Introdução: Os estudos com proteômica especificamente relacionada à nefrologia e urologia pediátrica são limitados. O diagnóstico do comprometimento da função renal e da sua deterioração na presença de uropatias obstrutivas congênitas (UOC) representa o desafio na rotina da nefro-urologia pediátrica. Novos biomarcadores com o potencial para detecção precoce da lesão renal surgiram recentemente, permitindo a escolha da melhor opção terapêutica no tempo hábil, e assim minimizando ou prevenindo o dano renal definitivo. Objetivos: Avaliar o perfil de dois biomarcadores renais séricos: creatinina (CrS) e cistatina C (CyCs); e seis biomaracadores renais urinários: lipocalina associada à gelatinase neutrofílica (NGAL), proteína ligadora de retinol (RBP), molécula de injúria renal 1 (KIM-1), cistatina C na urina (CyCu), fator transformador de crescimento-beta 1 (TGF-beta1) e microalbuminúria (uALB) durante o primeiro ano de vida em lactentes saudáveis; em relação à detecção precoce da lesão renal em lactentes com UOC; em relação à sua capacidade de prever a necessidade de intervenção cirúrgica em lactentes com UOC. Metodologia: 37 lactentes com UOC foram divididos em três subgrupos: 14/37 casos com hidroureteronefrose unilateral (HU), 13/37 com hidroureteronefrose bilateral (HB) e 10/37 com obstrução de vias urinárias baixas (OTUB); e comparados com 24 lactentes saudáveis. No grupo dos pacientes, as amostras de sangue e urina foram obtidas ao nascer e entre o 3º e 7º dia, 1º, 2º, 3º, 6º, 9º e 12º mês de vida. Grupo de controle seguia o mesmo cronograma, com exceção da coleta de sangue que ocorria ao nascer, entre o 3º e 7º dia, no 6º e 12º mês de vida. Todas as amostras foram armazenadas sob - 70 ºC, e analisadas posteriormente através de imunoensaio enzimático quantitativo (ELISA). Resultados: No grupo-controle, CrS, CyCs, CyCu e RBP refletiram a maturação glomerular e tubular. O ritmo de filtração glomerular pela CyCs atingiu os níveis estáveis no 6º mês de vida (93 ± 22 mL/min/1,73 m2). KIM-1 e TGF-beta1 mantiveram os níveis absolutos próximos ao limite de detecção pelo método. Os valores do NGAL no sexo feminino foram significativamente maiores (p=0,005) ao longo do 1º ano, quando comparados aos do sexo masculino. Em comparação aos controles, a coorte dos pacientes apresentou valores mais elevados para todos os biomarcadores urinários no 1º mês de vida (p <= 0,009), sendo que NGAL (p=0,005), TGF-betsa1 (p < 0,001) e ?ALB (p < 0,001) mostraram-se elevados desde o nascimento, em comparação aos controles. O RBP apresentou o melhor desempenho no subgrupo com HB e OTUB (AUC=0,844, sensibilidade >=83,3%, especificidade 94,3%), assim como o KIM-1 no HU (AUC=0,768, sensibilidade 70,7%, especificidade 82,7%). RBP em combinação com TGF-ß1 ou KIM- 1 e NGAL com CyCs e CyCu, atingiram os melhores resultados para detecção da lesão renal (AUC=0,934, sensibilidade 89,4%, especificidade 92,8%; AUC=0,896, sensibilidade 86,8%, especificidade 81,1%; AUC=0,867, sensibilidade 92,4%, especificidade 79,5%, respectivamente). Nos pacientes operados, os níveis elevados de RBP (p <= 0,043), NGAL (p <= 0,043), KIM-1 (p <= 0,03) e TGF-beta1 (p <= 0,034) baixaram significativamente após a cirurgia, no subgrupo com HU e OTUB. NGAL, isolado ou em combinação, com CyCs e CyCu demonstrou o melhor desempenho para determinar a necessidade cirúrgica (AUC=0,801, sensibilidade 63,6%, especificidade 96,7%; AUC=0,881, sensibilidade 87,7%, especificidade 82,2%, respectivamente). A analise do perfil dos biomarcadores indicou a necessidade da intervenção cirúrgica em 55,4% (7/13) dos casos não-operados e antecipou a decisão cirúrgica no mínimo 3 meses, em 58% (14/24) de todos os pacientes operados, baseada nas diretrizes atuais. Conclusão: A evolução dos valores normais dos biomarcadores no primeiro ano de vida, pode servir como a base para os próximos estudos de detecção precoce de afecções uro-nefrologicas. RBP, NGAL, KIM-1, TGF-beta1 e CyC, individualmente ou em combinação, demonstraram um forte potencial para identificar a lesão renal e servir como uma ferramenta de diagnóstico não-invasivo para diferenciar pacientes que necessitam de intervenção cirúrgica precoce daqueles que se beneficiariam de uma conduta conservadora / Introduction: The proteomics studies specifically related to pediatric nephrology and urology are limited. The diagnosis of renal function impairment and deterioration in congenital obstructive uropathies (COU) represents challenge in pediatric nephrourology routine. New renal biomarkers applied in this setting have potential for early renal injury detection, allowing reliable choice of optimal therapeutic options and thus preventing or minimizing definitive renal damage. Objectives: To analyze the first-year profiles of two serum renal biomarkers: Creatinine (CrS) and Cystatin C (CyCs); and six urinary renal biomarkers: Neutrophil Gelatinase-Associated Lipocalin (NGAL), Retinol- Binding Protein (RBP), Kidney Injury Molecule-1 (KIM-1), urine Cystatin C (CyCu), Transforming Growth Factor Beta 1 (TGF-beta1), and microalbuminuria (uALB) in a cohort of healthy infants; in relation to early detection of renal injury capability in a group of infants with COU; in relation to capability of predicting the need for surgery in a group of infants with COU. Methods: 37 infants with COU were divided in 3 subgroups: 14/37 cases with unilateral hydro(uretero)nephrosis (UH), 13/37 with bilateral hydro(uretero)nephrosis (BH) and 10/37 patients with lower urinary tract obstruction (LUTO), compared with 24 healthy infants matched by gestational age and birth weight. In the patient group, blood and urine samples were collected at birth, between 3rd-7th day, at 1st, 2nd, 3rd, 6th, 9th and 12th month of age. In the control group urine sampling followed the same routine with exception that blood sampling was obtained between 3rd-7th day, at 6th and 12th month of age. The samples were stored at -70 ºC, and thereafter analyzed by quantitative enzymatic immunoassay (ELISA). Results: In the group of healthy controls, the values of CrS, CyCs, CyCu and RBP reflected glomerular and tubular maturation. The glomerular filtration rate by CyCs reached steady-state levels at 6th month of life (93 ± 22 mL/min/1,73 m2). KIM-1 and TGF-beta1maintained very low absolute levels, near to the limit of detection by the method. NGAL levels in females were significantly higher (p=0,005) throughout the first year of life, when compared to male gender. In the cohort of patients, all the urinary biomarkers showed significantly higher values at the first month of life (p <= 0,009), while NGAL (p=0,005), TGF-beta1(p < 0,001) e uALB (p < 0,001) were high since birth, compared to control group. The best single biomarker performance was achieved by RBP in BH and LUTO subgroups (AUC=0,844, sensitivity >= 83,3%, specificity 94,3%), and by KIM-1 in UH subgroup (AUC=0,768, sensitivity 70,7%, specificity 82,7%). The best biomarker combination results for all subgroups were obtained by matching RBP with TGF-beta1 or KIM-1 and NGAL with CyC (AUC=0,934, sensitivity 89,4%, specificity 92,8%; AUC=0,896, sensitivity 86,8%, specificity 81,1%; AUC=0,867, sensitivity 92,4%, specificity 79,5%, respectively). In the operated group of patients, the levels of RBP (p <= 0,043), NGAL (p <= 0,043), KIM-1 (p <= 0,03) e TGF-beta1 (p <= 0,034) dropped significantly after surgery, in UH and LUTO subgroups. NGAL alone or in combination with CyCs and CyCu, demonstrated the best performance to determine the need for surgery (AUC=0,801, sensitivity 63,6%, specificity 96,7%; AUC=0,881, sensitivity 87,7%, specificity 82,2%, respectively). Biomarkers\' profile analysis indicated the need for surgical intervention in 55,4% (7/13) of non-operated cases and anticipated clinically based surgical decision for at least 3 months, in 58% (14/24) of all operated patients. Conclusions: The presented biomarkers\' normal values evolution during the first year of life can be of use as a base for future studies that will involve early detection of uronephrological disorders in infants. RBP, NGAL, KIM-1, TGF-beta1 and CyC, alone or in combination, demonstrated strong capability to identify renal injury and serve as a noninvasive diagnostic tool for differentiating between infants that require early surgical intervention from those who would benefit from conservative approach

Há poucos estudos relacionados com a caracterização dos componentes biológicos presentes no material particulado atmosférico, chamados bioaerossóis. Esses componentes biológicos podem assumir uma relação direta com a deflagração de doenças e também estão relacionados com os processos climáticos, em especial como núcleos de formação de gelo. Dentre os bioaerossóis, foram estudados, neste trabalho, os fungos anemófilos, sendo os principais os Basidiomycota, Ascomycota e os Fungos mitospóricos. O objetivo desse trabalho foi estimar a contribuição dos aerossóis fúngicos para a composição biogênica do material particulado atmosférico da Região Metropolitana de São Paulo. Foram realizadas medidas em dois sítios: na Cidade Universitária e no Pico do Jaguará. Para isso, foram caracterizados os tipos fúngicos utilizando-se um microscópio ótico e estimada a massa dos aerossóis de origem fúngica utilizando-se biomarcadores. Foram realizadas coletas durante 2013, 2014 e 2015 com o amostrador Burkard 7-day Recording Sampler, sendo que em 2015 também foram realizadas coletas com filtros de quartzo com o amostrador Airmetrics MiniVol portable Sampler. Foram identificados e obtida a concentração dos tipos fúngicos observados na atmosfera da RMSP, sendo encontrados 39 grupos principais de fungos, sendo os principais os Basidiomycotas. A concentração média total foi de 5736 (± 2459) esporos/m³ por dia. Os Ascomycota, Basidiomycota e os fungos mitospóricos se correlacionaram com as variáveis meteorológicas de forma diferente. Observou-se variação da concentração de esporos durante os diferentes períodos do dia, sendo que a maior concentração de esporos ocorreu na madrugada, possivelmente devido às condições meteorológicas (elevada umidade e temperaturas mais amenas), contudo a concentração dos fungos mitospóricos foi maior durante o período da tarde, principalmente devido aos mecanismos de liberação de esporos. No verão e na primavera foram obtidas as maiores concentrações de Ascósporos e Basidiósporos e no inverno e outono foram obtidas as maiores concentrações dos fungos mitospóricos. Além disso, observou-se uma correlação positiva e significativa entre a concentração de esporos totais e o material particulado inalável (MP10), indicando que ambos são influenciados da mesma forma pelas variáveis meteorológicas e/ou tem uma fonte em comum, como por exemplo a ressuspensão da poeira do solo. O Arabitol, Manitol, Treitol e os esporos de fungos são positivamente correlacionados, indicando que os três açucares podem ser traçadores de esporos de fungos na atmosfera. Utilizando-se um fator de conversão proposto na literatura foi possível estimar que 2% da concentração do MP10 e 8% da concentração do OC estão associados aos esporos de fungos, indicando a importância dos esporos fúngicos. Com o uso de modelos receptores foram observadas 6 fontes para o MP10: queima de biomassa resultante de processos industriais; queima de biomassa resultante da queima da vegetação; aerossóis fúngicos; formação de aerossol secundário; ressuspensão do solo e emissão veicular. Esses resultados constituem um avanço para a pesquisa em bioaerossóis no Brasil, já que muitas dessas análises ainda não haviam sido realizadas em áreas urbanas no Brasil / Although the biogenic components of Particulate Matter (PM) can have a direct relationship with the outbreak of respiratory diseases and can be linked to changes in climate processes there are very few studies related to its characterization in the atmospheric aerosols. Among the bioaerosols, the airborne fungi were studied, and the Basidiomycota, Ascomycota and Mitosporic fungi were the main characterized. The goal of this work was to estimate the contribution of the fungal aerosol to the RMSP atmosphere, with samplings in two sites: Cidade universitária (USP) and Pico do Jaraguá (PJ). For that, the fungi types were characterized using an optical microscope and it was estimated the mass of the fungal aerosol using biomarkers. Sampling was carried out during 2013, 2014 and 2015 with the \"Burkard 7-day Recording Sampler\", and in 2015, filters were sampled with the \"Airmetrics MiniVol portable Sampler. It was identified and determined the concentration of the fungi types observed in the RMSP atmosphere, and were found 39 main groups of fungi. The main group was the Basidiomycota. The mean concentration of the total fungi was 5736 (± 2459) spores/m³ per day. The Ascomycota, Basidiomycota and the mitosporic fungi correlated in different ways with the meteorological variables. A variation of the spores concentration during different times of the day was observed, with the occurrence of the highest concentration of spores at dawn, possibly due to weather conditions (high humidity and cooler temperatures), but the concentration of the mitosporic fungi was higher during the afternoon, mainly due to the spores release mechanisms. In the summer and spring were observed higher concentrations of Ascospores and Basidiospores and in the autumn and winter were obtained higher concentration of Mitospores. Besides that, it was also observed a positive and significant correlation between the fungal spore with the particulate matter, indicating that both are influenced in the same way by weather variables and/or has a common source such as soil resuspension. Arabitol, Mannitol, Threitol and the fungi spores are positive correlated, indicating that these three sugar alcohols can be a tracer for fungi spores in the atmosphere. Using the conversion factor proposed in the literature was possible to estimate that 2% of the PM10 concentration and 8% of the OC concentration it is due to fungi spores, indicating its importance. Using receptor modeling it was identified six sources for PM10, the biomass burning resulting from industrial processes, the biomass burning resulting from vegetation burning, the fungal aerosol, the secondary formation aerosol, the soil resuspension and vehicular emission. These results are a breakthrough for research on bioaerosols in Brazil, since many of this analyzes had never been performed in urban areas in Brazil

Submitted by Sandro Camargo (sandro.camargo@unipampa.edu.br) on 2015-03-08T18:38:21Z No. of bitstreams: 1 117110031.pdf: 1697065 bytes, checksum: 988fb2b0e3a730eaccfd605f9a8beb51 (MD5) / Made available in DSpace on 2015-03-08T18:38:21Z (GMT). No. of bitstreams: 1 117110031.pdf: 1697065 bytes, checksum: 988fb2b0e3a730eaccfd605f9a8beb51 (MD5) Previous issue date: 2013-07-19 / A leishmaniose visceral (LV) é uma zoonose parasitária com distribuição cosmopolita, causada pelo protozoário Leishmania spp. Diversos sistemas orgânicos podem ser afetados diretamente ou indiretamente pelo parasitismo, resultando em aspectos clínicos variáveis e inespecíficos em cães. Relatos isolados com indícios histológicos de acometimento miocárdico em cães com LV sugerem comprometimento deste sistema. Desta forma, objetivou-se com o presente estudo, avaliar os aspectos clínicos e histopatológicos do miocárdio de cães naturalmente infectados com leishmaniose visceral. Para tanto, as características físicas gerais, hematimétricas, bioquímicas (creatinina quinase-CK, fração MB da creatina quinase-CK-MB- e troponina I), radiográficas do tórax, eletrocardiográficas e histopatológicas do miocárdio foram avaliadas em 36 cães portadores de LV (grupo LV - GLV) e comparadas às variáveis médias de 15 cães hígidos (grupo controle- GC), com exceção da avaliação histopatológica, realizada apenas no GLV. Ao exame físico geral observou-se maior prevalência de cães assintomáticos no GC (100%) e polissintomáticos no GLV (66%). Dois cães do GLV apresentaram sopro sistólico de intensidade II/VI e III/VI, em região de foco mitral. Os valores médios de hemácia, hemoglobina e hematócrito foram inferiores nos cães do GLV (p<0,0001), associados a maiores valores de proteína total (p<0,0001), leucócitos totais (p=0,0151), neutrófilos (p=0,0027), CK (p=0,0153) e CK-MB (p=0,0038) quando comparado ao GC. Não foram observadas alterações radiográficas qualitativas ou diferenças quantitativas entre os grupos avaliados (p=0,1228). À eletrocardiografia foi detectada maior incidência de arritmia sinusal respiratória nos cães do GLV (75%) e ritmo sinusal nos cães do GC (60%), atestado pelo teste das proporções (p=0,016). A amplitude média da onda P foi inferior no GLV (p=0,008). Não foram observadas arritmias cardíacas em ambos os grupos, porém foi verificada no GLV a presença de episódios isolados de marcapasso migratório e sinus arrest associados à arritmia sinusal respiratória. A luz da histopatologia do miocárdio foi possível observar a presença de infiltrado inflamatório mononuclear em 77,8% (n=28) dos cães do GLV, sendo a maior prevalência de infiltrado linfoistioplasmocitário de intensidade leve. Conclui-se que os cães com LV do presente estudo apresentaram maior prevalência de aspectos clínicos polissintomáticos, associados à anemia normocítica normocrômica, acompanhada de leucocitose por neutrofilia e hiperproteinemia, elevação da atividade sérica de CK e CK-MB, sem alterações significativas da silhueta cardíaca à radiografia, maior prevalência de arritmia sinusal respiratória com episódios de marcapasso migratório e sinus arrest, relacionados à presença de infiltrado inflamatório mononuclear multifocal leve à histopatologia do miocárdio, com baixa incidência de vasculite necrosante e rara detecção de formas amastigotas da Leishmania sp. / Visceral leishmaniasis (VL) is a worldwide parasite zoonotic, caused by a protozoa Leishmania spp. Several organic systems may be affected directly or indirectly by the parasitism, resulting in variable and unspecific clinical signs in dogs. Myocardial damage in affected dogs is suggested by isolated reports with histological evidences of myocarditis. Therefore, the aim of this study was evaluate the clinical and myocardial histopathologic features of naturally infected dogs with VL. Therefore, general physical characteristics, hematological, biochemical (creatine kinase- CK, MB fraction of creatine kinase and troponin-I) radiographic of the thorax, electrocardiographic and histopathological myocardial were evaluated in 36 dogs with VL (VL group- VLG) and average variables compared to 15 healthy dogs (control group-CG), except for the histopathological evaluation. In the general physical examination was observed higher prevalence of asymptomatic dogs in the control group (100%) and polisymptomatic in GLV (66%). Two dogs of the GLV presented systolic murmur of intensity II / VI and III / VI, in the region of the mitral valve. The mean values of erythrocyte count, haemoglobin and haematocrit were lower in dogs GLV (p <0.0001), associated with higher total protein (p <0.0001), total leukocyte count (p = 0.0151), neutrophils (p = 0.0027), CK (p = 0.0153) and CK-MB (p = 0.0038) when compared to CG. Radiographic evaluation reveal no alterations or differences between groups (p = 0.1228). In the electrocardiography analysis, higher incidence of respiratory sinus arrhythmia with wandering pacemaker and sinus arrest in dogs of GLV (75%) and sinus rhythm in the CG (60%) was attested by the proportions test (p = 0.016). The mean P wave amplitude was lower in GLV (p = 0.008). Cardiac arrhythmias were not present in both groups, but sinus arrest was present in GLV. The histopathology of the myocardium displayed mononuclear cellinfiltration in 77.8% (n = 28) of the GLV dogs, with higher prevalence of mild lymphohistioplasmocytic inflammatory infiltrate. We conclude that enrolled dogs with LV presented aspect of polisymptomatic clinical features, associated with normochromic normocytic anemia, followed by leukocytosis, neutrophilia, hyperproteinemia, elevation of serum activity of CK and CK-MB, without significant changes of the cardiac silhouette in the radiography or heart rhythm by electrocardiogram, related to the presence of multifocal mild intensity mononuclear cell infiltration in the myocardial, with low incidence of necrotizing vasculitis and rare detection of amastigotes forms of Leishmania sp. on myocardium histopatology.

Renal cell carcinoma (RCC) is the most deadly of urological malignancies. While metastatic disease affects one third of patients at diagnosis, a further third of patients who undergo extirpative surgery with curative intent subsequently develop metastatic disease. Inconsistency in the clinical course ensures predicting subsequent metastasis is notoriously difficult, despite the routine use of prognostic clinico-pathological parameters in risk stratification. With greater understanding of pathways involved in disease pathogenesis, a number of biomarkers have been proposed to be of prognostic significance; however there are currently no molecular prognostic markers in clinical use. Genetic intra-tumoural heterogeneity (genetic ITH) has been described in clear cell RCC (ccRCC) and may limit the clinical translation of biomarkers. There has been no assessment of ITH at other molecular levels. The aim of this work was to define and compare proteomic, transcriptomic and DNA methylation ITH in ccRCC, and identify potential prognostic biomarkers. Using reverse phase protein arrays to study protein expression in multiple spatially separate regions of primary and metastatic ccRCC, proteomic ITH was demonstrated for the first time. Interestingly there was no significant difference in proteomic ITH in metastatic ccRCC tumour deposits compared to primary tumours. However, on analysis of differential protein expression between primary and metastatic ccRCC tissue using a tissue microarray and automated analysis of immunofluorescence, there was significantly greater expression of Ki67, p53, VEGFR1, SLUG and SNAIL in the metastases compared to the primary tumours. Subsequent profiling of gene expression and DNA methylation in multiple areas of the same primary tumours confirmed transcriptomic and methylomic ITH. On comparison of this multimolecular ITH, significantly greater proteomic ITH was seen compared to gene expression and DNA promoter methylation heterogeneity. Recent evidence suggests DNA methylation may be prognostically important in RCC and given the lower methylomic ITH in ccRCC, the identification of prognostic DNA methylation changes in ccRCC were pursued using the Infinium HumanMethylation450K Beadchip. Following development of an analysis pipeline, identification and validation of prognostic differentially methylated regions (DMR) was performed on an experimental cohort and published dataset respectively. Five DMRs, which were associated with disease recurrence in ccRCC, were identified. NEFM gene promoter methylation was the only DMR associated with cancer specific survival, independent of TNM stage and nuclear grade on multivariate analysis, which was confirmed on a third independent published dataset. This thesis therefore demonstrates multi-molecular ITH in ccRCC for the first time. Despite this, NEFM promoter methylation may be a useful independent prognostic marker of cancer specific survival.

Background: Breast and endometrial cancers are the most common gynaecological cancers in women in the UK. Early detection of tumours is crucial for improving patient survival rates. In breast cancer, mammography is the most reliable screening method for asymptomatic patients; however, its sensitivity is limited by breast density. Currently, there are no early screening assays for endometrial cancer. Thus, there is an urgent need to identify clinical biomarkers for improved non-invasive diagnosis of breast and endometrial cancer. Macrophages are abundant in the tumour microenvironment and their density has been associated with poor prognosis in breast cancer and decreased survival in endometrial cancer. Monocytes are precursors of macrophages and recent studies have shown an association with pro-tumoral functions. The aim of this study has been to examine the transcriptional profiles of human circulating monocytes and tumour associated macrophages (TAMs) in order to investigate their biological relevance and potential as biomarkers for cancer detection and prognosis. Methods: RNA-sequencing was performed on purified monocytes (22 healthy individuals, 21 breast cancer, 16 endometrial cancer samples), as well as purified normal macrophages, TAMs from breast tissue (4 breast cancer, 4 healthy breast) and endometrium tissue (5 endometrial cancer, 9 healthy endometrium). Results: A shift in the transcriptional profile of monocytes in cancer compared to controls was observed. Given these cancer-associated alterations circulating monocytes from cancer patients were called “Tumour Educated Monocytes” (TEMo). A TEMo-derived 13-gene signature was extracted that detected cancer, yielding an accuracy of 94%, a positive predictive value (PPV) of 92% and a negative predictive value (NPV) of 97%. External validation confirmed the ability of the signature to accurately identify cancer patients with perfect accuracy. Transcriptome profiling of TAMs revealed a significantly altered gene expression profile when compared to normal tissue resident macrophages. Furthermore, comparison of TAMs between breast and endometrial cancer also revealed differences suggesting that different tumour microenvironments induce different gene expression profiles in TAMs. Functional analysis of significant genes in breast cancer revealed similar biological pathways to those of murine studies suggesting that TAMs in humans and mice may have similar functions. A gene list of transmembrane receptors has been extracted by comparing breast cancer TAMs with publicly available datasets that could serve as markers for their identification. Finally, exploratory analysis identified a subset of 49 genes associated with recurrence-free and overall survival in publicly available datasets. Conclusion: To my knowledge this is the first genome-wide profiling study of human circulating monocytes and TAMs in breast and endometrial cancer. It provides evidence that monocytes and TAMs can alter their expression profile in the presence of cancer and, using bioinformatics tools I was able to identify biomarkers for diagnosis and prognosis of breast and endometrial cancer.

Microbial biodegradation is the primary mechanism by which petrogenic hydrocarbons (PHCs) are removed from the environment. Though hydrocarbon biodegradation is widely studied in marine systems, knowledge of how it occurs in freshwater systems is still lacking. The Peace-Athabasca Delta (PAD), located in northeastern Alberta, is an ideal location to study microbial hydrocarbon degradation since it has a long history of exposure to PHCs. What’s more, these PHCs are predominately sourced from bituminous deposits and are therefore relevant to the Canadian Oil Sands Industry. This thesis investigated the genetic potential for hydrocarbon degradation of PHCs via metagenomic reconstruction of microbial communities in lakes of the Peace and Athabasca Deltas, as well as reference lakes in the nearby boreal uplands. In order to properly evaluate the microbial community and its potential for hydrocarbon degradation, a comprehensive analysis of PHCs (including n-alkanes, polycyclic aromatic compounds (PACs), and petroleum biomarkers of terpanes, hopanes, and steranes) was performed. PHC analysis showed that n-alkanes in lake sediments from all three regions were highly similar and predominately biogenic, while PAC composition was significantly different in each region. Restricted-drainage lakes of the Athabasca Delta had the highest concentrations of PACs from petrogenic sources. Closed-drainage lakes in the Peace Delta had lower concentrations of PACs that likely originated from a mixture of pyrogenic and petrogenic sources. Closed-drainage lakes in the boreal upland region had the lowest concentrations of PACs likely sourced from pyrogenic wood combustion with traces of petrogenic PACs, possibly from atmospheric deposition of dust. Petroleum biomarkers of terpanes, hopanes, and steranes were successfully used to identify the long-range fluvial, and possibly atmospheric, transport of bituminous compounds more than one hundred kilometers from their potential source. This validates the future use of these biomarkers in environmental forensics. Microbial communities in all three regions under study were highly diverse, and their composition was significantly different in both sediment and water. Targeted gene analysis identified a total of 3885 genes involved in the degradation of n-alkanes and PACs in sediment and water. The results show that organic carbon, nitrogen, and sulfur content, as well as PAC and short-chain alkane concentrations were important chemical predictors of change in degradation gene composition. Furthermore, genes for anaerobic degradation of PHCs were identified in syntrophic bacteria, methanogens, nitrate and sulfate reducers, demonstrating the potential for syntrophic hydrocarbon degradation in PAD lakes. Though this thesis confirms the genetic potential for hydrocarbon degradation in PAD and boreal upland lakes, further research is necessary to determine whether these microbial communities can actively degrade the PHCs present in these lakes.

O Crack atua como um potente estimulante do sistema nervoso central, bloqueando a recaptação pré-sináptica de noradrenalina e dopamina, produzindo alto nível destes neurotransmissores nos receptores pós-sinápticos, ocasionando efeitos de prazer. O uso de drogas e a vulnerabilidade social podem resultar em desnutrição, causando danos à saúde. Além disso, peptídeos relacionados com o apetite podem estar regulando o uso de drogas. A leptina, um peptídeo produzido principalmente pelo tecido adiposo, informa ao cérebro a presença de energia, induzindo o bloqueio do neuropeptídio Y (NPY), diminuindo o apetite. Nossa hipótese é que a leptina possa estar regulando, além da ingestão de alimentos, o consumo de drogas. Não existem estudos na literatura sobre o estado nutricional de usuários de crack (com métodos bioquímicos e antropométricos), nem pesquisas que avaliam a relação da leptina com a gravidade do uso da droga. Assim, o objetivo desta tese foi avaliar o estado nutricional e os níveis séricos de leptina em usuários de crack e suas correlações com variáveis antropométricas, bioquímicas e gravidade de consumo da droga (crack). O artigo 1 da presente tese é uma análise transversal de 108 usuários, onde avaliamos o índice de massa corporal (IMC), composição corporal por bioimpedância (BIA), parâmetros bioquímicos e correlacionamos com a gravidade do uso da droga. O artigo 2 compreende uma análise dos níveis séricos de leptina, e suas correlações com IMC, BIA e gravidade do uso de crack em 40 indivíduos. Como principal achado do artigo 1, demonstramos que maioria dos indivíduos possui um IMC dentro da normalidade, no entanto, as análises bioquímicas indicaram alterações, mostrando que, embora os usuários de crack não possuam baixo peso, apresentam outras deficiências nutricionais específicas. No artigo 2, sugerimos que a leptina possa estar envolvida com a gravidade do uso de drogas, talvez em uma forma similar à modulação da ingestão de alimentos. / Crack is a potent central nervous system stimulant, inhibiting the presynaptic reuptake of noradrenaline and dopamine, producing high levels of these neurotransmitters at postsynaptic receptors, causing pleasure effects. Drug use and vulnerability can lead to malnutrition, causing a number of health consequences. In addition, appetite-related peptides may be regulating drug use. Leptin, a peptide produced primarily by adipose tissue, communicates the brain about energy reserves, inhibiting NPY and decreasing appetite. Our hypothesis is that leptin may be regulating, in addition to food intake, drug use. There are no studies that report crack users nutritional status (with biochemical and anthropometric methods), or about the relationship between leptin and the severity of drug use. Thus, the objective of this study was to evaluate the nutritional status and leptin serum levels, in crack users, and its correlations with anthropometric, biochemical and crack severity variables. The article 1 of the present study is a cross-sectional analysis, about nutritional status of 108 users. We evaluated BMI, body composition, biochemical parameters and correlated with the severity of drug use. Article 2 is a study about serum levels of leptin, and its correlations with BMI, BIA and severity of crack use in 40 individuals. As the main finding of article 1, we demonstrated that most individuals have a BMI within the normal range, however, biochemical analysis indicated below-expected parameters, showing that, Crack users are not underweight but have other specific nutritional deficiencies, which are also considered malnutrition. In paper 2, we suggest that leptin may be involved in the severity of drug use, perhaps in a similar way to food intake signaling.

A prevalência da obesidade em crianças e adolescentes tem aumentado em diversos países do mundo, inclusive no Brasil. Esse fato está fortemente relacionado à mudança no estilo de vida e aos hábitos alimentares. A transição nutricional é um fenômeno caracterizado pelo aumento da ingestão de calorias e o consumo alimentar tem sido relacionado à obesidade não somente quanto ao volume de ingestão alimentar como também à composição e qualidade da dieta. O Healthy Eating Index (HEI, índice de alimentação saudável) é uma medida de adesão às recomendações dietéticas americanas. O HEI é composto por um escore de 100 pontos, onde 10 componentes alimentares são avaliados e destinados a avaliar a qualidade da alimentação, com a finalidade de monitorar as mudanças na ingestão dietética ao longo do tempo e como um instrumento base para elaboração das atividades de educação nutricional e promoção de saúde na população. Muitos estudos avaliaram a qualidade da alimentação através desse índice, alguns mostraram associação positiva entre o HEI e a ingestão de nutrientes essenciais. Porém, poucos estudos correlacionaram o HEI com marcadores de perfil lipídico. Este estudo se preocupa com o surgimento de alguns fatores de risco cardiovascular já na infância, e uma das causas relacionadas a esse problema seria o tipo de alimentação que as crianças consomem. Para avaliar a qualidade da dieta e marcadores de perfil lipídico, realizou-se um estudo transversal com a finalidade de investigar a associação entre o HEI e marcadores de perfil lipídico e o índice de massa corporal em escolares de sete e oito anos de idade, de baixo nível socioeconômico. A amostra foi constituída de 305 escolares, sendo 173 meninos e 132 meninas, a média geral de idade foi de 7,7 ± 0,4 anos, o excesso de peso foi diagnosticado em 27,2% das crianças, sendo que 11,5% foram classificadas como obesas. A média geral da pontuação do HEI foi 58,8 ± 7,7. Neste estudo foi encontrada uma elevada proporção de crianças com consumo alimentar que precisa ser melhorado e nenhuma criança com qualidade boa da dieta. Além disso, não foi possível mostrar associação entre o HEI e os níveis de lipídeos séricos nas crianças estudadas. Os resultados desta pesquisa, aliados a estudos de intervenção já feitos, destacam a necessidade de elaborar e implementar políticas e programas de nutrição nas escolas que tenham como alvo as crianças e suas famílias, com o objetivo de aumentar a ingestão de legumes, verduras, frutas e reduzir a ingestão de produtos industrializados, sódio e gorduras. / The prevalence of obesity in children and adolescents has increased in several countries including Brazil. This fact is strongly related to changes in lifestyle and eating habits. The nutrition transition is a phenomenon characterized by increased calorie intake and food consumption has been linked to obesity not only in the amount of food intake as well as the composition and diet quality. The Healthy Eating Index – HEI – is a measure of adherence to U.S. dietary recommendations. HEI is composed of a score of 100 points, in which 10 food components are evaluated and are designed to assess the quality of food, in order to monitor changes in dietary intake over time and as a basic instrument for development activities nutrition education and promotion of health in the population. Many studies have assessed the quality of food through this index, some showed a positive association between the HEI and the intake of essential nutrients. However few studies HEI correlated with markers of lipid profile. This research is concerned with the emergence of some cardiovascular risk factors in childhood and one of the causes related to this problem would be the type of food that children consume. In order to assess the quality of diet and lipid profile markers a crosssectional study was conducted to investigate the association between the HEI and markers of lipid profile and body mass index in schoolchildren of seven and eight years old, low socioeconomic status. The sample consisted of 305 school children, 173 boys and 132 girls, the overall mean age was 7.7 ± 0.4 years, overweight was diagnosed in 27.2% of children, and 11.5 % were classified as obese. The general average HEI score was 58.8 ± 7.7. This study found a high proportion of children with food consumption that needs to be improved and no child with good quality diet. In addition, we could not show an association between the HEI and serum lipid levels in children. The results of this research combined with intervention studies ever made highlight the need to develop and implement policies and nutrition programs in schools that target children and their families in order to increase the intake of vegetables, fruits and reduce the intake of processed products, sodium and fats.

Os marcadores orgânicos moleculares (hidrocarbonetos alifáticos, esteróis e álcoois) em amostras de sedimentos superficiais e de testemunhos curtos coletados em diápiros e pockmarks localizados no talude sul do Brasil, foram utilizados para identificar as possíveis contribuições biogênicas (autóctones / alóctones). De modo geral, tanto nas amostras superficiais como ao longo dos testemunhos, o maior acúmulo de carbono orgânico total nos pockmarks foi observado, indicando o possível aprisionamento de sedimentos e de matéria orgânica no interior das concavidades. A presença de &#946;-sitosterol, campesterol, álcoois e n-alcanos pesados, indicaram contribuições terrígenas para a área de estudo, que pode ser advinda da descarga continental do Rio da Prata, uma vez que a predominância terrígena é proveniente das pradarias, típica vegetação do Uruguai e do sul do Rio Grande do Sul. O transporte de sedimentos terrígenos ocorre em direção ao norte, ao longo da plataforma continental sul brasileira, pela Corrente Costeira do Brasil. A presença de detritos de fitoplâncton e zooplâncton também ocorre devido à influência dos nutrientes do Rio da Prata. As possíveis liberações de gás/fluido, que sustentaram ecossistemas quimiossintéticos em diferentes intervalos de dois testemunhos em pockmark e diápiro foram constatadas através da presença relativamente maior de n-alcanos leves com número par de carbonos. / Organic molecular markers (aliphatic hydrocarbons, sterols and alcohols) in superficial and short cores sediment samples collected in diapirs and pockmarks located in the southern slope of Brazil were used in order to identify the possible biogenic contributions (autochthonous / allochthonous). In general, in surface samples and throughout the cores samples, the greatest accumulation of total organic carbon in the pockmarks was observed, indicating the possible entrapment of sediments and organic matter inside the concavities. The presence of &#946;-sitosterol, campesterol, alcohols and heavy n-alkanes indicated terrigenous contributions to the study area, which may be due to the continental discharge of Río de la Plata, since the terrigenous predominance is due to the prairies, a typical vegetation of Uruguay and the south of Rio Grande do Sul. The terrigenous sediments transport is carried through the North, along the Brazilian continental shelf, by the Coastal Stream of Brazil. The presence of phytoplankton and zooplankton debris is also due to the influence of the nutrients contribution from Río de la Plata. Possible gas / fluid releases, which sustained chemosynthetic ecosystems at different intervals of two pockmark and diaper cores, were verified by the relative predominance of even-numbered carbon n-alkanes.

Background The inflammatory bowel diseases (IBD) ulcerative colitis (UC) and Crohn's disease (CD) are chronic relapsing inflammatory disorders which have a rising incidence and cause significant morbidity. There are currently several treatment options with many more in the drug pipeline, but there are a lack of accurate biomarkers for decisions on treatment choice, assessment of disease activity and prognostication. There is a growing interest and desire for personalised or 'precision' medicine in IBD where novel biomarkers may help individualise IBD care in terms of diagnosis, choice of therapy, monitoring of response and detection of relapse. One class of functionally active biomarkers which have yet to be thoroughly investigated in IBD is damage-associated molecular patterns (DAMPs) including mitochondrial DNA (mtDNA). It has been recently shown that gut mitochondrial dysfunction can result in loss of epithelial barrier function and the development of colitis. Mitochondrial DAMPs have recently been described as elevated in several inflammatory diseases. Hypothesis The primary hypothesis of this thesis is that circulating levels of mtDNA is elevated in IBD. Secondary hypotheses are: (a) levels of other mitochondrial DAMPs are elevated in IBD, (b) circulating mtDNA can be used as a novel biomarker in IBD and (c) mtDNA is released locally at sites of inflammation in IBD. Methods Plasma and serum were collected prospectively from recruited IBD patients and non-IBD controls. Faeces and colonic tissue were collected from a subset of these patients. mtDNA in serum, plasma and faeces was measured using qPCR (amplifying COXIII/ND2 genes). Mass spectrometry was used to detect mitochondrial formylated peptides in the plasma of a subset of patients. IBD tissue was assessed for (a) mitochondrial damage using transmission electron microscopy (TEM) and (b) TLR9 expression, the target for mtDNA. Results 97 patients with IBD (67 UC and 30 CD), and 40 non-IBD controls were recruited. Plasma mtDNA levels were increased in UC and CD (both p < 0.0001) compared to non-IBD controls; with significant correlations with blood (CRP, albumin, white cell count), clinical and endoscopic markers of severity; and disease activity. In active UC, we detected significantly higher circulating mitochondrial formylated peptides and faecal mtDNA levels (vs. non-IBD controls [p < 0.01 and < 0.0001 respectively]) with demonstrable TEM evidence of intestinal mucosal mitochondrial damage. In active IBD, TLR9+ lamina propria inflammatory cells were significantly higher in UC/CD compared to controls (both p < 0.05). Conclusions Taken together, the findings suggest mtDNA is released during active inflammation in inflammatory bowel disease and is a potential novel mechanistic biomarker.