Aim: The aim of this thesis was to measure a large spectrum of biomarkers in serum or plasma of patients with operable stage of NSCLC and to evaluate and compare the clinical utility of these biomarkers in the three most important clinical applications for NSCLC: diagnosis, prognosis and postsurgery follow up care. Patients and methods: Total of 22 biomarkers with the most promising profiles were monitored: 8 standard tumor markers (cytokeratines Cyfra 21-1, TPA, TPS, and MonoTotal, CEA, SCC, TK, Chromogranin A) and 14 potential useful biomarkers including pro-inflammatory cytokines IL-6, IL-8, MCP-1, pro-angiogenic cytokine VEGF, matrix metaloproteinases MMP-1, MMP-2, MMP-7, MMP-9 and their inhibitors TIMP-1 and TIMP-2, adhesion molecules ICAM-1, VCAM-1, growth factor IGF-1, and PAI-1 stimulating tumor growth and angiogenesis. With a view of evaluating the clinical relevance of these markers for NSCLC we measured serum or plasma levels of these 22 markers in group of 93 patients with NSCLC undergoing radical surgery and in group of 20 patients with benign lung disease. For biomarker measurement were used conventional immunoanalytic routine methods (IRMA, REA, CLIA, MEIA, TRACE, ELISA) and multiplex immunoanalytic method. Results: Cyfra 21-1, MonoTotal, TPA, TPS, CEA, SCC, Chromogranin A, TIMP-1, MMP-1,...

O Kraft 36EC® (i.a. abamectina) e o Score 250EC® (i.a. difenoconazol) são agrotóxicos intensamente utilizados nas culturas de morango em regiões de clima tropical, embora sejam compostos classificados como extremamente tóxicos e muito perigosos ao ambiente. Misturas de agrotóxicos são aplicadas nas culturas agrícolas e a presença destes compostos na água pode potencializar os efeitos tóxicos a organismos aquáticos não alvos. A ocorrência do escorrimento superficial (runoff) contaminado com agrotóxico e a contaminação por aspersão direta (spray drift) em ecossistemas tropicais próximo aos corpos hídricos também podem comprometer o ecossistema aquático local devido à toxicidade desses compostos a organismos não alvos. Considerando os riscos ecológicos inerentes ao uso destes agrotóxicos, o objetivo principal desta pesquisa foi avaliar o efeito ambiental dos praguicidas Kraft 36EC® e Score 250EC® e de seus princípios ativos, abamectina e difenoconazol, respectivamente. Foram realizados testes de toxicidade aguda em laboratório a partir de exposições dos compostos isolados e em misturas utilizando como organismo teste embriões e adultos de Danio rerio. Experimentos in situ (mesocosmos) foram realizados a fim de avaliar os efeitos do runoff e spray drift contaminados com os agrotóxicos Kraft 36EC® e Score 250EC®, isolados e em misturas. Tanto nos mesocosmos quanto em experimentos de laboratório analisou-se, além da mortalidade, biomarcadores bioquímicos. Pelos resultados obtidos verifica-se que não foram observadas diferenças significativas entre a toxicidade isolada dos ingredientes ativos e suas respectivas formulações comerciais em testes agudos. Nos testes de toxicidade com as misturas dos ingredientes ativos, observou-se que a mistura de abamectina + difenoconazol promoveu um efeito tóxico sinérgico a adultos de Danio rerio em exposições agudas. Observou-se também a ausência do mesmo efeito nas exposições com as misturas das formulações comerciais neste mesmo estágio de vida. A exposição dos embriões às misturas de Kraft 36EC® e Score 250EC® mostrou um efeito antagônico nas baixas concentrações e efeito sinérgico nas mais altas. Verificou-se um aumento significativo na atividade da enzima de biotransformação 7-etóxiresorufina-0-deetilase (EROD) e nos níveis de malondialdeído MDA nas brânquias de peixes expostos à formulação comercial da abamectina. Houve um aumento na atividade da glutationa-S-transferase (GST), glutationa peroxidase (GPx) e níveis de MDA, e diminuição da glutationa redutase (GR) nas brânquias dos peixes expostos ao difenoconazol e sua formulação comercial. Também foi observado um aumento nas respostas dos biomarcadores analisados nas brânquias dos organismos expostos às misturas tanto dos princípios ativos quanto das formulações comerciais. A exposição ao Kraft 36EC® spray drift em campo promoveu os maiores efeitos deletérios no metabolismo de Danio rerio, seguido das exposições ao runoff contaminado com Kraft 36EC® e Score 250EC®. As formulações comerciais Kraft 36EC® e Score 250EC® e as misturas das mesmas promoveram alterações significativas no metabolismo de detoxificação, e causam estresse oxidativo em peixes. Diferenças no padrão de respostas dos biomarcadores entre os experimentos realizados em mesocosmos e em laboratório ficam evidentes devido à influência das concentrações utilizadas, das interações ecológicas entre as comunidades presentes no meio e das variáveis ambientais nos experimentos em mesocosmos. A avaliação da toxicidade de agrotóxicos, especialmente em países tropicais, e indicações para futuras pesquisas são discutidos. / Kraft 36EC® (a.s. abamectin) and Score 250EC® (a.s. difenoconazole) are intensely used pesticides in strawberry crops in tropical regions, even though they are classified as extremely toxic and very dangerous to the environment. Mixtures of agrochemicals are applied to agricultural crops and the presence of these compounds in water may potentiate toxic effects to non-target aquatic organisms. The contamination of edge-of-field water bodies through runoff and spray drift may compromise the local aquatic ecosystem due to the toxicity of these compounds to non-target organisms. Considering the potential ecological risks related with the use of these pesticides, the main objective of this research was to evaluate the environmental effects of the pesticides Kraft 36EC® and Score 250EC® and its active ingredients abamectin and difenoconazole, respectively. Acute toxicity tests were performed in the laboratory with the isolated compounds and their mixtures using zebrafish (Danio rerio) embryos and adults as test organism. A mesocosm experiment was also performed to evaluate the effects of simulated runoff and spray drift containing Kraft 36EC® and Score 250EC® individually and in mixtures. Besides lethality, selected biomarkers were also evaluated in the fish from the mesocosm and laboratory experiments. No significant differences were observed in effects of the active ingredients and their respective commercial formulations in acute fish tests after single exposure to each test compound. The acute mixture toxicity tests with the active ingredients showed that this mixture exerts a synergistic toxic effect to adult fish. The absence of the synergistic effect on exposures with the commercial formulations mixtures was also observed. Embryo exposure to Kraft 36EC® and Score 250EC® mixtures showed an antagonistic effect at low concentrations and synergistic effect at the highest. A significant increase was observed in the activity of the EROD biotransformation enzyme and in the levels of MDA in fish gills after exposure to Kraft 36EC®. An increase in GST activity, GPx and MDA levels, and a decrease of GR in fish gills was noted after fish exposure to difenoconazole and its commercial formulation. An increase in the responses of the biomarkers analyzed in the gills of the organisms exposed to the mixtures of both the active ingredients and the commercial formulations as compared to single-compound exposure was also observed. Exposure to simulated spray drift of Kraft 36EC® in the mesocosms promotes the greatest deleterious effects on metabolism of Danio rerio followed by exposures to the runoff contaminated with a Kraft 36EC® and Score 250EC® mixture. The commercial formulations Kraft 36EC® and Score 250EC® and their mixtures lead to significant changes in the detoxification metabolism resulting in oxidative stress to fish. Differences in biomarkers responses between the experiments performed in the mesocosms and the laboratory were noted and are due to the different concentrations tested; the ecological interactions between the communities present in the mesocosms and differences in environmental variables. The need and indications for future research related with the evaluation of pesticide toxicity, especially in tropical countries, are discussed.

Systemic sclerosis (SSc; scleroderma) is a chronic multisystem autoimmune disease that includes prominent skin involvement in all patients and is characterized by fibrosis, inflammation, and microvascular injury of the skin and internal organs. Clinical trial design for patients with systemic sclerosis (SSc) has been confounded by the heterogeneity of disease progression and lack of objective outcome measures. This has hampered identification of therapies for patients who have frequently fatal fibrotic complications. Direct pulmonary complications are the leading cause of death in SSc. For clinical trials in patients with diffuse cutaneous SSc, identification of a pharmacodynamic biomarker associated with clinical improvement would allow for alternative approaches to trial design. Furthermore, identification of a diagnostic biomarker for SSc complicated by pulmonary arterial hypertension (SSc-PAH) would provide a reliable non-invasive method for diagnosis of pulmonary arterial hypertension. Through the combination of high throughput technologies and clinical information we have identified three preliminary biomarkers for SSc: i) Two pharmacodynamic biomarkers for diffuse skin disease (dcSSc), one in using mRNA from skin biopsies and one using proteomic profiles from sera; ii) a serum based proteomic classifier for the screening and diagnostic evaluation of pulmonary arterial hypertension in systemic sclerosis. We show these biomarkers can be applied to assess changes in skin disease in dSSc patients over time and with further development could be used to supplement or replace the physical examination assessment (Modified Rodnan Skin Score, MRSS) as an outcome measure in clinical trials for dcSSc patients. Routine use of these biomarkers in SSc clinical trial design could expand treatment options for a patient population that currently has few if any treatment options that slow progression of disease. Furthermore we identified a serum biomarker for the major SSc pulmonary complication, SSc-PAH. This diagnostic SSc-PAH biomarker has the potential to be used as a screening tool in order to reduce the need for unnecessary invasive diagnostic procedures. This non-invasive screening method could lead to early diagnosis thus improving patient survival and aid in clinical management of a complication for which there are several treatments but which is still frequently fatal. / 2018-06-15T00:00:00Z

Persistent infection of the cervix with high risk (HR) types of Human Papilloma Virus (HPV) (HR-HPV) can result in precancerous lesions and cancers. However, most HPV infections can be cleared naturally by the immune response without causing disease. Although genetic variations have long been considered as the main explanation for individual heterogeneity in cancer susceptibility, the underlying mechanisms remain unclear. In this project, a panel of routinely taken clinical samples was assessed for 32 rationally selected SNPs with allele frequency related to disease outcome using the Taqman® OpenArray® system. The panel incorporated 475 HR-HPV negative, cytologically-normal cervical samples, 413 HR-HPV positive cervical high grade squamous intraepithelial lesion (HSIL) cases and 62 HR-HPV positive cervical cancers. Two SNPs, rs2234671 and rs2623047, were found with significant differences between HR-HPV negative, cytologically-normal samples and HR-HPV positive cervical HSIL cases. In the validation step, these two SNPs were further genotyped in the same set of samples using TaqMan® SNP genotyping assay and/or LightSNiP assay and in additional samples including 83 HR-HPV positive, cytologically-normal cervical samples, 21 HR-HPV positive cervical cancer cases, 129 HR-HPV positive vulval intraepithelial neoplasia cases and 23 HR-HPV positive vulval cancer cases. Statistical analysis was then performed based on pooled and re-grouped genotyping data of the above-mentioned samples under different genetic models so as to evaluate the associations with different stages in the disease process. After validation, SULF1 rs2623047 revealed a strong significant association with the susceptibility to HR-HPV infection but not with the development of high-grade squamous intraepithelial lesion and the progression to cervical cancer. CXCR1 rs2234671, by contrast, was associated with the progression of HR-HPV-related cancers and the minor allele CXCR1 827C was significantly enriched in HPV16 positive cancers. CXCR1 is a receptor for the chemokine CXCL8/IL-8 and CXCR1 rs2234671 leads to a serine to threonine change in an extracellular loop of the receptor. Functionally, the CXCR1 827C allele was shown to enhance cell motility in response to IL-8 stimulation in a chemotaxis assay with transiently transfected fibroblasts (HEK293 cells) and also in a wound healing assay with stably transduced cervical cancer (CaSki) cells. In addition, significantly increased cell proliferation upon IL-8 treatment was observed in two cervical cancer derived cell lines, CaSki and SiHa, transduced with CXCR1-827C allele, but not in their CXCR1 827G transduced counterparts. These findings suggest that SULF1 rs2623047 and CXCR1 rs2234671 may be genetic risk factors for HR-HPV-related cervical disease and CXCR1 rs2234671 might affect HR-HPV-related cancer susceptibility by functionally altering IL-8-CXCR1 signalling. This information has potential for use in the risk stratification of HR-HPV infected women and may also suggest new therapeutic targets to be exploited for treatment of cervical cancer patients.

There has been much interest in recent years about the potential of microRNA as a new source of biomarkers for the diagnosis of disease. The delivery of new diagnostic tools based on this potential has been limited by shortcomings in current microRNA detection techniques. This thesis explores the development of a new method of microRNA detection through the incorporation of conductive particles into oligonucleotide-functionalised polymers to form oligonucleotide cross-linked polymer composites. Such composites could provide a simple, rapid, and low-cost means of microRNA detection that could be easily multiplexed, providing a valuable tool for point-of-care medical diagnostics. This work presents oligonucleotide-functionalised carbon/polyacrylamide composites which demonstrate a selective swelling response in the presence of analyte oligonucleotide sequences and for which the electrical conductivity decreases with swelling. The composites were synthesised via UV-initiated free-radical polymerisation of carbon/- monomer mixtures upon custom electrode devices, consisting of interdigitated platinum electrodes fabricated upon a silicon substrate. The optimal cross-linker density and carbon loading concentration were determined as well as the best means of dispersing the carbon particles within the polymer. Various types of carbon particles, with differing sizes and aspect ratios, were compared and their performances as conductive additives for polymer swelling transduction evaluated. The swelling behaviour of these composites was evaluated by analysing images of composite microdroplets as they swell. The electrical characteristics of the composites were determined by measuring either the two-terminal resistance or the complex impedance of composite microdroplets on the electrode devices. Alternating and direct current measurement techniques were compared to determine the best approach for the transduction of composite swelling. The volumetric and electrical responses of oligonucleotide-functionalised carbon/polyacrylamide composites were analysed in solutions of analyte oligonucleotide and non-complementary controls. It has been demonstrated that, using carbon nanopowder composites and a direct current two-terminal resistance measurement, it is possible to differentiate between analyte and control solutions to concentrations as low as 10 nM, with single-base precision, in less than three minutes. However, the inability to detect at concentrations below this value, difficulties in differentiating between different analyte concentrations and thermal instability mean that, in their current form, oligonucleotide cross-linked polymer composites are unsuitable for the detection of circulating microRNA at clinically relevant concentrations. Potential avenues of work to address these challenges are discussed. Also presented are collaborative results for oligonucleotide-responsive polymers functionalised with morpholino nucleic acid analogues, in what is believed to be the first example of such a material. These morpholino-functionalised polymers offer significant advantages, in terms of stability and sensitivity, over their nucleic acid equivalents for bio-responsive polymer applications.

Philosophiae Doctor - PhD / Tuberculosis (TB) is an infectious disease with adverse effect on a global scale. The disease is one of the major causes of death in sub-Saharan Africa. Nearly 70% of TB-infected persons are co-infected by the human immunodeficiency virus (HIV). About 50% of TB/HIV patients are smear negative and up to 28% are sputum scarce, which is a significant problem in South Africa since sputum smear microscopy is the most widely used diagnostic test for TB. The detection of Mycobacterium tuberculosis (MTB) and resistance to the TB drug rifampicin (RIF) are the basis of the GeneXpert MTB/RIF protocol. The GeneXpert MTB/RIF is an automated nucleic acid amplification technique for detecting the DNA that originates from MTB. However, low sensitivity and low concentrations of MTB for DNA amplification are a serious issue associated with the protocol. Therefore, other TB diagnostic methods, such as the ones involving biochemical markers of TB, are becoming very important. / 2020-08-31

Biomarcadores tem se tornado uma parte essencial da pesquisa clínica. Na psiquiatria, diversos biomarcadores séricos têm sido estudados. No Transtorno Bipolar, considerável atenção tem sido dada a marcadores inflamatórios e entre esses destacam-se as citocinas. No entanto, o entendimento dos diferentes padrões de envolvimento desses marcadores ainda não foi suficientemente estudado. Também, até o momento, poucos foram os estudos que avaliaram seu papel como marcadores de atividade ou preditores de curso de doença. A presente tese é composta por dois estudos: 1) uma comparação de citocinas inflamatórias entre pacientes eutímicos com um diagnóstico de Transtorno Bipolar em relação a pacientes com Esquizofrenia e controles saudáveis. 2) um estudo prospectivo de pacientes com Transtorno Bipolar avaliando medidas de citocinas no baseline em relação a variáveis clínicas de um ano de seguimento. Como um todo, os resultados apresentados corroboram o involvimento de citocinas inflamatórias no Transtorno Bipolar e na Esquizofrenia, no entanto evidenciando um padrão diferenciado de envolvimento nos dois transtornos. Também a IL-6 mostrou-se alterada em pacientes eutímicos que viriam a apresentar um ou mais episódios depressivos durante um ano de seguimento e seu aumento correlacionou-se com o numero de dias com sintomas depressivos. Esses achados fornecem apoio adicional para a investigação de citocinas como possíveis biomarcadores para a atividade da doença ou preditores de recorrência. / Biomarkers are becoming an essential part of clinical research. In Psychiatry, several serum biomarkers are currently being studied. In Bipolar Disorder, increasing attetion has been given to inflammatory markers and among these cytokines has received special attention. However, the understanding of the different patterns of inflammation has not yet been fully clarified. Few studies have focused on its properties as markers of disease activity and predictors of outcome. This thesis contains two different studies: 1) a comparison of cytokine levels in patients with Bipolar Disorder, Schizophrenia and healthy controls. 2) a prospective study of euthymic patients with Bipolar Disorder assessing baseline cytokines and theis association with clinical variables during follow-up. These studies reinforce the idea of an inflammatory response in both Bipolar Disorder and Schizophrenia; however, the pattern of reponse seems to be different between the two disorders. Also, increased levels of IL-6 were observed in euthymic patients that would later present a depressive episode during follow-up and this increase was correlated with the numbers of days depressed. These findings five further support for the investigation of cytokines as possible biomarkers of disease activity and predictors of recurrence.

A esquizofrenia (SZ) é uma doença mental crônica e grave, que compromete o funcionamento psicossocial do indivíduo nos mais variados graus. Atinge 1% da população mundial, considerando todo seu espectro de sintomas (DSM-IV). O pobre funcionamento cognitivo é um dos principais fatores que explicam as elevadas taxas de prejuízos e encargos associados à esquizofrenia. A etiologia da SZ é desconhecida, tendo muitas hipóteses etiológicas como fatores genéticos, epidemias virais durante a gestação, época de nascimento, traumatismos de parto, infecções perinatais, condições neurológicas ou neuropsiquiátricas que geram sintomas tipo esquizofrênicos ou desenvolvimento anormal (avaliados por testes psicológicos, estudos de neuroimagem e neuropatológicos que sugerem alterações no desenvolvimento cerebral). A fisiopatologia da SZ pode ser resultante de uma desregulação na plasticidade sináptica por alterações de neurotofinas, radicais livres e processos inflamatórios. Existe uma larga evidência que os radicais livres podem ter um papel importante na fisiopatologia da SZ, podendo induzir danos na membrana celular, em proteínas e DNA. Problemas com estresse oxidativo, como o aumento da peroxidação lipídica foram relatados previamente em pacientes com SZ em primeiro episódio, virgens de tratamento e naqueles cronicamente medicados. As citocinas inflamatórias têm sido estudadas como importantes participantes na etiologia e desenvolvimento das doenças psiquiatricas. Seu papel ainda não é bem estabelecido, porém diversas alterações têm sido vistas nas doenças psiquiátricas. Dentre as citocinas, destacam-se as interleucinas (IL) e o fator de necrose tumoral alfa (TNF-α), que podem ter ação inflamatória e anti-inflamatória. Dentre as próinflamatórias, podemos destacar a IL-6 e o TNF-α. Alteração de IL na SZ tem sido relatada nesses últimos anos, relacionada à etiologia e à atividade da doença. Pacientes em episódio agudo da doença apresentaram aumento dos níveis séricos de IL pró-inflamatórias sugerindo atividade inflamatória sistêmica. Identificar, além dos sintomas clínicos, possíveis alterações bioquímicas e de neuroimagem em pacientes com SZ pode ajudar em futuras intervenções tanto para identificar, como para prevenir ou atenuar o curso da SZ. Estudos que permitam avançar no entendimento da psicopatologia deste grupo de pacientes são de grande importância, na medida em que proporcionarão futuras abordagens terapêuticas. Está bem estabelecido que a matéria cinzenta cortical e o volume de córtex préfrontal estão diminuídos em pacientes com SZ. Entretanto, os fatores que levam à perda de tecido não estão claras. Uma hipótese para esse fato é que o estado próinflamatório aumentado em SZ está relacionado com a diminuição volumétrica da massa cinzenta. O objetivo deste estudo piloto foi correlacionar os níveis séricos de IL-6 com o volume cortical total de pacientes com SZ e controles. Foram selecionados 36 pacientes com SZ (28 do sexo masculino, com idade média de 37,17 ± 12,05; anos de doença 15,56 ± 11,75), 35 controles pareados idade (21 do sexo masculino, idade média= 36,97 ± 13,04). As imagens foram adquiridas por um equipamento de ressonância magnética Philips Achieva 1.5T no Hospital de Clínicas de Porto Alegre, Brasil. Todas as imagens foram processadas usando o pipeline automatizado de FreeSurfer v5.1. Concluímos que a IL-6 está negativamente correlacionada com o volume cortical total (p= 0,027; rho= -0,370) nos pacientes com esquizofrenia, tal correlação não foi vista nos controles (p= 0,235, rho= -0,206). Nosso resultado sugere que a ativação inflamatória crônica em pacientes com SZ pode estar relacionada com a diminuição volumétrica total do córtex. / Schizophrenia (SZ) is a chronic and severe mental illness, which affects the psychosocial functioning of the individual in many degrees. It reaches 1% of the population, considering all its spectrum of symptoms (DSM-IV). Poor cognitive functioning is one of the main factors responsible for the high rates of disability and costs associated with schizophrenia. The etiology of SZ is unknown, and many etiological assumptions are taken, as genetic factors, viral epidemics during pregnancy, time of birth, birth trauma, perinatal infections, neuropsychiatric or neurological conditions that produce symptoms like schizophrenia or unnatural development (assessed by psychological tests, neuroimaging and neuropathological studies that suggest changes in brain development). The pathophysiology of SZ may be due to a deregulation in synaptic plasticity caused by changes in neurotrophins, free radicals and inflammatory processes. There is a wide evidence that free radicals may have a main role in the pathophysiology of SZ, and can induce damage into the membrane cell, in proteins and DNA. Problems with oxidative stress, such as increased lipid peroxidation have been previously reported in treatment virgem patients with SZ in first episodes and in those chronically treated. And inflammatory cytokines have been studied as important parts in the etiology of psychiatric diseases’ development. Its role is not well established, however a number of changes have been noticed in psychiatric illnesses. Among the cytokines, the Interleukins (IL) and the tumor necrosis factor alpha (TNF-α) stand out, these two may have inflammatory and anti-inflammatory action. Among the pro-inflammatory, we can highlight IL-6 and TNF-α. IL change in the SZ has been reported in these last few years, related to the etiology and disease activity. Patients with acute episode of the disease showed increased serum levels of IL proinflammatory suggesting systemic inflammatory activity. Identify not only the clinical symptoms, possible biochemical and neuroimaging abnormalities in patients with SZ can help in future interventions both to identify and prevent or slow down the course of SZ. Studies to enable progress in the understanding of psychopathology this group of patients are of great importance to the extent that provide future therapeutic approaches. It is well established that cortical gray matter and the prefrontal cortex volume are reduced in patients with SZ. However, the factors that lead to tissue loss are unclear. One possible explanation is that the increased proinflammatory state in SZ is related to the volumetric reduction of the gray matter. The objective of this pilot study was to correlate serum levels of IL-6 in the hole cortex volume of schizophrenic patients and controls. We selected 36 patients with SZ (28 male, average age 37.17±12.05; years of illness 15.56±11.75), 35 matched controls (21 male, average age= 36.97±13.04). Images were obtained by an MRI equipment, brand Philips Achieva 1.5T at Hospital de Clinicas de Porto Alegre, Brazil. All images were processed using automated pipeline FreeSurfer v5.1. We concluded thatIL-6 is negatively correlated with the total cortical volume in patients (p= 0.027, rho= -0.370), this correlation was not seen in controls (p= 0.235; rho= -0.206). Our results suggest that chronic inflammatory activation in patients with SZ can be related to the total volumetric reduction of the cortex.

Synapses are an early pathological target in a wide range of neurodegenerative conditions including adult-onset Alzheimer’s and Parkinson’s, and diseases of childhood such as spinal muscular atrophy and neuronal ceroid lipofuscinoses (NCLs). However, our understanding of the mechanisms regulating the stability of synapses and their exceptional vulnerability to neurodegenerative stimuli remains in its infancy. To address this, we have used the NCLs to model the molecular alterations underpinning synaptic vulnerability. Our primary objective is to identify novel regulators of synaptic stability as well as highlight novel therapeutic targets which may prove effective across multiple neurodegenerative conditions where synapses are an early pathological target. The NCLs, are the most frequent autosomal-recessive disease of childhood. There are currently 14 individual genes whose mutations result in similar phenotypes including blindness, cognitive/motor deficits, seizures and premature death. This suggests that despite the difference in the initiating mutation and the degenerative processes across this collective group are likely to impact on overlapping pathways. Focusing on two murine models of NCL; one with an infantile onset - CLN1 disease (Ppt1-/-) and one with a juvenile onset - CLN3 disease (Cln3-/-) we made use of the temporo-spatial synaptic vulnerability pattern in these mice to plan proteomic and in silico analyses. This pipeline was utilised to identify perturbed protein candidates and pathways correlating with differential regional synaptic vulnerability. This ultimately allowed the generation of a list of candidate proteins, some of which were relevant to human NCL as they were altered in post mortem brain samples. Interestingly, many of the correlative candidates also appear to show conserved alterations in both NCL forms examined and other neurodegenerative diseases. Next, candidates were genetically and/or pharmacologically targeted to study their modulatory effects on neuronal stability in vivo. This was done using CLN3 Drosophila as a rapid screening assay and led to the successful characterisation of a subset of candidates as either enhancers or suppressors of the CLN3-induced phenotype in vivo. As well as identifying regulators of neuronal stability, following a similar pipeline, we identified a set of putative biomarkers of disease progression in muscle and blood in the Ppt1- /- mice, a subset of which appeared conserved in Cln3-/- mice. One of these conserved candidates presented the same directionality of change in human post mortem brain samples, indicating its relevance to the human NCL. Following this workflow from spatio-temporal profiling of murine synaptic populations, to in silico analyses and in vivo phenotypic assessment, we demonstrate that we can identify multiple protein candidates capable of modulating neuronal stability in vivo and identified putative biomarkers that tracked disease progression.

Submitted by João Leandro Chaguri null (joaoleandro@ibb.unesp.br) on 2016-04-12T17:43:06Z No. of bitstreams: 1 Dissertação João Leandro.pdf: 2543552 bytes, checksum: 518f71110e7d2f26f7fab66e56f7a3b7 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-04-13T18:42:12Z (GMT) No. of bitstreams: 1 chaguri_jl_me_bot.pdf: 2543552 bytes, checksum: 518f71110e7d2f26f7fab66e56f7a3b7 (MD5) / Made available in DSpace on 2016-04-13T18:42:12Z (GMT). No. of bitstreams: 1 chaguri_jl_me_bot.pdf: 2543552 bytes, checksum: 518f71110e7d2f26f7fab66e56f7a3b7 (MD5) Previous issue date: 2016-03-04 / O fipronil é um inseticida fenilpirazol amplamente utilizado na Medicina Veterinária, na agricultura e domesticamente para o controle de pragas. O mecanismo responsável pela sua ação tóxica consiste na inibição seletiva dos receptores do ácido gama amino butírico GABA associado a canais de cloreto. Estudos recentes mostram que fipronil afeta organismos não-alvo, incluindo populações de espécies ambiental e, potencialmente, os seres humanos. O presente estudo verificou se a exposição a fipronil afeta a pressão arterial sistólica e biomarcadores relacionados. Assim, fipronil foi administrado oralmente a ratos (30 mg / kg / dia) durante 15 dias (grupo Fipronil) ou solução fisiológica (grupo controle). Fipronil aumentou significativamente a pressão arterial sistólica (158 ± 13 mmHg), em relação ao grupo controle (127 ± 3 mmHg). Significativamente, níveis mais elevados de fipronil no plasma foram observados no grupo Fipronil (0,46 ± 0.09μg / mL contra 0,17 ± 0.11μg / mL no grupo de controle). O grupo exposto ao Fipronil apresentou ganho de peso menor em comparação com o grupo controle. Enquanto o fipronil resultou num aumento das concentrações de endotelina-1, a redução da capacidade antioxidante e níveis menores de metaloproteinase de matriz 2 (MMP-2) e Nitrito/Nitrato em relação ao grupo controle, não foram observadas alterações em marcadores séricos renal e hepáticos. Portanto, este estudo sugere que o fipronil provoca hipertensão e a endotelina-1 desempenha um papel importante. Além disso, estes resultados sugerem que as reduções de tanto a MMP-2 e NO podem contribuir com a elevação da pressão sanguínea sistólica observada com fipronil. / Recent reports show that fipronil affects non-target organisms, including environmental species populations and potentially humans. We aimed to examine if fipronil exposure affects the systolic blood pressure and related biomarkers. Thus, fipronil was orally administered to rats (30 mg/kg/day) during 15 days (Fipronil group) or physiological solution (Control group). While fipronil increased significantly the systolic blood pressure (158 ± 13 mmHg), no significant changes were observed in Control group (127 ± 3 mmHg). Significantly, higher levels of fipronil in plasma were observed in Fipronil group (0.46 ± 0.09 μg/mL versus 0.17 ± 0.11 μg/mL in Control group). Fipronil group showed lower weight gain compared with Control group. While fipronil resulted in higher concentrations of endothelin-1, reduced antioxidant capacity and lower levels of circulating matrix metalloproteinase 2 (MMP-2) and nitric oxide (NO) metabolites compared to Control group, no alteration was observed in serum biomarkers of renal and hepatic/biliary functional abilities. Therefore, this study suggests that fipronil causes hypertension and endothelin-1 plays a key role. Also, these findings suggest that reductions of both MMP-2 and NO may contribute with the elevation of systolic blood pressure observed with fipronil.