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1
Wang, Meng. « Mutational analysis of DNA deaminases ». Thesis, University of Cambridge, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.611829.
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Beale, R. C. L. « DNA sequence specificity of APOBEC family deaminases ». Thesis, University of Cambridge, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.596493.
Texte intégralRésumé :
APOBEC family deaminases are capable of causing mutations by deaminating cytosine in DNA to uracil. This is exploited in diversification of the repertoire of antibodies by somatic hypermutation, and also in restricting the spread of retroviruses. APOBEC family induced mutations are not distributed entirely at random throughout the genes they deaminate; rather each APOBEC family member has its own preferred local sequence that will be preferentially targeted. Work presented in this thesis elucidates these preferred motifs for a number of different deaminases and investigates the structural basis of their specificity using viral and bacterial genetic assays. To determine the local sequence specificities of APOBEC proteins active in E. coli, a novel selection system was devised based on the conditional-lethal sacB gene. By varying the activity and orientation of promoters it was possible to target mutations to a chromosomally integrated sacB gene under certain conditions. Selecting for viable mutants generated mutation spectra for the AID, APOBEC1 and APOBEC3G deaminases. This enabled their preferred sequence motifs to be identified and correlated with particular mutation patterns found in vivo.
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Lembo, Gaia. « Substrate targeting and inhibition of editing deaminases ». Doctoral thesis, Università di Siena, 2021. http://hdl.handle.net/11365/1144295.
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Identification of small molecules against APOBEC3B
The APOBECs are deaminases that act on DNA and RNA to restrict exogenous nucleic acids. Yet, the signature of their mutagenic activity –especially that of APOBEC3A and APOBEC3B- has been observed in the cancer genomes and their ability to increase the genetic heterogeneity of tumours has been linked to the onset of drug resistance in cancer. As such inhibition of their enzymatic activity represents a potential target for anticancer therapies. During my PhD I worked at the identification of APOBEC3B small-molecule inhibitors. To this aim, I used a computational approach to perform a virtual screening on large library of molecules to block APOBEC3B enzymatic activity. I then tested selected molecules from the virtual screening using biochemical assays to quantify their effect on APOBEC3B activity and their capacity to interfere with APOBEC3B binding to DNA. Through this, I was able to identify two small molecules that reduce the activity of this protein, which could provide basis for the development of the first drug for anti-APOBEC activity.
Engineering ADAR2 to act on DNA
Genome editing technologies have revolutionized our ability to target and modify the genomes of living cells and organisms. The fusion of AID/APOBECs to genome editing tools such as Cas9 allowed the development the first base editor, molecules that can be targeted to mutate specific cytosines. The pool of available Base Editors is in constant expansion as new molecules are developed to target DNA with more specificity and efficiency. As the only adenine-targeting Base Editor is based on TadA- an RNA deaminase-, I focused on the development of a A•T base editor based on the catalytic domain of ADAR2. Adenosine Deaminases Acting on RNA (ADARs), are editing enzymes that catalyse the C6 deamination of adenosine (A) to produce inosine (I) in double-stranded RNA. As human ADAR2 is able to target DNA/RNA hybrids, I first tried -without success- to use chimeras of n/dCas9 and the deaminase domain of ADAR2 to induce mutations in a fluorescent reporter. I then used a bacterial screen to select for mutants of ADAR2 that act on DNA. I selected a mutant that induces a mutator phenotype in bacteria and DNA damage in mammalian cells. I am currently working to engineer this mutant into a Base Editor suitable for biotechnological applications such as gene therapy, antiviral treatment and cancer therapy.
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Hogg, Marion. « Characterising new roles for APOBEC4 and ADAR deaminases ». Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/4792.
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Deamination or the hydrolytic removal of one hydroxyl group from a base in DNA or RNA can lead to changes in the transcript and protein produced. Examples of this are the deamination of cytosine residues in DNA by activation induced deaminase (AID) during antibody diversification, or deamination of adenosine at the Q/R site in the GluR-B transcript by adenosine deaminase acting on RNA 2 (ADAR2), which regulates calcium permeability in neurons. The initial focus of my thesis was to characterise a putative novel deaminase APOBEC4. APOBEC4 was identified in a bioinformatic search for proteins containing the core catalytic residues common to the whole family of Cytidine Deaminase enzymes. The aim of the project was to express and purify recombinant APOBEC4 for in vitro characterization, however despite using different expression systems and purification conditions the majority of the recombinant protein was inherently insoluble and I could not isolate sufficient amounts of protein for further studies. Recombinant protein with a GST-tag was used to generate polyclonal antibodies which recognised recombinant protein but were unable to detect endogenous APOBEC4. The focus of my thesis then changed to the process of adenosine to inosine editing in RNA, which is a post-transcriptional mechanism for generating protein diversity. The enzyme family responsible for catalysing this reaction is known as ADAR, and Drosophila melanogaster has only one Adar gene. Flies lacking the Adar gene show locomotion defects and age-dependent neurodegeneration, however little is known about the molecular mechanism underlying these defects. To investigate this phenotype I performed microarray analysis on RNA isolated from heads of 5 day old flies lacking the Adar gene to characterize gene expression changes in the fly heads before neurodegeneration caused secondary effects. Analysis was also performed on Adar-null flies expressing either an active Adar gene or a catalytically-inactive Adar gene in cholinergic neurons to determine which transcripts could be directly regulated by Adar. I confirmed the microarray results by real-time PCR, and demonstrated that the changes in transcript level could be reversed by expression of either active or catalytically-inactive Adar. Expression of edited transcripts did not change dramatically. Filter-binding analysis and electrophoretic mobility shift assay revealed that recombinant ADAR could bind to all RNA transcripts analysed with similar affinity; both known substrates and potential new substrates for Adar, as well as transcripts that were chosen as negative controls due to their expression not altering in the expression microarray. Recombinant ADAR bound to dsRNA with a very high affinity; other transcripts investigated bound with considerably lower affinity, yet all transcripts investigated were bound by ADAR. Further analysis of transcript changes in Adar-null flies was investigated by performing microarray analysis with a custom-made splicing-sensitive microarray. Analysis revealed that a subset of transcripts were differentially spliced in Adar-null flies; however this group of transcripts was distinct from the group identified as being altered on the expression microarray, indicating that the splicing changes are independent of changes in expression. Analysis of exon-specific probes on the splicing array confirmed the transcript changes identified in the expression array. Real-time PCR confirmed the changes in splicing, and these transcripts were further examined by sequence analysis. This revealed several transcripts identified as altered by the AS array showed use of alternative polyadenylation sites indicating ADAR may have a role in detemining polyadenylation site selection.
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Torrini, Serena. « Physiological and pathological perspectives in the biology of APOBEC deaminases ». Doctoral thesis, Università di Siena, 2022. http://hdl.handle.net/11365/1194433.
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The thesis is focus on RNA editing mediated by two AID/APOBEC family members.
The aim of my work was the investigation of possible novel factors that regulate hAPOBEC1 expression or cofactors which help the deaminase to exert its activity. First, I characterised cellular models for their proliferation and clonogenic activities as well as cell cycle distribution evaluating a combinatorial effect of hAPOBEC1 and RBM47 which lead to a decrease in cell growth.
I investigated the role of RNA editing beyond the lipid transport by high-throughput sequencing which provided me information regarding new deamination events, RNA stability, and also a differential gene expression in presence or absence of the editosome components. By Differential expression analysis, I got a list of genes that are differentially expressed in clones with hAPOBEC1 and RBM47 which need to be analysed for their biological meaning.
From the mRNA-seq I got a consistent list of putative edited sites even though some of them were validated with no success. Moreover, I applied a genetic library screen to activate a high number of genes in cells expressing RBM47 to evaluate an eventual up-regulation of APOBEC1 and find factors which trigger its expression. The cells in which editing happened have been selected thanks to a specific fluorescent reporter containing ApoB target. The results have still to be analysed.
The second aim of my project was to study APOBEC3A regulation, by chemical and genetic screenings, through the development of a specific sensitive reporter system to detect APOBEC3A-mediated RNA editing. In this work I presented the design of an artificial fluorescent reporter containing a target of APOBEC3A like SDHB or DDOST properly built to produce a stop codon in the middle of the target and optimised for the levels of editing. I checked its specificity for APOBEC3A and not for other APOBEC proteins like APOBEC1 and APOBEC3B. This let me also detected a novel putative editing site mediated by APOBEC3A by Sanger sequencing. Moreover, I designed another fluorescent reporter system able to evaluate APOBEC3A RNA editing by fluorescent microscopy. I created stable cell lines expressing all the lentiviral reporter plasmids to further investigate induction of endogenous APOBEC3A and its regulation. In a future perspective the dual fluorescent reporter could be a useful tool to identify novel RNA editing targets upon the application of an activation library screen.
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Giorgio, Salvatore Di. « Computational approaches for the identification of APOBEC1- dependent RNA editing events in human tissues ». Doctoral thesis, Università di Siena, 2020. http://hdl.handle.net/11365/1096840.
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The AID/APOBECs are cytosine deaminases involved in diverse physiological contexts through their ability to edit DNA and RNA. This ability comes with a price: Activation-Induce cytidine Deaminase (AID), the main player in the antibody diversification processes, is responsible for some of the common genetic alterations in mature B-cells tumours; the APOBEC3 subgroup, important actors in virus defence, have been linked to different mutational processes in a number of cancers.
Also APOBEC1, an RNA/DNA editing enzyme, also able to restrict lentivirus and mobile elements, can act as a mutator in human cells, and its aberrant expression could be linked to the onset of alterations both at the genomic and transcriptomic level. APOBEC1 RNA editing is a post transcriptional process, its only well-characterized target is the Apolipoprotein B transcript (ApoB) in the small intestine where editing of C6666 induces formation of a stop codon and translation of a truncated protein.
Quite different is the situation in Rodents where, thanks to the availability of APOBEC1 knockout mice, hundreds of APOBEC1-dependent editing events, have been discovered beyond ApoB. To date, APOBEC1 deficiencies are not known and in humans and only a few transcripts have been added to the list of targets.
Despite the targets known in mice and in humans, we are far from understanding the overall physiological meaning of APOBEC1-induced C to U editing. If we exclude the efforts that have been made to identify and characterise C to U editing in rodents, only few computational approaches have been employed to identify and characterize human APOBEC1 targets.
This is the reason why I have used available human RNA-seq data to develop a computational strategy for the identification of APOBEC1 dependent RNA editing events.
I used The Cancer Genome Atlas (TCGA) and The Genotype-Tissue Expression (GTEX) to obtain datasets of samples in which APOBEC1 is expressed at different levels.
Using these datasets, I divided samples in high and low expression levels of APOBEC1, and through known tools and ad hoc scripts I built different pipelines to identify positions in the transcript that are differentially edited.
The pipeline includes several filters: removal of mapping artefacts, germline and somatic single nucleotide variants, removal of homopolymeric regions and so on.
Among the several strategies I used, the most promising are those applied to the GTEX small intestine data, where a strict analysis has shown the presence of at least 12 sites, including 3 known targets on the ApoB mRNA.
Surprisingly we found evidence of ApoB editing at canonical sites beyond the small intestine, even in absence of measurable APOBEC1 expression.
Considering the possible presence of APOBEC1 outside the gastric tissue, to improve our capacity to identify C to U editing in human tissues, I decided to create a database of C>U edited sites using RNA-seq from APOBEC1 -transfected Hek293T cell lines.
This database, despite not representing physiologically edited sites, it informs on all positions biologically editable. Crossing these positions with those obtained from the GTEX dataset results in the identification of hundreds of common edited sites.
Finally, I tested the hypothesis that APOBEC1 editing affects the transcript stability in Hek293T cells. Preliminary data suggest that APOBEC1 expression could shift the equilibrium between processed RNA and non-processed RNA towards the latter one.
The second part of the thesis centers on the study of RNA-off targets induced by Base editors (BEs).
In order to improve the safety of this powerful genome editing tool, another PhD student in the lab, Francesco Donati, selected several APOBEC1 mutant that are not able to edit the RNA while maintaining their mutagenicity on DNA. He investigated both the tumorigenicity of these mutants in mice and their use in genome editing. He obtained exonic and transcriptomic data from murine liver tumors and from cells overexpressing the mutant base editors, respectively. I performed the bioinformatic analyses to explore the mutational signature induced by APOBEC1 in mice, and to assess the off-targets effects on RNA and DNA of these base editors. I demonstrated that -contrarily to wild-type APOBEC1- these mutants provide the ability to perform genome editing in absence of detectable off-targets.The 2019-nCoV outbreak has become a global health risk. Editing by host deaminases is an innate restriction process to counter viruses, and it is not yet known whether it operates against coronaviruses. Here we analyze RNA sequences from bronchoalveolar lavage fluids derived from two Wuhan patients. We identify nucleotide changes that may be signatures of RNA editing: Adenosine-to-Inosine changes from ADAR deaminases and Cytosine-to-Uracil changes from APOBEC ones. A mutational analysis of genomes from different strains of human-hosted Coronaviridae reveals patterns similar to the RNA editing pattern observed in the 2019-nCoV transcriptomes. Our results suggest that both APOBECs and ADARs are involved in Coronavirus genome editing, a process that may shape the fate of both virus and patient.
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Periyasamy, Manikandan. « Cytidine deaminases are regulators of estrogen receptor activity in breast cancer cells ». Thesis, Imperial College London, 2012. http://hdl.handle.net/10044/1/9217.
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Breast cancer is the most common cancer worldwide, with 1.38 million women diagnosed with the disease each year. Estrogens play a critical role in the development and progression of breast cancer, their action being mediated by the estrogen receptors (ER), ERα and ERß, which are the members of the nuclear receptor superfamily of transcription factors. This understanding has led to the development of endocrine therapies aimed at inhibiting ER action by competitive binding to the ER (anti-estrogens), or using inhibitors of estrogen biosynthesis (aromatase inhibitors). Determining the mechanisms by which ER regulate gene expression will aid our understanding of the role of ER in breast cancer progression, response and resistance to endocrine therapies. Upon binding estrogen, ER drives the expression of estrogen responsive genes through the orderly recruitment of co-regulators that act by remodelling and modifying chromatin, ultimately promoting RNA polymerase II recruitment and transcription initiation. Previous work from our laboratory has shown that the APOBEC3B cytosine deaminase acts as an ERα transcriptional coactivator in reporter gene assays. Here, I have developed these initial observations and demonstrate that APOBEC3B is important for the regulation of estrogen responsive genes and breast cancer cell growth. I show that APOBEC3B is recruited to the promoters of estrogen-responsive genes and interacts with ERα. Studies carried out to identify the molecular mechanisms by which APOBEC3B regulates the expression of estrogen-responsive genes included its potential role in DNA demethylation and identified a role for APOBEC3B in DNA strand break formation at the promoter of the estrogen regulated pS2 gene. Together, these studies identify APOBEC3B as an important new coregulator of ERα that is required for the regulation of gene expression by estrogen in breast cancer cells.
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Katsuragi, Tohoru. « Microbial cytosine deaminases and their use in a new kind of cancer chemotherapy ». Kyoto University, 1990. http://hdl.handle.net/2433/78236.
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Li, Xianghua. « Physiological roles of Drosophila ADAR and modifiers ». Thesis, University of Edinburgh, 2013. http://hdl.handle.net/1842/12225.
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ADAR (Adenosine Deaminases acting on RNA) family proteins are double-strand RNA binding proteins that deaminate specific adenosines into inosines. This A-to-I conversion is called A-to-I RNA editing and is well conserved in the animal kingdom from nematodes to humans. RNA editing is a pre-splicing event on nascent RNA that may affect alternative splicing when the editing occurs in the exon-intron junction or in the intron. Also, editing may change biological function of small RNAs by editing the premicroRNAs or other noncoding RNAs. Editing also alters protein amino acid sequences because inosine in the mRNA base pairs with cytosine and is therefore read as guanosine. In mammals, there are three ADAR family proteins, ADAR1, ADAR2, and ADAR3, encoded by three different genes. So far, no enzymatic activity of ADAR3 is detected. The most frequently edited targets of ADAR1 and ADAR2 are regions covering copies of Alu transposable elements in primates. In addition, loss of some specific editing events leads to profound phenotypes when the editing does not occur correctly. For example, some human neural disorders – such as epilepsy, forebrain ischemia, and Amyotrophic Lateral Sclerosis – are known to be associated with abnormally edited ion channel transcripts. Drosophila has a single ADAR protein (encoded by the Adar gene) that is highly conserved with human ADAR2 (encoded by the ADARB1 gene). To date, 972 editing sites have been identified in 597 transcripts in Drosophila, and approximately 20% of AGO2-associated esiRNAs are edited. Similar to mammals, many ion channel-encoding mRNA transcripts undergo ADAR-mediated A-to-I editing in Drosophila. While Adar1 null mice die at the embryonic stage and Adar2 null mice die shortly after birth due to seizures, Adar null flies are morphologically normal and have normal life span under ideal conditions. However, Adar null flies exhibit severe neurodegeneration and locomotion defects from eclosion, whilst Adar overexpression (OE) is lethal. To better understand the physiological role of RNA editing and ADAR, and to shed light on ADAR-related human disease, I used Drosophila Adar mutant flies as a model organism to investigate phenotypes, and to find chromosomal deletions and specific mutations that rescue the neural-behavioural phenotype of the Adar null mutant flies. Using the publicly available chromosomal deletions collectively covering more than 80% of the euchromatic genome of Chromsome III, I performed a genetic screen to find rescuers of the lethality caused by Adar overexpression. I confirmed that mutation in Rdl (Resistant to dieldrin, the gene encoding GABAA receptor main subunit) rescues. This rescue was not likely caused by effects on Adar expression level or activity. Driven by the hypothesis that the rescue may be due to reduction in GABAergic input to neurons, I recorded spontaneous firing activity of Drosophila larval aCC motor neurons using in vivo extracellular current recording technique. As expected, the neurons overexpressing Adar had much less activities compared with wild type neurons. Also, I found that Adar null fly neurons fired much more and showed epilepsy-like increased excitability. Although feeding PTX (Picrotoxin), a GABAA receptor antagonist, failed to rescue the lethality, reducing the expression of GAD1 to reduce synthesis of GABA was able to rescue the ADAR overexpression lethality. These results suggest that ADAR may finetune neuron activity synergistically with the GABAergic inhibitory signal pathway. I used MARCM (mosaic analysis using a repressible cell marker) to detect cellautonomous phenotypes in Adar null cells in otherwise wild type flies. Although neurodegeneration, observed as enlarged vacuoles formation in neurophils, was detected both in histological staining and EM images, the Adar null neurons marked with GFP from early developmental stages were not lost with age. Nevertheless, swelling in the axons or fragmentation of the axon branches of Adar null neurons was sometimes observed in the midbrain. By comparing the Poly-A RNA sequencing data from Adar null and wild type fly heads, we detected significant upregulation of innate immune genes. I confirmed this by qRT PCR and found that inactive ADAR reduces the innate immune gene transcript levels almost as much as active ADAR does. Further, using the locomotion assay, I confirmed that reintroducing inactive ADAR into Adar null flies can improve the flies’ climbing ability. Based on the Adar null flies having comparatively low viability, I performed a second deficiency screen to find rescuers of Adar null low viability using the same set of deficiencies as in the lethality rescue screen described above. I found seven deletions removing 1 to 37 genes that significantly increased the relative viability of the Adar null flies. However, not all the rescuing deficiencies also improved the Adar null locomotion. One rescuing gene, CG11357 was mapped from one of the rescuing deficiencies, and some mutant alleles of cry, JIL-1 and Gem3 also showed significant effects on the Adar null fly viability. The single gene viability rescuers were also not necessarily locomotion or neurodegeneration rescuers. Although the initial aim was to find neural-behavioural rescuing genes from the viability screen, the viability rescuers found in the screen are more likely to play a role in different aspects of stress response for survival.
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Jaguva, Vasudevan Ananda Ayyappan [Verfasser]. « APOBEC3 DNA deaminases : A mechanistic study of A3A, A3C, and A3G action on retroviruses and counteraction by viral proteins / Ananda Ayyappan Jaguva Vasudevan ». Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2017. http://d-nb.info/1148720936/34.
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Mohammed, Essam Mohammed Ahmed. « The effect of APOBEC3 deaminases on HBV replication and the role of HBx in the inhibition of RNA interference and in the development of hepatocellular carcinoma ». Thesis, Imperial College London, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.485554.
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Hepatitis B is one of the world's major infectious diseases. Some 350 million people are chronic carriers of the virus (HBV), a significant minority go on to develop cirrhosis or cancer of the liver and over 1 million die annually from HBV liver disease. An effective vaccine has been available for nearly 20 years, however, vaccination cannot be used to treat established infections. The replication strategy of this virus has been described in detail, but virus-host interactions leading to acute and chronic disease are still poorly understood. The main objective of this thesis is to investigate the interaction ofHBV, in particular the HBx protein, with recently identified anti-viral pathways (RNA interference and APOBEC3 deaminases) and the relationship of HBV proteins to viral pathogenesis. The DNA editing enzyme APOBEC3G has been shown to cause lethal G to A mutations in replicating retroviruses during reverse transcription. HBV replication involves a reverse transcription step and recent evidence indicates that APOBEC3G can also interfere with HBV replication. I hav(fr.shown by RT-PCR that HepG2 and Huh7, liver cell lines, express very little APOBEC3G mRNA. Consequently, to investigate the effect ofAPOBEC3 deaminases on HBV, using transient and long term HBV replication models, I have constructed recombinant adenoviruses that express APOBEC3 enzymes. The expression of APOBEC3 deaminases by these recombinant adenoviruses has been shown, by in situ immunostaining, in HepG2, Huh7 and HepG2.2.15 (a HBV producing cell line) cells ,with a transduction efficiency of about 95%. I have shown that APOBEC3G can inhibit HBV replication by up to 90% and it causes extensive G to A mutations in the HBV genome. Other members of the APOBEC3 family -APOBEC3B, APOBEC3C and APOBEC3F- can also inhibit HBV replication to different levels. Using purified APOBEC3G' protein and DNA oligonucleotides corresponding to HBV precore/core sequences I have shown, in a cell free assay that a G at position 1896 can be converted to an A This corresponds to the G to A mutation which is observed in patients who are chronically infected with HBV but have become HBeAg negative. The results of these experiments suggest that APOBEC3G might be responsible for the in vivo precore 1896 G to A mutation. lhave also shown that the expression ofAPOBEC3G in hepatoma cell lines resulted in a 25-60 % increase in the intracellular HBsAg, which is the direct result of the inhibition ofHBV virion maturation. Other experiments indicate that HBx has some inhibitory effects on RNA interference. Finally as part of a collaborative study with Dr. Betty Slagle, Baylor College' of Medicine, Texas, USA, a double-transgenic HCV/HBx mouse, which is predisposed to liver disease (steatosis and hepatocellular carcinoma), has been shown to express HBx and HCV NS3 proteins by immunohistochemistry indicating that the co-expression of these proteins could have a synergistic effect leading to an increase in liver pathology.
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Kaiser, Shari Marie. « Control of retroviral replication by host cellular factors / ». Thesis, Connect to this title online ; UW restricted, 2007. http://hdl.handle.net/1773/5019.
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Spears, Jessica Lynn. « Trypanosoma brucei tRNA Editing Deaminase : Conserved Deaminase Core, Unique Deaminase Features ». The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1306848166.
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Silvas, Tania V. « Investigating the Structural Basis for Human Disease : APOBEC3A and Profilin ». eScholarship@UMMS, 2018. https://escholarship.umassmed.edu/gsbs_diss/955.
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Analyzing protein tertiary structure is an effective method to understanding protein function. In my thesis study, I aimed to understand how surface features of protein can affect the stability and specificity of enzymes. I focus on 2 proteins that are involved in human disease, Profilin (PFN1) and APOBEC3A (A3A). When these proteins are functioning correctly, PFN1 modulates actin dynamics and A3A inhibits retroviral replication. However, mutations in PFN1 are associated with amyotrophic lateral sclerosis (ALS) while the over expression of A3A are associated with the development of cancer. Currently, the pathological mechanism of PFN1 in this fatal disease is unknown and although it is known that the sequence context for mutating DNA vary among A3s, the mechanism for substrate sequence specificity is not well understood.
To understand how the mutations in Profilin could lead to ALS, I solved the structure of WT and 2 ALS-related mutants of PFN1. Our collaborators demonstrated that ALS-linked mutations severely destabilize the native conformation of PFN1 in vitro and cause accelerated turnover of the PFN1 protein in cells. This mutation-induced destabilization can account for the high propensity of ALS-linked variants to aggregate and also provides rationale for their reported loss-of-function phenotypes in cell-based assays. The source of this destabilization was illuminated by my X-ray crystal structures of several PFN1 proteins. I found an expanded cavity near the protein core of the destabilized M114T variant. In contrast, the E117G mutation only modestly perturbs the structure and stability of PFN1, an observation that reconciles the occurrence of this mutation in the control population. These findings suggest that a destabilized form of PFN1 underlies PFN1-mediated ALS pathogenesis.
To characterize A3A’s substrate specificity, we solved the structure of apo and bound A3A. I then used a systematic approach to quantify affinity for substrate as a function of sequence context, pH and substrate secondary structure. I found that A3A preferred ssDNA binding motif is T/CTCA/G, and that A3A can bind RNA in a sequence specific manner. The affinity for substrate increased with a decrease in pH. Furthermore, A3A binds tighter to its substrate binding motif when in the loop region of folded nucleic acid compared to a linear sequence. This result suggests that the structure of DNA, and not just its chemical identity, modulates A3 affinity and specificity for substrate.
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Picken, Nichola Caryl. « Structural studies of porphobilinogen deaminase ». Thesis, Birkbeck (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.314290.
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Pauklin, S. « Regulation of activation induced deaminase ». Thesis, University College London (University of London), 2009. http://discovery.ucl.ac.uk/18720/.
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Activation Induced Deaminase (AID) belongs to the protein family of DNA deaminases, which catalyse the deamination of the cytosine residues in single stranded DNA, resulting in the formation of deoxy-uracils. The enzymatic activity of AID is required for the immunoglobulin gene modifications by class switch recombination (CSR), somatic hypermutation (SHM) and gene conversion (iGC). While being essential for antibody diversification, the activity of AID can be harmful for the organism due to its direct mutagenic activities and induction of genomic instability. This thesis investigates AID regulation both, on the level of gene expression and its interaction partners, and the DNA repair pathways triggered by AIDmediated DNA deamination. Firstly, I have identified estrogen and progesterone as regulators of AID expression. This is achieved via direct binding of estrogen and progesterone receptors to AID promoter. Estrogen leads to an induction of AID expression and increase in AID-mediated downstream pathways – SHM, CSR as well as oncogenic translocations between Ig and c-myc loci. In contrast, progesterone results in a decrease in AID expression and an attenuation of its downstream pathways. Secondly, by generating DT40 cell lines with endogenously tagged AID, we used co-immunoprecipitation and subsequent mass spectrometry for identifying proteins that form a complex with AID in the cytoplasm, nucleoplasm and chromatin. The results of this approach gave us possible insight into the mechanistic process of AID-mediated DNA deamination in vivo, suggesting that chromatin bound AID resides in a complex with elongating RNA polymerase II. Thirdly, by expressing AID in meiotic recombination deficient fission yeast and nematode, we have established that a meiotic cell can process a base mismatch, using the base excision repair machinery, to give rise to meiotic recombination. This suggests that meiotic cells can process lesions other than Spo-11 induced DSBs for recombination.
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Abbott, C. M. « Adenosine deaminase in the wasted mouse ». Thesis, University of Reading, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.374695.
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Mosley, Julie Elizabeth. « Studies on recombinant ubiquitous and erythroid human porphobilinogen deaminase and mutational analysis of E. Coli porphobilinogen deaminase ». Thesis, University of Southampton, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.273856.
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Ahmed, R. A. A. « Rational design of inhibitors of porphobilinogen deaminase ». Thesis, University of Cambridge, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.595387.
Texte intégralRésumé :
The thesis describes the synthesis of new analogues (53-57) of PBG and their inhibition of and mechanistic studies on the enzyme PBG deaminase from E. coli. The analogues with an additional alkyl group, 53 and 54, were successfully obtained in good yields in 10 steps. The caged compound 97 was also obtained but the final step of hydrogenolysis of the benzyl protecting groups also caused reduction of the nitro group on the 2-nitrobenzyl substituent. Testing of analogues 53 and 54 with the enzyme (PBGD), however, showed no inhibition. A new route for the synthesis of the conformationally restricted analogue 6, 11-ethanoPBG 56 was developed in this work and one stereoisomer of 56 was obtained (the cis-isomer). When this analogue was tested as an inhibitor it also showed no inhibition. This was thought to be due to the stereochemistry of the compound since modelling studies had predicted that the trans-isomer would bind tightly to the enzyme not the cis-isomer. Therefore, the 11-hydroxy-6,11-ethanoPBG analogue 57 was prepared and tested as an inhibitor with the enzyme. The result was only a modest effect on the enzyme's kinetics. 6-Methyl-PBG 55 which has the extra methyl group on the acetate side chain of PBG, was prepared to test the importance of this chain. When the compound was tested as an inhibitor of PBG deaminase it showed strong inhibition and a K1 value of about 3mM was determined. This is the strongest inhibitor ever obtained for the enzyme. When this analogue was tested as a substrate for the enzyme it appeared by UV/visible spectroscopy to form a novel porphyrin, although due to the small quantity, it was not possible to isolate this porphyrin. In addition, it was possible to isolate the covalent enzyme/analogue complexes by the use of the FPLC technique. It was also possible to confirm the formation of complexes of the enzyme with one and two molecules of the substrate analogue bound for the first time by the use of the LC-mass spectrometry. This work also describes attempts to crystallise some of the enzyme/analogue intermediates, which were isolated by the FPLC. These attempts were successful and single crystals were obtained. These crystals diffract well and currently further work is going on to solve their structure. DesaminoPBG 26 has been previously prepared and gave a K1of 75 mM with PBG deaminase from human erythrocytes. In this project it has been tested with the PBG deaminase from E. coli and shown to be a less powerful inhibitor. A new approach for preparing 2,3-disubstituted pyrroles was developed and the 2-methyl-3-pentylpyrrole 59 was obtained in 70% yield. This compound is used as a building block in the biosynthesis of the red pigment prodigiosin 168, one of a class of naturally occurring polypyrroles which exhibit antimicrobial and cytotoxic properties.
20
Warren, M. J. « Investigations into the mechanisms of porphobilinogen deaminase ». Thesis, University of Southampton, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.233456.
Texte intégral
21
SICOURI, LARA. « TUMOR SUPPRESSOR MUTAGENESIS DRIVEN BY DNA DEAMINASE ». Doctoral thesis, Università degli Studi di Milano, 2016. http://hdl.handle.net/2434/365729.
Texte intégralRésumé :
Genomic instability is commonly associated with pathological disorders including cancer. The progressive accumulation of genetic abnormalities in cancer-associated genes can confer cellular autonomous proliferation, contributing to the oncogenic transformation. Although animal models have been instrumental to the understanding of the molecular mechanisms responsible for tumor progression, there are severe limitations for success. This thesis work aimed at the development of an innovative animal model that could recapitulate the ongoing lifelong accumulation of DNA lesions, leading to neoplastic transformation. To this end, the natural DNA mutating enzyme AID was fused to sequence specific DNA binding proteins. They were engineered to target tumor suppressor genes and to induce low-frequency mutagenesis in cell lines and in zebrafish. A TALE-Aid fusion protein was targeted to the p53 locus of mouse cell lines, and the Aid-dependent mutations were monitored by next-generation sequencing. The induced mutations occurred at a comparable frequency to those observed in Aid-induced non-immunoglobulin gene targets in B cells. Mutations were found mostly in the DNA binding domain of p53, possibly reflecting AID- hotspot residues in p53. Our approach also induced mutations that have not been characterized previously, and could provide further insight into p53 dependent oncogenesis. When TALE-AID were expressed and targeted to the p53 locus of zebrafish embryos, the activity induced developmental abnormalities with variable severity, leading to both an increased mortality, and impaired ovarian maturation and fertility. We have developed and initially characterized a novel tool for the in vitro / in vivo study of the accumulation of mutations in cancer-associated genes.
22
OhAinle, Molly. « Adaptive evolution and loss of function of a primate intrinsic immunity gene / ». Thesis, Connect to this title online ; UW restricted, 2007. http://hdl.handle.net/1773/4986.
Texte intégral
23
Wan, Li. « Insights into the length- and location-dependent deaminase activities of APOBEC3B/F and the deaminase activity determinants of APOBEC3F ». Kyoto University, 2017. http://hdl.handle.net/2433/228249.
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24
Kang, Jin-Ho. « The roles of threonine deaminase in Nicotiana attenuata ». Jena, 2006. http://deposit.ddb.de/cgi-bin/dokserv?idn=982750056.
Texte intégral
25
Miller, Andrew David. « Studies on the active site of PBG deaminase ». Thesis, University of Cambridge, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.235915.
Texte intégral
26
Lim, Saw Hoon. « Molecular analysis of porphobilinogen deaminase in higher plants ». Thesis, University of Cambridge, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.259764.
Texte intégral
27
SIGNA, SARA. « NETosis Dysregulation in Adenosine Deaminase 2 deficiency (DADA2) ». Doctoral thesis, Università degli studi di Genova, 2022. http://hdl.handle.net/11567/1080132.
Texte intégralRésumé :
Introduction: Deficiency of Adenosine deaminase 2 (DADA2) is a monogenic autoinflammatory disorder presenting a broad spectrum of clinical manifestations, including vasculitis, immunodeficiency and hematologic disease. The genetic mutations in ADA2 gene have been associated to an insufficient ADA2 activity leading to reduction in deamination of adenosine to deoxyadenosine, and a consequent accumulation of extracellular adenosine. The pathogenic mechanisms investigated so far have elucidated a skewed polarization from the M2 macrophage subtype to the proinflammatory M1 subtype with an increased production of inflammatory cytokines (TNF-α, Interferon IFN). More recently a chronic neutrophil activation and a dysregulation of NETosis, as process triggered by extracellular Adenosine, inducing TNFα secretion from Macrophages stimulated by NETs, has been implicated in the pathogenesis of DADA2.
Objectives: The aim of the project is to dissect NETosis directly in neutrophils isolated from DADA2 patients and healthy controls (HDs), and to quantify suicidal and vital NETosis induced by several stimuli. To determine if NET epitopes can change depending from the inflammatory microenvironment and if protein composition of NETs is disease specific, we used quantitative proteomics approach to characterize NET proteins, released from neutrophils after different stimuli in DADA2 patients, HDs and nongenetic vasculitis. Moreover, we investigated the mechanisms of NETs removal.
To verify if NETs can influence maturation or inflammatory phenotype of DCs, we characterized peripheral myeloid Dendritic cells (mDCs) and plasmocytoid DCs (pDC) in DADA2 patients and analyzed in vitro moDC maturation and cytokine production in presence of NETs.
Methods: We analyzed and quantified both suicidal and vital NETosis by Imaging Flow Citometry (IFC): neutrophils isolated from peripheral blood from DADA2 patients and HDs were stimulated in vitro with different stimuli (PMA, Adenosine and LPS) to induce NETosis and were analyzed in the ImageStreamXMark II IFC equipped with a MultiMag system. We evaluated also NETs remnants and DNAse in the plasma samples by ELISA assay. We used quantitative proteomics approach to characterize NET proteins, released from neutrophils after different stimuli in DADA2 patients, HDs and nongenetic vasculitis: LC-MS/MS analyses were conducted on Orbitrap Fusion Tribrid mass spectrometer and NET protein quantification was carried out using Label-Free Quantification method.
We isolated monocytes from peripheral blood with microbeads and we generated moDC after culture stimulation with LPS/NETs. On the 7th day we analyzed by flow cytometry the phenotype and the markers of differentiation. Quantification of cytokines was performed by flow cytometry bead array.
Results: We enrolled 14 patients with Adenosine Deaminase 2 deficiency diagnosis who underwent blood sampling at out Center, collecting also plasma samples from further 6 genetically confirmed DADA2 patients enrolled by other Italian Pediatric Rheumatology Units.
Neutrophils from DADA2 show a significant increased suicidal NETosis, identified as nuclear decondensation and Myeloperoxidase (MPO) colocalization with DNA, following PMA stimulation and we observed an increase also with LPS and Adenosine. Then we analyzed vital NETosis, identified as elongated shape of cells, nuclei polarized within the cell and not colocalized with MPO and we found an increased vital NETosis in DADA2 neutrophils. Accordingly, plasmatic levels of circulating nucleosomes (NET remnants) were elevated in patients; DNAse levels were normal but the activity was reduced. We set up experimental conditions for proteomic analysis of NETs, induced by PMA, Adenosine and TNFα, testing two patients, two HDs and two patients with non-genetic vasculitis: in total we identified 1770 proteins among which a hundred of proteins were significantly up or down-modulated in DADA2 NETs compared to controls NETs.
DC phenotype in DADA2 patients result concordant with HD, as well as moDCs cytokine production after LPS stimulation. We observed a stimulatory effect of NETs towards induction of TNF alpha, IL-6 production and IP-10 from moDCs in both HD and DADA2.
Conclusion: Our findings confirm a dysregulation in NETosis process in DADA2 patients, that can be induced by both LPS and adenosine. An increase of vital NETosis was also identified. Proteomic profile of NETs isolated from DADA2 is different from HD and PAN patients: NETs are qualitatively different between HD and DADA2. NETs in DADA2 may therefore interact differently with innate immunity compartment, stimulating DCs to produce citokines, contributing to the typical inflammatory phenotype.
28
Chung-Faye, Guy Allen. « Gene therapy strategies for colorectal cancer ». Thesis, University of Birmingham, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.246708.
Texte intégral
29
Molaudzi, Mulalo. « The usefulness of the adenosine deaminase assay for diagnosing tuberculosis pleuritis in immunocompromised patients at Dr George Mukhari tertiary laboratory, Pretoria ». Thesis, University of Limpopo (Medunsa Campus), 2012. http://hdl.handle.net/10386/671.
Texte intégralRésumé :
Thesis (MSc (Med)(Microbiology)) -- University of Limpopo, 2012.Mycobacterium tuberculosis is the most common cause of death world-wide and its incidence has been steadily increasing, which is more evident when comparing the global tuberculosis (T8) incidence of 9.24 million in 2006 to 9.27 million cases in 2007. African countries are the second most affected by the epidemic and South Africa is among the 22 highest burden countries most affected by T8 with a very high number of cases relative to the total population. The early diagnosis of tuberculosis and screening of contacts is the cornerstone for controlling spread of active T8 infection. T8 diagnosis becomes even more challenging in patients with immunosuppression (for example in human immunodeficiency virus (HIV) infected), in the case of latent infection and extra pulmonary T8 such as pleural T8. The definitive diagnosis of pleural T8 depends on the demonstration of M. tuberculosis in sputum, pleural fluid and pleural biopsy. Although acid fast bacilli (AF8) microscopy is a rapid, inexpensive and relatively simple method, it has low sensitivity. The culture method is more sensitive than AF8 microscopy, detecting 25-37% of all pleural tuberculosis cases however it takes 4 to 8 weeks for a visible growth on a solid medium. Therefore it is important to find a rapid and reliable test for the diagnosis of pleural T8 particularly in developing countries such as South Africa where there is a high T8 incidence and HIV infection rate.
30
Muramatsu, Masamichi. « Specific expression of activation-induced cytidine deaminase(AID), a novel member of the RNA editing deaminase family in germinal center B cells ». Kyoto University, 1999. http://hdl.handle.net/2433/181236.
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31
Penrose, Donna. « The role of ACC deaminase in plant growth promotion ». Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq53509.pdf.
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32
Xue, Kanmin. « Targeting of activation-induced deaminase to immunoglobulin switch regions ». Thesis, University of Cambridge, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.612056.
Texte intégral
33
Oliveira, Hugo Goulart de. « Auxilio diagnóstico da adenosina deaminase (ADA) no derrame pleural ». reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 1989. http://hdl.handle.net/10183/170972.
Texte intégral
34
Korkegian, Aaron. « Engineering yeast cytosine deaminase for improved efficacy in cancer gene directed enzyme prodrug therapy / ». Thesis, Connect to this title online ; UW restricted, 2007. http://hdl.handle.net/1773/5045.
Texte intégral
35
Meng, Xiangyu. « Insights in bladder cancer molecular biology and etiology through signature analysis of somatic mutations and functional annotation of genetic associations ». Thesis, université Paris-Saclay, 2022. http://www.theses.fr/2022UPASL051.
Texte intégralRésumé :
Le cancer de la vessie (BCa) est une tumeur fréquente (4ème cancer chez l'homme). Il présente une hétérogénéité importante et une charge mutationnelle somatique élevée. Une meilleure compréhension de sa biologie et de son étiologie devrait permettre d'améliorer sa prévention et sa prise en charge clinique.Cette thèse présente une série de travaux visant à faire progresser notre compréhension de la biologie et de l'étiologie du Bca, en utilisant deux approches: 1) l'analyse des signatures mutationnelles somatiques qui permet de mieux comprendre l'étiologie des mutations fréquentes dans les oncogènes (OG) et gènes suppresseurs de tumeurs (TSG) et d'identifier de nouveaux candidats translationnels; 2) l'annotation fonctionnelle des associations génétiques qui permet de passer des variants aux connaissances biologiques en priorisant les gènes et les voies associés à des effets génétiques forts ou conjoints. Après une description du contexte et des objectifs, les principaux résultats de cette thèse sont exposés sous forme de cinq travaux de recherche indépendants mais reliés. Dans le premier travail, l'analyse dans les BCa des signatures mutationnelles (du séquençage de l'exome) a démontré que la désamination médiée par APOBEC, principale source des mutations des BCa, représente un facteur étiologique clé de la prévalence élevée de la substitution S249C parmi toutes les mutations de FGFR3, l'un des oncogènes les plus importants des BCa. Dans le second travail, des analyses de l'ensemble de l'exome ont permis l'identification systématique des mutations "hotspot" induites par APOBEC, y compris celles des OG et des TSG, ainsi que l'estimation de leur caractère "driver" ou "passager". Le rôle d'un nouveau driver prédit, AHR Q383H, a été validé sur le plan informatique et fonctionnel. Dans le troisième travail, à partir des données du séquençage du génome, un certain nombre de mutations non codantes fréquentes associées à APOBEC ont été identifiées dans des tumeurs BCa, y compris les mutations du promoteur TERT, mutations driver non codantes les plus répandues dans les BCa. Il a également été démontré que les mutations codantes et non codantes fréquentes liées à APOBEC pouvaient être des biomarqueurs potentiels pour l'estimation de la charge mutationnelle liée à APOBEC et pour la prédiction de la réponse à l'immunothérapie dans les BCa. Dans le quatrième travail, une variante réminiscente de la signature mutationnelle de référence induite par le tabagisme a été identifiée dans les BCa. Elle était associée au sous-type moléculaire LumU, à une prolifération cellulaire accrue, à la suppression d'une réponse immunitaire, à la perte de la différenciation urothéliale et au gain d'une différenciation squameuse définies moléculairement, ainsi qu'à une réponse au traitement et à un pronostic médiocres. Un lien entre tabagisme et instabilité génomique complexe a également été noté, probablement responsable de la résistance au traitement et au mauvais pronostic chez les fumeurs. Dans le dernier travail, une annotation fonctionnelle des associations obtenues par GWAS des BCa (par une intégration des classements de gènes par plusieurs approches, suivie d'une analyse d'enrichissement des ensembles de gènes), a permis d'identifier de nouveaux gènes susceptibles de jouer un rôle dans le développement des BCa, et de découvrir des convergences intéressantes entre les variants germinaux et les altérations somatiques au niveau des gènes et des voies.En résumé, les travaux présentés dans cette thèse fournissent de nouveaux résultats importants concernant l'importance et la pertinence dans la biologie et l'étiologie des BCa des principaux facteurs endogènes et exogènes, à savoir la désamination médiée par APOBEC et le tabagisme, ainsi que du patrimoine génétique. Un cadre systémique unifié basé sur l'élucidation des interactions complexes entre ces facteurs et leurs conséquences devrait être la direction de futures recherchesBladder cancer (BCa) is one of the most common malignancy (the 4th in males). It is of significant heterogeneity and high somatic mutation burden. A better understanding of its molecular biology and etiology should lead to improvement of its prevention and clinical management.This thesis involves a series of efforts in advancing our understanding of BCa molecular biology and etiology, through two approaches: 1) somatic mutation signature analysis, which offers insights in the etiology of frequent mutations in oncogenes (OG) and tumor suppressor genes (TSG) and in the discovery of new translational candidates; 2) functional annotation of genetic associations, which enables translation from variants to insights through prioritization of the genes and pathways associated with stronger or joint genetic effects. Following a description of the background and objectives, the major findings of this thesis are organized in five independent and inter-correlated investigation reports. In the first report, mutation signature analysis in whole-exome sequenced BCa tumors demonstrated that the APOBEC-mediated deamination, the primary mutagenic source of BCa mutations, represented a key etiologic factor of the prominently high prevalence of S249C substitution across all mutations in FGFR3, one of the most important BCa oncogenes. In the second report, extended exome-wide analyses enabled systematic identification of APOBEC-induced hotspot mutations, including those in OGs and TSGs, plus estimation of the ‘driverness' or ‘passengerness' for those of undetermined functional relevance. The role of a predicted new driver, AHR Q383H, was validated computationally and functionally. In the third report, a number of APOBEC-associated frequent non-coding mutations were identified in whole-genome sequenced BCa tumors, including the TERT promoter mutations, the most prevalent non-coding driver mutations in BCa. It was further shown that APOBEC-related frequent coding and non-coding mutations could be potential biomarkers for estimation of APOBEC-related mutation burden and prediction of immunotherapy outcome in BCa. In the fourth report, a reminiscent variant of the tobacco-smoking induced reference mutation signature was identified in BCa tumors. It was associated with LumU molecular subtype, enhanced cell proliferation, suppressed immune signaling, loss of urothelial and gain of squamous differentiation defined molecularly, and poor treatment response and prognosis. A link between smoking and complex genomic instability features was also noted, likely responsible for treatment resistance and poor prognosis in smokers. In the last report, a functional annotation of BCa GWAS associations by integration of gene-rankings of multiple approaches followed by gene-set enrichment analysis identified novel genes likely playing a role in BCa development, and uncovered interesting convergences between germline variants and somatic alterations on both gene- and pathway-levels.In summary, the works presented in this thesis provide important new findings regarding the significance and relevance of the primary endogenous and exogenous factors, namely the APOBEC-mediated deamination and tobacco-smoking, so as the genetic makeup, in BCa molecular biology and etiology. A unified systems framework based on the elucidation of the complex interplays between these factors and their consequences should be the future direction of investigation
36
Wardle, Josephine. « Recognition of deaminated bases by DNA polymerases ». Thesis, University of Newcastle Upon Tyne, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.443025.
Texte intégral
37
Jeschke, Julia. « DNA-Schädigung beeinflusst die Regulation der Aktivierungs-induzierten Cytidin-Deaminase ». Diss., lmu, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:19-148005.
Texte intégral
38
Dinjens, Winandus Nicolaas Maria. « Distribution of adenosine deaminase complexing protein in normal and neoplastic cells ». Maastricht : Maastricht : Rijksuniversiteit Limburg ; University Library, Maastricht University [Host], 1988. http://arno.unimaas.nl/show.cgi?fid=5412.
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39
Björkegren, Emma Katia Madeleine. « Development of gene therapy for the treatment of adenosine deaminase deficiency ». Thesis, University College London (University of London), 2005. http://discovery.ucl.ac.uk/1444352/.
Texte intégralRésumé :
Adenosine deaminase (ADA) severe combined immunodeficiency (SCED) is a life-threatening condition resulting from lack of the ADA enzyme. Consequences include immunodeficiency and non-immunological symptoms such as neurological abnormalities. Bone marrow transplantation (BMT) from a haploidentical donor usually results in complete restoration of immune function. However, the majority of patients do not have a matched donor and are therefore treated with enzyme replacement therapy (PEG-ADA). This treatment is not always fully effective, it is expensive and needs to be administered throughout life. Gene therapy is an alternative treatment, and previous trials for ADA deficiency have shown that it can significantly improve immunological function. Immune recovery was assessed in three ADA-SCID patients treated with PEG-ADA by analysis of lymphocyte counts and emergence of naive T cells. One patient was not responding well to PEG-ADA and was enrolled in a Phase I clinical gene therapy trial. A gammaretroviral vector encoding ADA was constructed and tested extensively on cell lines and patient cells and a CD34+ cell transduction protocol was optimised. The gene therapy procedure was based on previous successful trials, and involved withdrawal of PEG-ADA prior to treatment to provide selective growth advantage for transduced cells, and mild conditioning to encourage engraftment. Assessments of immune function were then performed in a similar manner to patients treated with PEG-AD A. Recent evidence from studies of ADA deficiency indicates that it is a multi-organ disease. However, gene therapy using CD34+ cells may only correct the immunodeficiency without ameliorating non-immunological symptoms. Hence, studies were performed to develop systemic gene therapy for ADA-SCID, involving the use of CD34+ cells and mesenchymal stem cells (MSCs). MSCs were isolated from bone marrow, and their multipotential nature was assessed prior to and following gene transfer using a cloned ADA lentiviral vector. Transduced MSCs maintained their ability to undergo differentiation and transgene expression was not affected by this. These clinical and preclinical in vitro studies demonstrate that gene therapy holds therapeutic potential for treatment of ADA-SCID.
40
Woodcock, Sarah Catherine. « Studies on the mechanism of porphobilinogen deaminase using site directed mutagenesis ». Thesis, University of Southampton, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.385454.
Texte intégral
41
McNeill, Luke Alexander. « Studies of the self-assembly and catalytic mechanism of porphobilinogen deaminase ». Thesis, University of Southampton, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299364.
Texte intégral
42
Alishlash, O. A. « Role of activation-induced cytidine deaminase (AID) in follicular lymphoma biology ». Thesis, University of Liverpool, 2017. http://livrepository.liverpool.ac.uk/3009367/.
Texte intégralRésumé :
Follicular lymphoma is the second most common non-Hodgkin’s lymphoma (NHL). The clinical course of disease is heterogeneous, typically with multiple relapses. Most patients live 10 years or more. However, another group of patients deteriorate rapidly and may progress to death within two years. Activation induced cytidine deaminase (AID) is an enzyme that plays an important role in somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin genes (IG). It induces mutations in IG and non-IG genes leading to genomic instability and chromosomal breaks that are important in the pathogenesis of B-cell malignancies. In this study, we wanted to first measure AID mRNA and protein levels and its biological function in follicular lymphoma (FL) and then correlate each of these variables with clinical features. Our cohort consisted of 87 patients recruited into the Purine-Alkylator Combination In Follicular lymphoma Immuno-Chemotherapy for Older patients (PACIFICO) trial which is comparing alternative frontline chemoimmunotherapy regimens in older patients with FL. The patient samples were in the form of formalin fixed, paraffin embedded (FFPE) biopsies, which are notorious for nucleic acid degradation. We first chose the best kits for extracting RNA and DNA from FFPE biopsies then optimized the procedure to obtain higher quantity of RNA and DNA from the minimum amount of tissue. We then degraded RNA from an AID positive cell line by heating and compared the degraded material with intact material obtained from the same cells to identify a cut-off point for RNA degradation to be applied in a quantitative polymerase chain reaction (qPCR) experiments. This was followed by a qPCR experiment to identify AID mRNA expression in 59 patients. AID protein was then quantified by Immunohistochemistry (IHC) in all samples. We also aimed to measure the functional readout of AID, first by exploring the nuclear/cytoplasmic (N/C) ratio of AID 2 (AID is stored in the cytoplasm and translocates to the nucleus to function), which was calculated for 20 patients using confocal microscopy. A second AID functional measurement was applied using cloning and PCR to detect ongoing mutation and AID-induced mutation in the immunoglobulin heavy variable gene (IGHV) in 18 cases. Finally, we correlated AID expression and functional readouts with available baseline and longitudinal clinical data obtained from the Clinical Trials Unit. In summary, a significant positive correlation was found between AID mRNA and protein expression (P= 0.001). We also found a significantly higher AID N/C ratio in the patient group with higher total AID mRNA and protein expression (P= 0.025 and 0.023 respectively). No correlation was identified between AID mRNA or protein levels and baseline or longitudinal clinical data. However, AID functionality measured as N/C ratio of AID and AID-related or ongoing IGHV mutation was positively correlated with disease status, treatment response and patient survival times. In conclusion, we found that functional readouts of AID are more strongly associated with adverse clinical features in FL compared to AID mRNA or protein expression.
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Hou, Shurong. « Structural Mechanism of Substrate Specificity In Human Cytidine Deaminase Family APOBEC3s ». eScholarship@UMMS, 2020. https://escholarship.umassmed.edu/gsbs_diss/1079.
Texte intégralRésumé :
APOBEC3s (A3s) are a family of human cytidine deaminases that play important roles in both innate immunity and cancer. A3s protect host cells against retroviruses and retrotransposons by deaminating cytosine to uracil on foreign pathogenic genomes. However, when mis-regulated, A3s can cause heterogeneities in host genome and thus promote cancer and the development of therapeutic resistance. The family consists of seven members with either one (A3A, A3C and A3H) or two zinc-binding domains (A3B, A3D, A3D and A3G). Despite overall similarity, A3 proteins have distinct deamination activity and substrate specificity. Over the past years, several crystal and NMR structures of apo A3s and DNA/RNA-bound A3s have been determined. These structures have suggested the importance of the loops around the active site for nucleotide specificity and binding. However, the structural mechanism underlying A3 activity and substrate specificity requires further examination.
Using a combination of computational molecular modeling and parallel molecular dynamics (pMD) simulations followed by experimental verifications, I investigated the roles of active site residues and surrounding loops in determining the substrate specificity and RNA versus DNA binding among A3s. Starting with A3B, I revealed the structural basis and gatekeeper residue for DNA binding. I also identified a unique auto-inhibited conformation in A3B that restricts access to the active site and may underlie lower catalytic activity compared to the highly similar A3A. Besides, I investigated the structural mechanism of substrate specificity and ssDNA binding conformation in A3s. I found an interdependence between substrate conformation and specificity. Specifically, the linear DNA conformation helps accommodate CC dinucleotide motif while the U-shaped conformation prefers TC. I also identified the molecular mechanisms of substrate sequence specificity at -1’ and -2’ positions. Characterization of substrate binding to A3A revealed that intra-DNA interactions may be responsible for the specificity in A3A. Finally, I investigated the structural mechanism for exclusion of RNA from A3G catalytic activity using similar methods.
Overall, the comprehensive analysis of A3s in this thesis shed light into the structural mechanism of substrate specificity and broaden the understanding of molecular interactions underlying the biological function of these enzymes. These results have implications for designing specific A3 inhibitors as well as base editing systems for gene therapy.
44
Pienaar, Elaine. « Molecular and Kinetic Characterisation of wild type and mutant Porphobilinogen Deaminase ». Thesis, University Of Cape Town, 2016. http://hdl.handle.net/11427/29898.
Texte intégralRésumé :
The purpose of this dissertation was to provide an overview of acute intermittent porphyria,
focussing on the structure and function of the enzyme, porphobilinogen deaminase (PBGD),
as well as experimentally demonstrating the use of kinetic, structural and thermodynamic
approaches to shed light on the enzyme reaction. The key focus was to investigate the effect
of three mutations of the active site lysine 98 residue (K98) on the enzyme’s stability and
mechanism. Two clinically relevant PBGD mutants, the K98E and K98R were expressed. Both of these mutants have previously been described in patients. We engineered and expressed an additional mutant, K98A, in order to investigate the effect of charge at this residue. The K98E, K98R and K98A recombinant proteins were successfully engineered, expressed and purified. These mutations were kinetically characterised, and the low enzyme activity supports the fact that the K98E and the K98R are known-disease causing mutations. The negligible activity of the K98A and K98R mutants was predicted as a result of a loss of DPM co-factor binding, which was analysed and proved with a co-factor spectral shift assay. Further attempts to examine the interaction of co-factor binding involved removal of the bound cofactor from wild type enzyme, in order to investigate the possible interaction of the ‘apo’- enzyme with the DPM co-factor. However, no results were obtained to elucidate this
interaction, largely due to the highly unstable nature of the generated ‘apo’-enzyme.
Native polyacrylamide gel electrophoresis (PAGE) was performed in order to observe changes in enzyme-substrate complexes between the wild type and the different mutant proteins. The enzyme-substrate complexes for the wild type were clearly shown, however we could not do so in our mutant proteins. The secondary structure estimations as well as the conformational stability of the mutants were tested with the use of circular dichroism. Far- and near-UV analysis provided insight into the effect of each mutation on the enzyme’s secondary and tertiary structure respectively. Results indicate that the different mutations cause marginal alterations in secondary structure, and resulted in changes of aromatic ring conformations in the near-UV analysis. Finally, modelling of each mutation to known crystal structures of the human enzyme was done in order to provide a rationalisation of kinetic and conformational data. Although this provided only a static image and estimation of the structural effect of each mutation, it did allow for some speculation in order to rationalise the kinetic and conformational data obtained. Overall, this work illustrates how the characterisation of expressed, purified, AIP-associated mutant enzymes aids our understanding of the complex structure and mechanism of the PBGD enzyme.
45
Campbell, Bridget Genevieve. « Isolation and molecular characterisation of two Pseudomonas sp. ACC deaminase genes ». Master's thesis, University of Cape Town, 1995. http://hdl.handle.net/11427/9650.
Texte intégralRésumé :
Bibliography: p. 125-141.The phytohormone ethylene is essential to many plant developmental processes, of which the control of climacteric fruit ripening is among the best characterised. However this hormone eventually causes fruit rotting which results in a non-marketable product. One approach to reduce ethylene synthesis in plants is metabolism of its immediate precursor, 1-aminocyclopropane-1carboxylic acid (ACC). This can be achieved through degradation of ACC by the enzyme ACC deaminase to form α-ketobutyric acid and ammonia. ACC degrading soil microorganisms were identified by their ability to grow on ACC as a sole nitrogen source. Enzyme assays indicated that Pseudomonas had high ACC deaminase gene-specific primers and probes respectively revealed that only one bacterium, Pseudomonas fluerescens strain 17, had a gene with homology to previously sequenced ACC deaminase genes.
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Pienaar, Elaine. « Molecular and Kinetic Characteristics of wild type and mutant Porphobilinogen deaminase ». Master's thesis, Faculty of Health Sciences, 2015. http://hdl.handle.net/11427/30119.
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The purpose of this dissertation was to provide an overview of acute intermittent porphyria, focussing on the structure and function of the enzyme, porphobilinogen deaminase (PBGD), as well as experimentally demonstrating the use of kinetic, structural and thermodynamic approaches to shed light on the enzyme reaction. The key focus was to investigate the effect of three mutations of the active site lysine 98 residue (K98) on the enzyme’s stability and mechanism. Two clinically relevant PBGD mutants, the K98E and K98R were expressed. Both of these mutants have previously been described in patients. We engineered and expressed an additional mutant, K98A, in order to investigate the effect of charge at this residue. The K98E, K98R and K98A recombinant proteins were successfully engineered, expressed and purified. These mutations were kinetically characterised, and the low enzyme activity supports the fact that the K98E and the K98R are known-disease causing mutations. The negligible activity of the K98A and K98R mutants was predicted as a result of a loss of DPM co-factor binding, which was analysed and proved with a co-factor spectral shift assay. Further attempts to examine the interaction of co-factor binding involved removal of the bound cofactor from wild type enzyme, in order to investigate the possible interaction of the ‘apo’- enzyme with the DPM co-factor. However, no results were obtained to elucidate this interaction, largely due to the highly unstable nature of the generated ‘apo’-enzyme. Native polyacrylamide gel electrophoresis (PAGE) was performed in order to observe changes in enzyme-substrate complexes between the wild type and the different mutant proteins. The enzyme-substrate complexes for the wild type were clearly shown, however we could not do so in our mutant proteins. The secondary structure estimations as well as the conformational stability of the mutants were tested with the use of circular dichroism. Far- and near-UV analysis provided insight into the effect of each mutation on the enzyme’s secondary and tertiary structure respectively. Results indicate that the different mutations cause marginal alterations in secondary structure, and resulted in changes of aromatic ring conformations in the near-UV analysis. Finally, modelling of each mutation to known crystal structures of the human enzyme was done in order to provide a rationalisation of kinetic and conformational data. Although this provided only a static image and estimation of the structural effect of each mutation, it did allow for some speculation in order to rationalise the kinetic and conformational data obtained. Overall, this work illustrates how the characterisation of expressed, purified, AIP-associated mutant enzymes aids our understanding of the complex structure and mechanism of the PBGD enzyme.
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Kou, Tadayuki. « Expression of activation-induced cytidine deaminase in human hepatocytes during hepatocarcinogenesis ». Kyoto University, 2007. http://hdl.handle.net/2433/135722.
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Komori, Junji. « Activation-induced cytidine deaminase links bile duct inflammation to human cholangiocarcinoma ». Kyoto University, 2008. http://hdl.handle.net/2433/135857.
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Oliveira, Camila Belmonte. « Atividade das enzimas ntpdase, 5´-nucleotidase e adenosina deaminase em plaquetas de ratos infectados por Trypanosoma evansi ». Universidade Federal de Santa Maria, 2010. http://repositorio.ufsm.br/handle/1/10079.
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Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorNucleotide- and nucleoside-degrading enzymes are present in the surface of platelets, blood cells involved in clotting disturbances of Trypanosoma evansi-infected animals. Thus, this study was aimed at evaluating the activity of the enzymes NTPDase, 5 - nucleotidase and adenosine deaminase in platelets of rats experimentally infected by T. evansi. Animals were divided into four groups, according to the degree of parasitemia. Samples were collected at days 3 (group A: at the beginning of parasitemia), 5 (group B: high parasitemia) and 15 (group C: chronic infection). Group D (control group) was composed of non-infected animals. Blood samples with citrate as the anticoagulant were collected and used for platelet separation and enzymatic assays. NTPDase, 5 - nucleotidase and adenosine deaminase (ADA) activities were decreased (p<0.05) in platelets from rats of groups A and B, when compared to the control group. In group C, only NTPDase and 5 -nucleoside activities were decreased (p<0.001), observed by ADP and AMP hydrolysis. The correlation between platelet count and nucleotide and nucleoside hydrolysis was positive and statistically significant (p<0.05) in groups A and B. Platelet aggregation of all infected groups was decreased in comparison to the control group (p<0.05). Based upon the results, it is concluded that the alterations observed in the activity of the enzymes NTPDase, 5 -nucleotidase and adenosine deaminase in platelets of T. evansi-infected animals might be related to thrombocytopenia.
Nucleotide- and nucleoside-degrading enzymes are present in the surface of platelets, blood cells involved in clotting disturbances of Trypanosoma evansi-infected animals. Thus, this study was aimed at evaluating the activity of the enzymes NTPDase, 5 - nucleotidase and adenosine deaminase in platelets of rats experimentally infected by T. evansi. Animals were divided into four groups, according to the degree of parasitemia. Samples were collected at days 3 (group A: at the beginning of parasitemia), 5 (group B: high parasitemia) and 15 (group C: chronic infection). Group D (control group) was composed of non-infected animals. Blood samples with citrate as the anticoagulant were collected and used for platelet separation and enzymatic assays. NTPDase, 5 - nucleotidase and adenosine deaminase (ADA) activities were decreased (p<0.05) in platelets from rats of groups A and B, when compared to the control group. In group C, only NTPDase and 5 -nucleoside activities were decreased (p<0.001), observed by ADP and AMP hydrolysis. The correlation between platelet count and nucleotide and nucleoside hydrolysis was positive and statistically significant (p<0.05) in groups A and B. Platelet aggregation of all infected groups was decreased in comparison to the control group (p<0.05). Based upon the results, it is concluded that the alterations observed in the activity of the enzymes NTPDase, 5 -nucleotidase and adenosine deaminase in platelets of T. evansi-infected animals might be related to thrombocytopenia.
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Chielle, Eduardo Ottobelli. « EFEITO DA RUTINA SOBRE A ATIVIDADE DA ADENOSINA DEAMINASE EM RATOS DIABÉTICOS ». Universidade Federal de Santa Maria, 2012. http://repositorio.ufsm.br/handle/1/5921.
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Diabetes mellitus (DM) is a metabolic disorder of multiple etiology characterized by chronic hyperglycemia resulting from deficiency of insulin production and/or action. This state of hyperglycemia may cause a variety of cardiovascular, renal, neurological and eye complications. Adenosine deaminase (ADA) is an important enzyme responsible for regulation the levels of adenosine (ado) an important component of the system purinergic nucleoside. Changes in ADA activity has been demonstrated in several diseases, including DM. The Rutin (RT) is an abundant polyphenolic flavonoid found in food that exhibits multiple pharmacological activities including antibacterial, antitumoural, vasodilator and hepatoprotective activities. The objective of this study was to investigate the effect of RT on the activity of ADA in serum, tissues and biochemical parameters in models of diabetes induced by streptozotocin (STZ). Diabetes was induced in rats by an intraperitoneal injection of streptozotocin (STZ). RT (100 mg/kg/day) and glibenclamide (10mg/kg/day) were administered for 30 days, except for control groups (non diabetic and diabetic). Six groups of rats were used in the study and grouped based on fasting blood glucose levels after diabetes induction. The results showed an increase in ADA activity in serum and liver of diabetic rats, like transaminases (AST, ALT), -glutamyltransferase (-GT) and glucose. The RT at a concentration of 100 mg/kg was able to reduce the ADA activity in serum and liver tissue when compared with the diabetic control. The protective effect of RT was also observed increases the activity of enzymes ALT and -GT. Significant reductions were also observed in total cholesterol and LDL-cholesterol as well as in blood glucose levels in the diabetic group treated with RT. The results suggest that RT can improve hyperglycemia and hyperlipidemia, and restoring damaged liver function, as well as prevents the increase in ADA activity in serum and liver tissue on diabetic rats treated with this flavonoid.O Diabetes mellitus (DM) é uma disfunção metabólica de múltipla etiologia caracterizado por hiperglicemia crônica resultante da deficiência da produção e/ou ação da insulina. Esse estado de hiperglicemia pode provocar uma série de complicações cardiovasculares, renais, neurológicas e oculares. A Adenosina deaminase (ADA) é uma importante enzima responsável por regular os níveis de adenosina (ado), um importante nucleosídeo componente do sistema purinérgico. Alterações na atividade da ADA têm sido demonstradas em várias doenças, incluindo o DM. A rutina (RT) é um flavonoide polifenólico abundante nos alimentos que exibe múltiplas atividades farmacológicas como atividade antibacteriana, antitumoral, vasodilatadora e hepatoprotetora. O objetivo deste estudo foi verificar o efeito da RT sobre a atividade da ADA sérica e tecidual e parâmetros bioquímicos em modelos de diabetes induzidos por estreptozotocina (STZ). O diabetes foi induzido através de injeção única intraperitoneal (i.p.) de 55 mg/kg de STZ. A RT (100 mg / kg / dia) e a glibenclamida (10mg/kg/dia) foram administradas durante 30 dias, com exceção dos grupos controles (não diabéticos e diabéticos). Seis grupos de ratos foram utilizados no estudo e agrupados com base nos níveis de glicose em jejum após a indução de diabetes. Os resultados demonstraram um aumento na atividade da ADA no soro e no fígado de ratos diabéticos, assim como das transaminases (AST, ALT), -glutamiltransferase (-GT) e glicose. A RT na concentração de 100 mg/kg foi capaz de reduzir a atividade sérica e em tecido hepático da ADA quando comparado com o controle. O efeito protetor da RT também foi observado sobe a atividade das enzimas ALT e -GT. Reduções significativas foram observadas no colesterol total e LDL-colesterol, bem como, na concentração sérica de glicose no grupo diabético tratado com RT. Os resultados sugerem que a RT pode melhorar a hiperglicemia e dislipidemia, restabelecer danos à função hepática, bem como é capaz de prevenir o aumento da atividade da ADA no soro e no fígado de ratos diabéticos tratados com este flavonoide.