CHAPTER II: Objective: Our aim was to compare the performance of three serological assays in leprosy patients and their household contacts utilizing two quantitative ELISA tests using native PGL-I (PGL-1 ELISA), synthetic ND-O-HSA (ND-O-HSA ELISA), and the semi-quantitative lateral flow test (ML-Flow). Methods: Compare the performance of three immunological assays, PGL-I ELISA, ND-O-HSA ELISA, and ML-Flow were evaluated in 156 leprosy patients and 191 household contacts. Results: The sensitivity results of the PGL-1, ND-O-HSA, and ML-Flow were 68.83%, 63.65%, and 60.65%, respectively. The native and synthetic PGL-I ELISA assays detected antibodies in 22.73%, 31.82% of the paucibacillary (PB) patients, and the ML Flow test did not detect antibodies in this group. The ML-Flow test was able to discriminate patients into PB and multibacillary (MB) forms, while the native PGL-I and ND-O-HSA correlated with the bacillary load and the Ridley-Jopling clinical forms. In household contacts, the native PGL-I, ND-O-HSA, and ML-Flow assays detected seropositivity of 25%, 17%, and 10%, respectively. Conclusions: The use of ELISA and ML-Flow tests are thus recommended as additional tools in the diagnosis and classification of the clinical forms, aiding in prescribing the correct treatment regimen to prevent subsequent nerve damage and disability. CHAPTER III: Host pathogen interactions are mainly mediated by specialized molecules of the cell envelope. One of these essential mycobacterial cell wall components is the lipoarabinomannan (LAM). LAM has immunomodulatory roles, but its heterogeneity may be responsible for the differential immune response in leprosy patients and contacts. The research to structural motifs that could contribute as virulence factors and/or protective epitopes, and thereby derive effective biomarkers for diagnosis, drugs and/ or vaccines against leprosy has been very developed. Therefore, our aim was to develop specific mimetic peptides to this lipoglycan by using Phage Display of a random heptamer peptide library that may recognize a differential response in patients and contacts. We have used the anti-LAM CS-35 monoclonal antibody as a target for three rounds of selection. After sequencing and translation, peptides were pre-validated by ELISA and compared to the synthetic LAM-BSA antigen. The most reactive and repetitive peptide motif (A9) was subsequently tested against serum from 54 leprosy patients and 27 endemic controls by ELISA. The A9 phage-displayed peptide clone presented high levels of IgG antibodies in paucibacillary patients, from which 50% of them presented highly reactive sera. This reactivity has also been detected in tuberculoid, borderline-borderline and lepromatous patients. High levels of IgG1 were most frequent in endemic controls and reactional patients. On the other hand, the IgG profile and its subclasses in patients presented high levels of IgG and IgG2 and low levels of IgG1. The A9 clone presented a significant correlation with the synthetic LAM-BSA, for the IgG1 response. The highly reactive IgG response against the A9 clone was associated with the tuberculoid clinical form diagnosis, and detection of both IgG1 and IgG3 against this clone was associated with protection in endemic controls. CHAPTER IV: Heat Shock Proteins (HSPs), GroES and GroEL, are targets of strong human T-cell response, and a third of the cells responsive to M. leprae, recognize these proteins. Monoclonal antibodies mAbs CS-01 and CS-44 selected mimetic peptides that are ligands of their Fab portions, by phage display technique. Sera from 54 patients, 48 household contacts and 27 endemic controls were submitted to ELISA with B2 and A1 mimetic clones of the GroES and GroEL proteins, respectively, for detection of IgG and its subclasses. Using the mimetic clone of B2 GroES, the ELISA detected IgG antibodies present in sera of patients, contacts and endemic controls. The IgG antibodies were abundant in sera from multibacillary patients, especially in lepromatous (LL). A decline of IgG1 was found in patients and household contacts that became sick with leprosy and a raise of this subclass was present in sera of household contacts that did not develop the disease. With the mimetic clone of GroEL A1, the reactive antibodies were abundant in multibacillary patients, with a correlation with the bacillary load. In this study we observed that IgG antibodies against GroEL and GroES can be detected in the diagnosis of leprosy in serological tests produced with clones mimetics of these proteins. And the subclasses of IgG antibodies to GroES can demonstrate a targeting of antigenic molecules that induce the production of protective antibodies. / CAPÍTULO III: Objetivo: O objetivo do trabalho foi comparar a performance de três testes sorológicos em pacientes e seus contatos domiciliares utilizando dois testes quantitativos ELISA, um com o antígeno PGL-1 nativo (ELISA PGL-1), com o antígeno ND-O-HSA sintético (ELISA ND-O-HSA), e o teste semi-quantitativo do fluxo lateral (ML-FLow). Métodos: Os três testes imunológicos ELISA PGL-I, ELISA ND-O-HSA, e ML-Flow foram realizados utilizando soros de 156 pacientes com hanseníase e 191 contatos domiciliares. Resultados: Os resultados da sensibilidade dos testes ELISA PGL-1, ND-O-HSA e ML-FLow foram de 68.83%, 63.65%, e 60.65%, respectivamente. Os testes ELISA PGL-1 nativo e sintético detectaram anticorpos em 22,73% e 31,82% dos soros de pacientes paucibacilares (PB),e o teste ML-FLow não apresentou reatividade em nenhum soro. O ML-Flow foi capaz de discriminar entre os pacientes com as formas PB e multibacilares (MB), enquanto que o ELISA PGL-1 e ND-O-HSA correlacionaram com a carga bacilar e as formas clínicas de Ridley-Jopling. Em contatos domiciliares, os testes ELISA PGL-1 nativo, ND-O-HSA e Ml-FLow detectaram soropositividade de 25%, 17%, e 10%, respectivamente. Conclusões: O uso do teste ELISA e ML-Flow são recomendados no diagnóstico e classificação das formas clínicas, auxiliando na prescrição do tratamento correto e na prevenção do dano neural e da incapacidade. CAPÍTULO IV: Interação patógeno-hospedeiro é mediada principalmente por moléculas especializadas do envelope celular. Um dos componentes essenciais da parede celular das micobactérias é a lipoarabinomanana (LAM). A LAM tem um papel imunomodulador, mas a sua heterogeneidade pode ser responsável pela resposta imune diferenciada em pacientes com hanseníase e contatos. Espera-se que a pesquisa por motivos estruturais de M. leprae possam contribuir, como fatores de virulência ou epítopos de proteção, e assim, derivar biomarcadores eficazes para o diagnóstico, drogas e vacinas contra a hanseníase. Portanto, nosso objetivo foi desenvolver peptídeos miméticos específicos à LAM utilizando Phage Display de uma biblioteca aleatória de peptídeos heptameros que possam reconhecer uma resposta diferencial em pacientes com hanseníase e contatos. Nós utilizamos o anticorpo monoclonal anti-LAM, CS-35, como um alvo de três rodadas de seleção. Após seqüenciamento e tradução, os peptídeos foram pré-validados por ELISA e comparados com o antígeno sintético LAM-BSA. O motivo peptídeo mais reativo e repetitivo (A9) foi posteriormente testado contra o soro de 54 pacientes com hanseníase e 27 controles endêmicos por ELISA. O clone mimético A9 apresentou altos níveis de anticorpos IgG em pacientes paucibacibacilares (PB), sendo que 50% destes soros foram altamente reativos. Esta reação também ocorreu em pacientes tuberculóides, dimorfo-dimorfo e virchowiano. Controles endêmicos e pacientes reacionais apresentaram altos níveis de IgG1 no soro. Por outro lado, o perfil de IgG e suas subclasses em pacientes apresentou altos níveis de IgG e IgG2 e baixos níveis de IgG1. O clone A9 apresentou uma correlação positiva significativa com o antígeno LAM-BSA sintético para a resposta IgG1. A resposta de IgG altamente reativa contra o clone A9 foi associada com o diagnóstico da forma clínica tuberculóide, e a detecção de ambos, IgG1 e IgG3, contra esse clone foi associado à proteção nos controles endêmicos. CAPÍTULO V: Interação patógeno-hospedeiro é mediada principalmente por moléculas especializadas do envelope celular. Um dos componentes essenciais da parede celular das micobactérias é a lipoarabinomanana (LAM). A LAM tem um papel imunomodulador, mas a sua heterogeneidade pode ser responsável pela resposta imune diferenciada em pacientes com hanseníase e contatos. Espera-se que a pesquisa por motivos estruturais de M. leprae possam contribuir, como fatores de virulência ou epítopos de proteção, e assim, derivar biomarcadores eficazes para o diagnóstico, drogas e vacinas contra a hanseníase. Portanto, nosso objetivo foi desenvolver peptídeos miméticos específicos à LAM utilizando Phage Display de uma biblioteca aleatória de peptídeos heptameros que possam reconhecer uma resposta diferencial em pacientes com hanseníase e contatos. Nós utilizamos o anticorpo monoclonal anti-LAM, CS-35, como um alvo de três rodadas de seleção. Após seqüenciamento e tradução, os peptídeos foram pré-validados por ELISA e comparados com o antígeno sintético LAM-BSA. O motivo peptídeo mais reativo e repetitivo (A9) foi posteriormente testado contra o soro de 54 pacientes com hanseníase e 27 controles endêmicos por ELISA. O clone mimético A9 apresentou altos níveis de anticorpos IgG em pacientes paucibacibacilares (PB), sendo que 50% destes soros foram altamente reativos. Esta reação também ocorreu em pacientes tuberculóides, dimorfo-dimorfo e virchowiano. Controles endêmicos e pacientes reacionais apresentaram altos níveis de IgG1 no soro. Por outro lado, o perfil de IgG e suas subclasses em pacientes apresentou altos níveis de IgG e IgG2 e baixos níveis de IgG1. O clone A9 apresentou uma correlação positiva significativa com o antígeno LAM-BSA sintético para a resposta IgG1. A resposta de IgG altamente reativa contra o clone A9 foi associada com o diagnóstico da forma clínica tuberculóide, e a detecção de ambos, IgG1 e IgG3, contra esse clone foi associado à proteção nos controles endêmicos. / Doutor em Genética e Bioquímica

Made available in DSpace on 2014-12-17T13:53:20Z (GMT). No. of bitstreams: 1 JulianaRB.pdf: 735215 bytes, checksum: 0d417f3bbf4eadd09824101ceeb83227 (MD5) Previous issue date: 2008-08-29 / With the need of the companies in becoming more competitive within the market, it arises an incessant search for selective human potential, with a high level of capacity and low rotativity, which motivation results in production raise, quality optimization and waste reduction. This scenario requires a strategy development which advantages the Human Resources Quality Management. This way, the model of the Human System Audit (HSA), developed by the Spanish researchers Ouijano and Navarro, presents itself as an important tool to diagnosis and evaluation, contemplating the environment where the organization is inserted, its strategies, its organizational design, its processes and its organizational effectiveness. In this sense, the present study has identified the existent relation between the professional satisfaction and the Organizational Culture, based in the model HSA. The research has been a quantitative-descriptive one and has had as population the technical-administrative workers from the Federal Center of Technical Education of Rio Grande do Norte (CEFET RN). The data collection has occurred during May, 2008, by means of the application of a questionnaire in the HSA model. The sample was composed by 167 subjects, distributed among the Five units of the institution. It was used the factorial analysis, with the extraction method of main components and orthogonal rotation varimax, in order to extract the dimensions of the satisfaction and of the organizational culture and the calculation of Cronbach s Alpha coefficient, to evaluate the reliability of these dimensions. The factorial analysis of the satisfaction indicators has identified four factors,, all of them showing significance: gratefulness and relationship , self-realization , stability and security and physical conditions and social benefits . The result of the factorial analysis with the indicators of the organizational culture has extracted four factors and among them, three of them have obtained significance: Personal Satisfaction Style , Competitive-Denial-Power Style and the Conventional-Dependent Style . After identifying the dimensions of the satisfaction and culture found at CEFET-RN, it has been notice the existence or not of relation among them, through the application of Pearson s coefficient. It has been verified that all of the dimensions of the Professional satisfaction are correlated with some dimension of the organizational culture, having in outstand position, with higher intensity, the relation between the culture style of Personal Satisfaction and the satisfaction factor referring to the self-realization / A busca incessante de um potencial humano seleto, de n?vel de capacidade elevado e com baixa rotatividade, cuja motiva??o resulta em aumento na produ??o, otimiza??o da qualidade e redu??o de desperd?cios tem recebido maiores espa?os nas agendas, discuss?es e a??es das organiza??es neste mil?nio. Este cen?rio requer o desenvolvimento de estrat?gias que favore?am a Gest?o da Qualidade dos Recursos Humanos. Desta maneira, o modelo da Auditoria do Sistema Humano (ASH), desenvolvido pelos pesquisadores espanh?is Quijano e Navarro, apresenta-se como importante ferramenta, contemplando o ambiente onde a organiza??o est? inserida, suas estrat?gias, seu desenho organizacional, seus processos e sua efetividade organizacional. Neste sentido, o presente estudo identificou a rela??o existente entre a satisfa??o profissional e a Cultura Organizacional, com base no modelo ASH. A pesquisa foi de cunho quantitativo-descritivo e teve como popula??o os servidores t?cnico-administrativos do Centro Federal de Educa??o Tecnol?gica do Rio Grande do Norte. A coleta de dados ocorreu no m?s de maio de 2008, mediante a aplica??o de question?rio do modelo ASH. A amostra ficou composta por 167 sujeitos, distribu?dos entre as cinco unidades da Institui??o. Foi utilizada a an?lise fatorial, com m?todo de extra??o de componentes principais e rota??o ortogonal varimax, para se extrair as dimens?es da satisfa??o e da cultura organizacional e o c?lculo do coeficiente Alpha de Cronbach, para avaliar a confiabilidade destas dimens?es. A an?lise fatorial dos indicadores de satisfa??o identificou quatro fatores, todos eles demonstrando signific?ncia: reconhecimento e relacionamento , auto-realiza??o , estabilidade e seguran?a e condi??es f?sicas e benef?cios sociais . O resultado da an?lise fatorial com os indicadores da cultura organizacional extraiu quatro fatores e destes, tr?s obtiveram signific?ncia: Estilo de Satisfa??o Pessoal , Estilo Poder-Evita??o-Competitivo e o Estilo Convencional-Dependente . Ap?s identificar as dimens?es de satisfa??o e cultura encontradas no CEFET-RN, observou-se a exist?ncia ou n?o de rela??o entre elas, atrav?s da aplica??o do coeficiente de Pearson. Verificou-se que todas as dimens?es da satisfa??o profissional est?o correlacionadas com alguma dimens?o da cultura organizacional, destacando-se com maior intensidade a rela??o entre o estilo de cultura Satisfa??o Pessoal e o fator de satisfa??o referente ? auto-realiza??o

Resumo O presente trabalho teve como objetivo sintetizar e caracterizar um novo complexo trinuclear de rutênio, [Ru3O(CH3OO)6(3-pic)2(NO)]+, através de rotas sintéticas previamente descritas na literatura para complexos análogos. Para a obtenção deste complexo, foram necessárias cinco etapas de síntese, cada qual gerando o precursor da etapa seguinte. Os complexos precursores [Ru3O(CH3OO)6(3-pic)3]+, [Ru3O(CH3OO)6(3-pic)2(CO)] e [Ru3O(CH3OO)6(3-pic)2(H2O)]+, foram caracterizados por técnicas espectroscópicas e voltamétricas. Para o complexo de interesse, [Ru3O(CH3OO)6(3-pic)2(NO)]+, além da caracterização por técnicas espectroscópicas e voltamétricas, foram realizados estudos de fotólise e de interação com albumina de soro humano (HSA). Pelas técnicas espectroscópicas, pudemos ratificar as estruturas propostas tanto para o nitrosilo quanto para os precursores estudados. Para a caracterização do complexo [Ru3O(CH3OO)6(3-pic)2(NO)]+, utilizando a técnica de espectroscopia de infravermelho verificamos a coordenação do ligante NO ao centro metálico [Ru3O], devido ao estiramento característico deste ligante. Os espectros de absorção UV-vis e RMN mostraram que existe uma forte interação entre o elétron desemparelhado do centro metálico e o elétron do ligante NO. Pelos estudos de voltametria cíclica, observamos que os processos redox envolvendo o ligante NO, são compartilhados com o centro metálico. A fotólise do complexo [Ru3O(CH3OO)6(3-pic)2(NO)]+, mostrou-se eficiente, uma vez que a liberação fotoinduzida do NO ocorreu na região do visível e em pH fisiológico. Pelos estudos de supressão de fluorescência, observamos que o complexo realmente interage com a HSA na proporção de 1:1, na região em que se encontra o resíduo de triptofano. / The aim of the present study was synthesize and characterize a new trinuclear ruthenium complex, [Ru3O(CH3OO)6(3-pic)2(NO)]+, via synthetic routes previously described in the literature for analogous complexes. To obtain this complex, it took five synthetic steps, each one yielding a precursor for the next step. The precursor complexes [Ru3O(CH3OO)6(3-pic)3]+, [Ru3O(CH3OO)6(3-pic)2(CO)] and [Ru3O(CH3OO)6(3-pic)2(H2O)]+, were characterized by spectroscopic and voltammetric techniques. For the complex of interest, [Ru3O(CH3OO)6(3-pic)2(NO)]+, in addition to characterization by spectroscopic and voltammetric studies, it was carried out photolysis and interaction with human serum albumin. By spectroscopic techniques, we could confirm the proposed structures for both the nitrosyl and precursors. For characterization of the complex [Ru3O(CH3OO)6(3-pic)2(NO)]+, infrared spectroscopy allowed us to verify that the ligand coordination to the metal center [Ru3O], due to the characteristic stretching band of that ligand. The absorption spectra of UV-vis and NMR showed that there is a strong interaction between the unpaired electron of the metal center and the NO ligand. By cyclic voltammetry studies, we observed that the redox processes involving the NO ligand are shared with the metal center processes. The photolysis of the complex [Ru3O (CH3OO) 6 (3-pic) 2 (NO)] +, was efficient, since the photoinduced release of NO occurred in the visible region and at physiological pH. By fluorescence quenching studies, we observed that the complex actually interacts with the HSA in a 1:1 ratio, in the region which is the residue of tryptophan.

No description available.

Established biopharmaceuticals such as antibodies and derivatives thereof are relatively large. In cancer therapy, this creates a steep drug concentration gradient within tumors, leaving cells far from blood vessels effectively untreated. Continuous pseudo treatments should foster the development of drug resistance and might lead to eventual disease relapse. Drug concentration gradients can be operationalized as tissue penetration efficiencies, which are functions of molecular size. However, small particles are also subject to potent renal clearance, collapsing the therapeutic window beyond clinical applications. In this master’s thesis, spatial bispecificity was engineered into a single albumin binding domain (ABD). Resulting ABD derived affinity proteins (ADAPTs) are saved from urinary excretion by the grace of HSA, but in the more static microenvironment of tumors, following HSA dissociation, they are capable of tissue penetration efficiencies bestowed only upon smaller particles. To this end, phage display was used to raise ADAPTs against the cancer associated proteins human epidermal growth factor receptor 2 (HER2) and carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), but also the inflammation marker C-reactive protein (CRP). Via Sanger sequencing, 9 variants were picked for protein production and characterization, among which two spatially bispecific binders were found. ADAPTs were also evaluated for aggregation tendencies, structural conformity to library design, and thermal stability. One ADAPT, binding HER2, passed all tests of initial characterizations. Deep sequencing was used to analyze selection output, from which many more binders should be screened in future experiments. / Etablerade bioläkemedel liksom antikroppar och deras derivat är relativt stora protein. Som cancerterapeutiska skapar de således branta koncentrationsgradienter utgående från tumörpenetrerande blodkärl. Detta riskerar att lämna vissa cancerceller utanför det terapeutiska fönstret. Det svaga selektionstryck som således verkar i tumörperiferin fostrar cancerceller till att utveckla resistens mot detsamma. Koncentrationsgradienten beror på proteinets vävnadspenetrarande förmåga, vilken är en funktion av proteinets storlek. Mindre proteiner borde därmed lättare ackumuleras i hela tumören och förebygga resistensutveckling. Problemet med små proteiner är deras mycket korta halveringstid i serum, en följd av relativt obehindrad filtrering ut i urinen via njurarna. I det här examensarbetet utvecklades rumsbispecifika bindare av cancerassocierade protein med hjälp av fagdisplayselektioner från ett proteinbibliotek baserat på en enda albuminbindande domän (ABD). Resulterande ABD deriverade affinitetsprotein (ADAPT) undkommer ovan nämnda filtrering tack vare sin naturligt starka interaktion med humant serumalbumin (HSA). I den mer långsamt flödande tumörmikromiljön tillåts ADAPTerna efter albumindissociation sedan utöva en bland bioläkemedel överlägsen vävnadspenetration. Tre parallella selektionsspår utfördes mot de cancerassocierade målproteinerna human epidermal growth factor receptor 2 (HER2) och carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) samt den utsöndrade inflammationsmarkören C-reaktivt protein (CRP). Via Sangersekvensering kunde flera kandidater identifieras. Bland 6 karakteriserade ADAPTer uppvisade samtliga hög HSA-affinitet, tre konstaterades interagera specifikt med sitt målprotein, och två verkade binda även rumsbispecifikt. ADAPTer utvärderades även för sin benägenhet att bilda aggregat, strukturell överensstämmelse med experimentell design, och värmestabilitet. Endast en bindare, mot HER2, klarade sig genom alla prövningar som proteinkarakteriseringen innebar utan underkänt. Även en högparallel sekvensering utav selektionsresultat utfördes, men utanför de tidsramar som tillät ytterligare karakterisering.

In order to produce predictable and robust systems forprotein purification and detection, well characterized, small,folded domains descending from bacterial receptors have beenused. These bacterial receptors, staphylococcal protein A (SPA)and streptococcal protein G (SPG), possess high affinity to IgGand / or HSA. They are composed of repetitive units in whicheach one binds the ligand independently. The domains foldindependently and are very stable. Since the domains also havewellknown three-dimensional structures and do not containcysteine residues, they are very suitable as frameworks forfurther protein engineering. Streptococcal protein G (SPG) is a multidomain proteinpresent on the cell surface ofStreptococcus. X-ray crystallography has been used todetermine the binding site of the Ig-binding domain. In thisthesis the region responsible for the HSA affinity of ABD3 hasbeen determined by directed mutagenesis followed by functionaland structural analysis. The analysis shows that the HSAbindinginvolves residues mainly in the second α-helix. Most protein-based affinity chromatography media are verysensitive towards alkaline treatment, which is the preferredmethod for regeneration and removal of contaminants from thepurification devices in industrial applications. Here, aprotein engineering strategy has been used to improve thetolerance to alkaline conditions of different domains fromprotein G, ABD3 and C2. Amino acids known to be susceptibletowards high pH were substituted for less alkali susceptibleresidues. The new, engineered variants of C2 and ABD shownhigher stability towards alkaline pH. Also, very important forthe potential use as affinity ligands, these mutated variantsretained the secondary structure and the affinity to HSA andIgG, respectively. Moreover, dimerization was performed toinvestigate whether a higher binding capacity could be obtainedby multivalency. For ABD, binding studies showed that divalentligands coupled using non-directed chemistry demonstrated anincreased molar binding capacity compared to monovalentligands. In contrast, equal molar binding capacities wereobserved for both types of ligands when using a directed ligandcoupling chemistry involving the introduction and recruitmentof a unique C-terminal cysteine residue. The staphylococcal protein A-derived domain Z is also a wellknown and thoroughly characterized fusion partner widely usedin affinity chromatography systems. This domain is consideredto be relatively tolerant towards alkaline conditions.Nevertheless, it is desirable to further improve the stabilityin order to enable an SPA-based affinity medium to withstandeven longer exposure to the harsh conditions associated withcleaning in place (CIP) procedures. For this purpose adifferent protein engineering strategy was employed. Smallchanges in stability due to the mutations would be difficult toassess. Hence, in order to enable detection of improvementsregarding the alkaline resistance of the Z domain, a by-passmutagenesis strategy was utilized, where a mutated structurallydestabilized variant, Z(F30A) was used as a surrogateframework. All eight asparagines in the domain were exchangedone-by-one. The residues were all shown to have differentimpact on the alkaline tolerance of the domain. By exchangingasparagine 23 for a threonine we were able to remarkablyincrease the stability of the Z(F30A)-domain towards alkalineconditions. Also, when grafting the N23T mutation to the Zscaffold we were able to detect an increased tolerance towardsalkaline treatment compared to the native Z molecule. In allcases, the most sensitive asparagines were found to be locatedin the loops region. In summary, the work presented in this thesis shows theusefulness of protein engineering strategies, both to explorethe importance of different amino acids regarding stability andfunctionality and to improve the characteristics of aprotein. <b>Keywords:</b>binding, affinity, human serum albumin (HSA),albumin-binding domain (ABD), affinity chromatography,deamidation, protein A, stabilization, Z-domain, capacity,protein G, cleaning-in-place (CIP), protein engineering, C2receptor.

Noncovalent interactions are involved in many biological processes in which biomolecules bind specifically and reversibly to a partner. Often, proteins do not have a biological activity without the presence of a partner, a ligand. Biological signals are produced when proteins interact with other proteins, peptides, oligonucleotides, nucleic acids, lipids, metal ions, polysaccharides or small organic molecules. Some key steps in the drug discovery process are based on noncovalent interactions. We have focused our research on the steps involving ligand screening, competitive binding and ‘off-target’ binding. The first paper in this thesis investigated the complicated electrospray ionization process with regards to noncovalent complexes. We have proposed a model that may explain how the equilibrium between a protein and ligand changes during the droplet evaporation/ionization process. The second paper describes an evaluation of an automated chip-based nano-ESI platform for ligand screening. The technique was compared with a previously reported method based on nuclear magnetic resonance (NMR), and excellent correlation was obtained between the results obtained with the two methods. As a general conclusion we believe that the automated nano-ESI/MS should have a great potential to serve as a complementary screening method to conventional HTS. Alternatively, it could be used as a first screening method in an early phase of drug development programs when only small amounts of purified targets are available. In the third article, the advantage of using on-line microdialysis as a tool for enhanced resolution and sensitivity during detection of noncovalent interactions and competitive binding studies by ESI-MS was demonstrated. The microdialysis device was improved and a new approach for competitive binding studies was developed. The last article in the thesis reports studies of noncovalent interactions by means of nanoelectrospray ionization mass spectrometry (nanoESI-MS) for determination of the specific binding of selected drug candidates to HSA. Two drug candidates and two known binders to HSA were analyzed using a competitive approach. The drugs were incubated with the target protein followed by addition of site-specific probes, one at a time. The drug candidates showed predominant affinity to site I (warfarin site). Naproxen and glyburide showed affinity to both sites I and II. / QC 20100705

<p>In order to produce predictable and robust systems forprotein purification and detection, well characterized, small,folded domains descending from bacterial receptors have beenused. These bacterial receptors, staphylococcal protein A (SPA)and streptococcal protein G (SPG), possess high affinity to IgGand / or HSA. They are composed of repetitive units in whicheach one binds the ligand independently. The domains foldindependently and are very stable. Since the domains also havewellknown three-dimensional structures and do not containcysteine residues, they are very suitable as frameworks forfurther protein engineering.</p><p>Streptococcal protein G (SPG) is a multidomain proteinpresent on the cell surface of<i>Streptococcus</i>. X-ray crystallography has been used todetermine the binding site of the Ig-binding domain. In thisthesis the region responsible for the HSA affinity of ABD3 hasbeen determined by directed mutagenesis followed by functionaland structural analysis. The analysis shows that the HSAbindinginvolves residues mainly in the second α-helix.</p><p>Most protein-based affinity chromatography media are verysensitive towards alkaline treatment, which is the preferredmethod for regeneration and removal of contaminants from thepurification devices in industrial applications. Here, aprotein engineering strategy has been used to improve thetolerance to alkaline conditions of different domains fromprotein G, ABD3 and C2. Amino acids known to be susceptibletowards high pH were substituted for less alkali susceptibleresidues. The new, engineered variants of C2 and ABD shownhigher stability towards alkaline pH. Also, very important forthe potential use as affinity ligands, these mutated variantsretained the secondary structure and the affinity to HSA andIgG, respectively. Moreover, dimerization was performed toinvestigate whether a higher binding capacity could be obtainedby multivalency. For ABD, binding studies showed that divalentligands coupled using non-directed chemistry demonstrated anincreased molar binding capacity compared to monovalentligands. In contrast, equal molar binding capacities wereobserved for both types of ligands when using a directed ligandcoupling chemistry involving the introduction and recruitmentof a unique C-terminal cysteine residue.</p><p>The staphylococcal protein A-derived domain Z is also a wellknown and thoroughly characterized fusion partner widely usedin affinity chromatography systems. This domain is consideredto be relatively tolerant towards alkaline conditions.Nevertheless, it is desirable to further improve the stabilityin order to enable an SPA-based affinity medium to withstandeven longer exposure to the harsh conditions associated withcleaning in place (CIP) procedures. For this purpose adifferent protein engineering strategy was employed. Smallchanges in stability due to the mutations would be difficult toassess. Hence, in order to enable detection of improvementsregarding the alkaline resistance of the Z domain, a by-passmutagenesis strategy was utilized, where a mutated structurallydestabilized variant, Z(F30A) was used as a surrogateframework. All eight asparagines in the domain were exchangedone-by-one. The residues were all shown to have differentimpact on the alkaline tolerance of the domain. By exchangingasparagine 23 for a threonine we were able to remarkablyincrease the stability of the Z(F30A)-domain towards alkalineconditions. Also, when grafting the N23T mutation to the Zscaffold we were able to detect an increased tolerance towardsalkaline treatment compared to the native Z molecule. In allcases, the most sensitive asparagines were found to be locatedin the loops region.</p><p>In summary, the work presented in this thesis shows theusefulness of protein engineering strategies, both to explorethe importance of different amino acids regarding stability andfunctionality and to improve the characteristics of aprotein.</p><p><b>Keywords:</b>binding, affinity, human serum albumin (HSA),albumin-binding domain (ABD), affinity chromatography,deamidation, protein A, stabilization, Z-domain, capacity,protein G, cleaning-in-place (CIP), protein engineering, C2receptor.</p>

A new Laser Induced Fluorescence Capillary Zone Electrophoresis (LIF-CZE) bioassay to detect and study the catalytic activity of the sulfur assimilating enzyme commonly found in E. coli species; alkanesulfonate monooxygenase (EC 1.14.14.5) is described for the first time. This technique enables the possibility for direct injection onto a capillary for detection without the need for pre-concentration of sample and with minimal sample preparative steps prior to analysis. In this bioassay, a group of Fischer based cyanine dyes and two Oxazine (Nile red) derivatives were designed for further optimization as key Vis-NIR fluorescent substrate. In developing this technique, the test dyes were first assessed for their photophysical properties, based on four criteria; (1) photostable (2) solvatochromism (3) binding affinity towards both the monooxygenase active site and serum albumin and (4) chemical stability in strong electric field strength. Applying key dye characterization procedures including; molar absorptivity determination, quantum yield determination, photostability, solvatochromism and protein interaction studies it was determined that the Fischer indolium cyanine dyes were most suitable for the method development. The data revealed that under the test conditions, reduced flavin, the oxidative monooxygenase catalytically specifically converts the alkylsulfonate substituted cyanine dyes to the corresponding aldehyde. This new bioassay has proven to be quick, portable, sensitive, reliable and the exhibit the possibility of ‘on-the-spot’ detection; advantages not readily realized with other commonly applied techniques such as PCR, SPR, ELISA and GC used to study bacterial sulfur assimilation processes. In addition, recent literature results proposed by other research groups developing similar techniques showed strong reliance on GC analyses. Those assays involve the use of low molecular weight straight chain non-emissive alkanesulfonate substrates. Once enzyme catalysis occurs the aldehyde is formed becomes rather volatile and requires complex and tedious headspace sampling for GC analyses. This feature limits the in vitro applicability and eliminated the possibility in vivo development. Our goal is to further develop, optimize and present this CZE based bioassay as a suitable alternative to the current trends in the field while creating a more robust and sensitive in vitro monooxygenase detection method with the possibilities of in vivo application.

Marknaden för proteinbaserade läkemedel, de så kallade biologiska läkemedlen, är idag en industri som omsätter miljarder. Ett vanligt sätt att utveckla dessa läkemedel på är med hjälp av monoklonala antikroppar då de kan binda till sitt mål med hög specificitet. Däremot begränsas denna teknik av en lång och dyr produktion som dessutom kräver däggdjursbaserade uttrycksystem. En alternativ teknik till de monoklonala antikropparna är att använda små proteiner som enkelt kan produceras i bakterier till en låg kostnad. Dock begränsas denna metod av de små proteinernas korta cirkuleringstid i blodet. I ett tidigare projekt, har ett litet protein vid namnet ABDderived affinity ProTein (ADAPT) på cirka 7 kDa, utvecklats för att kunna binda till både humant serumalbumin (HSA) för att förlänga cirkulationstiden i blodet och Interleukin 17c (IL17c) som är ett pro-inflammatorisk cytokin. Studien visade dock att ADAPT proteinet inte samtidigt kunde binda till de båda molekylerna tillräckligt effektivt. Syftet med denna uppsats är därför att undersöka om det nämnda proteinet kan optimeras genom så kallad multimering och/eller manipulering av bindningssätet för HSA i syfte att åstadkomma en effektiv och mer långvarig cirkulationstid i blodet samtidigt som det binder sig till sitt mål, IL17c. Tio nya versioner av ADAPT proteinet har utvecklats genom att klona och transformera proteiner till en högt producerande Escherichia coli (E. coli) stam. Proteinerna har sedan producerats och renats fram. Det kunde observeras att proteinerna hade den önskade renheten för att kunna karaktäriseras. Vidare var det möjligt att se att proteinerna hade sin önskade molekylvikt och erhöll sin förväntade struktur som en alfahelix. Proteinernas smältpunkter hade förbättrats eller var liknande jämfört med det ursprungliga proteinet. Dessutom kunde alla proteiner återgå till sin ursprungliga struktur efter upphettning. Utvärderingen av proteinernas bindningskapacitet, med original proteinet som referens, visade på en ökad affinitet till sitt mål, IL17c, för två dimerer och trimeren samt en jämförbar affinitet för två av monomererna med ett manipulerat bindingssäte till HSA. Interaktion till HSA var jämförbar med den ursprungliga ADAPT molekylen för alla nya varianter förutom monomererna med ett manipulerat bindingssäte och dimeren med två manipulerat bindingssäten till HSA. Evaluering av de nya proteinernas kapacitet att binda samtidigt till HSA och IL17c visade att det var gynnsamt med en dimereiserad molekyl då det skapade en distans mellan molekylerna och dess bindningssäten. Vidare kunde det också visas att ordningen som molekylerna interagerade med varandra påverkade proteinernas simultana bindning. / The market for protein-based drugs, or the so-called biopharmaceuticals, is a multibillion-dollar industry today. In the development of protein-based drugs it is common to use monoclonal antibodies (mAbs) due to their ability to bind to its target with high specificity. However, therapeutical development of mAbs is limited by its long and expensive production in mammalian expression system. An alternative to mAbs are the so-called alternative scaffolds which are small proteins that can be produced in bacteria at lower costs. Although a drawback with the latter proteins is their short serum half-life. A small scaffold protein, ABD-Derived Affinity ProTein (ADAPT) of approximate 7 kDa was earlier engineered to obtain bispecific affinity, to Human Serum Albumin (HSA), to extend its half-life, as well as to the pro-inflammatory cytokine, Interleukin 17c (IL17c). Unfortunately, it was shown that the simultaneous binding was not efficient enough for its desired purpose. The aim with this project was therefore to investigate if the previous mentioned binder could be optimized by multimerization and/or manipulation of the HSA binding site for an efficient half-life extension. By generating ten new designs of the ADAPT variants, it was observed that the new variants had stable alpha helical structures and an improved or similar melting temperature as the original variant. The evaluation of the target binding displayed an improved affinity to the target, IL17c, for two of the dimeric versions as well as for the trimer and a comparable affinity for two of the monomers with a manipulated HAS binding site. The interaction to HSA was comparable to the original ADAPT for all binders except from the monomers with impaired HSA binding and the dimer with two impaired HSA binding sites. The evaluation of the simultaneous binding showed that it was favored by dimerization when a distance between the two molecule and their binding surfaces was added. Moreover, it could also be seen that the order of binding events had an impact on the simultaneous binding.