Recent occupational health studies have focused on dermal exposure at the hands, but have been unable to accurately express dose without knowing the HSA. There is no standard method to calculate HSA, though some researchers have derived HSA formulas based on dimensions from a Taiwanese population. This research paper describes a shortcut method to estimate the hand surface area (HSA) of a human hand from a two-dimensional hand tracing, and repeated a Taiwanese HSA study in order to explore the viability of its HSA formula in an American university population. A sample of nine adult men and nine adult women, each representing one third of the population percentile in hand length and hand breadth, were selected from a population within the University of South Florida in Tampa, FL. Hand length, breadth, a 2D hand tracing and a 3D light hand scan were collected from each participant. A linear regression was used to analyze the data sets and found a correlation (R=0.94) between 2D HSA and 3D HSA and slope of 2.6 (SD=0.2), with a regression equation of Y=2.6(X). A paired t-test was used to compare the Taiwanese HSA formula data against the 3D HSA. Results found that the Taiwanese data sets were significantly different from the 3D HSA (p<0.001), averaging 57 cm2 less than the 3D HSA. A jackknife analysis was implemented on the 2D HSA hand tracing data, and a paired t-test was performed between the jackknife estimate predictions and 3D HSA. Mean differences were not significantly different (p=0.97), with 0.87 cm2 difference between means. Results indicate that the USF Hand Tracing Method will provide a better estimate of HSA than the Taiwanese method, and can be used as a tool in HSA estimation.

No description available.

Biological development and progression are managed by a diverse macromolecular group called proteins. Protein structure results from a complex folding process that leads to a final active form. This protein state is susceptible to changes in the surrounding environment and an incorrect structure can be produced. Changes in the protein conformation can lead to the formation of protein aggregates. Adsorption of proteins onto surfaces is utilized in many research analyses, but is capable of irreversibly changing the protein structure and causing aggregation. Albumin is a plasma protein that adsorbs on many different surfaces because the structure easily rearranges. The structure of albumin once adsorbed has been shown to deteriorate; however, outcomes of both stabilization and aggregation have been found. A dynamic laser light scattering instrument will be utilized to measure the differences in size and determine the amount of aggregation. Our lab has developed a z-axis translating laser light scattering device (ZATLLS) that has been used to measure the sedimentation velocity of several different materials in solution. In this case, bovine serum albumin (BSA) will be adsorbed onto polystyrene particles and the particle settling velocity determined. The settling solution viscosity and density will also be ascertained, so Stoke's law can infer the average aggregate size of each experiment. BSA-coated polystyrene particles displayed a more controlled settling behavior compared to non-coated polystyrene particles. Although the BSA-coated particles had a smaller sedimentation velocity, a larger aggregate size was found due to the greater solution viscosity. Therefore, the ZATLLS instrument can be employed to measure sedimentation velocities of multiple interactions and the aggregation level inferred. Although most albumin molecules are remarkably similar, there are subtle differences in amino acid residues, length, and charge. Sedimentation velocities for human serum albumin (HSA) coated polystyrene particles and BSA-coated polystyrene particles only had a small difference. However an almost 50% higher solution viscosity was measured in BSA experiment solutions, and resulted in the slower settling of the larger aggregates compared to HSA-coated particles. Viscosity calibration curves for each albumin species were used to determine the amount of protein desorbed from the particles during the settling process. The larger solution viscosity for BSA-coated particle experiments led to a much larger degree of desorption. HSA was shown to be the more stable albumin species when adsorbed onto polystyrene particles. Temperature denaturing was performed to aid in the determination of the stability of BSA. Reversible and irreversible conformational changes in BSA were produced at 46ºC and 76ºC respectively. The solutions were cooled to room temperature before adsorption ontopolystyrene particles and the sedimentation velocities measured. A 50% difference in average viscosity between the reversibly and irreversibly changed BSA was found. This caused the larger aggregates formed in the 76ºC BSA experiments to have an almost equivalent sedimentation velocity to those in the reversibly denatured BSA experiments. Average aggregate size for reversibly denatured BSA was well within the ranges found for non-denatured BSA. In conclusion, irreversibly denatured BSA formed larger aggregates and was more likely to desorb from the polystyrene particles than reversibly changed BSA. / Master of Science

Neste trabalho, alguns novos complexos imínicos de cobre(II) com ligantes inspirados em biomoléculas como oxindóis, contendo grupos indólicos, imidazólicos ou pirrólicos com diferentes características estruturais, foram sintetizados e caracterizados por análise elementar, espectrometria ESI-MS e espectroscopias IV, UVNis e EPR. As possíveis interações desses complexos de cobre com a albumina humana (HSA) e com o plasma sanguíneo foram estudadas através das técnicas EPR, CD e SDS-PAGE, indicando que estas ocorrem principalmente no sítio N-terminal da proteína. Suas reatividades frente a compostos biológicos relevantes, tais como glutationa, ascorbato e peróxido de hidrogênio, também foram verificadas. Alguns dos complexos estudados podem ser ativados por glutationa, ascorbato ou peróxido de hidrogênio, sendo capazes de gerar espécies reativas de oxigênio em concentrações significativas, na presença desses redutores ou oxidantes biológicos. Adicionalmente, as propriedades pró-oxidantes de tais complexos foram investigadas, visando elucidar estudos prévios de suas atividades pró-apoptótica e antitumoral. Alguns destes complexos foram mais eficientes em causar danos oxidativos à 2-deoxi-D-ribose, enquanto outros foram mais eficientes em causar oxidantes na proteína HSA, com formação de grupos carbonílicos, principalmente em presença de H202. Experimentos de CD complementaram estes resultados, indicando que somente alguns complexos causaram modificações na &#945;-hélice da proteína. Experimentos de EPR com captador de spin, na presença de HSA e H202, mostraram a formação de quantidades apreciáveis de radicais hidroxil e radicais de carbono, em presença de peróxido de hidrogênio. Além disso, os complexos apresentaram notável habilidade de ligação ao DNA e conseqüente atividade nuclease, promovendo clivagens nas duas fitas. Experimentos de fluorescência, EPR, gel de eletroforese marcado com &#945;-32P-UTP e CD foram ainda realizados, visando elucidar o mecanismo de ação destes complexos no meio biológico. Estes experimentos indicaram que eles podem se associar ao DNA, através de suas bases ou pela interação com a deoxi-ribose, já que promoveram danos oxidativos nestes substratos. Entretanto, não catalisam a hidrólise dos grupos fosfato, atuando, portanto, predominantemente por um mecanismo oxidativo. Através de CD, poucas perturbações na elipsicidade do DNA foram observadas, o que indica que estes complexos provavelmente estão localizadas nas cavidades ou alças do ácido nucléico. / Some novel imine-copper(II) complexes with ligands inspired in biomolecules such as oxindoles, containing indole, pirrole or imidazole moieties with different structural features were synthesized, and characterized by elemental analysis, IV, UV/Vis and EPR spectroscopies, and ESI-MS spectrommetry. Interactions of these complexes with human serum albumin (HSA) and human plasma were verified by EPR, CD and SDS-PAGE techniques, showing that they occur mainly at the N-terminal site of the protein. Their reactivity towards biological relevant compounds, such as glutathione, ascorbate and hydrogen peroxide were also verified; some of them are capable of generating ROS in significant concentrations, in the presence of these reducing or oxidant agents. Additionally, the activity of such copper(II) complexes in promoting oxidative damage to different substrates was investigated, in order to elucidate previous studies on their pro-apoptotic and antitumoral activity. Some of these complexes were much more efficient to cause oxidative damage to 2-deoxy-D-ribose, especially in the presence of hydrogen peroxide. On the contrary, others were more active in causing damage to HSA protein, with the formation of carbonyl groups. Experiments by CD corroborated these results, since only some of the complexes caused modifications in the protein -helix. EPR spin trapping experiments, in the presence of HSA and H2O2, showed significant generation of hydroxyl as well as carbon centered radicals. Moreover, all the complexes showed remarkable ability to bind to DNA, promoting double-strand cleavage, upon H2O2 activation. In order to investigate their mechanism of action, fluorescence, EPR, gel-electrophoresis with labeled &#945;-32P-UTP and CD experiments were carried out. The results indicated that these complexes can bind to DNA through its bases or can interact with the deoxi-ribose rings, promoting oxidative damage to those substrates. On the contrary, they do not catalyze the hydrolysis of phosphate groups. By CD spectroscopy, little perturbations on the helicity conformation of the DNA were observed, indicating that these complexes are probably located in the grooves.

Neste trabalho, alguns novos complexos imínicos de cobre(II) com ligantes inspirados em biomoléculas como oxindóis, contendo grupos indólicos, imidazólicos ou pirrólicos com diferentes características estruturais, foram sintetizados e caracterizados por análise elementar, espectrometria ESI-MS e espectroscopias IV, UVNis e EPR. As possíveis interações desses complexos de cobre com a albumina humana (HSA) e com o plasma sanguíneo foram estudadas através das técnicas EPR, CD e SDS-PAGE, indicando que estas ocorrem principalmente no sítio N-terminal da proteína. Suas reatividades frente a compostos biológicos relevantes, tais como glutationa, ascorbato e peróxido de hidrogênio, também foram verificadas. Alguns dos complexos estudados podem ser ativados por glutationa, ascorbato ou peróxido de hidrogênio, sendo capazes de gerar espécies reativas de oxigênio em concentrações significativas, na presença desses redutores ou oxidantes biológicos. Adicionalmente, as propriedades pró-oxidantes de tais complexos foram investigadas, visando elucidar estudos prévios de suas atividades pró-apoptótica e antitumoral. Alguns destes complexos foram mais eficientes em causar danos oxidativos à 2-deoxi-D-ribose, enquanto outros foram mais eficientes em causar oxidantes na proteína HSA, com formação de grupos carbonílicos, principalmente em presença de H202. Experimentos de CD complementaram estes resultados, indicando que somente alguns complexos causaram modificações na &#945;-hélice da proteína. Experimentos de EPR com captador de spin, na presença de HSA e H202, mostraram a formação de quantidades apreciáveis de radicais hidroxil e radicais de carbono, em presença de peróxido de hidrogênio. Além disso, os complexos apresentaram notável habilidade de ligação ao DNA e conseqüente atividade nuclease, promovendo clivagens nas duas fitas. Experimentos de fluorescência, EPR, gel de eletroforese marcado com &#945;-32P-UTP e CD foram ainda realizados, visando elucidar o mecanismo de ação destes complexos no meio biológico. Estes experimentos indicaram que eles podem se associar ao DNA, através de suas bases ou pela interação com a deoxi-ribose, já que promoveram danos oxidativos nestes substratos. Entretanto, não catalisam a hidrólise dos grupos fosfato, atuando, portanto, predominantemente por um mecanismo oxidativo. Através de CD, poucas perturbações na elipsicidade do DNA foram observadas, o que indica que estes complexos provavelmente estão localizadas nas cavidades ou alças do ácido nucléico. / Some novel imine-copper(II) complexes with ligands inspired in biomolecules such as oxindoles, containing indole, pirrole or imidazole moieties with different structural features were synthesized, and characterized by elemental analysis, IV, UV/Vis and EPR spectroscopies, and ESI-MS spectrommetry. Interactions of these complexes with human serum albumin (HSA) and human plasma were verified by EPR, CD and SDS-PAGE techniques, showing that they occur mainly at the N-terminal site of the protein. Their reactivity towards biological relevant compounds, such as glutathione, ascorbate and hydrogen peroxide were also verified; some of them are capable of generating ROS in significant concentrations, in the presence of these reducing or oxidant agents. Additionally, the activity of such copper(II) complexes in promoting oxidative damage to different substrates was investigated, in order to elucidate previous studies on their pro-apoptotic and antitumoral activity. Some of these complexes were much more efficient to cause oxidative damage to 2-deoxy-D-ribose, especially in the presence of hydrogen peroxide. On the contrary, others were more active in causing damage to HSA protein, with the formation of carbonyl groups. Experiments by CD corroborated these results, since only some of the complexes caused modifications in the protein -helix. EPR spin trapping experiments, in the presence of HSA and H2O2, showed significant generation of hydroxyl as well as carbon centered radicals. Moreover, all the complexes showed remarkable ability to bind to DNA, promoting double-strand cleavage, upon H2O2 activation. In order to investigate their mechanism of action, fluorescence, EPR, gel-electrophoresis with labeled &#945;-32P-UTP and CD experiments were carried out. The results indicated that these complexes can bind to DNA through its bases or can interact with the deoxi-ribose rings, promoting oxidative damage to those substrates. On the contrary, they do not catalyze the hydrolysis of phosphate groups. By CD spectroscopy, little perturbations on the helicity conformation of the DNA were observed, indicating that these complexes are probably located in the grooves.

[ES] El cáncer es una de las principales causas de muerte a nivel mundial. Los tratamientos anticancerígenos generalmente usados tienen diversos efectos secundarios producidos por su baja especificidad. Esta es una de las razones por las que se sigue en continua búsqueda de nuevos tratamientos. Dentro de estas nuevas investigaciones se encuentra el extenso campo de la nanomedicina, es decir, el estudio de nuevos materiales a escala nanométrica. Esta permite reducir dichos efectos secundarios aumentando la selectividad y especificidad de los tratamientos. Dentro de los nanomateriales se encuentran las nanopartículas de upconversion que son capaces de absorber luz en el infrarrojo cercano y emitirla en la región ultravioleta-visible. Por otro lado, desde el principio de la historia de la medicina la luz se ha empleado como forma de tratamiento teniendo un rol muy importante. Un inconveniente para dichos tratamientos suele ser la necesidad de emplear luz de la región ultravioleta-visible, pues las biomoléculas son capaces de absorber y produce daño celular. En este contexto, la presente Tesis Doctoral se centra en el estudio de nuevas formas de tratamiento anticancerígeno combinando nanomedicina y luz. Para ello se han desarrollado nuevos fármacos fototóxicos y nuevos materiales capaces de ser activados mediante luz infrarroja cercana. En primer lugar, se sintetizaron nuevas fluoroquinolonas para explorar sus propiedades fototóxicas para su uso en fotoquimioterapia (Capítulo 3 de la Tesis). Se estudiaron las características fotofísicas y fotoquímicas de los nuevos compuestos, además de su capacidad para producir mayor fototoxicidad en las células en comparación con las fluoroquinolonas como la lomefloxacina mediante la aplicación de luz ultravioleta. En base a los resultados obtenidos se realizó un estudio para determinar las diferencias entre las interacciones de algunas fluoroquinolonas dihalogenadas, incluidas las comentadas anteriormente, y biomoléculas como ADN y proteínas. La reactividad de sus intermedios fotogenerados también se estudió en el Capítulo 4. Tras conocer en profundidad la capacidad fototóxica de los nuevos fármacos, en el Capítulo 5 se llevó a cabo el diseño de un nanosistema compuesto por fluoroquinolonas y nanopartículas de conversión ascendente. Se demostró la alta capacidad fototóxica de este nuevo nanosistema. De esta manera, se generó actividad fototóxica a partir de una fluoroquinolona sin el uso de luz ultravioleta Por otro lado, la formación de profármacos abre la puerta a la administración selectiva de fármacos contra el cáncer. Los profármacos consisten en la unión fotolábil de una molécula capaz de ser activada por la luz y el fármaco de interés. Sin embargo, un conocimiento profundo de las propiedades fotofísicas y fotoquímicas del fotodisparador y de los potenciales redox de ambos miembros de la diada puede ser crucial para obtener la fotoliberación deseada. Así, en el Capítulo 6, se destacó la relevancia de estos datos utilizando un profármaco formado por un derivado de cumarina como molécula fotoactivable y colchicina como fármaco. Finalmente, en el Capítulo 7 se exploró la síntesis de un nuevo nanosistema que contiene un profármaco formado por un derivado de cumarina unido al fármaco contra el cáncer clorambucilo y nanopartículas biocomatibles de conversión ascendente. La adición de albúmina de suero humano como recubrimiento de las nanopartículas cumple la doble función de obtener nanopartículas biocompatibles y ser el lugar de carga del profármaco. / [CA] El càncer és una de les principals causes de mort a nivell mundial. Els tractaments anticancerígens generalment usats tenen diversos efectes secundaris produïts per la seva baixa especificitat. Aquesta és una de les raons per les que se segueix en contínua recerca de nous tractaments. Dins d'aquestes noves investigacions es troba l'extens camp de la nanomedicina, és a dir, l'estudi de nous materials a escala nanomètrica. Aquesta permet reduir aquests efectes secundaris augmentant la selectivitat i especificitat dels tractaments. Dins dels nanomaterials es troben les nanopartícules de upconversion que són capaços d'absorbir llum en l'infraroig proper i emetre-la en la regió ultraviolada-visible. D'altra banda, des del principi de la història de la medicina la llum s'ha emprat com a forma de tractament tenint un paper molt important. Un inconvenient per aquests tractaments sol ser la necessitat d'emprar llum de la regió ultraviolada-visible, ja que les biomolècules són capaços d'absorbir-la i produïr dany cel·lular. En aquest context, la present Tesi Doctoral es centra en l'estudi de noves formes de tractament anticancerigen combinant nanomedicina i llum. Per això s'han desenvolupat nous fàrmacs fototòxics i nous materials capaços de ser activats mitjançant llum infraroja propera. En primer lloc, es van sintetitzar noves fluoroquinolones per explorar les seves propietats fototòxiques per al seu ús en fotoquimioteràpia (Capítol 3 de la Tesi). Es van estudiar les característiques fotofísiques i fotoquímiques dels nous compostos, a més de la seva capacitat per produir major fototoxicitat en les cèl·lules en comparació amb les fluoroquinolones com la lomefloxacina mitjançant l'aplicació de llum ultraviolada. En base als resultats obtinguts es va realitzar un estudi per determinar les diferències entre les interaccions d'algunes fluoroquinolones dihalogenades, incloses les comentades anteriorment, i biomolècules com ADN i proteïnes. La reactivitat de les seves intermedis fotogenerats també es va estudiar en el Capítol 4. Després de conèixer en profunditat la capacitat fototòxica dels nous fàrmacs, en el Capítol 5 es va dur a terme el disseny d'un nanosistema compost per fluoroquinolones i nanopartícules de upconversion. Es va demostrar l'alta capacitat fototòxica d'aquest nou nanosistema. D'aquesta manera, es va generar activitat fototòxica a partir d'una fluoroquinolona sense l'ús de llum ultraviolada D'altra banda, la formació de profàrmacs obre la porta a l'administració selectiva de fàrmacs contra el càncer. Els profàrmacs consisteixen en la unió fotolábil d'una molècula capaç de ser activada per la llum i el fàrmac d'interès. No obstant això, un coneixement profund de les propietats fotofísiques i fotoquímiques del fotodisparador i dels potencials redox de tots dos membres de la diada pot ser crucial per obtenir el fotoalliberament desitjada. Així, en el Capítol 6, es va destacar la rellevància d'aquestes dades utilitzant un profàrmac format per un derivat de cumarina com a molècula fotoactivable i colquicina com a fàrmac. Finalment, en el Capítol 7 es va explorar la síntesi d'un nou nanosistema que conté un profàrmac format per un derivat de cumarina unit a l'fàrmac contra el càncer clorambucilo i nanopartícules biocomatibles de upconversion. L'addició d'albúmina de sèrum humà com a recobriment de les nanopartícules compleix la doble funció d'obtenir nanopartícules biocompatibles i ser el lloc de càrrega del profàrmac. / [EN] Cancer is one of the leading causes of death worldwide. Generally used anticancer treatments have various side effects produced by their low specificity. This is one of the reasons why the search for new treatments continues. Within these new investigations is the extensive field of nanomedicine, which can be explained as the study of new materials on a nanometric scale. It can be translated in the reduction of these side effects by increasing the selectivity and specificity of the treatments. Among the nanomaterials are upconversion nanoparticles that are capable of absorbing light in the near infrared and emit it in the ultraviolet-visible region. On the other hand, since the beginning of the history of medicine, light has been used as a form of treatment, having a very important role. A drawback for such treatments is sometimes the need to use light from the ultraviolet-visible region since biomolecules are capable of absorbing and causing cell damage. In this context, this Doctoral Thesis focuses on the study of new forms of anticancer treatment combining nanomedicine and light. For this, new phototoxic drugs and new materials capable of being activated by near infrared light have been developed. First, new fluoroquinolones were synthesized to explore their phototoxic properties for using in photochemotherapy (Chapter 3 of the Thesis). The photophysical and photochemical characteristics of the new compounds were studied, in addition to their ability to produce greater phototoxicity in cells than fluoroquinolones such as lomefloxacin by applying ultraviolet light. Based on the results obtained, a study was carried out to determine the differences between the interactions of some dihalogenated fluoroquinolones including the above commented, and biomolecules such as DNA and proteins. The reactivity of their photo-generated intermediates was also studied in Chapter 4. After a deep knowledge of the phototoxic capacity of the new drugs, design of a nanosystem composed of fluoroquinolones and upconversion nanoparticles was carried out in Chapter 5. The high phototoxic capacity of this new nanosystem was demonstrated. In this way phototoxic activity was generated from a fluoroquinolone without the use of ultraviolet light. On the other hand, the formation of prodrugs opens a door to the selective administration of anticancer drugs. Prodrugs consist of the photolabile binding of a molecule capable of being activated by light and the drug of interest. However, a knowledge of the photophysical and photochemical properties of the phototrigger as well as the redox potentials of both members of the dyad can be crucial to obtain the desired photorelease. Thus, in Chapter 6, the relevance of these data was highlighted using a prodrug formed by a coumarin derivative as a photoactivatable molecule and colchicine as a drug. Finally, in Chapter 7 the synthesis of a new nanosystem containing a prodrug formed by a derivative of coumarin linked to the anticancer drug chlorambucil, and upconversion biocompatible nanoparticles was explored. The addition of human serum albumin as a coating for the nanoparticles fulfills the dual function of obtaining biocompatible nanoparticles and being the loading site for the prodrug. / Anaya González, C. (2021). Design of New Up-conversion Systems for Anticancer Therapies [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/172665 / TESIS

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.

Reliable analytical tools are important for time efficient and economical process development, production and batch release of pharmaceuticals. Therapeutics recovered from human plasma, called plasma protein products, involve a large pharmaceutical industry of plasma fractionation. In plasma fractionation of human immunoglobulin G (hIgG) and albumin (HSA) recommended analysis techniques are regulated by the European Pharmacopoeia and are including total protein concentration assays and zone electrophoresis for protein composition and purity. These techniques are robust, but more efficient techniques with higher resolution, specificity and less hands-on time are available. Surface plasmon resonance is an optical method to study biomolecular interactions label-free in real time. This technology was used in this master thesis to set up assays using Biacore systems for quantification of HSA and hIgG from all steps of chromatographic plasma fractionation as a tool for process development and in-process control. The analyses have simplified mass balance calculations to a high extent as they imply specific detection of the proteins compared with using total protein detection. The assays have a low hands-on time and are very simple to perform and the use of one master calibration curve during a full week decreases analysis time to a minimum. Quick, in-process control quantification of one sample is easily obtained within &lt;10 minutes. For final QC of hIgG or for process development, an assay to quantify the distribution of the IgG subclasses (1-4) was set up on Biacore and showed significantly lower hands-on time compared with a commercial ELISA. All assays showed reliable quantification and identification performed in unattended runs with high precision, accuracy and sensitivity.

Combinatorial protein engineering, taking advantage of large libraries of protein variants and powerful selection technology, is a useful strategy for developing affinity proteins for applications in biotechnology and medicine. In this thesis, two small affinity proteins have been subjected to combinatorial protein engineering to improve or redirect the binding. In two of the projects, a three-helix protein domain based on staphylococcal protein A has been used as scaffold to generate so called Affibody molecules capable of binding to key proteins related to two diseases common among elderly people. In the first project, Affibody molecules were selected using phage display technology for binding to Ab-peptides, believed to play a crucial role in Alzheimer’s disease, in that they can oligomerize and contribute to the formation of neural plaques in the brain. The selected Affibody molecules were found to efficiently capture Ab from spiked human plasma when coupled to an affinity resin. The structure of the complex was determined by nuclear magnetic resonance (NMR) and demonstrated that the original helix 1 in the two Affibody molecules was unfolded upon binding, forming intermolecular b-sheets that stabilized the Ab peptide as buried in a tunnel-like cavity. Interestingly, the complex structure also revealed that the Affibody molecules were found to homo-dimerize via a disulfide bridge and bind monomeric Ab-peptide with a 2:1 stoichiometry. Furthermore, Affibody molecule-mediated inhibition of Ab fibrillation in vitro, suggested a potential of selected binders for future therapeutic applications. In the second project, two different selection systems were used to isolate Affibody molecules binding to tumor necrosis factor alpha (TNF), which is involved in inflammatory diseases such as rheumatoid arthritis. Both selection systems, phage display and Gram-positive bacterial display, could successfully generate TNF-binding molecules, with equilibrium dissociation constants (KD) in the picomolar to nanomolar range. Initial characterization of the binding to TNF was evaluated by competitive binding studies between the Affibody molecules and clinically approved TNF antagonists (adaliumumab, infliximab and etanercept) and demonstrated overlapping binding sites with both adaliumumab and etanercept. Furthermore, linkers of different lengths were introduced between Affibody moieties, in dimeric and trimeric constructs that were evaluated for their ability to block the binding between TNF and a recombinant form of its receptor. In the dimeric constructs, a linker length of 20-40 amino acids seemed to have an advantage compared to shorter and longer linkers, and the tested trimeric construct could block the TNF binding at even lower concentration. The results provided valuable information for the design of future Affibody-based molecules that could be investigated in therapeutic or medical imaging applications. In the third project aiming to generate a protein domain with capacity to influence the pharmacokinetics of protein therapeutics, a natural serum albumin-binding domain (ABD) was subjected to an engineering effort aiming at improving the affinity to human serum albumin (HSA), a protein with an exceptional long half-life in serum (19 days). First-generation affinity improved ABD variants were selected using phage display technology from a constructed ABD library. After additional rational engineering of such first generation variants, one variant with a 10,000-fold improved affinity to HSA (KD ≈ 120 fM) was obtained. Furthermore, characterization of this molecule also demonstrated improved affinity to several other serum albumins. When used as a gene fusion partner, this affinity-maturated variant denoted ABD035, should have the potential to extend the half-life of biopharmaceuticals in humans, and several other animal species. / QC 20100722

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