Liver Biopsy

Liver biopsy is recommended as the gold standard method to determine diagnosis, fibrosis staging, prognosis and therapeutic indications in patients with chronic liver disease. However, liver biopsy is an invasive procedure with a risk of complications which can be serious. This book provides the management of the complications in liver biopsy. Additionally, this book provides also the references for the new technology of liver biopsy including the non-invasive elastography, imaging methods and blood panels which could be the alternatives to liver biopsy. The non-invasive methods, especially the elastography, which is the new procedure in hot topics, which were frequently reported in these years. In this book, the professionals of elastography show the mechanism, availability and how to use this technology in a clinical field of elastography. The comprehension of elastography could be a great help for better dealing and for understanding of liver biopsy

The Human ARF Tumor Suppressor Regulates Drosha Nucleolar Localization and rRNA Processing Activity

Ribosomes are vital to the survival of a cell, as they are directly responsible for the synthesis of proteins, which perform critical cellular functions. As such, majority of the energy reserves in a proliferating cell are expended towards synthesis of ribosomes. Cancer cells, with their enhanced proliferation rates, tend to upregulate ribosome biogenesis in order to meet the demand for increased protein synthesis necessary to sustain rapid proliferation. Many of the oncogenes and tumor suppressors known to be deregulated in cancers are capable of positively and negatively regulating ribosome biogenesis, respectively. The ARF tumor suppressor strongly suppresses ribosome biogenesis, particularly in presence of oncogenic signaling. Furthermore, ARF is capable of negatively regulating multiple oncogenes capable of driving tumorigenesis partly through the ribosome biogenesis pathway. As ARF loss is a frequent occurrence in cancer cells, delineating the ARF-regulatory network and determining the impact of ARF loss on this network can give significant insight into the biology of ARF-deficient tumor cells. Expression of the RNase III enzyme, Drosha, has been reported to have prognostic value in multiple cancers. However, Drosha expression appears to have a dual nature in tumorigenesis, as both overexpression and loss of Drosha have been reported to have tumorigenic functions. Although the mechanistic basis of this apparent duality are not yet known, gaining a deeper understanding of Drosha\u27s functional capabilities can give us an insight into its role in tumorigenesis. Drosha performs critical functions in biogenesis of multiple RNA species within the cell, including ribosomal RNA (rRNA), micro RNA (miRNA) and messenger RNA (mRNA). Drosha\u27s role in miRNA biogenesis is the most studied and characterized aspect of its functions and can explain the tumor suppressive aspect of its dual nature; a global decrease in miRNAs has been reported to be part of tumor progression, and loss of Drosha has the potential to significantly deplete mature miRNA population within the cell. However, how overexpression of Drosha can drive tumorigenesis remains to be studied. As enhanced ribosome biogenesis is another feature of cancer cells and Drosha has been shown to aid in processing of r RNA, Drosha\u27s role in ribosome biogenesis pathway has the potential to function in an oncogenic manner. Therefore, further characterization of Drosha\u27s role in ribosome biogenesis can significantly enhance our understanding of its contribution to tumorigenesis. Recent studies in mouse cell lines revealed that ARF tumor suppressor is capable of negatively regulating Drosha expression in a translation-dependent manner. Given the entrenched role of ARF in inhibiting ribosome biogenesis, I hypothesized that ARF\u27s ability to regulate Drosha could impact Drosha\u27s functions in ribosome biogenesis pathway. I further hypothesized that Drosha overexpression could function in a pro-proliferative manner through the ribosome biogenesis pathway. The data presented in this Dissertation reveals that human p14ARF is capable of regulating Drosha protein expression in a dynamic and localized fashion; loss of ARF increases over all cellular Drosha protein levels and also the localization of Drosha to the nucleolus. ARF potentially regulates nucleolar localization of Drosha by sequestering it away from nucleolus, as we found that ARF immunoprecipitated with Drosha in RNA-independent manner. Furthermore, loss of ARF enhances ribosome biogenesis both at the level of 47s rRNA transcription and processing. Association of Drosha with precursor rRNAs was also enhanced in absence of ARF, suggesting that enhanced nucleolar localization of Drosha upon ARF loss contributes to rRNA processing. Drosha overexpression by itself was able to increase ribosome biogenesis, with a modest increase in 47s rRNA transcription and a faster accumulation of 28s and 18s rRNAs. Drosha overexpression led to an increase in ARF expression, although this induction of ARF was not sufficient to inhibit Drosha\u27s ability to enhance ribosome biogenesis and cell proliferation. However, overexpression of ARF negated proliferative enhancement induced by Drosha overexpression. These results point towards a cross-regulatory loop between ARF and Drosha, with functional impact on ribosome biogenesis

Proteomics and dynamics of the human nucleolus

The nucleolus is the most prominent structure within the eukaryotic cell nucleus and it was established to be the site where the majority of ribosomal RNAs (5.8S, 18S and 28S) are transcribed, processed and assembled with ribosomal proteins to form ribosomal subunits. The sole role of ribosome biogenesis, however, cannot explain the specific nucleolar localisations of tumour suppressors, cell cycle-regulatory factors and viral proteins. Therefore, together with my colleagues in the laboratory of Prof. Angus Lamond, we carried out a proteomic approach with an aim to identify the core components of the human nucleolus isolated from HeLa cell nuclei. My role in this project includes verification of the newly identified components, database construction archiving the primary data and providing links to other related information in the public domain, and subsequent bioinformatics and microscopic analyses. So far, 400 proteins were identified in which -30% represents novel or uncharacterised proteins, partly reflecting the current poor status in the human genome annotation, but also reflecting the unknown complexity of the nucleolus. To facilitate the understanding of the functions of these novel proteins, I used deposited data of their gene activities and homologues across the species to identify in silico those novel proteins that are likely to be involved in ribosomal biogenesis. Like the nucleus, the nucleolus itself is subcompartmentalised into different domains, namely, the fibrillar centre, the dense fibrillar components and the granular components and these structures are disassembled and reassembled during mitosis in human cells. In order to understand the intricate mechanism behind these mitotic dynamics, I have generated a panel of 24 HeLa cell lines stably expressing one or more nucleolar marker to study the inter-relationships between these subnucleolar domains as well as their relationships with the chromosomes. The results suggest that (1) a core subunit of the RNA polymerase I dissociates from the chromosomes between prophase and metaphase and (2) the breakdown and reassembly are dependent on the dissociation and the recruitment of RNA polymerase I to the chromosomes respectively.As part of the follow-up to the nucleolar proteome identified, the study of one uncharacterised factor NHPX led to the discovery of a novel nucleolar targeting pathway that is observed in both primary and transformed cell lines. Although NHPX co-localises with the dense fibrillar component marker fibrillarin, NHPX transiently transits through the splicing speckles prior to the nucleolar accumulation whilst fibrillarin accumulates within the nucleolus immediately after the nuclear entry. The NHPX progression is dependent on pre-mRNA transcription and may link multiple RNA metabolic pathways that occur in distinct subnuclear domains.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Assessment of histopathological methods of evaluating response to neoadjuvant therapy in oesophageal and gastric adenocarcinoma

Upper gastrointestinal tract (GIT) cancers usually receive neoadjuvant therapy prior to surgery. The histological assessment of this response and if this can be predicted on the pre-treatment biopsy are the subject of this thesis. The first study assessed the inter- and intra-observer variation amongst pathologists in evaluating the degree of regression using the Mandard scoring system. The results showed that the reproducibility of this system was only fair to moderate in both cases of inter- and intra-observer testing. The second study examined the levels of expression of selected tumour markers before and after neoadjuvant chemotherapy. These included markers monitoring apoptosis (p53 and bcl-2), proliferation (Ki-67), angio- and lymphangio-genesis (VEGF, CD-31 and LYVE-1). The levels of expression in these markers were measured in the pre-treatment biopsies, to monitor if they could predict the response to neoadjuvant therapy. It was found that when the panel of chosen markers being used together, delivered a much higher power of prediction rather than adopting only one marker, where the collective power of prediction was 80.6%, whereas individually, the power of prediction ranged between 24.6% (VEGF) and 60.7% (Ki-67). The third study explored the use of digital image analysis in assessing the response to neoadjuvant therapy. It was found that while this technique paralleled the Mandard scoring system, it delivered a more objective and reproducible assessment. On the basis of these results I suggest that image analysis should be used to assess tumour regression especially in the context of clinical trials. In this retrospective study it has been shown that the pre-treatment biopsy can predict the degree of regression

From chromatin to protein synthesis: the role of glutamate, amyloid beta and tau in Alzheimer’s disease

Alzheimer’s disease (AD) is the most common form of dementia, which is characterised by extracellular Aβ plaques and intracellular neurofibrillary tangles, comprised of fibrils of Aβ42 and tau protein, respectively. A species of tau protein localised to the nucleus has been discovered, but its role in AD is still unclear. Glutamate excitotoxicity, oxidative stress, DNA damage, alteration of the chromatin and nucleolar stress are key features of AD. The early stages of the disease are characterised by minimal neurodegeneration and altered protein synthesis machinery. The culprit (s) and molecular link between these changes and the role of nuclear tau are unclear. This work utilised glutamate stress and Aβ42 oligomers to investigate the involvement of nuclear tau in the chromatin alteration, nucleolar dysfunction, and downstream protein synthesis impairment that occurs in AD. This revealed that glutamate stress in SHSY5Y neuroblastoma cells results in oxidative stress, a nuclear upsurge of phosphorylated tau and delocalisation of nucleolar tau, alongside, DNA damage, heterochromatin loss, nucleolar stress and protein synthesis inhibition, partly through eIF2α phosphorylation. Likewise, short incubation of SHSY5Y cells with Aβ42 oligomers led to significant oxidative stress, with gradual accumulation of nucleolar stress, which resulted in altered transcription and processing of 45S pre-rRNA and decrease in protein synthesis, without DNA damage. Although both glutamate and Aβ ultimately decreased protein synthesis, Aβ incubation led to an increase in heterochromatin formation and a reduction in RNA synthesis without DNA damage, pointing to a different mechanism of toxicity by the Aβ and glutamate stress. To characterise a nuclear role for tau, this work localised tau in the nucleolus and heterochromatin in the SHSY5Y cells and the human brain, where it associates with TIP5 – a key player in heterochromatin formation. Accordingly, tau knockdown destabilises the heterochromatin and increases rDNA transcription, indicating that tau is essential for silencing of the rDNA and heterochromatin stability, similar to TIP5. Overall, this thesis provides evidence that implicates glutamate and Aβ toxicity in some of the changes that occur in the disease and specifically implicates Aβ42 as a key culprit that drives changes in the early stage of the disease. It also reveals a new role for tau in the nucleus and points to its pathological involvement in AD

An immunohistologic study of biological parameters in prostatic intraepithelial neoplasia and adenocarcinoma.

by Kwan Yiu Wing.Thesis (M.Phil.)--Chinese University of Hong Kong, 1999.Includes bibliographical references (leaves 167-187).Abstracts in English and Chinese.ACKNOWLEDGMENTS --- p.IVABSTRACT --- p.1Chapter CHAPTER 1. --- INTRODUCTION --- p.5Chapter I. --- Epidemiology of Prostate Cancer --- p.5Chapter II. --- The Normal Prostate - Prostatic Anatomy --- p.9Chapter III. --- Pathology of Prostatic Cancers --- p.12Chapter CHAPTER 2. --- PROSTATIC INTRAEPITHELIAL NEOPLASIA --- p.16Chapter I. --- Introduction --- p.16Chapter II. --- "Definition, Characteristics and Grading" --- p.16Chapter III. --- Incidence and Prevalence of PIN --- p.26Chapter IV. --- Evidence Linking PIN with Prostatic Carcinoma --- p.27Chapter V. --- Conclusion --- p.37Chapter CHAPTER 3. --- HISTOLOGIC BIOMARKERS --- p.39Chapter I. --- p53 Protein --- p.39Chapter II. --- Proliferating Cell Nuclear Antigen (PCNA) --- p.44Chapter III. --- Ki-67 Antigen --- p.48Chapter IV. --- Epidermal Growth Factor Receptor --- p.49Chapter V. --- E-Cadherin --- p.51Chapter VI. --- CD44 --- p.54Chapter VII. --- nm23 --- p.58Chapter CHAPTER 4. --- OBJECTIVES OF STUDY --- p.62Chapter CHAPTER 5. --- MATERIALS AND METHODS --- p.63Chapter I. --- Materials --- p.63Chapter II. --- Methods --- p.71Chapter III. --- Interpretation of Immunostaining Results --- p.78Chapter IV. --- Statistical Analysis --- p.82Chapter CHAPTER 6. --- RESULTS --- p.83Chapter I. --- Immunohistochemical Results for p53 Protein --- p.83Chapter II. --- Results of Immunostaining of PCNA --- p.90Chapter III. --- Immunostaining and Quantitation of Ki-67 Expression --- p.97Chapter IV. --- Immunohistochemical Expression of EGFr --- p.105Chapter V. --- E-Cadherin --- p.110Chapter VI. --- CD44 --- p.115Chapter VII. --- Expression of nm23 in Prostatic Lesions --- p.122Chapter VIII. --- Correlation and Association of Expressions of All Biomarkers in Prostatic Lesions --- p.128Chapter CHAPTER 7. --- DISCUSSION --- p.132Chapter I. --- p53 Protein --- p.135Chapter II. --- PCNA --- p.137Chapter III. --- Ki-67 --- p.140Chapter IV. --- EGFr --- p.143Chapter V. --- E-Cadherin --- p.146Chapter VI. --- CD44 --- p.148Chapter VII. --- nm23 --- p.151Chapter VIII. --- Association between Biomarkers and Prostate lesions --- p.154Chapter CHAPTER 8. --- SUMMARY AND CONCLUSION --- p.157APPENDICES --- p.164Chapter I. --- Table of incidence and mortality rates of prostate cancer in the United States from 1973 to 1995 by race --- p.164Chapter II. --- Table of leading cancer deaths in Hong Kong from 1971 to 1996 --- p.165Chapter III. --- Table of incidence and mortality rate caused by prostate cancer in Hong Kong --- p.166Chapter IV. --- Reagents --- p.167REFERENCES --- p.16

Investigating the effects of tau mutations in induced pluripotent stem cell-derived neurons

Microtubule-associated protein tau (MAPT) is a neuronal protein which promotes microtubule assembly and stabilisation. The MAPT gene is alternatively spliced to give six tau isoforms: three with 3 microtubule-binding repeats (3R, excluding exon 10) and three with 4 microtubule-binding repeats (4R, including exon 10). MAPT mutations cause the progressive, degenerative disorder fronto-temporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17T), in which tau becomes abnormally phosphorylated and aggregates. FTDP-17T related MAPT mutations can either alter exon 10 splicing and the 3R:4R tau isoform ratio (such as N279K), or affect tau’s biochemical properties (such as P301L and V337M). In this project, I differentiated induced pluripotent stem cells (iPSCs) derived from control subjects and people with the N279K or V337M MAPT mutations to monolayer neurons. There was an increase in the proportion of neurons containing tau phosphorylated at the AT8 epitope in N279K cultures after 50 days, and more 4R tau was detected earlier in the N279K cultures than in controls, confirming the action of the N279K mutation. All monolayer neural cultures matured at similar rates in terms of synapse formation, neuronal marker expression and astrocyte production, and I did not detect other FTDP-17T-relevant phenotypes in MAPT-mutant cultures, perhaps due to the time points (50 and 80-100 days) at which I examined them. To investigate the effects of the N279K mutation independent of iPSC line genetic background, I attempted to create isogenic iPSC lines using CRISPR. This was not successful, but I obtained isogenic control and P301L iPSCs from Axol Bioscience, from which, along with the patient-derived P301L MAPT-mutant iPSCs, I made cerebral organoids (COs). Similar amounts of neuronal, astrocyte, and synaptic proteins were detected in the control and MAPT-mutant COs, as were cortical layer markers indicating a forebrain-like fate. Phosphorylation at the AT8 epitope was detected in the control and MAPT-mutant COs, as was tau misfolding, detected using the MC-1 antibody. In conclusion, some alterations similar to those observed in FTDP-17T brains can be observed in monolayer iPSC-derived neurons and perhaps in iPSC-derived COs. However, some phenotypes may be masked by experimental variability which needs to be addressed in the future.Funded by the MRC-DTP and the Sackler Foundatio

Proteomics

Biomedical research has entered a new era of characterizing a disease or a protein on a global scale. In the post-genomic era, Proteomics now plays an increasingly important role in dissecting molecular functions of proteins and discovering biomarkers in human diseases. Mass spectrometry, two-dimensional gel electrophoresis, and high-density antibody and protein arrays are some of the most commonly used methods in the Proteomics field. This book covers four important and diverse areas of current proteomic research: Proteomic Discovery of Disease Biomarkers, Proteomic Analysis of Protein Functions, Proteomic Approaches to Dissecting Disease Processes, and Organelles and Secretome Proteomics. We believe that clinicians, students and laboratory researchers who are interested in Proteomics and its applications in the biomedical field will find this book useful and enlightening. The use of proteomic methods in studying proteins in various human diseases has become an essential part of biomedical research