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A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfdment of the requirements for the degree of Doctor of Philosophy / Asthma is a complex syndrome which has a significant inflammatory basis which results from the complex interactions between heterogenous genetic and environmental factors. Although the environmental allergens are fairly well known, little information concerning the genetic differences between atopic and non-atopic individuals is available. Alpha-1 antitrypsin is the archetypal member of the serine proteinase inhibitor or serpin superfamily and the most important proteinase inhibitor in the lung with specificity to neutrophil elastase. Genetic deficiency of the protein is classically associated with early onset emphysema, bronchiecstasis, panniculitis, rheumatoid arthritis and glomerulonephritis. The S(E264V), Z(E342K), Ml (213 Ala) and M2 (R101H) variants of alpha-1 antitrypsin have been implicated in the pathogenesis of asthma. A novel finding was the identification of 2 new variants, the M1E(JOhannesburg) and the M IN(johannesburg) associated with asthma in individuals from South Africa / IT2018

by Chan Wah Lun. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 252-260). / Acknowledgments --- p.i / Abstract --- p.ii / Contents --- p.vi / Naming of TCS mutants and modified TCS protein --- p.x / Abbreviations --- p.xi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Physical and chemical properties of Trichosanthin --- p.1 / Chapter 1.2 --- Biological activities of Trichosanthin --- p.3 / Chapter 1.3 --- Renal tubular reabsorption and nephrotoxicity of Trichosanthin --- p.10 / Chapter 1.4 --- Objective and strategies of study --- p.11 / Chapter Chapter 2 --- Materials and methods --- p.19 / Chapter 2.1 --- General Techniques --- p.19 / Chapter 2.2 --- Site directed mutagenesis of Trichosanthin --- p.21 / Chapter 2.3 --- DNA sequencing --- p.37 / Chapter 2.4 --- Overexpression of modified Trichosanthin in E. coli --- p.42 / Chapter 2.5 --- Purification of modified Trichosanthin --- p.43 / Chapter 2.6 --- Breaking of Disulphide bridge between modified TCS --- p.44 / Chapter 2.7 --- Coupling of DX T40 to modified Trichosanthin --- p.44 / Chapter 2.8 --- Biological activities of modified Trichosanthin and Dextran-modified trichosantin conjugates --- p.46 / Chapter 2.9 --- Immunogenicity of modified Trichosanthin and Dextran-trichosanthin conjugates --- p.50 / Chapter 2.10 --- Nephrotoxicity of Trichosanthin and Dextran-trichosanthin conjugates --- p.53 / Chapter Chapter 3 --- Construction of TCS mutants --- p.61 / Chapter 3.1 --- Introduction --- p.61 / Chapter 3.2 --- Method --- p.61 / Chapter 3.3 --- Results --- p.62 / Chapter 3.3.1 --- Construction of K173C mutant --- p.62 / Chapter 3.3.2 --- Construction of R29C mutant --- p.64 / Chapter 3.3.3 --- Construction of K173C R29C double mutant --- p.65 / Chapter 3.4 --- Discussion --- p.66 / Chapter Chapter 4 --- "Expression,Purification and Ribosome- inactivating activities of Modified Trichosanthin proteins" --- p.87 / Chapter 4.1 --- Introduction --- p.87 / Chapter 4.2 --- Method --- p.87 / Chapter 4.3 --- Results --- p.88 / Chapter 4.3.1 --- "Expression, purification and ribosome-inactivating activity of K173C" --- p.88 / Chapter 4.3.2 --- "Expression ,purification and ribosome-inactivating activity of R29C" --- p.89 / Chapter 4.3.3 --- "Expression, purification and ribosome-inactivating activity of K173C R29C" --- p.90 / Chapter 4.4 --- Discussion --- p.91 / Chapter Chapter 5 --- Coupling of Dextran T40 to modified Trichosanthin --- p.108 / Chapter 5.1 --- Introduction --- p.108 / Chapter 5.2 --- Method --- p.109 / Chapter 5.3 --- Results --- p.109 / Chapter 5.3.1 --- Coupling of R29C --- p.109 / Chapter 5.3.2 --- Coupling of K173C --- p.111 / Chapter 5.3.3 --- Coupling of R29CK173C --- p.111 / Chapter 5.4 --- Discussion --- p.111 / Chapter Chapter 6 --- Biological Activities of modified Trichosanthin and Dextran-modified trichosanthin conjugates --- p.128 / Chapter 6.1 --- Introduction --- p.128 / Chapter 6.2 --- Method --- p.128 / Chapter 6.3 --- Results --- p.130 / Chapter 6.3.1 --- In vivo Biological activity- Mid-term abortifacient activity --- p.130 / Chapter 6.3.2 --- In vitro biological activities / Chapter 6.3.2a --- Ribosome-inactivating activity --- p.131 / Chapter 6.3.2b --- Anti-tumour activity --- p.132 / Chapter 6.4 --- Discussion --- p.133 / Chapter Chapter 7 --- Immunogenicity of Dextran-modified trichosanthin conjugates --- p.156 / Chapter 7.1 --- Introduction --- p.156 / Chapter 7.2 --- Method --- p.157 / Chapter 7.3 --- Results / Chapter 7.3.1 --- Immunogenicity without denaturation of protein --- p.158 / Chapter 7.3.2 --- Immunogenicity with denaturation of protein --- p.161 / Chapter 7.4 --- Discussion --- p.162 / Chapter Chapter 8 --- Nephrotoxicity of Trichosanthin and Dextran-Trichosanthin conjugates --- p.199 / Chapter 8.1 --- Introduction --- p.200 / Chapter 8.2 --- Method --- p.202 / Chapter 8.3 --- Results --- p.202 / Chapter 8.3.1 --- Functional study on nephrotoxicity of Trichosanthin --- p.202 / Chapter 8.3.2 --- Morphological study on the nephrotoxicity of Trichosanthin --- p.203 / Chapter 8.3.3 --- The effect of coupling of Dextran T40 on the nephrotoxicity of Trichosanthin --- p.206 / Chapter 8.4 --- Discussion --- p.207 / Chapter Chapter 9 --- General Discussion --- p.244 / References --- p.252

Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant genetic disorder that results from a mutation of one of two key signaling receptors for the transforming growth factor beta (TGFβ) superfamily: endoglin and activin receptor-like kinase 1 (ALK1). These mutations result in development of HHT Type 1 and HHT Type 2, respectively. Patients suffering from HHT experience spontaneous blood vessel growth that can lead to telangiectasia, arteriovenous malformation (AVM) development, and other related health problems. ALK1 is a serine/threonine kinase receptor found on the cell membrane of endothelial cells. ALK1 and its co-receptor endoglin, are activated by binding to the circulating BMP9 ligand. The ALK1-endoglin-BMP9 complex will then regulate endothelial proliferation by activating the SMAD pathway by phosphorylation. Mutations in the ACVRL1 gene can form a modified ALK1 protein that has a high potential to inhibit this function, causing the hyperproliferation of endothelial cells and the development of AVMs, and ultimately HHT Type 2. It is believed, however unproven, that ALK1 is heavily glycosylated in the extracellular domain. My thesis research was aimed at studying the glycosylation of ALK1 and at exploring the relevance of this glycosylation to the development of HHT. The glycosylation of ALK1 was investigated by using: (i) a computational prediction approach (NetNGlyc 1.0 bioinformatics server), (ii) a glycosylation inhibiting drug (tunicamycin), (iii) an in vitro enzymatic approach of glycosylation breakdown, and (iv) site-directed mutagenesis to identify the ASP residue glycosylated on ALK1. The bioinformatics software NetNGlyc predicted a N-linked glycosylation site on an asparagine (ASN) residue located at position 98 in the extracellular domain of ALK1. I further found that, based on western blot analysis, ALK1 proteins shifted to a lighter molecular weight (5-8 kDa) when treated with tunicamycin, as well as endo H and PNGase F enzymes, which represent two glycosidases able to remove N-linked oligosaccharides on proteins. Western blot analysis also revealed an identical shift in protein size (5-8 kDa) when comparing wild type ALK1 to an asparagine98-to-alanine (N98A) mutant ALK1 construct. The 5-8 kDa shift observed in the drug and enzymatic experiments indicate the removal of a bulky oligosaccharide from the wild type ALK1 protein. This 5-8 kDa shift observed in the mutagenesis experiment indicated that the same oligosaccharide addition could not occur on ALK1 when ASP98 was missing. Thus proving that the asparagine at the 98th position of ALK1 is involved in N-linked glycosylation. These important findings on ALK1 modification offer a greater understanding of the mechanisms behind ALK1 regulation and function, especially its role in controlling angiogenesis. Furthermore, this data provides grounds for further research into the importance of ALK1 glycosylation in the pathogenesis of HHT, as well as the investigation into new treatment regiments.

CRISPR/Cas9 is the gene editing tool of choice in basic research and poised to become one in clinical context. However, current studies on the topic suffer from a number of shortcomings. Mutagenesis is often assessed using bulk methods, which means rare events go undetected, unresolved or are discarded as potential sequencing errors. Many of the genotyping methods rely on short-range PCR, which excludes larger structural variants. Other methods, such as FISH, do not provide basepair resolution, making the genotype assessment imprecise. Furthermore, it is not well understood how Cas9 delivery format influences the dynamics of indel introduction. Finally, many studies of on-target activity were conducted in cancerous cell lines, which do not accurately model the mutagenesis of normal cells in the therapeutic context. In my thesis, I have investigated on-target lesions induced by Cas9 complexed with single gRNAs and no exogenous template. I have followed the time dynamics of Cas9-induced small indels as a function of reagent delivery methods, established an assay for quantification of Cas9-induced genomic lesions that are not small indels ("complex lesions") and used this assay to isolate and genotype complex lesions, many of which would be missed by standard methods. I found that DNA breaks introduced by single guide RNAs frequently resolved into deletions extending over many kilobases. Furthermore, lesions distal to the cut site and cross-over events were identified. Frequent and extensive DNA damage in mitotically active cells caused by CRISPR/Cas9 editing may have pathogenic consequences.

Orientador: Edson Luis Maistro / Banca: Maria Aparecida Marin Morales / Banca: Cláudia Aparecida Rainho / Resumo: Garcinia achachairu (GAC) é uma planta de origem boliviana que vem sendo utilizada na medicina popular para o tratamento de distúrbios gástricos, reumatismo, inflamações e como cicatrizante. A caracterização fitoquímica do extrato desta planta revelou que, uma benzofenona, a Gutiferona A (GA), é um dos seus compostos majoritários, que segundo estudos recentes, apresenta importante atividade antioxidante e antimicrobiana. Considerando o interesse em se aprofundar as análises do potencial farmacológico da GA e a inexistência de estudos que avaliem a sua toxicidade genética, o presente estudo foi elaborado visando investigar o potencial genotóxico e mutagênico da GA em diferentes células de camundongos in vivo, utilizando alguns dos testes tradicionais na área de mutagênese, como o Ensaio Cometa (EC) para a verificação da genotoxicidade e o Teste do Micronúcleo (TM) para a verificação da mutagenicidade. O experimento foi conduzido com camundongos Suíços albinos machos (Mus musculus) de 12 semanas, divididos em cinco grupos, constituídos cada um por seis animais. O grupo controle negativo recebeu, via gavagem, 0,3 mL de DMSO 1%. O grupo controle positivo, recebeu intraperitonealmente, 80 mg/kg de doxorrubicina. Os grupos tratados receberam, via gavagem, 0,3 mL da GA nas doses de 15, 30 e 60 mg/kg. Para a avaliação da genotoxicidade foi coletado sangue da veia caudal dos camundongos (4 e 24 horas após o tratamento), células do fígado, medula óssea, cérebro e testículos (coletadas 24 horas após o tratamento). Para a avaliação da mutagenicidade, foram coletadas células da medula óssea 24 horas após o tratamento. A citotoxicidade foi avaliada pela contagem de 200 eritrócitos policromáticos (PCE) e normocromáticos (NCE) e determinação de sua razão (PCE/NCE). Na amostra de sangue de 4h, analisadas pelo EC, os resultados obtidos mostraram que nas doses de 30 mg/kg e 60 mg/Kg. A análise ... / Abstract: Garcinia achachairu (GAC) is a native plant from Bolivia that has been used in folk medicine for the treatment of gastric disorders, rheumatism, inflammation and as a healing. The phytochemical characterization of this plant extract revealed that the benzophenone guttiferone A (GA) is one of its major compounds, which according to recent studies, has important antioxidant and antimicrobial activity. Considering the interest in deepening the analysis of the pharmacological potential of GA and the lack of studies assessing its genetic toxicity, the present study was designed in order to investigate the genotoxic and mutagenic effects of GA in different cells of mice in vivo, using some of the traditional tests in the mutagenesis area, the Comet Assay (CA) for genotoxicity evaluation and the Micronucleus Test (MT) for the mutagenicity assessment. The experiment was conducted in Swiss albino male mice (Mus musculus) with 12 weeks, divided into five groups with six animals each. The negative control group received, by oral gavage, 0.3 mL of 1% DMSO. The positive control group received, intraperitoneally, 80 mg/Kg of doxorubicin. The treated groups received 0.3 ml of GA at 15, 30 and 60 mg/kg, by gavage. For the genotoxicity evaluation, blood was collected from the tail vein of the mice (4 and 24 hours after treatment), and liver, bone marrow, brain and testicular cells were collected 24 hours after treatment. For the mutagenicity assessment, bone marrow cells were collected 24 hours after treatment. Cytotoxicity was assessed by scoring 200 consecutive polychromatic (PCE) and normochromatic (NCE) erythrocytes and their ratio (PCE/NCE) determined. For the 4 h blood sample, the results with GA at doses of 30 and 60 mg/kg showed that was a statistically significant increase in DNA damage in comparison to the negative control. For the 24 h blood sample, only 60 mg/kg dose showed significant genotoxicity. The analysis of ther ... / Mestre

F. tularensis is an intracellular pathogen, and is the causative agent of tularemia in humans. The ability of F. tularensis to parasitize host cells is largely dependent upon genes within a pathogenicity island (FPI), including those in the iglABCD operon. Specific mechanisms and gene products involved in regulation of the FPI are not well understood. I initiate the study of this regulatory system by creating an efficient Tn5-based mutagenesis system optimized for use in F. tularensis, and utilize this system to construct a lacZ reporter library. I identify genes differentially regulated in response to growth on two different media, including those in the iglABCD and fslABCD operons, and identify iron availability as a factor contributing to the differential regulation. One of these reporter strains, carrying a chromosomal iglB-lacZ fusion, is used as the basis for a secondary transposon mutagenesis to identify mutations that affect iglABCD expression. One such mutation is in FTL_1542 (migR), a hypothetical protein, and reduces expression of the iglABCD approximately 8-fold. The effect of this mutation on igl expression is likely through its effect on another known virulence regulator, fevR, as demonstrated by data from RT-PCR experiments. I compare the phenotypes of LVS fevR and migR mutant strains in primary macrophage and epithelial cell lines and in neutrophils. The mutation in migR effects growth and intracellular trafficking in macrophages but not epithelial cells, and reverses the ability of wild type F. tularensis to block the respiratory burst in neutrophils. When similar mutations were examined in the human virulent F. tularensis strain Schu S4, migR retained its regulatory role, but did not impair replication in macrophages. The migR mutation in Schu S4 did however have an attenuating effect when administered to mice intranasally. Comparison of LVS and Schu S4 in primary human airway epithelial cell infections revealed an inability of LVS to replicate within these cells, which is in contrast to the robust replication of LVS in cultured epithelial cell lines. Together, this work contributes to the understanding of regulatory mechanisms governing virulence gene expression in F. tularensis and highlights differences between LVS and Schu S4 strains.

Severe congenital neutropenia (SCN) is a rare blood disorder characterized by abnormally low levels of circulating neutrophils. Mutations in multiple genes like neutrophil elastase gene (ELANE) and granulocyte colony stimulating factor receptor (CSF3R) may cause SCN. The treatment of choice for SCN is the administration of granulocyte-colony stimulating factor (G-CSF) which elevates the neutrophil count and hence improves the survival and quality of life. Long term survivorship on G-CSF is however linked to development of MDS (myelodysplastic syndrome)/AML (acute myeloid leukemia). About 70% of MDS/AML patients acquire nonsense mutations affecting the cytoplasmic domain of CSF3R. In this project, we hypothesized that this coding region of CSF3R constitutes a hotspot, vulnerable to mutations resulting from excessive oxidative stress or endoplasmic reticulum (ER) stress. We used the murine Ba/F3 cell line to study the effect of induced oxidative or ER stress on the mutation rate in our hypothesized hotspot of the exogenous human CSF3R, the corresponding region in the endogenous Csf3r, and a leukemia-associated gene Runx1. Ba/F3 cells transduced with the cDNA for partial C-terminal of CSF3R fused in-frame with a Green Fluorescent Protein (GFP) tag was subjected to cellular stress inducing mutagen treatment for a prolonged period of time (30 days). The amplicon based targeted deep sequencing data for days 15 and 30 samples show that although there was increased mutagenesis observed in all genes, there were more mutations in the GFP region as compared to the GC-rich partial CSF3R region. Our findings also indicate that there is no correlation between the stress-inducing chemical treatments and mutagenesis in Ba/F3 cells. Thus, we conclude that there are other mechanisms to acquired mutations of CSF3R that help drive the evolution of SCN to MDS/AML. To test this hypothesis, further experiments using unique barcoding system are in progress to characterize the clonal competition between different mutant CSF3R and ELANE expressing cell lines. This study will shed further light on the selection advantage that is provided to cells because cooperativity between mutations in different genes.

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Doctor of Philosophy (PhD) / The field of epigenetics emerged primarily from studies in Drosophila, and is now being studied intensively by mammalian biologists. In order to increase our knowledge of epigenetic gene control in the mouse, I have studied modifiers of epigenetic gene silencing. My main method of investigation involved the characterisation of mutants from a sensitised ENU mutagenesis screen performed previously in our laboratory. The screen was carried out in an FVB/NJ strain carrying a variegating GFP transgene expressed in erythrocytes. To date we have recovered 12 dominant (D) and seven recessive (R) mutant mouse lines from this screen that display altered transgene expression. We have named these Mommes (Modifiers of murine metastable epialleles). I investigated the phenotype and attempted to identify the underlying causative mutation of two of these Momme mutants. MommeD6 is a semi-dominant, homozygous lethal mutation that acts as a suppressor of variegation with respect to the GFP transgene. This mutation has a large effect on the level of expression of the transgene in expressing cells, but little effect on the percentage of cells expressing the transgene. MommeD6 is linked to a 2.5 Mbp interval on chromosome 14. MommeD9 is a semi-dominant, homozygous lethal mutation that acts as an enhancer of variegation with respect to the GFP transgene. Mutants have a tendency to become obese as they age, show abnormal haematology profiles, and females develop infertility. MommeD9 is linked to a 17.4 Mbp region on chromosome 7. I produced and studied a strain carrying the same GFP transgene but in a new strain background, C57BL/6J. This strain provided an opportunity to look for strain-specific modifiers of expression of the GFP transgene. Several regions were mapped to chromosomal locations. Further work will be needed to identify the genes involved. This mouse will be useful in future mutagenesis screens of this type.