Killeen's (2005) Prep is an estimator of the replicability of an experiment. It is specifically defined as the probability of obtaining an effect of the same sign as that found in original experiment. Nevertheless, since it was announced, the validity and reliabliltiy of Prep has been challenged by a number of researchers. The present study aims at improving the performance of Prep by applying the nonparametric bootstrap method in its computation, and this bootstrap replication estimator is denoted as PBrep . A simulation study was carried out to compare the performance of Killeen's Prep and the proposed PBrep under different conditions. As expected, PBrep gives a more accurate estimation than Prep. However, PBrep occasionally fails to work properly when there is a zero population effect size, so there is still a room for improvement. / Killeen (2005) 發明的Prep是一種實驗重複估計量，它是指能夠獲得與最初實驗效應量一致方向的可能性。但自其發表以來，該系數的信度及效度仍受到不少學者的質疑。是次研究嘗試通過使用自助抽樣法以改善此系數的效能，並將改良的新系數命名為PBrep。不同環境下對兩個系數準確度的模擬測試結果顯示，PBrep比Prep能達到更準確的估計值。然而當目標總體不存在差別效應時，PBrep偶爾會出現較大的偏差，因此未來研究仍需在此方向作出改善。 / Chan, Man Lok. / Thesis M.Phil. Chinese University of Hong Kong 2014. / Includes bibliographical references (leaves 37-40). / Title from PDF title page (viewed on 14, September, 2016).

The thermophilic enzyme, Thermus aquaticus (Taq) DNA polymerase, is an essential tool in molecular biology because of its ability to synthesis DNA in vitro and its inherent thermal stability. Taq DNA polymerase is widely used in the polymerase chain reaction (PCR), an essential technique in a broad range of different fields from academic research to clinical diagnostics. The use of PCR-based tests in diagnostic testing is ever increasing; however, many of the samples being tested contain substances that inhibit PCR and prevent target amplification. Many attempts have been made to engineer polymerases not only to increase resistance to overcome the problem of inhibition, but also to enhance other characteristics such as fidelity, processivity and thermostability. Heparin, found in blood samples, and phytate, found in faecal samples, are two examples from a number of known PCR inhibitors. The mode of action of most PCR inhibitors is not well understood, but inhibition is thought to occur by enzyme binding or through the chelation of Mg2+ ions essential for PCR. In this project, a system of directed evolution by compartmentalised self-replication (CSR) was established and successfully employed to screen a mutant library for Taq DNA polymerase variants with enhanced resistance to the inhibitors heparin and phytate. CSR is a recently-established high-throughput method for the creation of novel polymerases, based on a feedback loop whereby polymerase variants replicate their own encoding gene. A mutant library of 106 variants was produced by random mutagenesis error-prone PCR, in which only the polymerase domain of Taq was mutagenised. Firstly, the CSR system was established and tested by performing a screen in the presence of heparin to select for heparin-resistant variants. Characterisation of selected variants revealed that a single round of CSR had produced a Taq variant (P550S, T588S) with a 4-fold increase in heparin resistance. The IC50 was increased from 0.012U/ml heparin to 0.050U/ml heparin. The study with heparin was followed by a phytate screen, in which two rounds of CSR were performed with an initial round of error-prone PCR followed by re-diversification (recombination) of the mutant library using the staggered extension process (StEP). The two rounds of CSR yielded a Taq variant with a 2-fold increase in phytate-resistance compared to the wild-type, with IC50 increased from 360μM phytate to 700μM phytate. The best phytate mutant (P685S, M761V, A814T) was further characterised and it was found that the catalytic activity, thermostability and fidelity of the mutant were comparable to the wildtype enzyme. The position of resistance-conferring mutations of the novel Taq variants evolved in this study provided some evidence for the inhibitors’ predicted modes of action in the case 2 of both phytate and heparin. As phytate’s mode of action is poorly understood, further investigations were performed to elucidate its role in PCR inhibition. A thorough investigation into the importance of relative phytate and Mg2+ levels on PCR was conducted and revealed for the first time convincing evidence that the primary mode of phytatemediated PCR inhibition is by chelation. Further work led to the successful crystallisation of Taq in the presence of phytate, although subsequent X-ray diffraction data to 2.5Å did not reveal phytate bound within the enzyme structure. Site-directed mutagenesis studies were used to probe cross-over between heparin and phytate-conferring mutations. Thus, in addition to providing valuable information for novel Taq variants with a potential application in fecal-based PCR diagnostic tests, this project has begun to provide insight into the fundamental aspects of the mode of action of phytate as a polymerase and PCR inhibitor.

Le virus de la mosaïque jaune du navet (TYMV) est un excellent modèle pour la réplication des virus à ARN simple brin de polarité positive. Ce petit virus de plante code l’ensemble desprotéines nécessaires à sa réplication sous forme d’un précurseur polyprotéine (206K). Du NauC-terminus, celui-ci porte une activité méthyltransférase, protéase à cystéine (PRO),hélicase et ARN-polymérase ARN dépendante (POL). Comme tous les virus à ARN(+)connus, son complexe de réplication est étroitement lié à des membranes cellulaires. Dans lecas du TYMV, c’est l’enveloppe des chloroplastes qui est impliquée. Un des acteurs clés de laréplication est la polymérase qui permet la synthèse de nouveaux génomes. La régulation de son activité implique dans un premier temps son clivage de la 206K par PRO. Ainsi libérée,elle est recrutée aux chloroplastes par une interaction directe avec le domaine PRO du produit de clivage 140K, afin de former le complexe de réplication. Un second clivage par PRO contenu dans 140K, à la jonction PRO-hélicase, permet de poursuivre le cycle de réplication.Récemment, il a également été montré que l’activité POL était régulée par la voie ubiquitine-protéasome durant le cycle viral. Ubiquitinilée par la cellule hôte, elle est adressée au protéasome où elle sera dégradée. Cependant, PRO, grâce à sa seconde fonction ubiquitine hydrolase, est capable de la protéger de cette dégradation. Afin de caractériser d’un point de vue structural ce mécanisme de régulation de la réplication, nous avons cristallisé, à l’aide d’un contaminant, PRO et avons résolu sa structure. L’empilement cristallin est tel que le Cterminus d’un domaine PRO est inséré dans la crevasse catalytique du domaine PRO suivant,nous fournissant ainsi des informations structurales sur son activité endopeptidase. Dans un second temps, afin d’avoir des informations sur sa seconde activité, nous avons réalisé un complexe stable entre PRO et une molécule d’ubiquitine afin de le cristalliser et résoudre sa structure. Enfin, nous avons initié l’étude cristallographique de la polymérase. / Turnip Yellow Mosaic Virus is an excellent model for positive-stranded RNA virus replication. It’s a small plant virus whose replication machinery is encoded in the viralgenome as a single polyprotein (206K). From N- to C-terminus, this 206K harbors amethyltransferase, a cysteine proteinase (PRO), a helicase and an RNA-dependent RNApolymerase (POL). As in all RNA(+) viruses known, the replication complex is bound to cellmembranes. For TYMV, it ,is the chloroplast envelope that is implicated. A key component inthe replication process is the polymerase, that allows the synthesis of new genomes. The regulation of its activity involves initially its cleavage by PRO from the 206K. Once liberated,the polymerase is recruited to the chloroplasts through a direct interaction with the PROdomain, contained in 140K, in order to form the replication complex. A second cleavage of140K by PRO at the PRO-helicase junction allows to continue the replication cycle. Recently,it has also been shown that the POL activity was regulated by the ubiquitin-proteasomesystem during the viral multiplication cycle. Ubiquitinilated by the host cell, POL is addressed to the proteasome where it is degraded. However, PRO, due to its second function as an ubiquitin hydrolase, is able to protect POL from its degradation. In order to characterizethis mechanism of replication regulation with a structural point of view, we crystallized,assisted by contaminant, PRO and resolved its structure. In the crystal packing, the C-terminalof a PRO domain is inserted into the catalytic cleft of the next PRO domain, thus providing us structural informations on its endopeptidase activity. In a second step, in order to obtain information about its second activity, we made a stable complex between PRO and amolecule of ubiquitin in order to cristallise it. Finally, we initiated the crystallographic studyof POL.Keywords:

The molecules of life are small to us—billionths of our size. They move fast too, and in the cell they crowd together impossibly. Bringing that strange world into ours is the trick of molecular biology. One approach is to harness many copies of a molecule and iterate a reaction many times to glimpse what happens at that small, foreign scale. This is a powerful way to do things and has provided major insights. But ultimately, the fundamental unit of molecular biology is the individual molecule, the individual interaction, the individual reaction. Single-molecule bioscience is the study of these phenomena. Eukaryotic DNA replication is particularly interesting from the single-molecule perspective because the biological molecules responsible for executing the replication pathway interact so very intricately. This work is based on replication in budding yeast—a model eukaryote. The budding yeast genome harbors several hundred sequence-defined sites of replication initiation called origins. Origins are bound by the Origin Recognition Complex (ORC), which recruits the ring-shaped Mcm2-7 complex during the G1 phase of the cell cycle. A second Mcm2-7 is loaded adjacent to the first in a head-to-head orientation; this Mcm2-7 double hexamer encircles DNA and is generally termed the Pre-Replicative Complex, or Pre-RC. Mcm2-7 loading is strictly dependent on a cofactor, Cdc6, which is expressed in late G1. Much less is known about the details of downstream steps, but a large number of factors assemble to form active replisomes. Origin-specific budding yeast replication has recently been reconstituted in vitro, with cell cycle dependence mimicked by the serial addition of purified Pre-RC components and activating kinases. This work introduces the translation of the bulk biochemical replication assay into a single-molecule assay and describes the consequent insights into the dynamics of eukaryotic replication initiation. I have developed an optical microscopy-based assay to directly visualize DNA replication initiation in real time at the single-molecule level: from origin definition, through origin licensing, to replisome formation and progression. I show that ORC has an intrinsic capacity to locate and stably bind origin sequences within large tracts of non-origin DNA, and that ordered Pre-RC assembly is driven by Cdc6. I further show that the dynamics of the ORC-Cdc6 interaction dictate the specificity of Mcm2-7 loading, and that Mcm2-7 double hexamers form preferentially at a native origin sequence. This work uncovers key variables that control Pre-RC assembly, and how directed assembly ensures that the Pre-RC forms properly and selectively at origins. I then characterize replisome initiation and progression dynamics. I show that replication initiation is highly precise and limited to Mcm2-7 double hexamers. Sister replisomes fire bidirectionally and simultaneously, suggesting that previously unidentified quality control mechanisms ensure that a complete pair of replisomes is properly assembled prior to firing. I also find that single Mcm2-7 hexamers are sufficient to support processive replisome progression. Moreover, this work reveals that replisome progression is insensitive to DNA sequence composition at spatial and temporal scales relevant to the replication of an entire genome, indicating that separation of the DNA strands by the replicative helicase is not rate-limiting to replisome function. I subsequently applied this replication assay to the study replisome-replisome collisions, a fundamental step in the resolution of convergent replication forks. I find that, surprisingly, active replisomes absolutely lack an intrinsic capacity to displace inactive replisomes. This result eliminates the simplest hypothesized mechanism for how the cell resolves the presence of un-fired replisomes and has prompted and guided the development of alternate testable hypotheses. Taken together, these observations probe the molecular basis of eukaryotic inheritance in unprecedented detail and offer a platform for future work on the many dynamic aspects of replisome behavior.

Deoxyribonucleic acid (DNA), like the hard drive in a computer, stores all the essential information for cell function and survival in nearly every single cell in our body. Four different bases are the building blocks of DNA that encode all the messages. As each cell divides, it must pass down its entire genomic DNA to both of its daughter cells. Given the vast amount of data that exists, many errors occur naturally every day and threaten the integrity of this biological hard drive. Normal cells are equipped with many repair tools to quickly and effectively respond to the lesions. When some of these errors disrupt the tightly regulated cell division, cells could undergo changes like an increase in the rate of division that eventually lead to cancer. One type of DNA damage that has a high propensity to cause genetic instability is the double-stranded break (DSB). Therefore, mechanisms that repair DSB are an important area of study in the fight against cancer and cancer causing syndromes. One of these repair pathways is homologous recombination (HR), which uses homologous sequences from either a sister chromatid or a homologue to fill in the information lost during a DSB. This homology pairing reaction requires a class of ATP-dependent proteins known as recombinases, with RAD51 being the one for humans. During HR, the early stages before pairing involve resection of the newly generated DSB ends to generate single-stranded DNA (ssDNA) overhangs, which are protected from degradation by replication protein A (RPA). RAD51 needs to displace the RPA from ssDNA and form a filament (the presynaptic complex) in order to initiate homology search. This process can be sped up by recombination mediators, which act to help RAD51 overcome the strong affinity of RPA for ssDNA that inhibits RAD51 binding and filament formation. Although Rad52 is the most important mediator in budding yeast, human RAD52 does not appear to have mediator function despite a high level of structural conservation. However, human RAD52 mediates ssDNA annealing and its deficiency is synthetic lethal with several important recombination proteins. Here, I use the single-molecule imaging technique of DNA curtains to visualize in real-time the competition and cooperativity between RPA, RAD52, and RAD51 on ssDNA through fluorescent labeling of RPA and RAD52. Using ssDNA curtains, I examine the conservation of facilitated dissociation from budding yeast to humans and show it does not require species-specific contacts. I also monitor the interactions of RAD52 with the RPA-ssDNA and find another point of conservation in the ability of RAD52 to upregulate the stability of RPA on ssDNA concerning facilitated dissociation. These RAD52-RPA-ssDNA complexes are long-lived; however, they are effectively displaced by RAD51 during filament assembly and do not re-bind appreciably to the RAD51 filament. Although RAD51 can still assemble on RAD52-RPA-ssDNA, I observe a significant inhibition on its nucleation (the first step in filament formation), but not elongation, by the presence of free RPA in solution. As DNA curtains allow efficient exchange of buffers in the micro-fluidic chambers while keeping ssDNA molecules tethered, I am able to follow individual DNA molecules overtime as they undergo different binding and filament assembly and disassembly reactions.

Cette thèse a été réalisée dans le cadre d’une cotutelle de thèse (3 ans) Franco-Libanaise entre l’Université Pierre et Marie Curie de Paris et l’Université Libanaise de Beyrouth. Nabil Tabaja a été entièrement financé par le Groupe de Recherche MAPE (Nanosized Porous Materials, Preparation, Advanced Characterization and Environmental Applications) de Beyrouth que nous tenons à remercier. Le but de ce travail était de tester et de valoriser des catalyseurs à base d’oxydes de fer et de ferrites pour la dépollution des eaux par photocatalyse sous lumière visible puis solaire. Les catalyseurs étudiés ont été préparés en employant des silices poreuses en tant que gabarits pour obtenir majoritairement : soit des nanoparticules, NP, d’oxydes de fer ayant cristallisé à l’intérieur des pores des silices (INTERNES, répliquées), soit des nanoparticules ayant cristallisé à l’extérieur des grains de silice et formées lors des traitements d’activation thermiques (EXTERNES). Nous avons employées des techniques faciles à transférer pour obtenir les silices (Chapitre 1). Notre objectif à ce niveau était d’obtenir plus de 50g d’au moins six silices ayant des diamètres de mésopores différents. La variation de ces diamètres s’accompagne de modifications des connections entre les mésopores principaux et des propriétés des surfaces des silices. Différents sels précurseurs de fer (chlorure, nitrate) ou des mélanges de métaux ont été déposées sur les différentes silices obtenues par des techniques de dépôt de type deux solvants (2S). Les échantillons ont été traités thermiquement à 700°C sous air pour obtenir des nanoparticules d’oxydes (Chapitre 2). Nous montrons que, si le diamètre des NP nanorépliquées est en général proche du diamètre des pores des silices initiales, les formes et la dispersion des nanoparticules internes dans les grains de silice dépendent de différents facteurs expérimentaux, des sels précurseurs, des solvants et du type de silice sélectionnée. Nous montrons également que la formation des particules externes est associée au traitement thermique imposé et peut être favorisée en choisissant le bon solvant et le bon sel précurseur pour une silice déterminée. Des premiers tests ayant démontrés un taux de lixiviation important dans le cas de catalyseur au fer, nous avons testé des ferrites de différentes compositions (cations (II) de différentes électronégativités, Ni(II), Co(II), Cd(II), Zn(II) ; cation (III), Cr(III)). Afin de comparer la réactivité catalytique des catalyseurs, deux types de réactions sont introduites successivement. Le premier type de réaction, l’oxydation photocatalytique du méthanol et du formaldéhyde, a été employé à titre fondamental. Dans ce cas, notre objectif était d’étudier la sélectivité de la réaction et d’identifier de façon inambigue quelles nanoparticules présentes dans la formulation des catalyseurs permettent, partant d’une espèce à un seul carbone, d’obtenir des produits à 2 ou plus atomes de carbone (Chapitre III). La seconde réaction, l’oxydation photocatalytique d’un pesticide, a été employée pour démontrer que les catalyseurs peuvent être utiles à titre appliqué. Le pesticide sélectionné a été le carbendazime (Chapitre IV) dont nous avons suivi la décomposition. Ces études n’ont été possibles que grâce à l’emploi de techniques de caractérisation avancées, de type TOC et GC-MS. Les meilleures activités catalytiques ont été analysées en fonction de la présence d’une majorité de NP internes et/ou externes et d’autres espèces plus dispersées et non visibles par DRX. / This thesis was carried out as part of a Franco-Lebanese collaboration thesis (3 years) between the University of Pierre et Marie Curie in Paris and the Lebanese University in Beirut. Nabil Tabaja was fully funded by the Research Group MAPE (nanosized Porous Materials, Preparation, Advanced Characterization and Environmental Applications) Beirut that we thank.The purpose of this study was to test and develop catalysts based on iron oxides and ferrites for decontamination of water by photocatalysis under visible and sunlight. The catalysts studied were prepared by using porous silica as templates to obtain predominantly either nanoparticles, NP, iron oxides having crystallized within the pores of the silica (INTERNAL, replicated), or nanoparticles having crystallized outside the silica grains formed and during the thermal activation treatments (external). We employed techniques easily transferable to obtain silicas (Chapter 1). Our goal at this level was more than 50 grams of at least six silicas having different diameters of mesopores. The change in these diameters is accompanied by changes in connections between major mesoporous silicas and the properties of surfaces. Various iron precursor salts (chloride or nitrate) or metal mixtures were deposited on the various silicas obtained by the two solvents techniques (2S). The samples were calcined at 700 ° C in air to obtain oxide nanoparticles (Chapter 2). We show that, if the diameter of NP nanoreplicated is generally close to the pore diameter of the initial silicas, and forms the dispersion of nanoparticles in the inner silica grain depends on various experimental factors of the precursor salts, solvents and the type of the selected silica. We also show that the formation of particles is associated with the external heat treatment can be promoted and imposed by choosing the right solvent and the right precursor salt for a specific silica. Initial tests have demonstrated an important release rate in the case of iron catalyst, we tested different compositions ferrites ((II) cations of different electronegativities, Ni (II), Co (II), Cd (II), Zn (II) cation (III), Cr (III)). In order to compare the catalytic activity of the catalysts, two types of reactions are successively introduced. The first type of reaction, the photocatalytic oxidation of methanol and formaldehyde was employed as fundamental. In this case, our objective was to study the selectivity of the reaction and identify what way inambigue nanoparticles in the formulation of catalysts allow, starting from a species to a single carbon, to obtain products with 2 or more carbon atoms (Chapter III). The second reaction, the photocatalytic oxidation of a pesticide, was used to demonstrate that the catalysts may be useful as applied. The pesticide was selected carbendazim (Chapter IV) which we have followed the breakdown. These studies have been possible thanks to the use of advanced characterization techniques, type TOC and GC-MS. The best catalytic activities were analyzed according to the presence of a majority of internal and / or external NP and other species more dispersed and invisible by XRD.

SbcCD has been shown to cleave a DNA hairpin formed by a palindromic DNA sequence on the lagging strand template of the E. coli chromosome. This activity was exploited to create a unique system for inducing a single site-specific DNA double-strand break (DSB) once per replication cycle. First, this work shows that the SOS response induced by this DSB is only essential for viability following multiple cycles of cleavage and repair. Next, the SOS-inducible inhibitor of cell division SfiA is shown to be dispensable for survival, despite demonstrating that cleavage of the palindrome causes both an increase in cell size and a delay in nucleoid segregation. A model of the E. coli cell cycle is presented to reconcile the observation that growth under chronic DSB induced conditions has no effect on generation time despite causing an increase in cell size. This system of DSB induction was then coupled with fluorescence markers on both sides of the palindrome to visualise the consequence of the DSB in vivo. Cleavage of the DNA hairpin by SbcCD in a recAmutant was used to selectively degrade the chromosome that replicated the palindrome on the lagging strand of replication, allowing two genetically identical sister chromosomes to be distinguished. This approach was used to show that chromosome segregation in E. coli is not random, but results in the segregation of lagging strand replicated DNA to mid-cell and leading strand replicated DNA to cell poles. Finally, this system for visualising the site of an inducible DSB was optimised for use in various other mutant backgrounds to allow the events of DSB repair to be dissected. This work provides a solid basis for further investigation into the relationship between replication, recombination and chromosome segregation in the model organism E. coli.

Earth’s crust and primordial ocean formed more than 4 billion years ago and life is believed to have originated on earth at least 3.6 billion years ago. This suggests that primitive cellular life must have evolved from non-living matter during that period of several hundred million years. To study the transition from chemistry to biology, a simple vesicular system called a protocell is an ideal model that is self-organized and contains informational or metabolic materials. This thesis starts by exploring the replication of a model genetic material under plausible prebiotic conditions. The non-enzymatic copying of RNA was found to be catalyzed by Fe2+, which used to be abundant in aqueous environments on the early anoxic earth. Fe2+ was found to be a better catalyst of non-enzymatic RNA copying and ligation in slightly acidic to neutral pH conditions than Mg2+, the divalent cation used to catalyze these reactions in previous studies. This finding suggests that ferrous iron could have facilitated the replication and evolution of RNA on the prebiotic earth. To gain a better understanding of the properties of protocell membranes, the impact of membrane composition and multi-bilayer structure on non-enzymatic and enzymatic biochemical reactions was studied. A fatty acid/phospholipid hybrid membrane system was proposed as a potential intermediate state in protocellular evolution. This membrane composition was investigated for its stability and permeability, two fundamental features of functional protocells. The system proved stable in the presence of divalent cations and retained permeability to small building block molecule. Vesicles with this composition were shown to host faster non-enzymatic RNA copying, and to enable enzymatic protein synthesis. To study the effects of multi-lamellarity, giant multilamellar vesicles (GMVs) were prepared by an extrusion-dialysis method. Compared with small unilamellar vesicles (SUVs), GMVs show slightly better ability to retain encapsulated RNA, while maintaining good permeability for small charged molecules. The multilamellar structure also promotes non-enzymatic RNA copying, providing preliminary evidence that membranes could also mediate catalytic functions as well as acting as a compartment. / 2020-07-02T00:00:00Z

Break Induced Replication (BIR), a mechanism by which cells heal one-ended double-strand breaks, involves the invasion of a broken strand of DNA into a homologous template, and the copying of tens to hundreds of kilobases from the site of invasion to the telomere using a migrating D-loop. Here we show that if BIR encounters an interstitial telomere sequence (ITS) placed in its path, BIR terminates at the ITS 12% of the time, with the formation of a new telomere at this location. We find that the ITS can be converted to a functional telomere by either direct addition of telomeric repeats by telomerase, or by homology-directed repair using natural telomeres. This termination and creation of a new telomere is promoted by Mph1 helicase, which is known to disassemble D-loops. We also show that other sequences that have the potential to form new telomeres, but lack the unique features of a perfect telomere sequence, do not terminate BIR at a significant frequency in wild-type cells. However, these sequences can cause chromosome truncations if BIR is made less processive by loss of Pol32 or Pif1. These findings together indicate that features of the ITS itself, such as secondary structures and telomeric protein binding, pose a challenge to BIR and increase the vulnerability of the D-loop to dissociation by Mph1, promoting telomere formation at the site.

Productive HIV-1 transcription yields a single ∼­9.2kb RNA. From this ∼9.2kb genomic RNA, greater than 40 different subgenomic mRNAs can be produced through alternative splicing using four 5' splice sites (ss) and seven 3'ss. Splice site utilization is governed by the inherent strength of the splice sites and by several identified cis acting elements. The HIV-1 Vif protein, required to overcome the cellular antiviral factor APOBEC3G, is encoded by a singly-spliced mRNA coupling 5'ss D1 to 3'ss A1. Alternatively, mRNAs spliced at A1 can utilize a downstream 5'ss, D2, resulting in inclusion of non-coding exon 2 in a small percentage of mRNAs. Expression of vif mRNA within infected cells is required but maintained at low levels. The purpose of studies described in this thesis was to identify and characterize elements within the HIV-1 genome regulating vif mRNA splicing. We identified an exonic splicing enhancer (ESE) within the 18nt downstream of HIV-1 3'ss A1, ESEVif. Mutation of ESEVif within the HIV-1 proviral clone pNL4-3 resulted in a dramatic decrease in vif mRNA, Vif protein, and undetectable levels of non-coding exon 2 inclusion. The cellular splicing factor SRp75 was found to selectively bind ESEVif in vitro. ESEVif mutant virus replicated in APOBEC3G-deficient T-cell lines as efficiently as wild-type virus. In APOBEC3G-producing T-cell lines, ESEVif mutant virus replicated to lower levels than wild-type virus. Other studies have identified additional mRNA splicing elements regulating splicing at A1: the downstream 5'ss D2 can promote or repress splicing, a G4 motif downstream of D2 represses splicing, and two ASF/SF2 dependent splicing enhancers, ESEM1 and ESEM2, promote splicing. Mutational analysis described in this thesis determined that loss of both ESEVif and the G4 motif resulted in wild-type levels of splicing at A1. Mutations of each identified ESE influenced vif mRNA splicing in the order ESEVif>ESEM2>ESEM1. The data presented in this thesis support a model of vif mRNA splicing regulation in which exon 2 ESE act to overcome the negative G4 motif insuring sufficient levels of Vif production for efficient replication in the presence of APOBEC3G.