QC 20100618

Alzheimer’s disease is the most common cause of dementia and was responsible for over 2% of all deaths in Sweden 2012. One of the pathological hallmarks is amyloid plaques built by fibrillated Amyloid β. Luminescent conjugated oligothiophenes are known to stain and give characteristic fluorescence spectra when staining amyloid fibrils. Little is however known about the interactions between LCOs and fibrils. Studies have been performed on molecules more traditionally known to stain amyloid fibrils. Studies have also been performed on fibrils using limited proteolysis. So far no studies have been performed using LCOs combined with limited proteolysis in order to study the interaction pattern between LCOs and fibrils. Amyloid β is expressed and purified using a simple few step purification protocol. The amyloid β peptide was then fibrillated in several generations in order to select for a homogenous fibril structure. This purification protocol also has the ability to purify different oligomers of Amyloid β that are interesting from a toxicity point of view. In this thesis optical characteristics and limited proteolysis with mass spectrometry are being used to studies the interactions between LCOs and fibrillated amyloid β. The proteolytic pattern was suggestive of an accessible N-terminal and a hidden C-terminal of Amyloid β M1-42 in the fibril. It was also shown that the proteolysis cleavage pattern of Chymotrypsin is not disrupted when the LCO pKTAA was used to stain fibrils. The emission spectra from the two LCOs pATAA and pKTAA changes differently when subjected to continuous excitation indicative of conformational changes or chemical modification.

Submitted by VICTOR AUGUSTO DOMINGOS DIAS null (victordiaszootecnista@gmail.com) on 2016-08-22T15:46:32Z No. of bitstreams: 1 TESE - VICTOR AUGUSTO DOMINGOS DIAS.pdf: 1882002 bytes, checksum: 9487fafaeb94e582e81be00a1501e16f (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-08-23T18:12:58Z (GMT) No. of bitstreams: 1 dias_vad_dr_jabo.pdf: 1882002 bytes, checksum: 9487fafaeb94e582e81be00a1501e16f (MD5) / Made available in DSpace on 2016-08-23T18:12:58Z (GMT). No. of bitstreams: 1 dias_vad_dr_jabo.pdf: 1882002 bytes, checksum: 9487fafaeb94e582e81be00a1501e16f (MD5) Previous issue date: 2016-08-02 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A atuação das enzimas proteolíticas durante o post-mortem é um dos principais fatores que determinam a qualidade da carne, sendo as enzimas calpaína e calpastatina uma das principais atuantes neste processo. O objetivo deste estudo foi avaliar a relação entre a expressão gênica dos genes CAPN1, CAPN2 e CAST, a quantificação das enzimas µ-calpaína, m-calpaína e calpastatina, dentro de grupos contrastantes para maciez e crescimento animal. Foram utilizados dados de 90 animais machos não castrados da raça Nelore, com idade aproximada de 24 meses, permanecendo em confinamento por 95 dias. Após o abate foram colhidas amostras do músculo Longissimus thoracis entre a 9ª e 13ª costelas, da meia-carcaça esquerda. As amostras coletadas foram utilizadas para medir a área de olho de lombo (AOL), espessura de gordura subcutânea (EGS), índice de marmorização (IM), perdas por cozimento, coloração instrumental da carne e força de cisalhamento (FC). A partir do restante da carne, foram coletadas alíquotas para a realização das análises de índice de fragmentação miofibrilar (MFI) e lipídeos totais (LT). Foram coletadas alíquotas para a análise da expressão gênica e quantificação das enzimas. Os dados de força de cisalhamento foram utilizados como critério para a separação em carne macia e dura. Também foram utilizadas o ganho de peso para a separação quanto ao crescimento animal. Os seguintes grupos (n=10) foram formados: Leve, Pesado, Duro e Macio. As análises estatísticas foram realizadas utilizando-se o procedimento GLM e MIXED do programa SAS. No presente trabalho não foi encontrada diferença nas expressões dos genes CAPN1 e CAPN2 entre os grupos. Para o grupo contrastante para maciez também não foi encontrada diferença significativa (p<0,05 para a quantidade das enzimas µ e m-calpaína, e da calpastatina. Não foi encontrada correlação da quantidade das enzimas µ e m-calpaína, e calpastatina com as características PF e GP. Não foi encontrada diferença significativa (p<0,05) para o grupo contrastante para maciez da quantidade das enzimas µ e m-calpaína. Porém foi encontrada diferença significativa (p<0,05) para a quantidade da enzima calpastatina, onde os animais do grupo de carne dura apresentaram maiores valores em relação aos de carne macia. Além de apresentarem uma alta correlação entre a quantidade de calpastatina e as características FC (0) e FC (7) (r= 0,81 e 0,74, respectivamente). A diferença entre os grupos contrastantes e a alta correlação entre os níveis de calpastatina com as características de maciez demonstra a importância do sistema das calpainas na proteólise das miofibrilas, sendo os níveis de calpastatina um alvo potencial para a seleção de animais, com o objetivo de melhorar as características da qualidade da carne dos animais da raça Nelore. / The role of proteolytic enzymes during the post-mortem is one of the main factors that determine the quality of the meat, and the enzymes calpain and calpastatin are one of the main factors in this process. The aim of this study was to evaluate the gene expression of the genes CAPN1, CAPN2 and CAST, and quantify the enzymes μ-calpain, m-calpain and calpastatin within contrasting groups animal growth, and thougness. 90 animals data were used uncastrated male Nellore, aged approximately 24 months remaining in confinement for 95 days. After slaughter were harvested Longissimus muscle samples thoracis between the 9th and 13th ribs, the left half-carcase. The samples were used to measure the ribeye area (REA), subcutaneous fat thickness (SFT), marbling index (MI), cooking loss, instrumental color of the meat and shear force (SF). From the rest of the meat, aliquots were collected to perform the myofibrillar fragmentation index analysis (MFI) and total lipids (TL). Were collected aliquots for the analysis of gene expression and quantification of enzymes. The data of shear force were used as criteria for the separation of tender and tough meat. The characteristics of weight gain for the separation on the animal growth were also used. The following groups (n=10) were formed: lightweight, Heavy, tough meat and tender meat. Statistical analyzes were performed using the GLM and MIXED procedure of Statistical Analysis System program. In this study there was no difference in the expression of CAPN1 and CAPN2 genes between the groups. In this study there was no difference in the expressions of CAPN1 and CAPN2 genes between the groups. For the contrasting group for tenderness meat was also no significant difference (p<0.05) for the amount of enzymes μ and m-calpain, and calpastatin. There was no correlation between the amount of μ and m-calpain enzymes, and calpastatin with PF and GP. There was no significant difference (p<0.05) for the contrasting group for meat tenderness of the amount of enzyme μ and mcalpain. However, a significant difference (p<0.05) for the amount of enzyme calpastatin where animals of tough meat group had higher values on the tender meat. In addition to having a high correlation between the amount of characteristics calpastatin and SF (0) and SF (7) (r = 0.81 and 0.74, respectively). The difference between the contrasting groups and the high correlation between the calpastatin levels with tenderness characteristics demonstrates the importance of the system of calpains proteolysis of myofibrils, and the calpastatin levels of a potential target for the selection of animals for the purpose of improve the quality characteristics of the meat of Nellore.

Made available in DSpace on 2017-07-10T17:48:09Z (GMT). No. of bitstreams: 1 Lariane_Souza_da_Silva.pdf: 1189397 bytes, checksum: 1e10edda08abaad18f95d44a9235040e (MD5) Previous issue date: 2015-09-10 / Fundação Araucária / The growing demand of milk to supply the population has promoted the evolution of this chain, especially with regard to aspects related to food safety. Thus, this study traced psychrotrophic and proteolytic microorganisms, which are responsible for the separation of the main milk protein, k-casein, with emphasis on identifying and preserving Pseudomonas and Pseudomonas aeruginosa. This species presents high virulence, thus, it is very important to public health and to produce biofilm on several surfaces. The tracing samples were collected from the milking phases and were collected with hand swabs on udder, bucket, cooler, blower, raw milk collection and of milking water. The nonparametric Kruskal-Wallis analysis was used to screening of microorganisms in milking phases, while CMP evaluation results were obtained by the Principal Component Analysis. The results indicated that as the psychrotrophic bacteria counting increased (p <0.05), CMP values decreased. This has indicated lower levels of proteolysis, and it could possibly be related to the fact that psychrotrophic microorganisms present in samples did not have the proteolytic ability. The highest point of contamination indicated in tracing was the surface of the animals udder due to the adopted management, since most producers did not carry out a correct hygiene during this phase / A crescente demanda por leite para a abastecimento da população promoveu a necessidade de evolução dessa cadeia, principalmente no que diz respeito aos aspectos relacionados à segurança dos alimentos. O presente estudo rastreou microrganismos psicrotróficos e proteolíticos que realizam a quebra da principal proteína leiteira, a k-caseína, com destaque para a identificação e preservação de Pseudomonas e Pseudomonas aeruginosa. Essa espécie é de alta virulência, com importância em saúde pública e também em formação de biofilmes em superfícies diversas. As amostras coletadas para rastreamento provieram das etapas de ordenha e foram coletadas com swabs de mão, em teto, balde, resfriador, insuflador, e a coleta do leite cru e da água da ordenha. Utilizou-se a análise não paramétrica de Kruskal-Wallis para o rastreamento dos microrganismos nas etapas de ordenha e os resultados da avaliação do CMP foram obtidos pela Análise de Componentes Principais. Os resultados indicaram que: à medida que a contagem de bactérias psicrotróficas aumentou (p<0,05), diminuíram-se os valores para o CMP, indicando menor nível de proteólise, o que possivelmente poderia estar relacionado ao fato dos microrganismos psicrotróficos presentes nas amostras não possuíam a capacidade proteolítica. O ponto de maior contaminação indicado no rastreamento foi a superfície de teto dos animais, devido ao manejo adotado, pois a maioria dos produtores não realizavam a higienização correta

Orientador: Mirna Lucia Gigante / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-04T02:38:22Z (GMT). No. of bitstreams: 1 Henrique_VivianeSoccioMonteiro_D.pdf: 1578287 bytes, checksum: 701e35fa0f63acfe70512383a04328f3 (MD5) Previous issue date: 2004 / Resumo: Os objetivos deste trabalho foram avaliar o comportamento da fase aquosa do queijo Prato durante sua maturação e o efeito do pH sobre a proteólise, a firmeza e a capacidade de derretimento do queijo. Para avaliar o efeito do tempo de maturação sobre a fase aquosa do queijo, após a fabricação, amostras foram aleatoriamente escolhidas diariamente, até o quinto dia de maturação, e avaliadas quanto à quantidade de fase aquosa liberada por centrifugação. A fase aquosa foi avaliada quanto aos teores de sólidos totais, nitrogênio total, frações nitrogenadas e perfil eletroforético. Os resultados indicaram que a fase aquosa liberada por centrifugação diminuiu significativamente durante os primeiros 5 dias de maturação evidenciando um rápido aumento da capacidade de retenção de água da matriz protéica. Através do perfil eletroforético evidenciou-se o aumento de frações caseicas (as1-I-caseína e g-caseína) e de proteínas intactas (b-caseína e as1-caseína) na fase aquosa do queijo com 5 dias de maturação. Estes resultados sugerem que, além da proteólise, o comportamento da fase aquosa do queijo no período inicial da maturação pode ser também responsável pela melhoria da funcionalidade do queijo, uma vez que afeta a hidratação da matriz protéica. Para estudar o efeito do pH sobre a proteólise, a firmeza e a capacidade de derretimento do queijo Prato durante a maturação utilizou-se um método de alteração do pH pós-fabricação. Os queijos utilizados para a troca de pH e posterior avaliação da proteólise foram ralados e os utilizados para a avaliação da firmeza e da capacidade de derretimento foram fatiados (6,5 x 5,0 x 3,0 cm). Em ambos os casos, as amostras foram divididas em 3 porções que foram submetidas aos tratamentos de alteração do pH utilizando-se um dessecador com prateleiras. A primeira porção foi exposta a atmosfera de amônio para aumentar o pH; a segunda porção foi exposta ao vapor de ácido acético para abaixar o pH; a terceira porção serviu de controle. Após a alteração do pH as amostras foram imediatamente embaladas a vácuo e armazenadas a 12 °C. Para avaliar a proteólise amostras foram aleatoriamente escolhidas após 1, 8, 15, 22, 29, 44 e 58 dias de maturação e avaliadas quanto ao pH, sólidos totais, nitrogênio total, nitrogênio solúvel em pH 4,6, nitrogênio solúvel em TCA 12% e perfil eletroforético. A avaliação do efeito do pH sobre a firmeza e a capacidade de derretimento foi realizada após 8 dias de maturação. A melhor condição do teste de derretimento para o queijo Prato foi previamente definida como sendo 130 °C/ 10 minutos. Os resultados indicaram que os índices de extensão e profundidade de maturação aumentaram significativamente ao longo do tempo, porém, aumentaram menos para o pH mais baixo, quando, comparado aos demais tratamentos. O perfil eletroforético evidenciou que nos queijos de menor pH a degradação da *S1 e da *-caseínas ocorreu mais lentamente e menos extensivamente do que nos queijos controle e de mais alto pH. Além disso, observou-se que existe uma correlação linear positiva entre pH e a firmeza dos queijos. Não se observou associação significativa entre o pH e a capacidade de derretimento dos queijos / Abstract: The objectives of this study were to evaluate the evolution of the aqueous phase of Prato cheese in the initial stages of ripening and the independent effect of pH on proteolysis, firmness and melting ability of the cheese. To evaluate the effect of ripening time on the aqueous phase of the cheese after manufacture, samples were randomly taken every day up to the fifth day of ripening and subsequently evaluated for the amount of aqueous phase separated by centrifugation. The aqueous phase was evaluated for the level of total solids, total nitrogen, nitrogen fractions and electrophoretic profile. The results indicated that the aqueous phase separated by centrifugation significantly decreased over the first five days of ripening, thereby evidencing a rapid increase of the water binding capacity of the protein matrix. The electrophoretic profile evidenced an increase of the levels of casein fractions (as1-I-casein and g-casein) and intact proteins (b-casein and as1-casein) in the aqueous phase of cheese after 5 days ripening. These results suggest that, in addition to proteolysis, the evolution of the aqueous phase of cheese in the initial stages of ripening may also contribute to improving the functionality of cheese, as a result of the effect the aqueous phase has on the degree of hydration of the protein matrix. A post-manufacture pH change methods was used to study the independent effect of pH on proteolysis, firmness and melting ability of Prato cheese during ripening. The cheeses submitted to post-manufacture pH change and subsequent evaluation of proteolysis were grated, whereas those used for evaluation of firmness and melting ability were sliced (6,5 x 5,0 x 3,0cm). In both cases, the cheese samples were divided into 3 equal portions and submitted to pH change treatments in a desiccator with shelves. The first portion was exposed to ammonium hydroxide to raise the pH; the second portion was exposed to acetic acid to lower the pH; the third portion was used as control (no treatment). Immediately after the pH change procedure was completed, the samples were vacuum packed and stored at 12°C. To evaluate the effect on proteolysis, randomly selected samples were submitted to analysis after 1, 8, 15, 22, 29, 44 and 58 days ripening and evaluated for pH, total solids, total nitrogen, nitrogen soluble at pH 4,6, nitrogen soluble in 12% trichloroacetic acid (TCA) and electrophoretic profile. Evaluation of the effect of pH on firmness and melting capacity was performed after 8 days ripening. 130°C/10 minutes had previously been determined as the optimal temperature/time test condition for the melting test of Prato cheese. The results indicated that both the ripening extension index and the ripening depth index significantly increased with time, but the increase was less intense in the case of the low pH cheeses. The electrophoretic profile showed that degradation of *S1 and *-caseins occurred at a slower rate and less extensively in the low pH cheeses as compared to the cheese samples of the control and high pH groups. Furthermore, statistical analysis showed that there is a positive linear correlation between pH and the degree of firmness of the cheeses investigated. No significant association was found between pH and the melting ability of the cheeses / Doutorado / Tecnologia de Alimentos / Mestre em Tecnologia de Alimentos

Burkholderia cepacia is a common soil bacterium of significance in agriculture and bioremediation. B. cepacia is also an opportunistic pathogen of humans causing highly communicable pulmonary infections in cystic fibrosis and immunocompromized patients. The pyrB gene encoding ATCase was cloned and ATCase was purified by the glutathione S-transferase gene fusion system. The ATCase in B. cepacia has been previously classified as a class A enzyme by Bethell and Jones. ATCase activity gels showed that B. cepacia contained a holoenzyme pyrBC complex of 550 kDa comprised of 47 kDa pyrB and 45 kDa pyrC subunits. In the course of purifying the enzyme, trimeric subunits of 140 kDa and 120 kDa were observed as well as a unique proteolysis of the enzyme. The 47 kDa ATCase subunits were cleaved to 40 kDa proteins, which still demonstrated high activity as trimers. The proteolysis site is between Ser74 and Val75 residues. To confirm this, we converted the Ser74 residue to an Ala and to an Arg by site-directed mutagenesis. After this primary sequence changed, the proteolysis of ATCase was not observed. To further investigate the characteristics of B. cepacia pyrB gene, a pyrB knock-out (pyrB-) was constructed by in vitro mutagenesis. In the assay, the 550 kDa holoenzyme and 140 kDa and 120 kDa trimers disappeared and were replaced with a previously unseen 480 kDa holoenzyme pyrB- strain. The results suggest that B. cepacia has two genes that encode ATCase. ATC1 is constitutive and ATC2 is expressed only in the absence of ATC1 activity. To check for the virulence of these two strains, a eukaryotic model virulence test was performed using Caenorhabditis elegans (C. elegans). The pyrB1+pyrB2+ (wild type) B cepacia killed the nematode but pyrB1-pyrB2+ B. cepacia had lost its virulence against C. elegans. This suggests that ATC1 (pyrB1) is involved in virulence in B.cepacia and ATC2 (pyrB2) is not.

O proteassomo é o componente do sistema Ubiquitina-Proteassomo (UPS), responsável pela degradação de proteínas intracelulares marcadas com cauda de ubiquitina. No entanto, a unidade catalítica do proteassomo (20SPT), destituída de unidades regulatórias, é capaz de degradar proteínas de maneira ubiquitina-independente. Diversas modificações pós-traducionais já foram descritas para o 20SPT, incluindo a S-glutatiolação. De acordo com Demasi e col., (2003) o 20SPT da levedura Saccharomyces cerevisiae possui a atividade tipo-quimiotripsina modulada por glutationa e o mecanismo de glutatiolação implica na formação do intermediário ácido sulfênico. No presente trabalho, identificamos por espectrometria de massas (MS/MS) um total de sete resíduos diferentes de cisteína glutatiolados no 20SPT, sendo seis in vitro por incubação com GSH e três in vivo, extraído de células crescidas até atingir fase estacionária tardia em meio rico. Analisando a estrutura 3D do 20SPT, observou-se que os resíduos de cisteína glutatiolados não estão localizados na entrada da câmara catalítica nem próximos aos sítios-ativos, indicando um mecanismo alostérico da modulação da atividade proteassomal. O proteassomo glutatiolado extraído de leveduras é capaz de degradar proteínas oxidadas de maneira mais eficiente que o proteassomo reduzido por DTT, e ainda, esta degradação gera perfis peptídicos diferenciados por utilizar distintamente as atividades sítio-especificas, como visualizado por análises de HPLC e MS/MS. Por microscopia eletrônica verificamos a conformação aberta da câmara catalítica do proteassomo glutatiolado, sendo esta imediatamente fechada pela remoção da glutationa do 20SPT na presença de DTT. Caracterizamos ainda, enzimas reponsáveis pela desglutatiolação do 20SPT, capazes de recuperar as atividades proteassomais que haviam sido diminuídas pela glutatiolação: as oxidoredutases glutarredoxina 2 e as tiorredoxinas citosólicas. O mecanismo ainda inclui a hidrólise dessas oxidorredutases, fenômeno também verificado para diversas proteínas da suprafamília tiorredoxina, provavelmente devido a propriedades estruturais desta família. A glutatiolação do proteassomo apresenta-se como uma nova modificação pós-traducional de ocorrência fisiológica dependente do estado redox celular. Esta modificação promove aumento da atividade proteolítica, sugerindo uma função antioxidante atuante na remoção de proteínas oxidadas durante desafios oxidativos / The proteasome is the protease of the Ubiquitin-Proteasome System (UPS) responsible for the breakdown of intracellular ubiquitin-tagged proteins. However, the catalytic particle of the proteasome (20SPT) is capable of hydrolyzing some substrates in an ubiquitin-independent fashion. The S-glutathiolation of the 20SPT was described among several post-translational modifications and according to Demasi et. al. (2003), the chymotrypsin-like activity of proteasome from yeast Saccharomyces cerevisiae is regulated by glutathione. The mechanism of S-glutathiolation is dependent on the formation of the sulfenic acid intermediate in the cisteine residues of the 20SPT. In this present work, we identified in vitro and in vivo, a total of seven different S-glutathiolated proteasomal cysteine residues by mass spectrometry studies (MS/MS) and, by analyzing the 3D structure of the 20SPT, the modified cysteine residues are not located either on the entrance of the catalytic core or near to the active sites, indicating an allosteric mechanism of proteasomal modulation. During protein degradation, the natively S-glutathiolated 20SPT produces different patterns of peptide products when compared to the DTT-reduced particle through distinct site-specific cleavage of the protein substrates, as herein demonstrated by HPLC and MS/MS analyses. Furthermore, by electron microscopy, we showed that the entrance of the natively glutathiolated 20SPT is in the open conformation that immediately shifts to the closed conformation in the presence of DTT. We have also characterized the deglutathiolase role of the oxidoreductases Glutaredoxin 2 and Citosolic Thioredoxins 1 and 2 which recover the partially inhibited 20SPT activities. The deglutathiolation mechanism also includes the oxidoreductase degradation dependent on the 20SPT activation. The proteasome Sglutathiolation emerges as a new physiological post-translational modification correlated to the cellular redox state. Moreover, the S-glutathiolation of the 20SPT increases its proteolytic activity suggesting an antioxidant role by removing oxidized proteins generated during oxidative challenges.

Organic co-solvent-induced secondary conformation of α17-40 of human hemoglobin facilitates the splicing of E30-R31 in a mixture of its complementary segments by V8 protease. The amino acid sequence of α17-40 has been conceptualized by the general structure FR1-EALER-FRII and the pentapeptide sequence EALER playing a major role in inducing the α-helical conformation. The primary structure of α17-40 has been engineered in multiple ways to perturb one, two, or all three regions and the influence of the organic co-solvent-induced conformation and the concomitant resistance of E30-R31 peptide bond to V8 protease digestion has been investigated. The central pentapeptide (EALER), referred to here as splicedon,3 appears to dictate a primary role in facilitating the splicing reaction. When the same flanking regions are used, (1) splicedons that carry amino acid residues of low α-helical potential, for example G at position 2 or 3 of the splicedon, generate a conformational trap of very low thermodynamic stability, giving an equilibrium yield of only 3%-5%; (2) splicedons with amino acid residues of good α-helical potential generate a conformational trap of medium thermodynamic stability and give an equilibrium yield of 20%-25%; (3) the splicedons with amino residues of good α-helical potential and also an amino acid that can generate an i, i + 4 side-chain carboxylate-guanidino (amino) interaction, a conformational trap of maximum thermodynamic stability is generated, giving an equilibrium yield of 45%-50%; and (4) the thermodynamic stability of the conformational trap of the spliced peptide is also influenced by the amino acid composition of the flanking regions. The V8 protease resistance of the spliced peptide bond is not a direct correlate of the amount of α-helical conformation induced into the product. The results of this study reflect the unique role of the splicedon in translating the organic co-solvent-induced product conformation as a site-specific stabilization of the spliced peptide bond. It is speculated that the splicedon with higher α-helical potential as compared to either one of the flanking regions achieves this by integrating its potential with that of the flanking region(s). Exchange of flanking regions with the products of other V8 protease-catalyzed splicing reactions will help to establish the general primary structural requirements of this class of splicing reactions and facilitate their application in modular construction of proteins.

No description available.

Acyldepsipeptide (ADEP1) is an antibiotic that binds to Escherichia coli ClpP, mimicking the interaction that the protease typically establishes with ClpA/ClpX ATPases in bacterial cells. Binding of ADEP1 causes the N-terminal end of the ClpP to adopt a structured β-hairpin and triggers opening of the axial gate in the tetradecameric ClpP. Open conformation of the axial gate causes translocation of the substrates into the catalytic chamber of ClpP and the resultant uncontrolled proteolysis renders cellular death making ADEP1 a potent antibiotic. Our current understanding about the ADEP1-induced open conformation of the axial gate is limited. Based on the existing X-ray structures, it is unclear whether the mechanism of ADEP1-mediated activation of ClpP is conserved in Gram-positive and Gram-negative bacteria. To understand the activation mechanism of ClpP by ADEP1, we obtained Bacillus subtilis ClpP variants with amino acid substitutions in the N-terminal region and tested the effect of these mutations on substrate translocation using fluorescence-based proteolytic assays and cryo-electron microscopy. We found that compromising the integrity of the β-hairpin adopted by the N-terminal region prevented translocation of the substrate into the catalytic chamber of B. subtilis ClpP. These results suggest that the structural requirements for a functional axial channel are conserved in Gram-positive and Gram-negative bacteria. This study defines the structural requirements for ADEP1-mediated activation of the ClpP protease and serves as a model for the functioning of ClpP in the context of the ClpAP and ClpXP complexes. / Thesis / Master of Science (MSc)