Modelling the genomic structure, and antiviral susceptibility of Human Cytomegalovirus

Human Cytomegalovirus (HCMV) is found ubiquitously in humans worldwide, and once acquired, the infection persists within the host throughout their life. Although Immunocompetent people rarely are affected by HCMV infections, their related diseases pose a major health problem worldwide for those with compromised or suppressed immune systems such as transplant recipients. Additionally, congenital transmission of HCMV is the most common infectious cause of birth defects globally and is associated with a substantial economic burden. This thesis explores the application of statistical modelling and genomics to unpick three key areas of interest in HCMV research. First, a comparative genomics analysis of global HCMV strains was undertaken to delineate the molecular population structure of this highly variable virus. By including in-house sequenced viruses of African origin and by developing a statistical framework to deconvolute highly variable regions of the genome, novel and important insights into the co-evolution of HCMV with its host were uncovered. Second, a rich database relating mutations to drug sensitivity was curated for all the antiviral treated herpesviruses. This structured information along with the development of a mutation annotation pipeline, allowed the further development of statistical models that predict the phenotype of a virus from its sequence. The predictive power of these models was validated for HSV1 by using external unseen mutation data provided in collaboration with the UK Health Security Agency. Finally, a nonlinear mixed effects model, expanded to account for Ganciclovir pharmacokinetics and pharmacodynamics, was developed by making use of rich temporal HCMV viral load data. This model allowed the estimation of the impact of immune-clearance versus antiviral inhibition in controlling HCMV lytic replication in already established infections post-haematopoietic stem cell transplant

Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) represents one of the most significant global health issues, given its high prevalence and the challenging nature and physiology of the liver and hepatic surgery, in its many forms. This means that the most appropriate management for HCC should incorporate a multidisciplinary approach, combining the expertise from several different specialties. This book showcases the various steps in the development, diagnosis, staging, and management of HCC and provides views and thoughts from true experts in the field. As such, it is a useful resource for any physician or surgeon, whether training or practicing, who is interested in caring for patients with HCC

GENOME-WIDE ANALYSIS OF CHICKEN MIRNAS AND DNA METHYLATION AND THEIR ROLES IN MAREK'S DISEASE RESISTANCE AND SUSCEPTIBILITY

Marek's disease (MD) is a T cell lymphoma in chickens and causes high mortality and morbidity in productive chickens. Two inbred chicken lines, resistant line 63 and susceptible line 72, with the same MHC haplotype, showed distinct disease outcomes after MDV infection. The current studies aimed to illustrate the role of microRNA (miRNAs) and DNA methylation in MD resistance and susceptibility in chickens. First, to ascertain the function of miRNAs, miRNA microarray experiments were used to identify miRNAs sensitive to MDV infection in the 2 lines. Most miRNAs were repressed in line 72 after MDV infection, while their transcription was steady in line 63. The miRNA target genes were identified in chickens. Cellular miRNA gga-miR-15b and gga-let-7iwere reduced in infected line 72 chickens and MD tumors. The downregulation of the two miRNAs increased the expression of ATF2 (activating transcription factor 2) and DNMT3a (DNA methyltransferase 3a) in infected line 72. These results indicated that miRNAs may play antiviral functions through modulating target gene expression. Next, to characterize the role of miRNAs in MDV infection, the selected chicken miRNAs were overexpressed in MDV infected DF-1 cells. The overexpressions of chicken miRNA gga-miR-15b and gga-let-7i, by using the retroviral based vector, significantly restricted MDV replications in vitro. MDV oncoprotein was repressed, suggesting that chicken miRNAs may limit MDV propagation. Finally, we found deregulation of transcription of DNA methyltransfereases (DNMTs) in lines 63 and 72 after MDV infection, which coordinated with the methylation alterations in the 2 lines. Infection induced differential methylation regions (iDMRs) that were identified through genome-wide DNA methylation quantification. Genes overlapping line-specific iDMRs were related with pathways of different functions in these two lines, implying the involvement of DNA methylation in MD- resistance and susceptibility. An in vitro study showed that DNA methylation inhibitor repressed viral spread and viral replication. In conclusion, the observed variations of miRNA expression and DNA methylation may be associated with disease predisposition in chickens

Analysis of HIV-host interaction on different scales

The human immunodeficiency virus depends on molecular pathways of the host for efficient replication and spread. The intricate network of host-virus interactions shapes the virus\u27; evolution by driving the pathogen to evade immune recognition and constraining it to maintain its capacity to replicate. Study of the HIV-host interactions provides important insights into viral evolution, pathogenicity and potential treatment strategies. This thesis presents an analysis of HIV-host interactions on several scales, ranging from individual protein interactions to whole genomes. On the scale of individual interaction we analyze structural and physical determinants of the interaction between host TRIM5alpha and virus capsid — an interaction of potential therapeutic interest due to the capacity of TRIM5alpha to block retroviral infections. On the scale of viral population we present two studies of a highly variable region of the virus genome involved in the interaction with host cell coreceptors upon virus cell entry. The studies provide insights into the virus evolution and the physicochemical and structural properties related to its interaction with cellular coreceptors. On the scale of the single cell we develop models of HIV cell entry involving virus, host and environmental factors. The models represent a comprehensive picture of the virus phenotype and allow one to view the variability of virus phenotypes on 2D phenotype maps. On the genomic scale we perform a large-scale analysis of all HIV-host interactions. This study reveals insights into general patterns of the host-pathogen evolution and suggests candidate host proteins involved in interactions potentially important for the infection and interesting for further study on other scales. Interactions and processes crucial for the HIV infection reemerge across the scales pointing to the importance of integrative, multi-scale studies of host-pathogen biology.Das Humane Immundefizienz-Virus hängt von molekularen Mechanismen des Wirts für seine effiziente Replikation und Ausbreitung ab. Das komplizierte Netzwerk von Wirt-Virus Interaktionen formt die Evolution des Virus, indem es den Erreger dazu bringt, sich der Erkennung durch das Immunsystem zu entziehen und seine Replikationskapazität aufrecht zu erhalten. Das Studium der HIV-Wirt Interaktionen erlaubt wichtige Einblicke in die viralen Evolution, die Pathogenität des Virus, sowie mögliche Behandlungsstrategien. Diese Arbeit stellt eine Analyse der HIV-Wirt-Interaktionen in mehreren Größenordnungen vor, von einzelnen Protein-Interaktionen bis hin zur Analyse ganzer Genome. In Hinblick auf einzelne Interaktionen untersuchen wir strukturelle und physikalische Determinanten der Interaktion zwischen dem Wirtfaktor TRIM5alpha; und dem viralen Kapsid - eine Interaktion, die von therapeutischem Interesse ist wegen der Fähigkeit von TRIM5alpha, retrovirale Infektionen zu blockieren. In Hinblick auf virale Populationen präsentieren wir zwei Studien einer hochvariablen Region des viralen Genoms, die in der Interaktion des Virus mit zellulären Rezeptoren des Wirts beim viralen Zelleintritt involviert sind. Diese Studien geben Einblick in die virale Evolution und die physikalisch-chemischen und strukturellen Eigenschaften des Virus bezüglich dessen Interaktion mit zellulären Ko-Rezeptoren. Auf der Skala der einzelnen Zelle entwickeln wir Modelle des HIV Zelleintritts welche das Virus, den Wirt und Umgebungsfaktoren berücksichtigen. Diese Modelle bieten ein umfassendes Bild des viralen Phänotyps und erlauben es, die Variabilität des Virus auf 2D-Phänotyp-Karten zu visualisieren. Im genomweiten Maßstab führen wir eine groß angelegte Analyse aller HIV-Wirt-Interaktionen durch. Diese Studie erlaubt Einblicke in allgemeine Muster der Wirt-Pathogen-Evolution und identifiziert Kandidaten für Wirtsproteine, deren Interaktionen potenziell wichtig für die virale Infektion sind und deren weitere Untersuchung in anderen Größenordnungen von Interesse ist. Interaktionen und Prozesse, die von entscheidender Bedeutung für die HIV-Infektion sind zeigen sich wiederholt in allen untersuchten Maßstäben und unterstreichen die Bedeutung einer integrativen und multi-skalaren Untersuchung der Wirt-Pathogen-Biologie

Clinical Studies, Big Data, and Artificial Intelligence in Nephrology and Transplantation

In recent years, artificial intelligence has increasingly been playing an essential role in diverse areas in medicine, assisting clinicians in patient management. In nephrology and transplantation, artificial intelligence can be utilized to enhance clinical care, such as through hemodialysis prescriptions and the follow-up of kidney transplant patients. Furthermore, there are rapidly expanding applications and validations of comprehensive, computerized medical records and related databases, including national registries, health insurance, and drug prescriptions. For this Special Issue, we made a call to action to stimulate researchers and clinicians to submit their invaluable works and present, here, a collection of articles covering original clinical research (single- or multi-center), database studies from registries, meta-analyses, and artificial intelligence research in nephrology including acute kidney injury, electrolytes and acid–base, chronic kidney disease, glomerular disease, dialysis, and transplantation that will provide additional knowledge and skills in the field of nephrology and transplantation toward improving patient outcomes

Current Perspectives on Viral Disease Outbreaks

The COVID-19 pandemic has reminded the world that infectious diseases are still important. The last 40 years have experienced the emergence of new or resurging viral diseases such as AIDS, ebola, MERS, SARS, Zika, and others. These diseases display diverse epidemiologies ranging from sexual transmission to vector-borne transmission (or both, in the case of Zika). This book provides an overview of recent developments in the detection, monitoring, treatment, and control of several viral diseases that have caused recent epidemics or pandemics

Rational Vaccine Design by Reverse & Structural Vaccinology and Ontology

Vaccination is one of the most successful public health interventions in modern medicine. However, it is still challenging to develop effective vaccines against many infectious diseases such as tuberculosis, HIV, and malaria. There are challenges in integrating the high volume, variety, and variability of vaccine-related data and rationally designing effective and safe vaccines efficiently. In my thesis study, I systematically and comprehensively analyzed manually annotated protective vaccine antigens in the Protegen database and identified these protective antigens' enriched patterns. I then created Vaxign-ML, a novel machine learning-based reverse vaccinology method based on the curated Protegen data for rational vaccine design. Vaxign-ML was used to successfully predict vaccine antigens for tuberculosis and Coronavirus Disease 2019 (COVID-19). I also developed a new structural vaccinology design program that optimizes COVID-19 spike glycoprotein as a vaccine candidate for enhanced vaccine protection via T cell epitope engineering. The vaccine antigens selected and optimized by Reverse and Structural Vaccinology in this dissertation are subjected to future experimental verification. Furthermore, I created a community-based Ontology of Host-Pathogen Interactions (OHPI), which served as a platform to semantically represent the interactions between host and virulence factors that are also protective antigens. I developed the Vaccine Investigation Ontology (VIO) for standardized metadata representation for high throughput vaccine OMICS data analysis. Overall, my thesis research aims to uncover protective antigen patterns, create methods/tools to effectively develop vaccines against infectious diseases of public health significance, and strengthen our understanding of vaccine protection mechanisms. These works can be further expanded and integrated with other technologies such as epitope prediction, molecular epidemiology, and high-throughput sequencing to build the foundation of precision vaccinology.PHDBioinformaticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/167997/1/edong_1.pd

Alternations of microRNAs, the microbiome, and gut-host interactions in gastrointestinal diseases

Over the past few decades, an ageing population combined with a shift towards a Western lifestyle has predisposed many individuals towards inter-connected gastrointestinal (GI) diseases, including inflammatory bowel disease (IBD), colorectal cancer (CRC), gastric cancer (GC) and Clostridioides difficile (C. difficile) infection (CDI). anti-TNF-α treatment for IBD patients has a high unresponsive rate, by using bioinformatics approaches, I identified neutrophil chemotaxis may contribute to the treatment resistance and IL13RA2 is the best predictor to identify treatment unresponsive patients. On the other hand, in the intestinal tract, colonocytes consistently exfoliate and shed into the lumen, affecting gut microbiota composition. These molecular/microbial changes involved in disease pathogenesis can be detected in faeces. By using Taqman probe-based real-time polymerase chain reaction (RT qPCR) assay, several non-coding microRNAs (such as miR-18a, miR-20a, miR-221 and miR 135b) and gut microbes (including Fusobacterium nucleatum, Parvimonas micra, Gemella morbillorum, Peptostreptococcus anaerobius, Clostridium hathewayi and Lachnoclostrium sp.) are highly expressed/enriched in faeces in CRC individuals compared to control subjects. The use of a faecal immunological test (FIT) in combination with these biomarkers may improve the non-invasive CRC screening accuracy. Furthermore, Epstein-Barr virus (EBV) is an oncogenic virus and EBV-driven GC accounts for roughly 10% of total GC cases. GC cells infected with EBV alter the molecular aspect at whole-genome, transcriptome, and epigenome levels. For instance, AKT2 activated by mutation in EBV-positive GC cells affecting downstream MAPK and focal adhesion signalling pathways; AKT2 mutation associates with poor patient survival in EBV-positive GC. Furthermore, once patients have received GI treatments, it may suppress/interfere with the patients’ immune system, disrupt the gut flora homeostasis and trigger CDI. Faecal microbiota transplantation (FMT) has been demonstrated as an effective and alternative treatment strategy for CDI patients. However, it is still in clinical trials due to safety concerns. My study revealed that serum miRNAs such as miR-23a-3p, miR-150-5p, miR-26b-5p and miR-28-5p could be used to monitor FMT treatment in CDI patients, and these markers inversely correlate with IL-12B, IL-18, FGF21 and TNFSRF9 at serum protein and mRNA levels, respectively. Furthermore, miR-23a and miR-150 showed cytoprotective effects against C. difficile Toxin B (TcdB)

Non-Coding RNAs in Ovarian Cancer

Ovarian cancer (OC) is the most lethal form of gynaecological cancer, with high- grade serous ovarian carcinoma (HGSOC) being the most common and the deadliest subtype. Non-coding RNAs are a recently discovered species of RNAs that do not code for proteins, yet play a crucial role in both normal physiology and disease. The overall goal of this thesis was to apply the power of non-coding RNAs to OC with the following aims: (1) to identify novel small non-coding RNAs present in serum that could separate patients with HGSOC from healthy women as well as predict their surgical outcome, (2) to assess the role of long non-coding RNAs (lncRNAs) in promoting cisplatin resistance in cell line models of OC, and (3) to study the effects of mutant-p53 on mRNAs and lncRNAs using a small compound known as APR-246 as well as investigating the drug’s mechanisms of action. Firstly, the lethality of OC could partially be attributed to the lack of specific symptoms, leading this disease to be termed the ‘silent killer’, as well as low inci- dence rate of 9.4 cases per 100,000 individuals, both requiring a highly accurate test for population screening that remains an ongoing challenge. Measuring the levels of small non-coding RNAs, known as microRNAs, in serum, experiments described in this thesis aimed to identify novel microRNAs that could separate pa- tients with HGSOC from healthy women as well as predict their surgical outcome, one of the most important factors influencing overall patient survival. Because serum microRNAs can be affected by pre-analytical factors such as haemolysis, the sensitivity of four methodologies to detect low levels of haemolysis was first determined. This work is published in Plos One. The work described in this thesis identified a novel serum microRNA, miR-375, that could improve the accuracy of CA-125, a routinely used biomarker in diagnosing OC, in separating patients with HGSOC from healthy women. Next, serum microRNA miR-34a-5p was found to predict the surgical outcome of patients with HGSOC. In fact, miR-34a-5p was found to be superior to CA-125 for this purpose. Although the standard therapy for treating OC consists of surgical removal of the tumour followed by chemotherapy containing platinum/taxane agents, this regimen may be too aggressive for a sub- set of patients who might benefit from neoadjuvant chemotherapy, i.e. chemother- apy followed by the surgery. A pre-operative expectation of the the surgical out- come could help surgeons decide on optimal timing for surgery. Both miR-375 and miR-34a-5p were also unaffected by haemolysis. Secondly, although OC is initially sensitive to chemotherapy, most patients develop resistance within two years, resulting in recurrent disease that is difficult to treat. To identify novel lncRNAs that could promote drug resistance, expression of ninety lncRNAs was profiled in cell line models of cisplatin resistance. Five lncRNAs were found to have the potential to promote cisplatin resistance in vitro, and lncRNA Urothelial Cancer Associated 1 (UCA1) was selected for further investigations. Despite its role in promoting cisplatin resistance in bladder cancer, UCA1 was not found to promote cisplatin resistance in cell line models of OC. Lastly, the tumour suppressor TP53 plays a central role in the biology of cancer and it is almost universally mutated in HGSOC. Recent evidence suggests that p53, the protein encoded by TP53, can significantly influence the expression of both small and long non-coding RNAs. Experiments described in this thesis aimed to investigate the effect of mutant-p53 on protein-coding and non-coding RNAs by using a small compound known as APR-246 which has been reported to restore wild-type p53 activities in multiple cancers by stabilising the structure of mutant- p53. Despite currently undergoing a phase Ib/II clinical trial for potential treatment of recurrent HGSOC, the ability of APR-246 to restore wild-type p53 activities in HGSOC has not been tested. A global transcriptomic analysis conducted in this thesis discovered that p53-responsive mRNAs and lncRNAs were not robustly induced following APR-246 treatment in two cell line models of HGSOC, but indicated that APR-246 could function by inducing high levels of reactive oxidative species (ROS). Overall, data presented in this thesis demonstrated the utility of small non- coding RNAs in identifying patients with HGSOC from healthy women as well as predicting their surgical outcome. This thesis also implicated that lncRNAs, in general, could have a role in promoting cisplatin resistance in OC as well as suggested that APR-246 could, based on evidence obtained from the expression of p53-responsive mRNAs and lncRNAs, act independently of mutant-p53. Together, this research raises novel ways for clinical management of patients with HGSOC and addresses the challenge of drug resistance using non-coding RNAs, as well as questions the assumed mechanisms of action of the ‘p53-activating’ drug APR- 246