The human brain is a complex and powerful organ, directing every aspect of life from somatosensory and motor function to visceral responses to higher order cognition. Neurological and psychiatric disorders often disrupt normal functioning. While the clinical symptoms of such disorders are known, their biological underpinnings are not as clearly characterized. Structural neuroimaging is a powerful, non-invasive tool that can play a critical role in finding biomarkers of these illnesses. Currently, variations in pre-processing techniques yield inconsistent and conflicting results. As neuroimaging is a nascent branch of medical research, gold standards in imaging methodologies have not yet been established. Quantitatively validating and optimizing the way these images are preprocessed is the first step towards standardization. Voxel-based morphometry (VBM) is one technique that is commonly used to compare whole-brain structural differences between groups. Statistical tests are used to compare intensities of voxels throughout all brain scans in each group. In order to ensure that comparable voxels are being tested, the images must be fitted into a common space, which is done through image preprocessing. Spatial normalization to templates is an early pre-processing step that is executed unreliably as many options for both templates and normalization algorithms exist. To determine the effect variations in template usage may cause, we utilized a VBM approach to detect simulated lesions. Template performance was analyzed by comparing the accuracy with which the lesion was detected.

The Apolipoprotein E (APOE) ε4 allele is the best-established genetic risk factor for sporadic Alzheimer's disease (AD) while the ε2 allele confers a reduced risk compared with the most common ε3 allele. Neuroimaging studies using magnetic resonance imaging (MRI) have shown that APOE genotype affects brain structure and function. The aims of the research presented in this DPhil thesis were twofold: 1) to investigate the effect of APOE genotype on brain function in healthy adults using magnetoencephalography (MEG), which is a direct measure of neuronal activity and 2) to explore interactions between the AD risk factors APOE ε4 allele, age and female gender and their effects on brain structure and function using resting-state functional MRI and diffusion tensor imaging. MEG revealed similar neuronal activity at rest for APOE ε2 and ε4 carriers, i.e. those with opposite AD risk, indicating a more general effect on the functional architecture of the brain that is not directly linked to AD risk. However, differences between APOE ε2 and ε4 carriers became apparent when reactivity to stimuli was explored using the excellent temporal resolution of MEG. APOE ε4 carriers showed faster sensory pro- cessing and APOE genotype effects were found for functional networks associated with attention. In the second part of this project, female APOE ε4 carriers showed overall significantly reduced functional connectivity between the hippocampus and precuneus and a significant age-related decrease in connectivity of these regions. Increased vulnerability of this connection might be one reason for increased AD risk and interventions targeting hippocampal connectivity might be especially effective in at-risk populations. The research presented in this DPhil thesis showed a complex pattern of APOE genotype effects on brain structure and function. While global APOE genotype effects on functional and structural connectivity do not follow patterns of AD risk, more specific measures of connectivity and task-related brain function could be of use in the development of preclinical markers for AD development.

INTRODUÇÃO: A sepse é uma resposta inflamatória sistêmica causada por infecção suspeita ou confirmada. As avaliações clínicas são essenciais para a sua detecção e tratamento precoce. Hemoculturas podem demorar até dois dias para produzir um resultado, e nem sempre são confiáveis. No entanto, estudos recentes sugeriram que a expressão de CD64 de neutrófilos pode ser uma alternativa sensível e específica para o diagnóstico de uma infecção sistêmica. OBJETIVO: Analisar a diferença de valores entre CD64 de indivíduos com síndrome de resposta inflamatória sistêmica (SIRS), e sepse suspeita ou confirmada, que satisfazem os critérios de diagnóstico para SIRS ao chegar na unidade de emergência. MÉTODO: Este foi um estudo de coorte prospectivo observacional. A amostra foi composta de 109 pacientes com idade de 18 anos ou mais, com critérios de SIRS na chegada ao serviço de emergência. Expressão CD64 foi medida no prazo de 6 horas de internação, e novamente após 48 h. RESULTADOS: A análise da curva ROC sugeriu que um corte de 1.45 dos níveis de CD64 poderia diagnosticar sepse com uma sensibilidade de 0,85, especificidade de 0,75, uma precisão de 82,08%, um valor preditivo positivo de 0,964, um valor preditivo negativo de 0,375 e uma razão de verossimilhança de 3,3381. A área sob a curva foi de 0,832. CONCLUSÃO: CD64 parece ser útil como biomarcador, sensível e específico para discriminar entre SRIS e sépsis. / INTRODUCTION: Sepsis is a systemic inflammatory response to suspected or confirmed infection. Clinical evaluations are essential for its early detection and treatment. Blood cultures may take as long as two days to yield a result, and are not always reliable. However, recent studies have suggested that neutrophil CD64 expression may be a sensitive and specific alternative for the diagnosis of systemic infection. OBJECTIVE: To analyze the difference in CD64 values between subjects with systemic inflammatory response syndrome (SIRS), suspected or confirmed sepsis, who meet diagnostic criteria for SIRS upon arriving at an emergency unit. METHOD: This was a prospective observational cohort study. The sample consisted of 109 patients aged 18 years with criteria for SIRS on arrival to Emergency department. CD64 expression was measured within 6 hours of hospital admission, and once again after 48 h. RESULTS: ROC curve analysis suggested that a cutoff of 1.45 for CD64 expression could diagnose sepsis with a sensitivity of 0.85, a specificity of 0.75, an accuracy of 82.08%, a positive predictive value of 0.964, a negative predictive value of 0.375 and a positive likelihood ratio of 3.3381. The area under the curve was 0.832. CONCLUSION: CD64 appears to be a useful, sensitive and specific biomarker in discriminating between SIRS and sepsis.

Background: Reliable biomarkers are needed to evade the risk of injury, invasiveness and discomfort of endoscopies, which are required for inflammatory bowel disease (IBD) diagnosis and extent of disease assessment in ulcerative colitis (UC) patients. The need for biomarkers is accentuated in children, wherein the most frequently used IBD biomarker yields low specificity. Proteomics of clinical samples or their enriched components is a means to evaluate and identify alterations in proteins reflective of disease, with the potential for use as biomarkers and for providing insight on disease pathogenesis. Methods: Proteins were isolated from the intestinal mucosal-luminal interface (MLI), collected from the ascending and descending colon of pediatric treatment-naive patients. The intestinal MLI proteomes of 42 IBD and 18 control patients were analyzed by high resolution mass spectrometry (HRMS). Multivariate analysis and receiver operating characteristics curves were performed to develop protein biomarker panels to discriminate IBD from control, and for UC extent of disease. ELISAs were used to assess a subset of biomarker candidates in stool samples from an independent pediatric cohort (n=24). Extracellular vesicles (EVs) were isolated by ultracentrifugation from the intestinal MLI of 11 IBD and seven control patients, and analyzed by electron microscopy, nanoparticle tracking analysis and HRMS. Results: A biomarker panel of four proteins classified patients as either controls or active IBD with 97.5% accuracy. A second biomarker panel correctly classified 100% of UC patients as presenting with pancolitis or non-pancolitis. The differential protein expression of two biomarker candidates (catalase and leukotriene A-4 hydrolase) identified from the intestinal MLI was comparable in stool samples. Comparison of EV proteomes isolated from IBD patients and controls identified differential expression of processes related to host defense and immunity. Conclusions: Proteomic analysis of clinical samples identified differentially expressed proteins that can classify IBD patients from non-IBD controls and distinguish UC patients with pancolitis from those without pancolitis; proteins identified in intestinal aspirates displayed consistent differential expression in stool. Furthermore enrichment of EVs from the intestinal MLI indicates that these may contribute to the dysregulated host response against the intestinal microbiota which is observed in IBD.

Made available in DSpace on 2014-06-11T19:35:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-06-30Bitstream added on 2014-06-13T21:07:32Z : No. of bitstreams: 1 silva_lm_dr_botfm.pdf: 518955 bytes, checksum: e691275d6c953c74d7e4df49bba41750 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Estudar a função renal de pacientes arteriopatas submetidos a cirurgia vascular, avaliando a concordância entre as estimativas do ritmo de filtração glomerular (RFG) obtidos pela aferição da creatinina e cistatina C plasmática, verificando se diabetes, hipertensão e função renal pré-operatórias apresentam relação com função tubular no pós-operatório e investigando a possível influência da hemodiluição na avaliação da função renal por meio da cistatina C. Trata-se de estudo de coorte, prospectivo, incluindo 144 pacientes consecutivos submetidos à anestesia para cirurgia arterial e distribuídos em 4 grupos, sendo (GDH), diabéticos e hipertensos, (GD), diabéticos, (GH), hipertensos e (GN), sem hipertensão ou diabetes. Foram obtidos urina para dosagens laboratoriais de creatinina urinária (Ucr) (mmol⁄L), fosfatase alcalina (FA) (U⁄L), -glutamiltransferase ( GT) (U⁄L) e sangue para dosagem de albumina (g/dL), globulina (g/dL) uréia (mg/dL), creatinina (mg/dL), cistatina C (mg/L) e aferida a osmolaridade plasmática (mOsm/L) no pré-operatórios (M1) e após 24 horas do término da cirurgia (M2). As estimativas do RFG foram comparadas pelo método de Bland-Altman. Os limites de concordância entre as equações para estimativa do RFG pela creatinina e cistatina C estudadas foram amplos tanto no pré como no pós-operatório e a diferença entre as médias das equações analisadas aumentou no período pós-operatório. Em 76,39 % dos pacientes analisados houve diminuição dos valores de cistatina C no pós-operatório. Houve correlação moderada entre a variação de cistatina C e a variação da osmolaridade plasmática (r=0,41; p<0,0001). No modelo de regressão linear múltipla somente a variação da osmolaridade plasmática esteve implicada na variação da cistatina C. Houve aumento dos valores de GT, GT/Ucr e FA x GT/Ucr em M2 no grupo GN... / The aim of this study was to study the renal function of patients submitted to anaesthesia for arterial surgery, evaluating the agreement between GFR equations by cystatin C and creatinine, checking whether preoperative diabetes, hypertension, and renal function had any relationship with postoperative tubule function, and investigating possible hemodilution influence in cystatin C GFR based equations. Prospective cohort study including 144 patients submitted to anaesthesia for arterial surgery enrolled consecutively and divided into four groups: (GDH), diabetes and hypertension, (GD), diabetes, (GH), hypertension, and (GN), without hypertension or diabetes. Urine was obtained for laboratory analysis of urinary creatinine (Ucr) (mmol⁄L), alkaline phosphatase (AP) (U⁄L), -glutamyltransferase ( GT) (U⁄L), and blood for albumin (g/dL), globulin (g/dL), urea (mg⁄dL), creatinine (mg⁄dL), cystatin C (mg⁄L), and the plasma osmolarity (mOsm/L), before (M1) and 24h after the end of surgery (M2). Bland and Altman analysis was used to assessing agreement between two methods of GFR of measurements. The limits of agreement between creatinine and cystatin C GFR based equations were large and the mean difference in postoperative period was considerably higher than in preoperative period. In 76,39% of the patients analyzed, there was a decrease in the cystatin C value in the postoperative period. There was a moderate correlation between the cystatin C variation and the plasma osmolarity variation (r=0,41; p< 0,0001). In the multiple linear regression model, only the plasma osmolarity variation was implicated in the cystatin C variation. Values of GT, GT/Ucr, and AP x GT/Ucr increased at M2 in GN. Patients without renal function compromise (GFR > 90mL/min/1,73m2) presented increased GT/Ucr and AP x GT/Ucr values at M2 and those with slightly compromised renal function (60-90mL/min/1,73m2)... (Complete abstract click electronic access below)

Reusability and reliability of published data are fundamental requirements for translating results from animal experiments into reliable clinical biomarkers. Current success rates for biomarker discovery are poor, and we need to develop new tools to collate, integrate and analyse datasets, such as knowledge bases, that would enable more effective translation from mouse to human or across disciplines. However, concerns about the validity, reproducibility and replicability of existing data must be addressed first. Here, I interrogate the quality of methods reporting in experimental models of infection and inflammation. Despite evidence that most of the assessed parameters, such as sex and age, can influence the experimental results, the quality of methods reporting was poor. Inadequate methods reporting means that it is not always possible to confirm whether findings were due to improper experimental conditions that biased the results. Thus, such inaccuracies would have an impact on the construction of knowledge base tools that require appropriate annotation. However, I provide reusable checklists that could improve the quality of methods reporting prior to publication and can be used to verify papers post-publication to enable researchers from different fields to interrogate published data. Another reason that biomarkers may fail is that it can be difficult to determine the causative pathways that will better predict disease outcome in a chronically inflamed tissue where multiple pathways are happening simultaneously. I conducted novel research to identify and verify whether investigating animal models long before onset of colitis would identify potentially causative biomarkers for colitis in an animal model. Previous studies have shown early influx of dendritic cells are associated with resistance to Trichuris muris-induced colitis in mice, suggesting early biomarkers may be detectable that might predict disease outcome in inflammatory diseases, such as inflammatory bowel disease (IBD).In the T. muris colitis model, I identified differences in gene expression of multiple components of the receptor for advanced glycation end-products (RAGE) activation pathway between colitis-resistant and colitis-susceptible mice, occurring just 24 hours post infection; before any observable clinical symptoms were present. RAGE is a receptor that binds products of damage, such as calprotectin, and initiates pro-inflammatory cascades. However, RAGE can be cleaved from the cell membrane to form a soluble receptor (sRAGE) that cannot mediate proinflammatory signals, yet can bind to damage products, effectively rendering them harmless. During a longer infection timecourse, colitis-resistant mice produced significantly more sRAGE during infection, consistent with an increased ability to prevent inflammatory ligands from activating membrane-bound RAGE. These findings were also supported by additional experiments using T. muris infection in Il-10-/- mice. In summary, I have carried out an analysis of methods reporting quality in immunology research that can help improve the reliability of existing data relevant to the further study of IBD and beyond. I have also identified sRAGE as a potential biomarker for the onset of colitis in the T. muris infection model, with implications for diagnosis and treatment of IBD in a clinical setting.

INTRODUÇÃO: A sepse é uma resposta inflamatória sistêmica causada por infecção suspeita ou confirmada. As avaliações clínicas são essenciais para a sua detecção e tratamento precoce. Hemoculturas podem demorar até dois dias para produzir um resultado, e nem sempre são confiáveis. No entanto, estudos recentes sugeriram que a expressão de CD64 de neutrófilos pode ser uma alternativa sensível e específica para o diagnóstico de uma infecção sistêmica. OBJETIVO: Analisar a diferença de valores entre CD64 de indivíduos com síndrome de resposta inflamatória sistêmica (SIRS), e sepse suspeita ou confirmada, que satisfazem os critérios de diagnóstico para SIRS ao chegar na unidade de emergência. MÉTODO: Este foi um estudo de coorte prospectivo observacional. A amostra foi composta de 109 pacientes com idade de 18 anos ou mais, com critérios de SIRS na chegada ao serviço de emergência. Expressão CD64 foi medida no prazo de 6 horas de internação, e novamente após 48 h. RESULTADOS: A análise da curva ROC sugeriu que um corte de 1.45 dos níveis de CD64 poderia diagnosticar sepse com uma sensibilidade de 0,85, especificidade de 0,75, uma precisão de 82,08%, um valor preditivo positivo de 0,964, um valor preditivo negativo de 0,375 e uma razão de verossimilhança de 3,3381. A área sob a curva foi de 0,832. CONCLUSÃO: CD64 parece ser útil como biomarcador, sensível e específico para discriminar entre SRIS e sépsis. / INTRODUCTION: Sepsis is a systemic inflammatory response to suspected or confirmed infection. Clinical evaluations are essential for its early detection and treatment. Blood cultures may take as long as two days to yield a result, and are not always reliable. However, recent studies have suggested that neutrophil CD64 expression may be a sensitive and specific alternative for the diagnosis of systemic infection. OBJECTIVE: To analyze the difference in CD64 values between subjects with systemic inflammatory response syndrome (SIRS), suspected or confirmed sepsis, who meet diagnostic criteria for SIRS upon arriving at an emergency unit. METHOD: This was a prospective observational cohort study. The sample consisted of 109 patients aged 18 years with criteria for SIRS on arrival to Emergency department. CD64 expression was measured within 6 hours of hospital admission, and once again after 48 h. RESULTS: ROC curve analysis suggested that a cutoff of 1.45 for CD64 expression could diagnose sepsis with a sensitivity of 0.85, a specificity of 0.75, an accuracy of 82.08%, a positive predictive value of 0.964, a negative predictive value of 0.375 and a positive likelihood ratio of 3.3381. The area under the curve was 0.832. CONCLUSION: CD64 appears to be a useful, sensitive and specific biomarker in discriminating between SIRS and sepsis.

INTRODUÇÃO: A sepse é uma resposta inflamatória sistêmica causada por infecção suspeita ou confirmada. As avaliações clínicas são essenciais para a sua detecção e tratamento precoce. Hemoculturas podem demorar até dois dias para produzir um resultado, e nem sempre são confiáveis. No entanto, estudos recentes sugeriram que a expressão de CD64 de neutrófilos pode ser uma alternativa sensível e específica para o diagnóstico de uma infecção sistêmica. OBJETIVO: Analisar a diferença de valores entre CD64 de indivíduos com síndrome de resposta inflamatória sistêmica (SIRS), e sepse suspeita ou confirmada, que satisfazem os critérios de diagnóstico para SIRS ao chegar na unidade de emergência. MÉTODO: Este foi um estudo de coorte prospectivo observacional. A amostra foi composta de 109 pacientes com idade de 18 anos ou mais, com critérios de SIRS na chegada ao serviço de emergência. Expressão CD64 foi medida no prazo de 6 horas de internação, e novamente após 48 h. RESULTADOS: A análise da curva ROC sugeriu que um corte de 1.45 dos níveis de CD64 poderia diagnosticar sepse com uma sensibilidade de 0,85, especificidade de 0,75, uma precisão de 82,08%, um valor preditivo positivo de 0,964, um valor preditivo negativo de 0,375 e uma razão de verossimilhança de 3,3381. A área sob a curva foi de 0,832. CONCLUSÃO: CD64 parece ser útil como biomarcador, sensível e específico para discriminar entre SRIS e sépsis. / INTRODUCTION: Sepsis is a systemic inflammatory response to suspected or confirmed infection. Clinical evaluations are essential for its early detection and treatment. Blood cultures may take as long as two days to yield a result, and are not always reliable. However, recent studies have suggested that neutrophil CD64 expression may be a sensitive and specific alternative for the diagnosis of systemic infection. OBJECTIVE: To analyze the difference in CD64 values between subjects with systemic inflammatory response syndrome (SIRS), suspected or confirmed sepsis, who meet diagnostic criteria for SIRS upon arriving at an emergency unit. METHOD: This was a prospective observational cohort study. The sample consisted of 109 patients aged 18 years with criteria for SIRS on arrival to Emergency department. CD64 expression was measured within 6 hours of hospital admission, and once again after 48 h. RESULTS: ROC curve analysis suggested that a cutoff of 1.45 for CD64 expression could diagnose sepsis with a sensitivity of 0.85, a specificity of 0.75, an accuracy of 82.08%, a positive predictive value of 0.964, a negative predictive value of 0.375 and a positive likelihood ratio of 3.3381. The area under the curve was 0.832. CONCLUSION: CD64 appears to be a useful, sensitive and specific biomarker in discriminating between SIRS and sepsis.

Tumor biopsies may help to reliably distinguish hepatocellular carcinoma (HCC) from other tumors, mostly cholangiocarcinoma as well as to identify the patient populations who most benefit from target-driven HCC treatments, in order to improve the success rate of experimental therapies. Clarifying tumor biology may also lead to identify biomarkers with prognostic role and/or enabling to predict response or resistance to therapies. Recently, clinical trials have more efficiently included biomarker endpoints and increasingly collected tumor tissue from enrolled patients. Due to their frail status and sometimes fast-progressing disease, the performance status of patients with HCC progressing on first-line therapy can deteriorate quickly, preventing their enrollment in clinical trials. However, the challenge of identifying the proper patient at the proper time can be overcome by periodic inter-department meetings involving the key specialists taking care of HCC patients, and solid networks between research centers and referring institutions. An early planned biopsy would also facilitate timely inclusion of patients in biology-driven clinical trials. Ultimately, institution of multidisciplinary teams can optimize treatment choice, biopsy timing, and quick enrollment of patients in clinical trials, before their performance status deteriorates.

Philosophiae Doctor - PhD / The human epidermal growth factor receptor (HER)-family of receptor tyrosine kinases; human epidermal growth factor receptor 1, human epidermal growth factor receptor 2, human epidermal growth factor receptor 3 and human epidermal growth factor receptor 4 (EGFR/HER1, ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4) plays a major role in the pathogenesis of many solid tumours, in approximately 25 - 30% of breast cancers. Breast cancer is the second most common type of cancer and affects around 3000 women annually in South Africa alone. While the benefits of treatment and cancer progress to enhance therapeutic effectiveness for the patient are well documented, it is also important to employ or fabricate methods in which cancer can be screened at an early stage. A number of gene and protein based biomarkers have shown potential in the early screening of cancer. One specific biomarker that is over-expressed in 20 - 30% of human breast cancers is the human epidermal growth factor receptor 2 (Her2/neu). Several methods have been developed for detection of Her2/neu oncogene including immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), fluorescent in situ hybridisation (FISH) and polymerase chain reaction(PCR). However, these methods are subjected to interference problem. For these reasons an ultrasensitive, cheap and easy to use genosensor has been developed for early detection of the Her2/neu oncogene using electrochemical and spectroscopic methods. Due to their high surface-to-volume ratio, electro-catalytic activity as well as good biocompatibility and novel electron transport properties quantum dots are highly attractive materials for ultra-sensitive detection of biological macromolecules via bio-electronic or bio-optic devices. In this study a quantum dots (QDs)-based genosensor was developed in which Ga2Te3-based quantum dots were synthesised using a novel aqueous solution approach by mixing 3-mercaptopropionic acid (3MPA)-capped gallium metal precursor with reduced tellurium metal. The morphological, compositional and structural characterisation of the QDs was investigated prior to their utilization in DNA sensor construction.