Nuisance odor levels produced by solid waste management operations are subject to regulatory standards due to their impacts on the quality of life of the residents living nearby the facility. Failure to meet regulatory standards may result in fines, litigation, inability to acquire permits, mitigation, and re-siting operations. Since measurement of environmental nuisance odors is currently limited to subjective techniques, monitoring odor levels to meet such standards is often problematic. This is becoming more acute as increasing residential populations begin to encroach on properties adjacent to landfills. In order to ensure that nuisance odor issues are minimized, it is necessary to provide an objective measurement. The objective of the current research is to develop a biosensor for providing an objective, standard measurement of odors. The approach is to modify the human odorant binding protein (hOBPIIa), isolated using published biomolecular techniques, by fluorescently tagging it with a chromophore functional group. When this protein is tagged with a fluorophore marker and excited in a spectrofluorometer, it emits light of a certain wavelength that can be detected and quantified. Once odorant molecules are exposed to this complex, they start replacing the fluorophore, and as a result, the emitted light intensity decreases in proportion to the number of odorant molecules. Since the protein response depends on odorant concentration, following an inverse Beer’s Law relationship, the odorants can be quantified accurately and rapidly using fluorometric measurements. The results establish quantitation ranges for different pure and mixture of odorant gases as well as the amount of gas that can be quantified across various flow rates. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2020. / FAU Electronic Theses and Dissertations Collection

Submitted by Ricardo Cedraz Duque Moliterno (ricardo.moliterno@uefs.br) on 2017-02-14T00:41:33Z No. of bitstreams: 1 PPGBiotec - Disserta??o corrigida - Isis Bugia.pdf: 6811384 bytes, checksum: 2380cbb790d35324858de90e106415fc (MD5) / Made available in DSpace on 2017-02-14T00:41:33Z (GMT). No. of bitstreams: 1 PPGBiotec - Disserta??o corrigida - Isis Bugia.pdf: 6811384 bytes, checksum: 2380cbb790d35324858de90e106415fc (MD5) Previous issue date: 2016-03-11 / The Visceral Leishmaniasis (VL) is the second most important vector-borne disease in the world, transmitted in the Americas by Lutzomyia longipalpis, vector control is essential for the prevention of the disease. But since it is not possible to identify the oviposition sites, the fight is directed to adult insects, using traps impregnated with chemical attractants. Whereas the Odorant Binding Proteins (OBPs) act in the first level of odor selection, this work used in silico methodology to identify putative vector olfactory chemical modulators based on the structure of OBPs and known ligands. For this, tridimensional (3D) structure of L. longipalpis OBPs were predicted by three comparative modeling methods. The best model, predicted by I-Tasser, was refined by Molecular Dynamics on Gromacs. Then, in a hierarchical virtual screening approach, natural compounds of ZINC12 closer to the typical OBP ligands in global chemical space, provided by ChemGPS-NP, were evaluated and staggered concerning affinity with the orthosteric site from the OBP, by molecular docking on DOCK6. The compounds were scored by GRIDSCORE, then the 100 best classified were submitted to AMBERSCORE, which took into account the flexibility from both OBP and the docked ligands. The lowest energy conformations interacted with a hydrophobic pocket through residues Met6, Gly10, Glu11, Ala9 Arg14, Leu74, Met53, Phe118, Phe119, Pro120, amino groups and formed ionic interaction with carboxyl of Glu11, Furthermore, Phe119, Asn29 and Gln69 formed hydrogen bonds, this last formed donor and acceptor H-bonds. / A Leishmaniose Visceral (LV) ? a segunda doen?a vetorial mais importante do mundo, transmitida nas Am?ricas por Lutzomyia longipalpis, o controle do vetor ? indispens?vel ? preven??o da doen?a. Mas como n?o ? poss?vel identificar onde ocorre a oviposi??o, o combate ? direcionado aos insetos adultos, utilizando armadilhas impregnadas com atrativos qu?micos. Considerando que as Prote?nas Ligadoras de Odor (OBPs) atuam no primeiro n?vel de sele??o dos odores, este trabalho utilizou uma metodologia in silico para identificar potenciais moduladores qu?micos olfativos do vetor baseando-se na estrutura das OBPs e de ligantes conhecidos. Para isso, foram preditas as estruturas tridimensionais (3D) de OBPs de L. longipalpis por tr?s m?todos de modelagem comparativa. O melhor modelo, predito pelo I-Tasser, foi refinado por Din?mica Molecular no Gromacs. Ent?o, numa abordagem hier?rquica da triagem virtual, os compostos naturais do ZINC12 mais pr?ximos dos t?picos ligantes de OBPs no espa?o qu?mico global, fornecido pelo ChemGPS-NP, foram avaliados e escalonados quanto ? afinidade com o s?tio ortost?rico da OBP, pelo acoplamento molecular no DOCK6. Os compostos foram pontuados pelo Gridscore, em seguida, os cem melhores classificados foram submetidos ? pontua??o pelo Amberscore, que levou em conta a flexibilidade tanto da OBP como dos ligantes acoplados. As conforma??es de menor energia interagiram com um bols?o hidrof?bico atrav?s dos res?duos Met6, Ala9, Gly10, Glu11, Arg14, Met53, Leu74, Phe118, Phe119, Pro120; grupamentos amino formaram pontes salinas com a carboxila do Glu11. Al?m disso, os res?duos Phe119, Asn29 e Gln69 formaram liga??es hidrog?nio, sendo que, este ?ltimo res?duo formou liga??es-H aceptoras e doadoras.

Embora os insetos sejam essenciais para a vida na Terra, alguns afetam negativamente a sociedade humana atuando como vetores de doenças, como por exemplo a doença de Chagas. De acordo com a Organização Mundial de Saúde, no Brasil, o método mais eficaz para prevenir a propagação do agente etiológico, Trypanosoma cruzi, é por controle vetorial. Rhodnius prolixus é um importante vetor de doença de Chagas no Brasil. Como outros insetos, seu comportamento depende de semioquímicos percebidos por um sistema olfativo sofisticado. As proteínas que realizam a comunicação entre ambiente externo e os receptores de odorantes são chamadas de proteínas ligadoras ao odorante (Odorant Binding Protein, OBPs), e são proteínas solúveis, extracelulares e pequenas (15-17 kDa). Visando aumentar o conhecimento sobre essas proteínas para possivelmente desenvolver estratégias de manipulação comportamental de R. prolixus, foram selecionados três genes putativos de OBPs, RproOBP 8, 11 e 19. Os genes foram selecionados através de ferramentas de bioinformática e foram clonados e expressos em diferentes vetores linhagens celulares de Escherichia coli. A proteína recombinante RproOBP 8 e 11 foram expressas em E. coli BL21(DE3). A RproOBP 19 obteve os melhores resultados quando expressa em E. coli BL21(DE3)pLysS. As proteínas recombinantes foram estudadas usando Espalhamento Dinâmico da Luz (DLS), cujos resultados sugeriram melhores soluções tamponantes para trabalhar e armazenar essas proteínas. Esses tampões também foram utilizados para rastrear as condições de cristalização. Embora não tenham sido obtidos cristais para serem usados em coleta de dados de difração de raios X, algumas condições se mostraram promissoras para serem refinadas posteriormente. A RproOBP11 apresentou uma cor vermelha quando purificada. O espectro de UV-Visível mostra um máximo de absorção em 413 nm, o que sugere a presença de um grupo heme no estado oxidado. Os espectros obtidos por Dicroísmo Circular (CD) para a RproOBP11, caracterizam uma proteína rica em α- hélice devido aos picos negativos em 208 e 222 nm. Na presença de 5 possíveis ligantes: pentanol, hexanol, heptanol, octenol e octanol, os espectros de CD não mostraram mudança, podendo representar falta de interação ou interações fracas. A Calorimetria de Titulação Isotérmica (ITC) foi realizada com os mesmos compostos, nenhum deles mostrou interação com a RproOBP11 em tampão 20 mM fosfato pH 7,0. Os mesmos resultados de ITC foram obtidos para o RproOBP19 em tampão 20 mM HEPES pH 7,0. Os resultados do CD sugerirem que o RproOBP19 é uma proteína com regiões intrinsecamente desordenadas. Quando os espectros de CD foram obtidos na presença de TFE a RproOBP19 apresenta espectro mais semelhante às proteínas de α- hélice. Estes resultados representam a primeira investigação molecular de OBPs de R. prolixus. Testes adicionais são necessários para descobrir a estrutura e função dessas OBPs e assim, realizar uma abordagem direcionada do controle vetorial e a possibilidade de usar essas pequenas proteínas como ferramentas biotecnológicas. / Although insects are essential to life on earth, some can affect negatively human society acting as vectors of diseases. One of those is Chagas disease. According to the World Health Organization, in Brazil the most effective method to prevent the spread of the etiologic agent, Trypanosoma cruzi, is by vector control. Rhodnius prolixus is important Chagas disease vector in Brazil. It is a blood-feeding triatomine whose behavior, like other insects, originally depends of signals given by the environment as semiochemicals perceived by a sophisticated olfactory system. The proteins that perform the communication between external environment and the odorant receptors are called Odorant Binding Proteins (OBPs), and are soluble, extracellular and small proteins (15-17 kDa). Aiming to increase knowledge about these proteins to possibly develop strategies for behavioral manipulation of R. prolixus , three putative OBP genes, RproOBP 8, 11 and 19, were selected. The genes were selected using bioinformatics tools and were cloned and expressed in different vectors of Escherichia coli cell lines. Recombinant protein RproOBP 8 and 11 were expressed in E. coli BL21(DE3). RproOBP 19 obtained the best results when expressed in E. coli BL21(DE3)pLysS. Recombinant proteins were studied using Dynamic Light Scattering (DLS), whose results suggested better buffering solutions for working and storing conditions. Such buffers were also used to track crystallization conditions. Although no crystals have been obtained for use in X-ray diffraction data collection, some conditions have proved promising for further refinement. RproOBP 11 showed a red color when purified. The UV-Visible spectrum shows a maximum absorption at 413 nm, which suggests the presence of a heme group in the oxidized state. The spectra obtained by Circular Dicroism (CD) for RproOBP11, characterize an α-helix rich protein due to negative peaks at 208 and 222 nm. In the presence of 5 possible ligands: pentanol, hexanol, heptanol, octenol and octanol, the CD spectra did not show change, being able to represent lack of interaction or weak interactions. Isothermal Titration Calorimetry (ITC) was performed with the same compounds, none of them showed interaction with RproOBP 11 in 20 mM phosphate buffer pH 7.0. The same ITC results were obtained for RproOBP19 in 20 mM HEPES buffer pH 7.0. CD results suggest that RproOBP19 is a protein with intrinsically disordered regions. When CD spectra were obtained in the presence of TFE RproOBP19 presents a spectrum more similar to the α-helix proteins. These results represent the first molecular investigation of OBPs of R. prolixus. Further tests are needed to uncover the structure of these OBPs and provide insight into the function allowing a more targeted approach of vector control and the possibility to use such small proteins as biotechnological tools.

Les OBP sont des petites protéines solubles qui se lient avec des molécules odorantes et phéromonales. Le rôle des OBPs n’est pas complètement compris. Une hypothèse suggère que l’OBP solubilise et transporte les ligands aux récepteurs olfactifs et la liaison entre les molécules odorantes et l’OBP est non spécifique. Une autre hypothèse suggère que le complexe formé est une liaison spécifique entre une molécule odorante donnée et une OBP spécifique. Ce travail de thèse montre que les OBPs sont impliquées dans la première étape de la discrimination des odeurs. Dans un premier temps, nous avons montré l’implication de la Phe35 et la Tyr 82 dans la sortie du ligand par l’OBP. Dans un second temps, nous avons mis en évidence la présence de différentes isoformes d’OBP et de VEG qui diffèrent par les modifications post-traductionnelles (phosphorylation et GlcNAcylation) a la fois sur les protéines natives extraites de la muqueuse respiratoire et sur les protéines recombinantes produites par P.pastoris et CHO. Ces isoformes sont capables de discriminer des molécules odorantes et phéromonales. Les OBPs ne sont pas des transporteurs passifs car elles assurent un fin codage des molécules odorantes ou phéromonales avant l’interaction de ce complexe avec un récepteur spécifique. / OBPs are small soluble proteins that bind with odorant molecules and pheromones. The role of OBP is not completely understood. A hypothesis suggests that OBP solubilize and transport the ligands to olfactory receptors and the binding between odorant molecule and OBP is unspecific. An other hypothesis suggest that the complex formed is the specific binding between a given odorant molecule and a specific OBP. This work of thesis show that OBP are involved in the first step of odorant discrimination. Initially, we have showed the involvement of the Phe35 and Tyr 82 in the uptake of ligands by OBP. Second, we have given rise to the presence of various isoform of OBP and VEG that differ by post-translational modifications (phosphorylation and GlcNAcylation) both on natives proteins extract of respiratory mucosa and on recombinants proteins produce by P. pastoris and CHO. These isoforms are able to discriminate of odorant molecules and pheromones. OBPs are not passives carriers because they ensure a fine coding of odorant molecules and pheromones before interaction of this complex with specific receptor.

The development of an inexpensive, portable, stable, sensitive and selective biosensor for detection of odorants is a daunting task. Here, we hypothesized the development of a detector layer composed of the protein groups; the olfactory receptors (ORs) and the odorant binding proteins (OBPs), known to bind odorants in animal sensing. We report the design of 13 OR gene-vector constructs, and their subsequent transformation into Escherichia (E.) coli (BL21 (DE3)-STAR-pLysS) strain. Moreover, we report the expression of several ORs into an in vitro wheat germ extract using three separate detergent mixes for protein solubilization.   In addition, we describe the design of an odorant binding protein from the Aenopheles gambiae mosquito PEST strain (OBP-PEST) gene-vector construct under an IPTG (Isopropyl β-D-1-thiogalactopyranoside) inducible promoter. OBP-PEST was heterologously expressed in E.coli with an 8 amino acid long sequence (WSPQFEK) attached C-terminally, via a thrombin cleavage site and a flexible linker (GSSG). The WSPQFEK sequence, commonly referred to as a Strep-tag, enabled subsequent affinity chromatography purification of the protein, via binding to an engineered Streptavidin equivalent. Surprisingly, the OBP-PEST was found to contain a signal sequence leading to its truncation and secretion when expressed in E.coli.   Biophysical analyses were established using Circular Dichroism (CD) for the analysis of two lipocalins: Beta-lactoglobulin (BLG) and OBP-PEST. We studied the solubility, refoldability and the conformational transitions of BLG, as a result of change in solvent, pH and temperature. The secondary structure of OBP-PEST and its thermal stability was investigated.   In conclusion, this thesis work has enabled biophysical analyses of OBP-PEST and future analogs of interest to the development of a stable protein detector layer. Although further experiments are needed to fully characterize the biophysical properties, and to find odorant substrates of OBP-PEST, it was found to be a suitable alternative to ORs in a biosensor detector layer application. More importantly, an inherent OBP-PEST signal sequence was found to mediate protein secretion when expressed heterologously in E.coli. To the best of our knowledge this is the first lipocalin discovered to be secreted upon heterologous expression in E.coli.   We hypothesize that this signal peptide could be used as a means for targeted secretion and, hence, efficient protein purification.

Insect Odorant-Binding Proteins (OBPs) are small, water-soluble molecules that solubilize hydrophobic odorant molecules in the sensillum lymph and transport them to their cognate receptors in the olfactory receptor neurons. With the availability of the genome sequence of the African malaria mosquito, Anopheles gambiae, there has been a profound interest in the characterization and functional analyses of Obp genes in order to understand the molecular basis of mosquito host-seeking behavior. However, no direct evidence has been found for specific functions of any mosquito OBPs. In this study, I describe the comparative genomics and expression analyses on two mosquito Obp genes (Obp1 and Obp7) as well as efforts to determine their functions. Both of these Obp genes were identified in Anopheles stephensi and only Obp7 gene was identified in Anopheles quadriannulatus by screening bacterial artificial chromosome (BAC) libraries of these species. Comparative analyses revealed several interesting features including segments of conserved non-coding sequences (CNSs) that contain potential regulatory elements relevant to olfactory tissue development and blood-feeding. The expression profiles of these genes were examined in detail in the Asian malaria mosquito An. stephensi. Obp1 and Obp7 transcripts were significantly higher in females than male mosquitoes and they were predominantly found in the antenna, which is the primary olfactory organ of mosquitoes. Twenty-four hours after a blood meal, mRNA levels of these two genes were significantly reduced in the maxillary palp and proboscis, referred to as secondary olfactory organs of mosquitoes. These findings collectively indicate that Obp1 and Obp7 genes in An. stephensi likely function in female olfactory response and may be involved in behaviors related to blood-feeding. To investigate the function of these Obp genes more directly, a Sindbis virus based expression system is established to knockdown the two Obp gene orthologs in Aedes aegypti. The effective knockdown of Obp1 and Obp7 genes (8 and 100-fold, respectively) is accomplished in female mosquito olfactory tissues. The potential for a systematic analysis of the molecular players involved in mosquito olfaction using this newly developed technique is discussed. Such analysis will provide the foundation for interfering with mosquito host-seeking behavior for the prevention of disease transmission. / Ph. D.

Rhipicephalus micropulus, conhecido como carrapato dos bovinos, é um dos parasitas mais importantes para a pecuária, pois causa enormes prejuízos ao produtor. Esses prejuízos se estendem desde a ação espoliativa que o carrapato exerce nos seus hospedeiros até a transmissão de uma diversidade de patógenos. Os carrapatos utilizam vários estímulos térmicos, sonoros, visuais, gustativos, táteis e olfativos na fase de busca pelo hospedeiro. Essas substâncias são conhecidas como semioquímicos e podem atuar em indivíduos de uma mesma espécie como os ferormônios ou entre indivíduos de espécies diferentes como os alomônios e cairomônios. Nos bovinos, já se sabe que uma de suas lipocalinas pode atrair insetos, porém não é sabido se essa atração ocorre para R. microplus. Já é sabido que o carrapato consegue distinguir odores liberados entre bovinos das raças Nelore e Holandês Preto e Branco (HPB), sendo mais atraído para bovinos da raça Holandês. A lipocalina por ser uma proteína globular e estar associada com o transporte de pequenas moléculas hidrofóbicas, tais como odorantes e esteroides, e pode favorecer a atração do R. microplus, uma vez que essa proteína bovina pode ligar a odorantes e desempenhar um papel fundamental na liberação de odores para o ambiente, que por sua vez pode atrair, ou não, o R. microplus. Existem diferentes níveis de infestação de carrapatos entre bovinos das raças Nelore e Holandês, bem como entre os sexos da mesma raça. Além disso, o período do ciclo de vida do bovino também pode influenciar na susceptibilidade a infestação, por exemplo, as vacas no período de lactação são consideradas mais susceptíveis ao parasito. Assim, o objetivo do presente estudo foi avaliar o papel da lipocalina bovina na resistência e suscetibilidade ao carrapato, através da quantificação dessa proteína em líquidos corporais dos bovinos, tais como soro, saliva, urina, suor, secreção nasal e biopsia de pele obtidos de bovinos que apresentam fenótipos contrastantes de infestações, a saber: hospedeiros macho (touros) e fêmea (vacas em lactação) das raças resistentes (Bos indicus) e suscetíveis (Bos taurus). Os resultados deste trabalho tem demonstrado que a lipocalina bovina está presente em todos os fluídos investigados, exceto a urina, como observado por Western blot. Com os resultados deste trabalho podemos concluir que a bcOBP apresenta 9 diferença significativa nos fluidos de saliva e secreção nasal de touros e vacas em lactação da raça HPB quando comparados com touros e vacas em lactação da raça Nelore. Em amostras de biópsia de pele também ocorreu uma maior marcação da bcOBP na raça susceptível (HPB), demonstrando que a bcOBP possivelmente esta auxiliando na susceptibilidade destes bovinos ao carrapato, através do transporte de um maior número de odorantes que estariam atraindo um maior número de carrapatos. Ao analisar os fluidos entre bovinos da mesma raça, porém de sexo diferente, observou-se uma maior quantidade de bcOBP em vacas no período de lactação HPB nos fluidos de saliva e secreção nasal, visto que as vacas estão no período de lactação e são mais susceptíveis a infestações. Consequentemente, a bcOBP poderia estar colaborando para a sua maior susceptibilidade quando comparadas com touros HPB, e o mesmo ocorreu para biopsia de pele. Porém no fluído soro o aumento foi significativo para touro HPB quando comparado às vacas em lactação da raça HPB. Acredita-se que esse fenômeno ocorra devido ao período de lactação nas fêmeas uma vez que a produção da lipocalina bovina esta intimamente relacionada com a produção do leite. Os resultados deste trabalho demonstram que possivelmente a bcOBP na raça HPB está carreando odorantes para o ambiente os quais estariam atraindo mais carrapatos, e auxiliando na susceptibilidade destes bovinos. / Rhipicephalus micropulus, known as bovine tick, is one of the most important parasites for livestock, as they cause enormous damage to the producer. These demages extend from the spoliation action that the tick exerts on the skin of its hosts until the transmission of a diversity of pathogens. Ticks use various thermal, sound, visual, gustatory, tactile and olfactory stimuli in the search phase to the host. These substances are known as semiochemicals and can act within individuals of the same species as the pheromones or among individuals of different species such as alomones and cairomones. In the cattle, it is known that the lipocalin protein may attract insects, but this attraction is not known to influence the R. microplus. It is known that the tick can distinguish released odors between Nelore and Holstein cattle, being more attracted to cattle of the Holstein breed. Lipocalin, being a globular protein and associated with the transport of small hydrophobic molecules, such as odorants and steroids, can favor the attraction of R. microplus, since this bovine protein can bind to odorants and play a key role in the release of odors into the environment, which in turn may or may not attract R. microplus. There are different levels of tick infestation among cattle of the Nelore and Holstein breeds, as well as between the sexes of the same breed. In addition, the life cycle period of the bovine can also influence the susceptibility to infestation. For example, cows in the lactation period are considered more susceptible to the parasite. Thus, the objective of the present study was to evaluate the role of bovine lipocalin in the resistance and susceptibility to tick by quantifying that protein in bovine body fluids such as serum, saliva and urine, sweat, nasal secretions and skin biopsies obtained from resistant (Bos indicus) and susceptible (Bos taurus) cattle breeds. The results of this work have demonstrated that bovine lipocalin is present in all investigated fluids except urine, as observed by Western Blot. With the results of this work we can conclude that bcOBP presents a significant difference in fluids saliva and nasal secretion of bulls and cows in lactation breed HPB when compared to bulls and lactating cows of the Nelore breed. In skin biopsy specimens, there was also a greater labeling of bcOBP in the susceptible strain (HPB), which could then aid in the susceptibility of these cattle to the 11 tick, by transporting a larger number of odorants that would be attracting a greater number of ticks. When analyzing the fluids between bovines of the same breed, but of different sex, a greater amount of bcOBP was observed in cows during the period of HPB lactation in the fluids saliva and nasal secretion, since the cows are in the lactation period and are more susceptible to infestations. Consequently, bcOBP could be contributing to its greater susceptibility when compared to HPB bulls, and the same was done for skin biopsy. However, in the fluid serum, the increase was significant for the bull when compared to the lactating cows of the HPB race. It is believed that this phenomenon occurs due to the lactation period in females since the production of bovine lipocalin is closely related to milk production. The results of this work demonstrate that possibly Lipocalin in Holstein cattle is carrying odorants to the environment which would be attracting more ticks, and enhancing in the susceptibility of these cattle.

Rhipicephalus micropulus, conhecido como carrapato dos bovinos, é um dos parasitas mais importantes para a pecuária, pois causa enormes prejuízos ao produtor. Esses prejuízos se estendem desde a ação espoliativa que o carrapato exerce nos seus hospedeiros até a transmissão de uma diversidade de patógenos. Os carrapatos utilizam vários estímulos térmicos, sonoros, visuais, gustativos, táteis e olfativos na fase de busca pelo hospedeiro. Essas substâncias são conhecidas como semioquímicos e podem atuar em indivíduos de uma mesma espécie como os ferormônios ou entre indivíduos de espécies diferentes como os alomônios e cairomônios. Nos bovinos, já se sabe que uma de suas lipocalinas pode atrair insetos, porém não é sabido se essa atração ocorre para R. microplus. Já é sabido que o carrapato consegue distinguir odores liberados entre bovinos das raças Nelore e Holandês Preto e Branco (HPB), sendo mais atraído para bovinos da raça Holandês. A lipocalina por ser uma proteína globular e estar associada com o transporte de pequenas moléculas hidrofóbicas, tais como odorantes e esteroides, e pode favorecer a atração do R. microplus, uma vez que essa proteína bovina pode ligar a odorantes e desempenhar um papel fundamental na liberação de odores para o ambiente, que por sua vez pode atrair, ou não, o R. microplus. Existem diferentes níveis de infestação de carrapatos entre bovinos das raças Nelore e Holandês, bem como entre os sexos da mesma raça. Além disso, o período do ciclo de vida do bovino também pode influenciar na susceptibilidade a infestação, por exemplo, as vacas no período de lactação são consideradas mais susceptíveis ao parasito. Assim, o objetivo do presente estudo foi avaliar o papel da lipocalina bovina na resistência e suscetibilidade ao carrapato, através da quantificação dessa proteína em líquidos corporais dos bovinos, tais como soro, saliva, urina, suor, secreção nasal e biopsia de pele obtidos de bovinos que apresentam fenótipos contrastantes de infestações, a saber: hospedeiros macho (touros) e fêmea (vacas em lactação) das raças resistentes (Bos indicus) e suscetíveis (Bos taurus). Os resultados deste trabalho tem demonstrado que a lipocalina bovina está presente em todos os fluídos investigados, exceto a urina, como observado por Western blot. Com os resultados deste trabalho podemos concluir que a bcOBP apresenta 9 diferença significativa nos fluidos de saliva e secreção nasal de touros e vacas em lactação da raça HPB quando comparados com touros e vacas em lactação da raça Nelore. Em amostras de biópsia de pele também ocorreu uma maior marcação da bcOBP na raça susceptível (HPB), demonstrando que a bcOBP possivelmente esta auxiliando na susceptibilidade destes bovinos ao carrapato, através do transporte de um maior número de odorantes que estariam atraindo um maior número de carrapatos. Ao analisar os fluidos entre bovinos da mesma raça, porém de sexo diferente, observou-se uma maior quantidade de bcOBP em vacas no período de lactação HPB nos fluidos de saliva e secreção nasal, visto que as vacas estão no período de lactação e são mais susceptíveis a infestações. Consequentemente, a bcOBP poderia estar colaborando para a sua maior susceptibilidade quando comparadas com touros HPB, e o mesmo ocorreu para biopsia de pele. Porém no fluído soro o aumento foi significativo para touro HPB quando comparado às vacas em lactação da raça HPB. Acredita-se que esse fenômeno ocorra devido ao período de lactação nas fêmeas uma vez que a produção da lipocalina bovina esta intimamente relacionada com a produção do leite. Os resultados deste trabalho demonstram que possivelmente a bcOBP na raça HPB está carreando odorantes para o ambiente os quais estariam atraindo mais carrapatos, e auxiliando na susceptibilidade destes bovinos. / Rhipicephalus micropulus, known as bovine tick, is one of the most important parasites for livestock, as they cause enormous damage to the producer. These demages extend from the spoliation action that the tick exerts on the skin of its hosts until the transmission of a diversity of pathogens. Ticks use various thermal, sound, visual, gustatory, tactile and olfactory stimuli in the search phase to the host. These substances are known as semiochemicals and can act within individuals of the same species as the pheromones or among individuals of different species such as alomones and cairomones. In the cattle, it is known that the lipocalin protein may attract insects, but this attraction is not known to influence the R. microplus. It is known that the tick can distinguish released odors between Nelore and Holstein cattle, being more attracted to cattle of the Holstein breed. Lipocalin, being a globular protein and associated with the transport of small hydrophobic molecules, such as odorants and steroids, can favor the attraction of R. microplus, since this bovine protein can bind to odorants and play a key role in the release of odors into the environment, which in turn may or may not attract R. microplus. There are different levels of tick infestation among cattle of the Nelore and Holstein breeds, as well as between the sexes of the same breed. In addition, the life cycle period of the bovine can also influence the susceptibility to infestation. For example, cows in the lactation period are considered more susceptible to the parasite. Thus, the objective of the present study was to evaluate the role of bovine lipocalin in the resistance and susceptibility to tick by quantifying that protein in bovine body fluids such as serum, saliva and urine, sweat, nasal secretions and skin biopsies obtained from resistant (Bos indicus) and susceptible (Bos taurus) cattle breeds. The results of this work have demonstrated that bovine lipocalin is present in all investigated fluids except urine, as observed by Western Blot. With the results of this work we can conclude that bcOBP presents a significant difference in fluids saliva and nasal secretion of bulls and cows in lactation breed HPB when compared to bulls and lactating cows of the Nelore breed. In skin biopsy specimens, there was also a greater labeling of bcOBP in the susceptible strain (HPB), which could then aid in the susceptibility of these cattle to the 11 tick, by transporting a larger number of odorants that would be attracting a greater number of ticks. When analyzing the fluids between bovines of the same breed, but of different sex, a greater amount of bcOBP was observed in cows during the period of HPB lactation in the fluids saliva and nasal secretion, since the cows are in the lactation period and are more susceptible to infestations. Consequently, bcOBP could be contributing to its greater susceptibility when compared to HPB bulls, and the same was done for skin biopsy. However, in the fluid serum, the increase was significant for the bull when compared to the lactating cows of the HPB race. It is believed that this phenomenon occurs due to the lactation period in females since the production of bovine lipocalin is closely related to milk production. The results of this work demonstrate that possibly Lipocalin in Holstein cattle is carrying odorants to the environment which would be attracting more ticks, and enhancing in the susceptibility of these cattle.