INTRODUÇÃO E OBJETIVO: Recidiva da hepatite C crônica com progressão acelerada, embora imprevisível, da fibrose é responsável por piora no prognóstico após o transplante de fígado (Tx). Biópsia hepática protocolar é considerada o padrão ouro para estadiamento da fibrose na recidiva da hepatite C pós-Tx. Para superar as limitações da biópsia, principalmente custo e complicações, marcadores simples e não invasivos de fibrose hepática têm sido propostos para pacientes imunocompetentes, porém com escassos estudos disponíveis no contexto pós-Tx. O objetivo desse estudo é avaliar o desempenho diagnóstico dos marcadores não-invasivos para estadiar fibrose hepática em pacientes pós-Tx. MÉTODOS: Pacientes consecutivos receptores de Tx com recidiva da hepatite C (n=45) que foram submetidos a 118 biópsias hepáticas foram incluídos. Variáveis laboratoriais dentro de trinta dias de cada biópsia foram consideradas. Índice da razão AST-plaqueta (APRI), razão AST/ALT, Escore discriminativo de Bonacini (EDB), Escore de Pohl e índice idade-plaqueta foram calculados para cada biópsia. Fibrose significante foi definida como estágio METAVIR 2. RESULTADO: A área sob a curva ROC (receiver operating characteristic) do Escore discriminativo de Bonacini para predizer fibrose significante foi 0,68, superior aos outros testes avaliados. Utilizando-se o melhor ponto de corte, um valor de Escore discriminativo de Bonacini 8 foi 42% sensível e 95% específico, com razão de verossimilhança positiva e negativa de 7,98 e 0,62, respectivamente. Análise multivariada identificou razão AST/ALT como preditor independente de fibrose significante (OR=4.2; CI 95%=1.5-11.4; p-valor=0.005, ponto de corte 0,89). Análise adicional considerando apenas uma biópsia por paciente confirmou o desempenho superior do Escore discriminativo de Bonacini em relaçãoaos outros testes avaliados, com uma área sob a curva de 0,76. CONCLUSÃO: Escore discriminativo de Bonacini foi o marcador laboratorial não invasivo com melhor desempenho diagnóstico para predizer fibrose hepática significante em pacientes com recidiva de hepatite C crônica pós-Tx / BACKGROUND AND AIM: Recurrent hepatitis C with accelerated, although unpredictable, fibrosis progression accounts for a poor prognosis after liver transplantation (LT). Per protocol liver biopsy is considered the gold standard for fibrosis staging in recurrent hepatitis C after LT to overcome the limitations of liver biopsy, mainly cost and complications, simple non-invasive liver fibrosis tests have been proposed for immunocompetent patients, butfew data are available in the post-transplant setting. The aim of this study was to evaluate diagnostic performance of noninvasive tests to stage liver fibrosis in LT setting. METHOD: Consecutive LT patients with recurrent hepatitis C (n=45) who have undergone 118 liver biopsy were included. Laboratory variables at the time of biopsies were recorded. AST to platelet ratio index (APRI), AST/ALT ratio, Bonacini discriminant score (BDS), Pohl score and age-platelet index were calculated at the time of biopsies. Significant fibrosis was defined as METAVIR stage 2. RESULT: The area under the receiver operating characteristic (ROC) curve (AUC) of Bonacini discriminant score for predicting significant fibrosis was 0,68, better than the other non-invasive liver fibrosis tests. Using the best cutoff value, Bonacini discriminant score value 8 was 42% sensitive and 95% specific, with positive and negative likelihood ratio of 7,98 and 0,62, respectively. Multivariate analysis identified AST/ALT ratio as an independent predictor of significant fibrosis (OR=4.2; CI 95%=1.5-11.4; p-value=0.005, cutoff point 0,89). Additional analysis considering only one biopsy per patient confirmed the superior performance of Bonacini discriminant score compared to the other non-invasive liver fibrosis tests, with an AUC of 0,76. CONCLUSION: Bonacini discriminant score was the non-invasive liver fibrosis test with the best performance for significant liver transplant patients with recurrent hepatitis C

A insuficiência cardíaca é uma síndrome clínica de alta morbimortalidade e por este motivo é crescente o interesse em se estudar novos biomarcadores da doença visando buscar caminhos para novas estratégias terapêuticas. Neste contexto, a análise do ar exalado pode ser promissora. Baseado nestes dados e na observação de que pacientes com insuficiência cardíaca grave exalam odor peculiar, ainda em estudo piloto, nós identificamos acetona no ar exalado de pacientes com insuficiência cardíaca. Assim, nosso estudo teve por objetivo primário avaliar o papel da acetona exalada como biomarcador do diagnóstico de insuficiência cardíaca e de insuficiência cardíaca descompensada. Como objetivo secundário, avaliar sua relação com a classe funcional segundo a classificação da New York Heart Assocation (NYHA) e sua correlação com o peptídeo natriurético do tipo B (BNP). Entre maio de 2009 e setembro de 2010, pacientes consecutivos com disfunção sistólica (grupo IC) admitidos na emergência (insuficiência cardíaca descompensada grupo ICDESCOMP) e pacientes estáveis nos últimos três meses encaminhados para o teste cardiopulmonar (insuficiência cardíaca compensada grupo ICCOMP) foram submetidos à coleta de ar exalado (extração em água) para posterior análise qualitativa por cromatografia gasosa acoplado à espectrometria de massas e quantificação por espectrofotometria de absorção, através da reação com salicilaldeído. Entre os 235 pacientes avaliados, 89 foram incluídos (59 com insuficiência cardíaca descompensada e 30 com insuficiência cardíaca compensada), 61% do sexo masculino e com mediana de idade de 52 anos. Vinte indivíduos saudáveis (grupo controle) pareados por idade participaram do estudo. O valor mediano (intervalo interquartil) de acetona exalada foi maior no grupo IC em relação ao controle [3,70 g/L (1,69-10,45 g/L) versus 0,39 g/L (0,30-0,79 g/L), p < 0,001]. O valor mediano de acetona em pacientes com insuficiência cardíaca descompensada foi maior do que no grupo com insuficiência cardíaca compensada [7,80 g/L (3,60-15,20 g/L) versus 1,22 g/L (0,682,19 g/L), p < 0,001]. A acurácia do método tanto para o diagnóstico de insuficiência cardíaca (acetona > 1,16 g/L; área sob a curva = 0,94) quanto para o diagnóstico de insuficiência cardíaca descompensada (acetona > 2,50 g/L; área sob a curva = 0,93) foi aproximadamente 85 %, semelhante à acurácia do BNP (BNP > 42 pg/mL; área sob a curva = 0,97 e BNP > 424 pg/mL; área sob a curva = 0,94, respectivamente). Houve correlação positiva entre acetona exalada e BNP (r = 0,772, p < 0,001). Observamos aumento progressivo nas concentrações de acetona exalada de acordo com a piora da classe funcional segundo NYHA (p < 0,001). Assim, podemos concluir que nosso estudo revelou a acetona exalada como um novo biomarcador do diagnóstico de insuficiência cardíaca e de insuficiência cardíaca descompensada, que está associado à maior gravidade da doença e que apresenta correlação positiva com BNP. Sua dosagem é um novo método de diagnóstico não invasivo que pode ser realizado à beira leito, cuja acurácia é semelhante à do BNP / Heart failure (HF) is a condition associated with high mortality and frequent hospital admissions. In this context, multiple biomarkers of heart failure severity have emerged recently. However, the usefulness of most of these biomarkers has not been fully established. Exhaled breath has been considered a suitable tool (biomarker) to evaluate different diseases. Based on the clinical observation that patients with acute decompensated heart failure (ADHF) exhale a distinct odor, in a pilot study we have identified acetone in exhaled breath of heart failure patients and this study aimed to evaluate the role of acetone as a new biomarker of heart failure and ADHF disease. As secondary aims, we intended to analyze the relation to New York Heart Association (NYHA) class and the correlation with B-Type Natriuretic Peptide (BNP). Patients with systolic dysfunction (HF group) admitted consecutively at the emergency room (ADHF group) and stable patients referred to the cardiopulmonary test (chronic HF CHF group) between May 2009 and September 2010 were submitted to exhaled breath collection (extraction into water). Acetone identification was done by gas chromatography-mass spectrometry (GC-MS) and its determination by absorption spectrophotometry after reaction with salicylaldehyde. Twenty healthy subjects matched for age were enrolled (control group). Among 235 patients with HF, 89 were included in the study (59 ADHF and 30 CHF), 61% male, with median age of 52 years. Median exhaled breath acetone value (interquartile range) was higher in the HF group when compared to control group [3.7 g/L (1.69-10.45 g/L) versus 0.39 g/L (0.30-0.79 g/L), p < 0.001] and also higher in ADHF when compared to CHF group [7.80 g/L (3.60-15.20 g/L) versus 1.22 g/L (0.682.19 g/L), p < 0,001]. The accuracy of the method to diagnose CHF (Acetone > 1.16 g/L; AUC = 0.94) and ADHF (Acetone > 2.5 g/L; AUC = 0.93) was similar to the accuracy of BNP (BNP > 42 pg/mL; AUC = 0.97 and BNP > 424 pg/mL; AUC = 0.94, respectively). There was a positive correlation between exhaled breath acetone and plasmatic BNP (r = 0.772, p < 0.001). Levels of exhaled breath acetone were different among the four different NYHA classes (p<0.001). In summary, we can conclude that our study showed exhaled breath acetone as a new biomarker of heart failure and ADHF. It is associated with heart failure severity and has a good correlation with BNP. This is a promising non-invasive diagnostic method of heart failure, whose accuracy is equivalent to BNP

O tumor odontogênico queratocístico é um tumor odontogênico benigno recentemente classificado como tal pela Organização Mundial de Saúde. O alto índice de recidiva, a similaridade com outras lesões odontogênicas císticas e mutações genéticas associadas, estimulam continuamente estudos com finalidade de aprimorar o diagnóstico e o entendimento do comportamento desta lesão. As citoqueratinas, principal componente do citoesqueleto epitelial, têm sido utilizadas como possíveis marcadores no diagnóstico do tumor odontogênico queratocístico, apesar da discrepância dos resultados publicados. O gene PTCH1, com mutação já relatada associada ao tumor odontogênico queratocístico, expressa proteína de mesmo nome que parece estar associada com a etiologia ou com o prognóstico do tumor odontogênico queratocístico. Vinte casos de tumor odontogênico queratocístico foram submetidos à técnica de imunoistoquímica para detecção da expressão das citoqueratinas 10, 13, 17 e 19 e da proteína PTCH1. Cada caso foi representado por dois momentos distintos da mesma lesão, sendo metade dos casos representados por lesões sem história de recidiva e a outra metade constituída de casos com história de recidiva. A marcação obtida em cada um dos momentos foi comparada, verificando assim a estabilidade de expressão. A influência da inflamação na expressão imunoistoquímica também foi avaliada. As citoqueratinas 10 e 17 se mostraram com maior porcentagem de positividade (82, 5% e 97,5%, respectivamente) e com maior estabilidade entre os momentos (65% e 95%, respectivamente). A proteína PTCH1 foi positiva em todos os momentos, apresentando assim estabilidade total para os casos estudados. Não houve diferença estatisticamente significante, para nenhum dos anticorpos utilizados, entre os grupos sem ou com história de recidiva ou de expressão nas áreas de inflamação. A estabilidade das citoqueratinas 10 e 17 sugere que estas possam ser utilizadas associadamente como auxiliar de diagnóstico do tumor odontogênico queratocístico. A proteína PTCH1 demonstrou alta positividade e estabilidade; porém não pôde ser relacionado ao comportamento do tumor odontogênico queratocístico. / The keratocystic odontogenic tumor is a benign odontogenic tumor recently classified as such by the World Health Organization. The high recurrence rate, the similarity with other odontogenic cystic lesions and the genetic associated mutations continuous stimulate studies intending diagnostic enhancement and behavior understanding of such lesion. The citokeratins, main component of the epithelial cytoskeleton, have been used as possible diagnostic markers of the keratocystic odontogenic tumor, in spite the discrepancy of the published results. The PTCH1 gene, with already reported mutation associated with the keratocystic odontogenic tumor, expresses a protein with the same name that seems to be associated with the etiology or the prognosis of the keratocystic odontogenic tumor. Twenty cases of keratocystic odontogenic tumor were submitted to the immunohistochemical technique for detection of the expression of citokeratins 10, 13, 17 and 19 and the protein PTCH1. Each case was represented by two distinct moments of the same lesion, being half of the cases represented by lesion without recurrence history and the other half constituted of lesion with recurrence history. The obtained staining in each moment was compared, thus verifying the expression stability. The influence of inflammation in the immunohistochemical expression was also evaluated. The cytokeratins 10 and 17 demonstrated higher positivity percentage (82.5% and 97.5%, respectively) and greater stability among the moments (65% and 97.5%, respectively). The PTCH1 protein was positive in all moments, thus presenting total stability for the studied cases. There was no statistical difference, for none of the antibodies, either among the groups without or with history of recurrence or in the expression in areas with inflammation. The stability of the cytokeratins 10 and 17 suggests that they can be used together as auxiliary for the diagnosis of the keratocystic odontogenic tumor. The protein PTCH1 demonstrated high positivity and stability; however it could not be related to the behavior of the keratocystic odontogenic tumor.

Shen, Jiayun. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 166-199). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Introdução A poluição do ar é um fator de risco associado com descompensação e mortalidade em pacientes com insuficiência cardíaca (IC). Objetivo Avaliar o impacto de um filtro de polipropileno sobre desfechos cardiovasculares em pacientes com IC e voluntários saudáveis durante exposição controlada à poluição. Métodos Ensaio clínico duplocego, controlado e cruzado, incluindo 26 pacientes com IC e 15 voluntários saudáveis, expostos a três protocolos diferentes de inalação randomizados por ordem: Ar Limpo; Exposição à Partículas de Exaustão do Diesel (ED); e ED filtrada. Os desfechos estudados foram função endotelial por índice de hiperemia reativa (RHi) e índice de aumento (Aix), biomarcadores séricos, variáveis de teste cardiopulmonar submáximo (caminhada de seis-minutos [tc6m]; consumo de oxigênio [VO2]; equivalente ventilatório de gás carbônico [VE/VCO2 slope]; consumo de O2 por batida [PulsoO2]) e variabilidade da frequência cardíaca (VFC). Resultados No grupo IC, a ED piorou o RHi [de 2,17 (IQR: 1,8-2,5) para 1,72 (IQR: 1,5-2,2); p=0,002], reduziu o VO2 [de 11.0 ± 3.9 para 8.4±2.8ml/Kg/min; p < 0.001], o tc6m [de 243,3±13 para 220,8 ± 14m; p=0,030] e o PulsoO2 [de 8.9 ± 1.0 para 7.8±0.7ml/bpm; p < 0.001]; e aumentou o BNP [de 47,0pg/ml (IQR: 17,3-118,0) para 66,5pg/ml (IQR: 26,5-155,5); p=0,004]. O filtro foi capaz de reduzir a concentração de poluição de 325±31 para 25±6?g/m3 (p < 0,001 vs. ED). No grupo IC, o filtro foi associado com melhora no RHi [2,06 (IQR: 1,5-2,6); p=0,019 vs. ED); aumento no VO2 (10.4 ± 3.8ml/Kg/min; p < 0.001 vs. ED) e PulsoO2 (9.7±1.1ml/bpm; p < 0.001 vs. ED); e redução no BNP [44,0pg/ml (IQR: 20,0-110,0); p=0,015 vs. ED]. Em ambos os grupos, a ED reduziu o Aix, sem efeito do filtro. O uso do filtro foi associado com maior ventilação e reinalação de CO2. Outras variáveis pesquisadas como VE/VCO2 slope e VFC não sofreram influências entre os protocolos. Conclusão A poluição do ar afetou adversamente o desempenho cardiovascular de pacientes com IC. Este é o primeiro ensaio clínico demonstrando que um simples filtrorespiratório pode prevenir a disfunção endotelial, a intolerância ao exercício e o aumento do BNP associados à poluição em pacientes com IC. O uso de máscaras com filtro tem o potencial de reduzir a morbidade associada à IC. Identificador ClinicalTrials.gov: NCT01960920 / Background Air pollution is considered a risk factor for heart failure (HF) decompensation and mortality. The effects of respiratory filters on patients with HF exposed to air pollution have not been established. Objective To test the effects of a respiratory filter intervention (filter) during controlled pollution exposure Methods Double-blind, randomized to order and 3-way crossover study with 26 HF patients and 15 control volunteers. Participants were exposed in three separate sessions to: clean air, diesel exhaust exposure (DE) or filtered-DE. Endpoints were endothelial function via reactive hyperemia index (RHi), and arterial stiffness (Aix), serum biomarkers, variables from submaximal cardiopulmonary exercise test (sixminute walk test [6mwt]; oxygen uptake [VO2]; ventilation and carbon dioxide production ratio [VE/VCO2 slope]; oxygen uptake per heart beat [O2Pulse]), and heart rate variability (HRV). Results In patients with HF, DE was associated with a worsening in RHi [from 2.17 (IQR: 1.8-2.5) to 1.72 (IQR: 1.5-2.2); p=0.002]; a decline in VO2 [from 11.0±3.9 to 8.4±2.8ml/Kg/min; 0.001], 6mwt [from 243.3 +- 13.0 to 220.8±13.7m; p=0.030] and O2Pulse [from 8.9±1.0 to 7.8±0.7ml/beat; 0.001] and a rise in BNP [from 47.0pg/ml (IQR: 17.3-118.0) to 66.5pg/ml (IQR: 26.5-155.5); p=0.004]. Filtration reduced the particulate concentration (from 325±31 to 25±6?g/m3; 0.001 vs. DE). In the HF group, filter was associated with an improvement in RHi [2.06 (IQR: 1.5-2.6); p=0.019 vs. DE]; an increase in VO2 (10.4 ± 3.8ml/Kg/min; p < 0.001 vs. DE) and O2Pulse (9.7 ± 1.1ml/beat; p < 0.001 vs. DE); and also a decrease in BNP [44.0pg/ml (IQR: 20.0-110.0); p=0.015 vs. DE]. In both groups DE decreased Aix, however filtration did not change these responses. In both groups, filtration was associated with higher pulmonary ventilation and CO2 rebreathing. Other variables as VE/VCO2 slope and HRV did not differ between exposure protocols. Conclusion Air pollution adversely affects cardiovascular performance in HF patients. To our knowledge, this is the first trial demonstrating that a simple respiratory-filter can prevent endothelial dysfunction; exercise intolerance and BNP rise in patients with HF during DE. Given these potential benefits, the widespread use of filters in HF subjects exposed to traffic-derived air pollution may have beneficial public health impacts and reduce the burden of HF ClinicalTrials.gov Identifier: NCT01960920

Firstly, a cell line derived from the liver of tilapia, Hepa-T1, was used as a model and exposed to two sub-lethal concentrations of waterborne copper for 96 h. The proteins expressed in Hepa T1 were investigated by differential protein profiling using two-dimensional gel electrophoresis (2-DE). It was found that Cu2+ (120 microM and 300 microM) caused differential expression of 93 different proteins, 18 of which were further verified by real-time quantitative polymerase chain reaction (PCR) analysis. Following analysis with ingenuity pathway software, several proteins were found to be involved in lipid metabolism, tissue connective development and cell cycle control, thus indicating that copper toxicity affects these cellular functions. / Fourthly, to further reveal the mechanism of copper tolerance and sensitivity in tilapia and zebrafish, two important copper transporters (ATP7A & B) and metallothionein (MT) were chosen for studying. Until now, a full length of ATP7A and partial length of ATP7B were obtained in tilapia. Then a real time quantitative PCR was conducted to study the different regulations of these three genes in tilapia and zebrafish. It was found that Cu2+ could induce more MT and ATP7A & B in tilapia than zebrafish both in vivo and in vitro. These results help us to understand that the copper tolerance of tilapia is possibly due to higher expression level of both copper transporters and MT. / Last but not least, I also compared the toxicity and biomarker gene expression in zebrafish exposed to Cu2O nanoparticle (NP) and CuCl2, respectively. It was found that the toxicity of CuCl2 is much higher than that of Cu2O NP. Then seven genes, including MT, ATP7A & B, copper transporter 1 (Ctr1), metal regulatory transcription factor 1 (MTF1), glutathione sulfur transferase (GST), Cu/Zn superoxide dismutase (Cu/Zn SOD), were chosen for studying. It was found that both Cu2O NP and CuCl 2 up-regulated the mRNA levels of MT, Cu/Zn SOD, and Ctr1, ATP7A & 7B, but down-regulated the mRNA levels of GST. Interestingly, the inductions of MT, Ctr1, ATP7A & B in the Cu2O NP exposure groups were much higher than that of CuCl2 exposure groups in vivo . Furthermore, as determined by using Ctr1, ATP7A and ATP7B gene expression, the no observable effect levels (NOELs) of CuCl2 and nano-Cu2O were 11 ppb and 50 ppb, whereas the lowest observable effect levels (LOELs) of CuCl2 and nano-Cu2O were 43 ppb and 125 ppb. (Abstract shortened by UMI.) / Secondly, the high copper contents in the liver of the tilapia make this fish a suitable model for the study of copper binding proteins. Liver was dissected from tilapia injected with Cu2+ and cytosolic fractions were separated by using Superdex 75 column chromatography followed with atomic absorption spectrometry. Fractions containing copper-binding proteins were found in two major peaks, analyzed using differential proteomic approaches, and loaded on a Cu chelating ion-immobilized affinity column (Cu-IMAC). Of the 113 differentially expressed proteins in these two peaks, some well-characterized copper binding proteins were found, including copper transporter ATP7A, cytochrome c oxidase, metallothionein, collagen, catalase, and vitellogenin. These proteins are mainly involved in endocrine disruption, mitochondria dysfunction, ion competition, lipid metabolism, copper transfer, and cytoskeleton disruption. In addition, a more concrete image about copper transportation pathway was hypothesized according to the function of the novel copper binding proteins identified. / The aims of this study are to identify some novel copper binding proteins and proteins related to Cu2+ toxicity or detoxification mechanisms in the tilapia (Oreochromis niloticus) and the zebrafish (Danio rerio) using a proteomic approach, and to reveal the mechanism of copper tolerance and copper sensitivity by comparing the different biochemical responses to copper exposures between the two model species. / Thirdly, zebrafish liver cell line (ZFL) was also used as a model to study the mechanism of copper toxicity. After processing similar experimental procedures of previous Hepa T1 experiment, 72 different proteins were identified to be regulated by Cu2+ (100 microM and 200 microM). More than 50 % of these proteins were also found differentially expressed in the tilapia. The results suggested that the toxicity mechanism between zebrafish and tilapia was generally conserved. Although, in ZFL, the regulation of several proteins, related to ROS effect, mitochondrion copper transportation, and stress response, was quite different from that in tilapia. / Chen, Dongshi. / Adviser: Chun Ung Ming. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 173-190). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Circulating RNA was previously demonstrated through the identification of tumour-derived transcripts in the plasma and serum of cancer patients. This finding inspired the detection of cell-free fetal mRNA in maternal plasma which in turn facilitated the development of promising non-invasive prenatal assessment strategies applicable to pregnancies regardless of fetal sex. / Finally, in the last study, I implemented what I have learnt from the analysis of circulating fetal RNA into the development of brain-derived RNA transcripts for detection in the plasma of patients who had sustained brain injuries. A systematic approach based on gene expression microarray analysis was adopted to search for circulating brain-specific mRNA markers. Transcripts showing high expression levels in brain tissues but low expression levels in peripheral blood were identified. However, the detectability of these brain-derived mRNA markers in both peripheral and jugular plasma was found to be low. Instead, concentrations of these mRNA markers were found to be higher in cerebral spinal fluid (CSF) from brain-injured than non-brain-injured patients. / In section IV of this thesis, I reviewed and modified the blood sample processing and plasma RNA extraction protocols that were previously practised, in an attempt to enrich circulating fetal RNA in maternal plasma. Besides mRNA, extraction protocols for microRNAs (miRNAs), a new class of circulating nucleic acid markers, were also evaluated. The modifications in the protocols that I introduced led to significant improvements in RNA yield and enhanced the accuracy and reliability of circulating RNA detection in plasma, especially for those marginally detectable transcripts. / Recently, in addition to maternal plasma, there have been studies by other research groups reporting the presence of placental/fetal mRNA in maternal whole blood. In the first part of this thesis, I studied a list of previously identified placental mRNA transcripts, including chorionic somatomammotropin hormone 1 (CSH1), KiSS-1 metastasis-suppressor (KISS1), placenta-specific 4 (PLAC4) and placenta-specific 1 (PLAC1) in maternal whole blood and determined if this whole blood-based approach offered advantages over maternal plasma analysis. The presence of KISS1, PLAC4 and PLAC1 in non-pregnant and post-partum blood samples as well as the confirmed maternal contribution of PLAC4 mRNA in maternal blood proved that most of the detected 'placental' mRNA molecules in maternal whole blood were of maternal origin. To explore if more pregnancy-associated circulating mRNA markers could be developed for maternal whole blood analysis, candidates were mined after performing gene expression microarray comparison of whole blood samples from pregnant and non-pregnant individuals. The pregnancy-specificity of the identified gene candidates was further investigated. However, their presence in non-pregnant whole blood and lack of clearance after pregnancy indicated that they were not "pregnancy-specific" markers. These data suggested that pregnancy specific transcripts could be more readily identified from maternal plasma than whole blood. / The results presented in this thesis have not only provided a foundation facilitating the precise and accurate detection of fetal-specific RNA markers but have also improved the current understanding of the biology of circulating RNA. / Heung, May Sze. / Advisers: Dennis Lo; Rossa Wk Chiu. / Source: Dissertation Abstracts International, Volume: 72-11, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 212-239). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Fresh isolated erythrocyte ghost membranes and erythrocyte suspensions were incubated with an organic hydroperoxide, tert-butyl hydroperoxide, to establish the in vitro oxidative stress models. Circulating erythrocytes from normal individuals were fractionated into subpopulations of different ages by ultracentrifugation and used as an in vivo model. In these models, membrane iPF2alpha-III content accumulation was proportional to oxidative stress and correlated with decreased membrane fluidity. In circulating erythrocytes, membrane iPF2alpha-III increased with age and inversely correlated with membrane fluidity only in the core region. / Oxidative stress is involved in the pathophysiology of a wide variety of human diseases. Isoprostanes, a family of prostaglandin derivatives, are mainly derived from free radical peroxidation of specific polyunsaturated fatty acids (PUFA). Measurement of F2-isoprostanes (F2-iPs) or one specific biologically active isomer (iPF2alpha-III) is considered to be a reliable lipid peroxidation marker in human diseases. However, the association observed between increased plasma/urine F2-iPs and diseases does not necessarily reflect tissue oxidative damages. Circulating erythrocytes, a tissue with limited biosynthetic capacity and poor repair mechanism, would offer a number of advantages for assessment of in vivo oxidative damages. In this thesis, human erythrocyte membrane iPF2alpha-III content was investigated as a new marker for in vivo oxidative stress assessment. Membrane fluidity was used as an indirect marker of cellular function. / To use membrane iPF2alpha-III in a human disease with known oxidative stress burden, 49 Chinese patients on long-term haemodialysis and 31 healthy Chinese subjects were recruited. Both plasma and membrane iPF 2alpha-III showed that haemodialysis patients had increased oxidative stress. Only membrane iPF2alpha-III, but not the conventional used plasma iPF2alpha-III, correlated with membrane fluidity. Furthermore, the significant inverse correlation between membrane iPF 2alpha-III and the core region of membrane fluidity was observed for this group of patients too. Since membrane iPF2alpha-III was shown to provide a link between oxidative stress and erythrocyte function, it would be considered as a new marker of in vivo erythrocyte oxidative stress assessment. (Abstract shortened by UMI.) / Yu Xiongwen. / "July 2005." / Advisers: Wai Kei Christopher Lam; Chung Shun Ho. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3724. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 198-223). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

In conclusion, gene expression profiling in blood may have potential to be an adjuvant marker for early detection of AD. Expression marker panel is more informative than single gene expression signature. Further validation in prospective studies will substantiate its clinical application and explore its potential to differentiate AD from other dementias and to predict the progression from MCI to AD. (Abstract shortened by UMI.) / In the study, the profile of 12 target gene expression levels in peripheral blood cells were determined in 96 AD, 145 MCI and 167 normal controls (NC) by quantitative real-time RT-PCR. The genes were identified with (i) high expression in blood and brain; (ii) differential expression between AD and control; (iii) AD related candidate genes. Then, a list of genes were selected including CTSB, CTSD, DDT, ITPKB, NDUFA6, NRD1, PIN1, SNX2, TSC1, UQCRC1, CNR2, GSTM3. Seven genes were found to be differentially expressed between AD and NC group, with upregulation of CTSB, CTSD, DDT, TSC1 and UQCRC1, and downregulation of ITPKB and PIN1 in AD patients. Expression levels of two genes were increased in the MCI compared with NC group, including CTSB and CTSD. In addition, an upregulation of CTSD, UQCRC1, NRD1 and downregulation of ITPKB were observed in AD subjects in comparison to the MCI group (Mann-Whitney U test, p&lt;0.05). After adjusting for confounding factors of age, gender, education level, ApoE4 status and the total CIRS score, expression level of any single gene was not associated with the classfication of AD or MCI (Logistic regression, p>0.05). A five gene biomarker panel, including DDT, ITPKB, PIN1, TSC1 and UQCRC1 was identified with logistic regression analysis. The function of Logit(P)= ln(P/(1-P))= b0+b1RatioDDT+ b2RatioITPKB + b3Ratio PIN1 +b4 RatioTSC1+b5Ratio UQCRC1 were defined as the probability of a subject to be diagnosed as "AD" or "MCI' by using 5-gene biomarker panel. ROC analysis showed that the AUC for the 5-gene biomarkers panel in differentiating between AD and NC, between MCI and NC, between AD and MCI were 0.79 (95% CI, 0.72-0.86; p&lt;0.001), 0.61 (95% CI, 0.53-0.69; p=0.007) and 0.68 (95% CI, 0.60-0.76; p&lt;0.001) respectively. The 5-gene combination was found to discriminate AD subjects from normal controls with good sensitivity and specificity of 70.7% and 86.7% respectively at an optimal cut-off point of 0.486. Low sensitivity (42.4%) and acceptable specificity (76.2%) were observed at a cut-off threshold of 0.505 when differentiating MCI from NC subjects. Between AD and MCI subjects, gene combination showed a sensitivity of 61% and specificity of 73.7% at a cut-off value of 0.496. / Several genes including CTSD, DDT, NDUFA6, TSC1 and UQCRC1 were found in association with the cognitive and psychiatric symptoms, indicating the role of genetic factors in moderating the presence of cognitive and NP profiles in demented individuals. / The aim of the present study is to evaluate the gene expression profiling of peripheral leukocytes in Chinese subjects with Alzheimer's disease (AD) and explored its potential of clinical application. Behavioral phenotypes of cognitive performance and neuropsychiatric assessment were also investigated in association with gene expression in AD. Persons with mild cognitive impairment (MCI), as an at-risk state between normal aging and clinical dementia, was also assessed in consideration that the information may provide a better understanding of the mechanisms involved in clinical progression of AD. / The genes identified in this study were involved in processes implicated in neurodegneration, including protein isomerization (PIN1), calcium disequilibrium and mitochondria insufficiency (ITPKB and UQCRC1), increased inflammatory response (DDT), apoptosis (CTSB and CTSD) and neurogeneration (NRD1 and TSC1). / Fu, Yan. / Adviser: Chiu Wa Lam. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 132-168). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

INTRODUÇÃO E OBJETIVO: Recidiva da hepatite C crônica com progressão acelerada, embora imprevisível, da fibrose é responsável por piora no prognóstico após o transplante de fígado (Tx). Biópsia hepática protocolar é considerada o padrão ouro para estadiamento da fibrose na recidiva da hepatite C pós-Tx. Para superar as limitações da biópsia, principalmente custo e complicações, marcadores simples e não invasivos de fibrose hepática têm sido propostos para pacientes imunocompetentes, porém com escassos estudos disponíveis no contexto pós-Tx. O objetivo desse estudo é avaliar o desempenho diagnóstico dos marcadores não-invasivos para estadiar fibrose hepática em pacientes pós-Tx. MÉTODOS: Pacientes consecutivos receptores de Tx com recidiva da hepatite C (n=45) que foram submetidos a 118 biópsias hepáticas foram incluídos. Variáveis laboratoriais dentro de trinta dias de cada biópsia foram consideradas. Índice da razão AST-plaqueta (APRI), razão AST/ALT, Escore discriminativo de Bonacini (EDB), Escore de Pohl e índice idade-plaqueta foram calculados para cada biópsia. Fibrose significante foi definida como estágio METAVIR 2. RESULTADO: A área sob a curva ROC (receiver operating characteristic) do Escore discriminativo de Bonacini para predizer fibrose significante foi 0,68, superior aos outros testes avaliados. Utilizando-se o melhor ponto de corte, um valor de Escore discriminativo de Bonacini 8 foi 42% sensível e 95% específico, com razão de verossimilhança positiva e negativa de 7,98 e 0,62, respectivamente. Análise multivariada identificou razão AST/ALT como preditor independente de fibrose significante (OR=4.2; CI 95%=1.5-11.4; p-valor=0.005, ponto de corte 0,89). Análise adicional considerando apenas uma biópsia por paciente confirmou o desempenho superior do Escore discriminativo de Bonacini em relaçãoaos outros testes avaliados, com uma área sob a curva de 0,76. CONCLUSÃO: Escore discriminativo de Bonacini foi o marcador laboratorial não invasivo com melhor desempenho diagnóstico para predizer fibrose hepática significante em pacientes com recidiva de hepatite C crônica pós-Tx / BACKGROUND AND AIM: Recurrent hepatitis C with accelerated, although unpredictable, fibrosis progression accounts for a poor prognosis after liver transplantation (LT). Per protocol liver biopsy is considered the gold standard for fibrosis staging in recurrent hepatitis C after LT to overcome the limitations of liver biopsy, mainly cost and complications, simple non-invasive liver fibrosis tests have been proposed for immunocompetent patients, butfew data are available in the post-transplant setting. The aim of this study was to evaluate diagnostic performance of noninvasive tests to stage liver fibrosis in LT setting. METHOD: Consecutive LT patients with recurrent hepatitis C (n=45) who have undergone 118 liver biopsy were included. Laboratory variables at the time of biopsies were recorded. AST to platelet ratio index (APRI), AST/ALT ratio, Bonacini discriminant score (BDS), Pohl score and age-platelet index were calculated at the time of biopsies. Significant fibrosis was defined as METAVIR stage 2. RESULT: The area under the receiver operating characteristic (ROC) curve (AUC) of Bonacini discriminant score for predicting significant fibrosis was 0,68, better than the other non-invasive liver fibrosis tests. Using the best cutoff value, Bonacini discriminant score value 8 was 42% sensitive and 95% specific, with positive and negative likelihood ratio of 7,98 and 0,62, respectively. Multivariate analysis identified AST/ALT ratio as an independent predictor of significant fibrosis (OR=4.2; CI 95%=1.5-11.4; p-value=0.005, cutoff point 0,89). Additional analysis considering only one biopsy per patient confirmed the superior performance of Bonacini discriminant score compared to the other non-invasive liver fibrosis tests, with an AUC of 0,76. CONCLUSION: Bonacini discriminant score was the non-invasive liver fibrosis test with the best performance for significant liver transplant patients with recurrent hepatitis C