that resemble the ocluding loop from cathepsin B.

Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2018-03-12T17:36:57Z No. of bitstreams: 1 PEDRO HENRIQUE RODRIGUES DE ALENCAR AZEVEDO.pdf: 15048741 bytes, checksum: a121d29e5dc4898c7b8e4a85def01e12 (MD5) / Made available in DSpace on 2018-03-12T17:36:57Z (GMT). No. of bitstreams: 1 PEDRO HENRIQUE RODRIGUES DE ALENCAR AZEVEDO.pdf: 15048741 bytes, checksum: a121d29e5dc4898c7b8e4a85def01e12 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / As calicreínas teciduais humanas (KLKs) compreendem uma família de 15 enzimas serina proteases (KLKs 1-15) amplamente encontradas nos tecidos humanos. Em diversas patologias como a dermatite atópica, psoríase, síndrome de Netherton, câncer de ovário, mama e testículos, as KLKs encontram-se em concentrações elevadas. Por exemplo, as KLKs 5 e 7 estão mais abundantemente expressadas na pele, na qual estão envolvidas com o processo de descamação da mesma, e também presentes em alguns tipos de carcinomas. Dessa forma, as KLKs 5 e 7 são consideradas importantes alvos terapêuticos para o tratamento de doenças onde elas encontram-se superexpressadas, enfatizando a existência de somente um fármaco comercialmente disponível como inibidor de KLK. Nesse contexto, o trabalho descreve a síntese de 3 séries de compostos peptideomiméticos, incorporando o cerne estatina e diferentes resíduos de aminoácidos, planejados como candidatos a inibidores das enzimas serina proteases do KLKs 5 e 7. Os compostos finais foram obtidos utilizando uma rota sintética eficiente tendo como reação-chave a formação da ligação peptídica entre o cerne estatina e cloridratos de aminoésteres, previamente sintetizados. Os compostos sintetizados foram identificados por técnicas de Ressonância Magnética Nuclear, Infravermelho e Espectrometria de massas de alta resolução e os produtos finais serão avaliados em testes in vitro de inibição das enzimas KLKs / Human tissue kallikreins (KLKs) comprise a family of 15 serine protease enzymes (KLKs 1-15) widely found in human tissues. In several pathologies such as atopic dermatitis, psoriasis, Netherton syndrome, ovarian, breast and testis cancer, KLKs are in high concentrations. For example, KLKs 5 and 7 are more abundantly expressed in the skin, in which they are involved in the desquamation process, and also present in some types of carcinomas. Thus, KLKs 5 and 7 are considered important therapeutic targets for the treatment of diseases where they are over expressed, emphasizing the existence of only one commercially available drug as a KLK inhibitor. In this context, the work describes the synthesis of three series of peptideomimetic compounds incorporating the statin core and different amino acid residues, designed as candidates for inhibitors of the serine protease enzymes of KLKs 5 and 7. The final compounds were obtained using an efficient synthetic route based on the reaction of formation of the peptide bond between the statin core and previously synthesized amino acid hydrochlorides. The synthesized compounds were identified by Nuclear Magnetic Resonance, Infrared and High Resolution Mass Spectrometry techniques and the final products will be evaluated in in vitro inhibition assays of the KLKs enzymes