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No. of bitstreams: 2 Dissertação - Émelin Alves dos Santos - 2016.pdf: 1617820 bytes, checksum: 16ea9a34990cb6d247fd464223a95256 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-04-04 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Strongyloidiasis is mainly predominant in regions of tropical and subtropical climates may present asymptomatic limited to the gastrointestinal tract. However, immunosuppression can cause hyperinfection and dissemination of larvae. Diabetes Mellitus (DM) causes immune dysregulation, became the individual more susceptible to secondary infections as strongyloidiasis. The chronicity of the disease in this group patients difficult the diagnosis, this fact conduct to research new methodologies such as circulating immune complex detections. Currently, there are few reports in literature on the evaluation immunological profile of strongyloidiasis in patients with DM and epidemiological research this disease important for monitoring the number of cases. In this context, this study had as objective determine serum levels of specific IgG antibodies and circulating immune complex in serum samples of infected patients and control individuals negative by ELISA. The survey was conducted with patients attended at the diabetes ambulatory in Jataí - GO and nondiabetic individuals residing in the city. The authorization for collect blood samples was obtained by signature Term of Informed Consent (TIC) it is characterized individuals in two groups: Group I patients with DM1 and 2, Group II individuals negative control for S. stercoralis and DM. Serum samples were tested by ELISA for detection of IgG and circulating immune complexes. We analyzed 50 serum samples from Group I and 50 in Group II, 30% were male and 70% female, for both groups. A total of 19 (38%) serum sample were positive for IgG in Group I and 2 (4%) for Group II, the majority were female age between 57 and 69 years. For detection circulating immune complexes only one sample was positive in Group I (2%) and Group II (0%). The seroprevalence of IgG anti-Strongyloides detection in diabetic patients was high when compared to the control group, detection of circulating immune complexes in Group I was low. In this context, we emphasize the importance the realization of immune complex research by ELISA as complementary tool in diagnosis of active infection S. stercoralis. / A estrongiloidíase é predominante principalmente em regiões de climas tropicais e subtropicais, podendo apresentar-se assintomática quando limitada ao trato gastrointestinal. No entanto, a imunodepressão pode levar a quadros de hiperinfecção e disseminação das larvas. O Diabetes Mellitus (DM) provoca disfunção do sistema imune, tornando o indivíduo mais susceptível a infecções secundárias, como a estrongiloidíase. A cronicidade da doença neste grupo de pacientes dificulta o seu diagnóstico, fato este que tem levado a pesquisa de novas metodologias, como a detecção de imuncomplexos circulantes. Atualmente, existem poucos relatos na literatura sobre a avaliação do perfil imunológico da estrongiloidíase em pacientes com DM, sendo a investigação epidemiológica desta doença importante para o acompanhamento do número de casos. Neste contexto, o presente estudo tem como objetivo determinar os níveis séricos de anticorpos IgG específicos e imunocomplexos circulantes em amostras de soros de pacientes infectados e de indivíduos controle negativo, por ELISA. A pesquisa foi realizada com pacientes atendidos no ambulatório de diabetes da prefeitura de Jataí - GO e indivíduos não diabéticos residentes no município. A autorização para coleta das amostras de sangue foi obtida pela assinatura do Termo de Consentimento Livre e Esclarecido (TCLE), sendo os indivíduos caracterizados em dois grupos: Grupo I pacientes portadores de DM1 e 2, Grupo II indivíduos controle negativo para S. stercoralis e DM. As amostras de soro foram testadas pela técnica imunoenzimática ELISA para detecção de IgG e imunocomplexos circulantes. Foram analisadas 50 amostras de soro do Grupo I e 50 do Grupo II, sendo que 30% eram do sexo masculino e 70% do sexo feminino, para ambos os grupos. Um total de 19 (38%) amostras de soro foram reagentes para IgG no Grupo I e 2 (4%) para o Grupo II, sendo a maioria do sexo feminino com faixa etária entre 57 e 69 anos. Para detecção de imunocomplexos circulantes, apenas uma amostra foi reagente no Grupo I (2%) e nenhuma no Grupo II (0%). A soroprevalência para detecção de IgG anti-Strongyloides em pacientes diabéticos foi elevada quando comparado ao grupo controle, enquanto que a detecção de imunocomplexos circulantes no Grupo I foi baixa. Neste contexto, ressalta-se a importância da realização da pesquisa de imunocomplexos por ELISA como ferramenta complementar para o diagnóstico da infecção ativa por S. stercoralis.

Neste trabalho foi feito uma investigação sorológica em moradores na região amazônica e moradores rurais do sudeste brasileiro, através do teste de inibição da hemaglutinação (HI) para a cepa vacinal da Febre Amarela mais 17 sorotipos diferentes de arbovírus, sendo quatro do gênero Alphavirus (ALP), nove do gênero Flavivirus (FLA) e cinco do gênero Orthobunyavirus (ORT), para verificar qual desses vírus estão circulando nessas regiões. Os resultados do HI foram divididos em quatro áreas: na cidade Machadinho dOeste (Rondônia) (N=285) tivemos para ALP=50 (17,54%), FLA=270 (94,74%), ORT=58 (20,35%) e negativos (NEG)=12 (4,20%). Nas pessoas que moram ao longo do Rio Machado (Rondônia) (N=343) tivemos ALP=99 (28,86%), FLA=229 (66,76%), ORT=41 (11,95%) e NEG=93 (27,10%). Na cidade Teodoro Sampaio (São Paulo) (N=78) tivemos FLA=55 (70,51%) e NEG=23 (29,5%). Na cidade Jacupiranga (São Paulo) (N=149) tivemos ALP=7 (4,7%), FLA=50 (33,56%), ORT=20 (13,42%) e NEG=84 (56,4%). Concluímos que os resultados obtidos sugerem a circulação desses vírus nas regiões estudadas. / In this work we performed a serological investigation in residents of Amazon region and country people who lives in Southeastern of Brazil, by hemagglutination inhibition (HI) for Yellow fever vaccine and 17 different serotype of arboviruses: four from the genus Alphavirus (ALP), nine from the genus Flavivirus (FLA), and five from the genus Orthobunyavirus (ORT), to try to ascertain which of these arboviruses are circulating in these areas. The results of HI were divided into four areas: In Machadinho dOeste city (Rondônia) (N=285) we had to ALP=50 (17,54%), FLA=270 (94,74%), ORT=58 (20,35%) and negatives (NEG)=12 (4,20%). In people who live along the Machado River (Rondônia) (N=343) we had to ALP=99 (28,86%), FLA=229 (66,76%), ORT=41 (11,95%) and NEG=93 (27,10%). In Teodoro Sampaio City (São Paulo) (N=78) we had to FLA=55 (70,51%) and NEG=23 (29,5%). In Jacupiranga City (São Paulo) (N=149) we had to ALP=7 (4,70%), FLA=50 (33,56%), ORT=20 (13,42%) and NEG=84 (56,4%). In conclusion, the obtained data suggest the circulation of these viruses in these regions.

Abstract Bladder cancer when superficial has a good prognosis but it has a high recurrence risk and about 10–15% of the superficial carcinomas will progress into muscle invasive or metastatic type. The most powerful factor for predicting the behavior of bladder carcinoma is the stage of the tumor. Invasion to the lamina propria increases the risk of recurrence and progress to muscle-invasive tumor. Also grade of the tumor and tumor multiplicity associates with high risk for recurrence. New markers are still needed to find those patients who need more and better treatments to avoid the recurrence and progress. The need for new non-invasive markers to diminish the need for frequent cystoscopy in follow-up is also obvious. Gelatinases MMP-2 and MMP-9 are known to associate to tumor invasion and progression. Also their tissue inhibitors TIMP-1 and TIMP-2 take part in these diversified processes and metastasis formation. In the present work the expression and clinical value of gelatinases MMP-2 and MMP-9 and their tissue inhibitors TIMP-1 and TIMP-2 were evaluated in bladder carcinoma. Primary tissue samples of 121 patients were analyzed for expression of MMP-2 and/or MMP-9 using immunohistochemistry. The serum samples of 87 patients who were treated in the Oncology Department of Oulu University Hospital were collected and studied with ELISA. The control group consisted of 44 healthy volunteers. Overexperssion of MMP-2 protein correlated significantly to disease-specific survival and showed an independent prognostic value as a biomarker. High MMP-9 expression instead correlated to favorable overall survival of bladder cancer patients. Circulating proMMP-2, TIMP-2 and MMP-2:TIMP-2 complex levels were lower in cancer patients than in healthy volunteers in control group. High levels of all these three markers correlated with better prognosis in bladder cancer patients.

Memoria para optar al Título Profesional de Médico Veterinario / Se realizó la prueba de ELISA para evaluar la respuesta serológica de gallinas ponedoras comerciales según el plan de vacunación contra la Bronquitis Infecciosa (BI) aplicado en un plantel comercial, consistente en 8 vacunas vivas aplicadas al día de edad, 4, 7, 10, 14, 17, 20 y 70 semanas de edad. Un total de 500 gallinas constituyó la muestra de 25 lotes de gallinas de diferentes edades y número de vacunas aplicadas (20 sueros de cada lote). Estos lotes de gallinas estaban alojados en distintos sectores de esta empresa. Los lotes de gallinas con 6, 7 y 8 vacunas aplicadas, presentaron títulos de anticuerpos contra BI con una Media Geométrica (MG) de 1137 a 6612. El análisis estadístico según el número de vacunas aplicadas, demostró que hubo un incremento significativo de los títulos de anticuerpos después de 6 vacunas, al obtenerse una Media Aritmética (MA) de 2341; y vuelve a haber un incremento significativo a una MA de 4711 con 7 vacunas. Sin embargo, con 8 vacunas se logra una MA de 4434, que es estadísticamente similar a la anterior. El análisis estadístico de los sectores de gallinas con 7 vacunas, demuestra títulos de anticuerpos contra BI con una MA de 2887 a 6193, que estadísticamente se puede diferenciar en dos grupos principales. Los sectores de gallinas con 8 vacunas presentaron títulos de anticuerpos con una MA en un rango de 3799 a 5622, que también se diferencian, en dos grupos estadísticamente diferentes. Se concluyó que los títulos de anticuerpos obtenidos de diferentes lotes de gallinas, son compatibles con la información bibliográfica y manuales técnicos de la prueba de ELISA. Además, la diversidad de títulos de anticuerpos obtenidos entre los distintos lotes o sectores de gallinas, con o sin número de vacunas similares, pueden estar influenciadas por las diferencias temporales de la aplicación de las vacunas, diferencias estructurales de los galpones y los sectores, diferencias de las vacunas, diferencias de manejo y personal de vacunación, entre otros factores. / ELISA test was performed to evaluate serological response of commercial laying hens according to a vaccination plan against Infectious Bronchitis (IB) applied in a commercial farm. Five hundred laying hens were sampled from 25 flocks of different ages and number of vaccines given (20 sera from each flock). Flocks were located in different sectors of the farm. Flocks of hens with 6, 7 and 8 vaccines given, showed antibody titers against IB with a Geometric Mean (GM) from 1137 to 6612. The statistical analysis according to the number of applied vaccines, showed that there was a significant increase in antibody titers after 6 vaccines with an Arithmetic Mean (AM) of 2341; and again there was a significant increase of the AM to 4711 with 7 vaccines. However, with 8 vaccines, the AM of 4434 was statistically similar to the previous group. Statistical analysis of the hen sectors with 7 vaccines showed antibody titers against IB with an AM of 2887 to 6193, which statistically can be differentiated into two main groups. Sectors of hens with 8 vaccines, exhibited antibody titres with an AM in a range of 3799 to 5622, which also differ in two statistical different groups. It was concluded that antibody titers obtained from different flocks of hens are consistent with literature and technical guidelines for ELISA test. In addition, the diversity of antibody titers obtained among flocks or sectors of birds, with or without number of similar vaccines, may be influenced by temporary differences in the application of vaccines, structural differences of the facilities and building sectors, differences associated to vaccines, differences of management and vaccination staff, among others.

Oxytocin (OT) and vasopressin (AVP) are neuropeptides with diverse effects on social behavior, cognition and stress responses. Recent studies suggest that OT facilitates and responds to affiliative forms of human-animal interaction (HAI). However, previous studies measuring OT and AVP in dogs have been limited to measures from blood or urine, which present concerns related to the invasiveness of sample collection, the potential for matrix interference in immunoassays, and whether samples can be collected at precise time points to assess event-linked endocrine responses. Previous studies from our laboratory validated salivary measures of OT and AVP in dogs, however, it is currently unknown whether these measures respond dynamically to aspects of HAI. Here, we investigated the effects of affiliative forms of HAI on both plasma and salivary OT and AVP in dogs. We employed a within-and between-subjects design with a group of Labrador retrievers and Labrador retriever x golden retriever crosses (23 females, 15 males). Half of the dogs engaged in 10 min of free-form friendly interaction with a human experimenter (HAI condition), and the other half rested quietly in the same environment, without human interaction (control condition). We collected blood and saliva samples before, and immediately following both experimental conditions, and all samples were analyzed using enzyme-linked immunosorbent assays (ELISAs) following previously validated protocols. Dogs participating in HAI exhibited a significant increase in both salivary OT (+ 39%) and plasma OT (+ 5.7%) whereas dogs in the control group did not. Salivary AVP showed no change in the HAI group but increased significantly (+ 33%) in the control group. Plasma AVP decreased significantly following HAI (13%) but did not change across time in the control condition. Within the dogs exposed to HAI, increases in salivary OT, and decreases in plasma AVP, were predicted by the extent of affiliative behavior between the dog and human (indexed by scores from a principal components analysis of social behaviors between the dog and human). Collectively our results suggest that measures of salivary OT and AVP provide useful biomarkers in studies of HAI, and afford a flexible and non-invasive toolkit than can be employed in diverse research contexts.

Thesis advisor: Thomas C. Chiles / Rapid diagnosis of infectious disease at the site of the patient is critical for preventing the escalation of an outbreak into an epidemic. This is particularly true for cholera, a disease known to spread swiftly within resource-limited populations. A device suited to point-of- care (POC) diagnosis of cholera must not only demonstrate laboratory levels of sensitivity and specificity, but it must do so in a highly portable, low-cost manner, with a simplistic readout. Here, we report novel proof-of-concept lab-on-a-chip (LOC) electrochemical immunosensors for the detection of cholera toxin subunit B (CTX), based on two nanostructured architectures: the gold dendritic array, and the extended core coax (ECC). The dendritic array has an ~18x greater surface area than a planar gold counterpart, per electrochemical measurements, allowing for a higher level of diagnostic sensitivity. An electrochemical enzyme-linked immunosorbant assay (ELISA) for CTX performed via differential pulse voltammetry (DPV) on the dendritic sensor demonstrated a limit-of detection of 1 ng/mL, per a signal-to-noise ratio of 2.6, which was more sensitive than a simple planar gold electrode (100 ng/mL). This sensitivity also matches a currently available diagnostic standard, the optical ELISA, but on a miniaturized platform with simple electrical readout. The ECC was optimized and explored, undergoing several changes in design to facilitate sensitive LOC electrochemical detection. The ECC matched the off-chip sensitivity towards CTX demonstrated by a previous non-extended core coaxial iteration, which was comparable to a standard optical ELISA. In contrast to the previous coaxial architecture, the ECC is amenable to functionalization of the gold core, allowing for LOC detection. ECCs were functionalized using a thiolated protein G, and CTX was detected via an electrochemical ELISA. While this work is ongoing, the ECC shows promise as a platform for LOC electrochemical ELISA. The ability to potentially meet POC demands makes biofunctionalized gold dendrites and ECCs promising architectures for further development as LOC sensors for the detection of infectious disease biomarkers. / Thesis (PhD) — Boston College, 2018. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Due to the pandemic, research concerning SARS-CoV-2 became of the utmost importance. In this research, we aimed to find and synthesize a library of peptide epitopes that carry functional properties of the ACE-2 receptor binding to the virus protein for the purpose of creating a therapeutic treatment (i.e. viral inhibition). In order to do this, we used MST to determine binding affinity. After that, we validated the binding properties of our peptide epitopes and applied them as SARSCoV-2 antibody indicators using ELISA. We, then, functionalized gold nanoparticles with the peptide epitopes to assess its utility as a potential SARS-CoV-2 competitive inhibitor. From the set of peptides in the library, P25 showed the most functional properties in both MST and serological ELISA, while P1 successfully conjugated to the gold nanoparticles in different forms (PEG-P1, linker-P1, and mutated P1). Finally, P1 was validated to have antibody binding through sandwich ELISA. In the future, these findings can be applied to inhibit viral activity through drug delivery.

The aim of this master‘s thesis is to prepare a monoclonal antibody against cyclin-dependent kinase 13 (CDK13). The theoretical part focuses on antigen-antibody binding, which is essential for the use of monoclonal antibodies in the determination of CDK13 as well as the transcription that this kinase affects. This section is also devoted to Western blot and ELISA methods for detection of newly generated antibodies. Furthermore, the antibodies and the antigen definition are stated, which are later on discussed. The practical part is devoted to the preparation of antigen - its isolation and purification on a peristaltic pump. It also addresses immunization, its course, and the amount of antigen used to immunize mice. After immunization, the work focuses on fusion of sp-2 cells and splenocytes, which were first removed from the immunized mouse and purified. After the fusion alone, selection of hybridomas on HAT selection medium is mentioned, followed by detection first by ELISA and later by Western blotting. The resulting hybridomas with positive ELISA response are frozen for further testing at the Veterinary Research Institute in Brno, where the entire practical part of this thesis was carried out. These frozen hybridomas are further tested by immunoprecipitation to conclude this thesis.

Global lifespans are longer than ever before and there is an increasing shift towards a more aged global population. Also, the majority of severe disease and deaths in the recent and ongoing COVID-19 pandemic is found in individuals over 60 years of age. Therefore, there is an urgent need to gain insight into how the immune system changes with age; specifically: (1) what are the drivers of chronic, systemic inflammation (‘inflammaging’) that occur in some but not all older individuals and (2) how, in turn, numerical aging and chronic inflammation collide to impact anti-viral immunity and lead to poor infection outcomes. In this body of work, two focused research questions were addressed as part of an overarching goal to determine the links between numerical age, chronic inflammatory status, and immune cell function. First, the role of iNKT cells in the inflammation found with ART-suppressed HIV infection and aging was studied, and second, connections between age and pre-existing immunity to SARS-CoV-2 were investigated. Invariant natural killer T (iNKT) cells are a unique, innate-like T cell subset known to bridge innate and adaptive immune responses and can exert inflammatory and immunosuppressive effector functions. The role of iNKT cells in the chronic inflammation found with ART-suppressed HIV infection and/or normal aging is unclear. Therefore, iNKT cell frequencies and surface phenotypes were measured from a HIV and Aging cohort comprised of ART-suppressed HIV+ subjects and matched uninfected controls stratified by age into younger and older groups and iNKT cell signature were correlated with plasma markers of chronic inflammation. Specific characteristics of iNKT cells were associated with aviremic HIV infection and/or advanced age, and distinct links between iNKT cell signatures and markers of chronic inflammation were found. Further, multivariate analysis (PLS-DA) of the collected dataset revealed that iNKT cell and plasma markers stratified younger from older subjects within both the uninfected and aviremic HIV+ groups. Older age is arguably the strongest predictor of severe clinical outcomes and mortality after SARS-CoV-2 infection. The role of pre-existing, cross-reactive immunity in COVID-19 outcomes is unclear to date. A newly developed, highly sensitive serological assay (the BU ELISA) was used to elucidate links between pre-existing immunity to SARS-CoV-2 and age. We found SARS-CoV-2 receptor binding domain (RBD) and/or nucleocapsid protein (N) reactive antibodies (IgG, IgM, and/or IgA isotypes) in all pre-pandemic subjects tested, with a wide range in antibody levels. SARS-CoV-2 reactive immunoglobulin levels tracked with antibodies specific for analogous viral proteins from endemic coronavirus strains and were lowest in subjects over 70 years of age compared with younger counterparts. In sum, these findings provide evidence of lower pre-existing immunity to SARS-CoV-2 in elderly individuals, and this may account for their poor infection outcomes. In conclusion, the findings in this work provide new insight into the impact of age and chronic inflammation on productive and protective immune responses. These results underscore the need for further investigations into the immune cell mechanisms and inflammaging pathways that subvert healthy aging.

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