Dissertations / Theses on the topic 'Mutants de type-1'

Dans cette thèse l’expression du récepteur CB1 dans l’hippocampe de souris mutantes ré-exprimant spécifiquement le gène spécifiquement dans certains types cellulaires du cerveau tels que : les neurones glutamatergiques du télencéphale dorsal, et les neurones GABAergiques a été analysé. De plus, dans le but de connaître la distribution anatomique exacte des récepteurs CB1 astrogliaux par rapport aux synapses excitatrices et inhibitrices, j’ai étudié l’expression des récepteurs CB1 dans les astrocytes de souris exprimant le récepteur CB1 seulement dans les astrocytes et une souris mutante ciblée pour exprimer la protéine cytoplasmique hrGFP diffusible dans les cellules astrogliales, ce qui permet une meilleure détection des prolongements astrocytaires. Les conclusions de ce travail de thèse sont les suivantes : la distance la plus commune entre le récepteur CB1 astroglial et la synapse la plus proche est de 400 à 800 nm. La majorité des synapses entourées par des astrocytes immunopositifs pour le récepteur CB1 dans l’hippocampe, est de nature excitatrice. Les souris mutantes ré-exprimant le récepteur CB1 caractérisées dans ce travail de thèse montrent : 1) l’expression du récepteur CB1 dans différents types cellulaires, 2) la réexpression est limitée à une population neuronale particulière ou aux astrocytes, 3) les niveaux endogènes de récepteurs CB1 sont maintenus dans les différents types cellulaires ré-exprimant le récepteur CB1. De façon générale, ces résultats nous montrent que les souris mutantes ré-exprimant le récepteur CB1 sont d’excellents outils pour l’étude fonctionnelle et translationnelle sur le rôle de ce récepteur dans le cerveau sain ou pathologique<br>The Cannabinoid Type I receptor protein (CB1) expression in the hippocampus of rescue mice modified to express the gene exclusively in specific brain cell types: such as dorsal telencephalic glutamatergic neurons, or GABAergic neurons have been analyzed. Furthermore, aiming at knowing the exact anatomical distribution of the astroglial CB1 receptors with respect to the excitatory and inhibitory synapses, the CB1 receptor expression in astrocytes of mouse expressing CB1 receptor only in astrocytes and mutant mouse expressing the protein hrGFP into astrocytes (that allows for better detection of the astrocytic processes) have been also investigated. The results showed that the majority of the hippocampal synapses surrounded by CB1 receptor immunopositive astrocytes in the 400-800 nm range are of excitatory nature. Moreover, the CB1 receptor rescue mutant mice characterized in this Doctoral Thesis have proven 1) to express CB1 receptors in specific brain cell types; 2) the re-expression is limited to the particular brain cell populations; 3) the endogenous levels of CB1 receptors are maintained in the brain cell types re-expressing the receptor. Which makes this mutant mice excellent tools for functional and translational investigations on the role of the CB1 receptors in the normal and diseased brain

The herpes simplex virus type 1 (HSV-1) UL12 gene encodes an alkaline nuclease. Although the UL12 gene is not absolutely essential for replication, UL12 null mutants produce 100-1000 fold less viable virus than wt HSV-1. It has been suggested that the alkaline nuclease functions to remove branched structures from high molecular weight concatemeric DNA prior to its cleavage into monomeric genomes that are packaged into viral capsids. Failure to remove the branches results in unstable packaging of DNA into capsids which fail to egress from the nucleus. This thesis describes detailed characterisation of the HSV-1 mutant, ambUL12 (Patel et al., 1996) which fails to express the alkaline nuclease due to the insertion of an amber stop codon into the UL12 open reading frame. The ability of ambUL12 to replicate and package both viral genomic DNA and amplicons (plasmids containing the HSV-1 origin of replication and DNA encapsidation signal) was examined. In contrast to results obtained with other alkaline nuclease mutants, which replicate and package DNA with close to wt HSV-1 efficiency (Shao et al., 1993; Martinez et al., 1996b), ambUL12 displayed a 3-5 fold drop in replication and a 15-20 fold drop in packaging of genomic DNA. Similar reductions were observed in the replication and packaging of amplicon DNA. The replication and packaging of amplicons by ambUL12 in these transient assays could be partially complemented when UL12 was supplied in trans. Close inspection of the DNA molecules synthesised during transient assays demonstrated that amplicon replication intermediates are complex high molecular weight concatemers that undergo intermolecular recombination, analogous to viral DNA replication intermediates.

Alpha-synuclein is a major protein constituent of neuronal Lewy bodies (LB) in Parkinson's disease (PD). Astroglial over-expression of the heme-degrading enzyme, heme oxygenase-1 (HO-1) in the PD substantia nigra promotes mitochondrial iron sequestration and may sensitize dopaminergic neurons to oxidative injury (51). The effects of transient hHO-1 transfection on levels of endogenous or co-transfected alpha-synuclein (WT or A30P) were measured by Western blot in M17 cells in the presence or absence of the HO and proteasome inhibitors, SnMP and lactacystin, respectively. Deferoxamine and bilirubin were administered to sister cultures to delineate the role(s) of iron and bilirubin (heme breakdown products) on altered alpha-synuclein metabolism. hHO-1 transfection significantly decreased endogenous and transgenic WT alpha-synuclein levels, effects reversed by SnMP, lactacystin or deferoxamine (but not bilirubin), but had no effects on A30P levels. Thus, iron liberated by HO-1 over-expression may induce proteasomal degradation of WT alpha-synuclein, thereby promoting synaptic dysfunction. Conversely, failure of HO-1 to suppress mutant alpha-synuclein and its putative toxic gain of function may predispose to familial PD.

Human complex diseases, like Diabetes and Cancer, affect many people worldwide today. Despite existing knowledge, many of these diseases are still not preventable. Complex diseases are known to be caused by a combination of genetic factors, as well as environmental and life style factors. The scope of this investigation covered the genomics of Type 1 Diabetes (T1D). There are 49 human genomic regions that are known to carry markers (disease-associated single nucleotide mutations) for T1D, and these were extensively studied in this research. The aim was to find out in how far this disease may be caused by problems in gene regulation rather than in gene coding. For this, the genetic factors associated with T1D, including the single point mutations and susceptibility regions, were characterised on the basis of their genomic attributes. Furthermore, mutations that occur in binding sites for transcription factors were analysed for change in the conspicuousness of their binding region, caused by allele substitution. This is called SNP (Single nucleotide polymorphism) sensitivity. From this study, it was found that the markers for T1D are mostly non-coding SNPs that occur in introns and non-coding gene transcripts, these are structures known to be involved in gene regulatory activity. It was also discovered that the T1D susceptibility regions contain an abundance of intronic, non-coding transcript and regulatory nucleotides, and that they can be split into three distinct groups on the basis of their structural and functional genomic contents. Finally, using an algorithm designed for this study, thirty-seven SNPs that change the representation of their surrounding region were identified. These regulatory mutations are non-associated T1D-SNPs that are mostly characterised by Cytosine to Thymine (C-T) transition mutations. They were found to be closer in average distance to the disease-associated SNPs than other SNPs in binding sites, and also to occur frequently in the binding motifs for the USF (Upstream stimulatory factor) protein family which is linked to problems in Type 2 diabetes.

Le concept d'interféronopathie de type I émerge en 2011 et fait référence à un ensemble de pathologies Mendéliennes caractérisées par une hyperactivation des interférons (IFN) de type I. Tous les gènes associés au syndrome d'Aicardi-Goutières (SAG), la première interféronopathie de type I décrite, sont impliqués dans la détection ou le métabolisme des acides nucléiques. Les autres protéines mutées associées aux interféronopathies de type I modifient toutes la voie de signalisation des acides nucléiques, de manière directe, indirecte ou encore non définie. Les IFN de type I se fixent à un récepteur unique et activent la Janus kinase 1 (JAK1) et la tyrosine kinase 2 conduisant à l'expression de gènes stimulés par les IFN (IFN-stimulated genes, ISGs) via la phosphorylation des facteurs de transcription STAT1 et STAT2. Notre équipe a développé des outils diagnostiques des interféronopathies de type I, comprenant la signature IFN, analyse combinée de l'expression de 6 ISGs, et, plus récemment, une méthode de détection de l'IFN alpha à l'aide de la technologie «single molecule array». Les mutations monogéniques associées jusqu'à présent aux interféronopathies de type I causent des phénotypes variables. Leurs points communs sont une morbidité et une mortalité importantes, notamment en raison de leur réponse faible aux immunomodulateurs classiques. Les mutations activatrices de TMEM173 codant pour STING (Stimulator of IFN genes) sont responsables d'une inflammation sévère, connue sous le nom de STING-associated vasculopathy with onset in infancy (SAVI), et caractérisée par une vascularite cutanée et une atteinte interstitielle pulmonaire conduisant à une insuffisance respiratoire terminale. STING, une protéine du réticulum endoplasmique (RE), agit comme un adaptateur cytosolique de la détection de l'ADN, permettant la synthèse d'IFN de type I via la phosphorylation d'IRF3. Une cohorte internationale de 20 patients SAVI est décrite dans cette thèse soulignant l'hétérogénéité clinique de cette maladie. Nous avons également étudié le lien entre des mutations hétérozygotes de COPA et une activation de la voie des IFN de type I. COPA code pour la sous-unité alpha du complexe du coatomère I, impliqué dans le transport rétrograde entre le RE et le Golgi. Les mutations hétérozygotes de COPA sont à l'origine d'un phénotype proche du SAVI et entraînent une hausse du stress du RE et une réponse immunitaire de type Th17. Cependant, la physiopathologie de cette maladie reste peu connue. Nous avons étudié un groupe de 8 patients qui illustre l'hétérogénéité phénotypique de cette affection nouvellement décrite. Nous avons observé des similitudes entre l'histologie pulmonaire du syndrome COPA et du SAVI, ainsi qu'une signature IFN, des taux élevés d'IFN alpha dans le sérum et une phosphorylation de STAT1 dans les lymphocytes des patients. Dans un modèle cellulaire, la coexpression de COPA muté et de STING sauvage entraîne la phosphorylation d'IRF3 et à une induction d'ISGs, suggérant que les mutations de COPA conduisent à une activation dépendante de STING de la voie des IFN de type I. Nous avons mené avec succès le premier essai clinique d'un inhibiteur de JAK1, le ruxolitinib, dans le contexte du SAVI. L'amélioration clinique remarquable a été confirmée in vitro et ex vivo. La gravité de la maladie nous a également poussé à chercher des alternatives thérapeutiques pour contrôler la voie des IFN de type I. Nous avons montré que l'inhibition d'IKK bloquait efficacement la production et la signalisation des IFN de type I dans les cellules de patients STING in vitro. Devant les résultats prometteurs de l'inhibition de JAK1 dans le SAVI, nous avons ensuite testé le ruxolitinib dans le cadre d'autres interféronopathies de type I monogéniques (COPA, TREX1) mais aussi chez une enfant ayant une dermatomyosite sévère, une maladie pour laquelle le rôle pathogénique de l'IFN de type I a été suggéré<br>The term 'type I interferonopathies', first coined in 2011, refers to a set of Mendelian disorders associated with constitutive up-regulation of type I interferon (IFN) signalling. All of the genes associated with Aicardi-Goutières syndrome (AGS), the first Mendelian type I interferonopathy described, have been implicated in either the processing or sensing of nucleic acids. Beyond AGS, the other mutated proteins associated with type I interferonopathies have a direct, indirect, or currently undefined action on nucleic acid signalling. Type I IFNs drive the expression of IFN-stimulated genes (ISGs) through the engagement of a common receptor and the subsequent activation of Janus kinase 1 (JAK1) and tyrosine kinase 2, and phosphorylation of STAT1 and STAT2. Our team has developed diagnostic tools to identify type I interferonopathies, comprising a so-called IFN signature, involving the assessment of mRNA expression of 6 ISGs and, more recently, a high sensitivity assay of IFN alpha protein using single molecule array technology. Monogenic mutations so far recognised as type I interferonopathies are associated with a wide spectrum of phenotype. The hallmark of these diseases is their significant morbidity and mortality, associated with an apparent absence of response to conventional immunosuppressive therapies. Activating mutations in TMEM173, encoding stimulator of IFN genes (STING), cause a severe inflammatory condition referred to as STING-associated vasculopathy with onset in infancy (SAVI), characterised by skin vasculopathy and interstitial lung disease leading to end-stage respiratory failure. The endoplasmic reticulum (ER) protein STING is a central component of DNA sensing that induces type I IFNs through phosphorylation of IRF3. An international cohort of 20 STING patients is reported in this thesis, emphasising the clinical heterogeneity of this condition. We also investigated the link between heterozygous mutations in COPA and type I IFN signalling. COPA encodes the alpha subunit of the 7 member coatomer complex I, involved in retrograde transport from the golgi to the ER. Heterozygous mutations in COPA cause a phenotype showing some overlap with SAVI, and are associated with increased ER stress and priming of a Th17 response. However, the precise pathophysiology of this disease is so far undefined. We have studied a group of 8 patients illustrating the phenotypic variability of this emerging disease. We observed commonalities in the lung pathology in COPA and SAVI, as well as an IFN signature, raised levels of IFN alpha in the serum and phosphorylation of STAT1 in patient T cells. In a cellular model, phosphorylation of IRF3 and increased ISG expression were observed in cells co-transfected with wild type STING and mutant COPA plasmids, suggesting that mutations in COPA lead to constitutive activation of type IFN signalling through STING. We reported, for the first time, the successful use of a JAK1 inhibitor, ruxolitinib, in the context of SAVI. We observed a marked clinical effect, which was mirrored by the results of in vitro and ex vivo experiments. Because of the severity of SAVI, we also aimed to evaluate alternative therapeutic approaches to block type I IFN signalling and showed that IKK inhibition efficiently abrogated in vitro constitutive activation of type I IFN production and signalling in cells from STING patients. Considering the promising results of JAK1 blockade in SAVI, we then trialled ruxolitinib in other monogenic type I interferonopathies (TREX1, COPA) and in a child with severe dermatomyositis, a disease where type I IFN has been suggested to play a key pathogenic role

L'exportine-1 (ou XPO1) joue un rôle clé dans le transport de nombreux ARN et près de 200 protéines cargos. La mutation « hot-spot » XPO1-E571K est présente chez près de 25% des patients atteints par le lymphome primitif du médiastin (PMBL) et la forme classique du lymphome de hodgkin (cHL), et à plus faible fréquence dans la leucémie lymphoïde chronique (LLC) (3%). Des mutations touchant la voie JAK2/STAT6 sont aussi retrouvées dans le PMBL et le cHL. C’est pourquoi nous avons étudié l’implication de XPO1 dans la voie JAK2/STAT6. Nous avons montré que STAT6 est une protéine cargo de XPO1 par les technique immunofluorescence et PLA (Proximity Ligation Assay). Nous avons montré que le traitement au selinexor induit une accumulation nucléaire de STAT6 sauvage et mutée et que STAT6 peut interagir avec les formes mutée et sauvage de XPO1.Afin de rechercher l’impact fonctionnel de la mutation E571K de XPO1 nous avons comparé plusieurs paramètres physiologiques entre les trois lignées de PMBL. Aucune différence n’a été observée. En effet, la présence de l’amplification de l’allèle sauvage dans la lignée présentant la mutation E571K (MedB1) pourrait masquer les éventuels effets de la mutation. De plus, dans la cohorte de patients que nous avons étudiée la mutation n’est jamais retrouvée à l’état homozygote ou hémizygote indiquant l’importance du dosage génique. Nos expériences de CRISPR-Cas9 sur la lignée U2940 dont le gène XPO1 est sauvage ont confirmé notre hypothèse. De manière intéressante, la présence de la mutation E571K modifie l’affinité de XPO1 pour le selinexor. En effet, le KO de l’allèle muté dans la lignée de cHL UH-01 rend les cellules moins sensibles au selinexor. Nous avons conclu que la balance entre l’allèle sauvage et l’allèle muté est un élément clé pour définir les propriétés oncogéniques de XPO1. Dans une étude préliminaire, une étude protéomique dans les lignées PMBL indique que XPO1-E571K est liée à l’importine-β1 ce qui pourrait modifier la localisation subcellulaire de XPO1.Nous avons montré que la localisation cytoplasmique de cycline D1 contrôle le mécanisme d’invasion et de migration dans cellules de lymphome à cellules du manteau (MCL). Cycline D1 étant une protéine cargo de XPO1 nous avons recherché d’éventuelles anomalies de XPO1 dans les lignées de MCL. Aucune anomalie d’expression de localisation ou de fonction de n’a été observée. Il pourrait être intéressant de déterminer la localisation subcellulaire de cycline D1 au moment du diagnostic afin de proposer une meilleure prise en charge pour les patients. De plus, bien que le selinexor soit toujours en essai clinique, son utilisation pour le traitement du MCL pourrait être envisagée dans les cas les plus agressif où cycline D1 est cytoplasmique<br>Exportin-1 (or XPO1) plays a key role in the nuclear export of several RNAs and more than 200 proteins. The XPO1-E571K "hot-spot" mutation is present in nearly 25% of patients with primary mediastinal B-cell lymphoma (PMBL), and the classical form of Hodgkin lymphoma (cHL) but at a lower frequency (3%) in chronic lymphocytic leukemia (CLL). Mutations affecting the JAK2/STAT6 pathway are common in PMBL and cHL. We first studied the role of XPO1 in the nuclear export of STAT6 in PMBL cell lines. Using immunofluorescence and proximity ligation assay (PLA) techniques, we showed that STAT6 is a cargo of XPO1. We also showed that a selinexor treatment induced a nuclear accumulation of wild-type and mutant STAT6 whatever the XPO1 status.In order to investigate the functional impact of the XPO1-E571K mutation, we compared several physiological parameters between the three PMBL cell lines bearing or not the mutation. No differences were observed despite the expression of the XPO1E571K allele. However, in the cell line harboring the XPO1 mutation (MedB1), the wild-type (wt) allele was amplified possibly masking the effects of the mutation. In addition, in the cohort of patients we studied, the mutation was never found in the homozygous or hemizygous state indicating the importance of the gene dosage. CRISPR-Cas9 experiments allowing the introduction of the mutation in the U2940 wt cell line confirmed our hypothesis. Interestingly, the presence of the E571K mutation changed the affinity of XPO1 for selinexor. Indeed, the knockout of the mutated allele in the cHL UH-01 line decreased its sensitivity to selinexor. We concluded that the balance between the wt and the mutated alleles is a key element in defining the oncogenic properties of XPO1. In a preliminary study, we conducted a proteomic analysis to identify XPO1E571K partners in the PMBL lines. Our results showed that XPO1E571K interacts with the importin-β1 which could modify the subcellular localization of XPO1.Mantle cell lymphoma (MCL) cells are characterized by the overexpression of cyclin D1. Cyclin D1 being an XPO1 cargo protein, we looked for possible XPO1 abnormalities in several MCL cell lines. No abnormalities in the expression, localization neither function of XPO1 were observed. But, we showed that the cytoplasmic portion of cyclin D1 controlled the invasion and migration of MCL cells. It might be interesting to determine the subcellular localization of cyclin D1 at the time of diagnosis in order to offer a better treatment management for MCL patients. In addition, although selinexor is still in clinical trials, its use for the treatment of MCL could be considered in the most aggressive cases where cyclin D1 is cytoplasmic

碩士<br>國立成功大學<br>微生物暨免疫學研究所<br>92<br>Herpes simplex virus type 1 infects and establishes latent infection in most humans for life. Acyclovir is the best drug for therapy to inhibit viral replication now, but drug-resistant [thymidine kinase negative (TK-)] mutants are reported frequently in the clinic. Interferons (IFNs), including IFN-alpha, IFN-beta and IFN-gamma, are potent antiviral cytokines. Based on the previous study, combination of IFN-beta and IFN-gamma has the best efficacy to inhibit the replication of wild type virus. In this study, we tested this combination on TK- mutants in vitro using Vero cells and found that IFN-beta + IFN-gamma reduced the plaque formation of TK- mutants by 50-folds, which they are resistant to acyclovir. In vivo, we infected mice with 2×106 PFU of TK- mutant virus (294dltk) by corneally inoculation, which can result in the reactivation of 294dltk under this condition. The pretreatment of IFN-beta and IFN-gamma reduced the viral replication in eyes about 1000-folds on day 1 post infection (p.i.) during acute phase and the ratio of viral reactivation in trigeminal ganglia from 70% to 0% at day 30 p.i. during latent phase. The posttreatment of IFNs also had the same effect but was not effective as pretreatment. In addition, we tested this on tkLTRZ1 TK- mutant, which reactivates when co-infects with wild type virus. We found that the viral replication in eyes at day 1 p.i. and in ganglia at day 3 p.i. and the viral reactivation form ganglia, spinal cord and brain stem were reduced after treatment. In order to mimic the immunocompromiced hosts, we used 294dltk to infect ICR-nu mice. Then we compared the viral growth in eyes and ganglia. The preliminary evidence showed that IFNs are able to efficiently inhibit viral growth in immunocompromised hosts. These data suggest that combination of IFN-beta and IFN-gamma may be an alternative therapy for drug-resistant herpes simplex virus type 1.

碩士<br>國立成功大學<br>微生物及免疫學研究所<br>92<br>Herpes simplex virus type 1 infects and establishes latent infection in most humans for life. Acyclovir is the best drug for therapy to inhibit viral replication now, but drug-resistant [thymidine kinase negative (TK-)]mutants are reported frequently in the clinic. Interferons (IFNs), including IFN- , IFN- and IFN- , are potent antiviral cytokines.Based on the previous study, combination of IFN- and IFN- has the best efficacy to inhibit the replication of wild type virus.In this study, we tested this combination on TK- mutants in vitro using Vero cells and found that IFN- + IFN- reduced the plaque formation of TK-mutants by 50-folds,which they are resistant to acyclovir. In vivo, we infected mice with 2×106 PFU of TK- mutant virus (294dltk) by corneally inoculation, which can result in the reactivation of 294dltk under this condition. The pretreatment of IFN- and IFN- reduced the viral replication in eyes about 1000-folds on day 1 post infection (p.i.) during acute phase and the ratio of viral reactivation in trigeminal ganglia from 70% to 0% at day 30 p.i. during latent phase. The posttreatment of IFNs also had the same effect but was not effective as pretreatment. In addition, we tested this on tkLTRZ1 TK- mutant, which reactivates when co-infects with wild type virus. We found that the viral replication in eyes at day 1 p.i. and in ganglia at day 3 p.i. and the viral　reactivation form ganglia, spinal cord and brain stem were reduced after treatment. In order to mimic the immunocompromiced hosts, we used 294dltk to infect ICR-nu mice.Then we compared the viral growth in eyes and ganglia. The preliminary evidence showed that IFNs are able to efficiently inhibit viral growth in immunocompromised hosts. These data suggest that combination of IFN- and IFN- may be an alternative therapy for drug-resistant herpes simplex virus type 1.

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctor of Philosophy in Medicine Johannesburg 2015<br>The currently approved integrase strand transfer inhibitors (INSTIs), raltegravir (RAL) and elvitegravir (EVG) effectively halt HIV-1 replication but their use is limited by their low genetic resistance barrier and cross resistance. For instance, integrase amino acids N155 and Q148 represent genetic pathways selected by both drugs and are associated with considerable cross resistance to both RAL and EVG. Dolutegravir (DTG) is a second generation drug manufactured to exhibit a more robust resistance profile than RAL and EVG, and retains activity against RAL and EVG resistant isolates. Most research on drug resistance patterns have been carried out with emphasis on HIV-1 subtype B and inadequately assessed in HIV-1 subtype C. Thus, the aim of this study was to establish the drug resistance mutation profiles of HIV-1 subtype C primary virus isolates that evolve/emerge under selective pressure of the INSTIs RAL, EVG and DTG, and evaluate their impact on strand transfer. In vitro selection experiments were carried out using six primary virus isolates (three wild-type, FV, and three reverse transcriptase drug resistant, MR, viruses) grown in peripheral blood mononuclear cells in the presence of increasing concentrations of RAL, EVG and DTG, and monitored to beyond virus break-through. Viral RNA was extracted from various time points and the pol region was RT-PCR amplified and sequenced using conventional Sanger-based sequencing and next generation sequencing (Illumina MiSeq). HIV-1 subtype C FV6 wild-type and mutant recombinant integrase (generated by site-directed mutagenesis) were expressed, purified and used in strand transfer assays and surface plasmon resonance (SPR) experiments to establish the binding affinities of IN-DNA. Wild-type FV primary viruses were successfully grown in the presence of increasing concentrations of RAL, EVG and DTG, up to 266 nM, 66 nM and 32 nM, respectively. Drug resistant MR viruses were successfully grown in the presence of increasing concentrations of RAL, EVG and DTG, up to 266 nM, 16 nM and 8 nM, respectively. Sequence analysis on both platforms revealed the presence of the previously described drug resistance mutations T66IK, E92Q, F121Y, Q148R, N155H and R263K in some viruses, and additionally H114L was detected. RAL was observed to select for substitutions Q148R and N155H/H114L in isolates FV6 and MR69, respectively. EVG selected F121Y, T66I/R263K, T66K and T66I in FV3, FV6, MR69, MR81, and MR89, respectively. DTG selected substitutions E92Q and M50I in FV3 and MR81, respectively. In silico data exhibited changes in hydrophilicity, hydrophobicity and side chain changes as well as changes in polarity, and all substitutions displayed acceptable minimisation energies and distances between the atoms. Seven IN mutants were expressed and purified, and thereafter tested for efficiency in strand transfer. All mutant FV6T66I, FV6E92Q, FV6H114L, FV6F121Y, FV6Q148R, FV6N155H and FV6R263K IN enzymes demonstrated an overall loss in strand transfer capacity of 37.1%, 21.5%, 66.1%, 63.2%, 60.2%, 30.5% and 3.4%, respectively. This is the first report on loss of strand transfer activity associated with H114L. The loss in strand transfer capacity in all the mutants was not reflected by their overall binding affinities to donor DNA, as determined by surface plasmon resonance, likely attributed to the role of different residues associated with DNA and drug binding in the IN quaternary structure. In conclusion, this is the first report describing IN drug selection experiments using primary HIV-1 subtype C isolates, and a detailed genotypic and biochemical characterisation of the associated mutations.

Thesis (Ph. D.)--University of Virginia, 2000.<br>Spine title: HIV-1 variants resistant to RevM10. Includes bibliographical references (p. 207-247). Also available online through Digital Dissertations.

碩士<br>國立臺灣大學<br>微生物與生化學研究所<br>94<br>Sucrose synthase (SuS) catalyzes the reversible conversion of sucrose and UDP into fructose and UDPG. The enzyme is encoded by at least six RSus genes in rice. In this study, recombinant RSuS1 expressed and purified from E. coli was used to study on the structure – function relationship of the enzyme. The results of sequence analysis and 3D structure modeling suggest that the RSuS1 is a GT-B fold glycosyltransferase of the GT4 family in CAZy classification. Mutants analysis show that the Asp303, Gln307, His463 and His502 play important roles in substrate binding or active sit. Tyr310, His463 and Arg94 are important in tetrameric structure. Divalent cations, Mg2+, Mn2+, Ca2+, and Co2+ stimulate the recombinant RSuS1 synthetic activity, but inhibit the cleavage activity. Metabolites do not affect the recombinant RSuS1 activity significantly. Triton X-114 partition analysis shows that recombinant RSuS1 is not an integral membrane protein. The phospholipids do not affect the RSuS1 activity.

The use of tissue- or tumor-selective promoters in targeted gene therapy for prostate cancer depends on strong and selective activity. The -426 to +28 bp probasin (PB) promoter has the potential to target selected genes to the prostatic epithelial cells. We showed that in the assay of luciferase reporter gene driven by PB promoter, activation of luciferase expression was 1.5-fold increase in human prostate cancer cell LNCaP relative to non-small cell lung cancer cell H460. Significant increase of luciferase activity was observed in both cell lines when infection with ICP27 (-) HSV-1 was added to PBluciferase plasmid transfected LNCaP and H460 cells. In addition, an expression vector (PB-ICP27) was generated in which one of the herpes simplex virus type 1 (HSV-1) immediate early (IE) genes required for viral replication~ICP27 was regulated by PB promoter. This vector was transfected into LNCaP, H460 and one other nonprostate cancer cell HepG2 (hepatoma), the cell lines were then infected with ICP27 (-) HSV-1 and resulting viral stock from these cells were used to infect ICP27 expression 2-2 cell. The plaque yields from infected 2-2 cells were counted and reflected the production of ICP27 (-) HSV-1 replication in PB-ICP27 vector transfected cells that can express ICP27 protein. Although we found that the profound viral replication was observed in LNCaP cells compared to that of HepG2 cells, the production of viral replication in H460 cells was surprisingly significant higher than that in LNCaP cells and was contrasted to our PB-driven luciferase assay result. Our study demonstrates that (1). -426 to +28 bp PB promoter shows relative tissue-specific activity; (2). Infection with ICP27 (-) HSV-1 can enhance the expression levels of reporter gene driven by PB promoter and ICP27 (-) HSV-1 can only replicate in the cells expressing ICP27 protein; (3). The vector construct containing ICP27 gene driven by PB promoter may not be an ideal delivery system for the purpose of prostate tissue-specific gene expression.

碩士<br>臺北醫學大學<br>醫學科學研究所<br>96<br>Salmonella is an important food-borne pathogen of public health concern. Sal-monella enterica contains more than 2,500 serotypes among which Typhimurium is an important etiology of gastroenteritis. Adhesion of bacteria to the host epithelial cells is a prerequisite step in establishing infection, while fimbriae are the primary structures implicated in such an adherent event. The type 1 fimbriae are the most commonly found fimbrial appendages on the outer membrane of Salmonella enterica serotype Typhimurium. The strongly fimbriate-phase bacteria can be isolated following serial passage every 48 hours in static broth culture, while non-fimbriate bacteria are obtained by growth on solid agar media. The phenotypic expression of type 1 fimbriae in S. Typhimurium is the result of the interaction and cooperation of several genes in the fim gene cluster. A transposon insertion mutagenesis has revealed that several genes outside the fim gene cluster may also involve in the expression of type 1 fimbriae both in static broth and on solid agar culture conditions. One S. Ty-phimurium mutant ubiB constitutively produced type 1 fimbriae in both culture condi-tions. The ubiB gene could encode a NAD(P)H-flavin reductase. Southern hybri-dization indicated that the transposition event occurred once in this mutant strain. Transforming the plasmid pTA-ubiB that contained the coding sequence of ubiB con-ferred the ubiB mutant back to the type 1 fimbrial phenotype as seen in the parental strain as indicated by yeast agglutination and electron microscopy. Reverse tran-scription polymerase chain reaction (RT-PCR) indicated that the fimA expression of the ubiB mutant strain obtained from the static broth was about 1.2% of that obtained from the solid agar culture. When ubiB strain harbored the plasmid pTA- ubiB, the fimA expression from broth was about 34 fold of that from the solid agar. As a con-trol, 16S rRNA was constantly expressed in all the strains tested. Growth curve analysis suggested that ubiB mutant had a slower growth rate in the log phase than the parental strain and the ubiB (pTA- ubiB) strain. How ubiB gene product affects type 1 fimbrial expression in S. Typhimurium is of interest and warrants further investiga-tion.