Trichomonas vaginal is is the sexually transmitted agent of trichomoniasis. Although the organism is believed to only reproduce via binary fission, genes specific to 5 Meiosis and Homologous Recombination have been identified in its genome. It is unclear whether the organism has the ability to undergo sexual reproduction or if it has lost that ability over time. Aside from meiosis, these genes could be expressed for use in antigenic variation and in the creation or transfer of resistance genes to other cells. In this study, we induced the expression ofDMCl and Rad51 homologues-key players in Homologous Recombination-using a system of tetracycline induction. We localized DMCl to both the cytoplasm and the nucleus, while Rad51 is localized to the nucleus. We performed a DNA strand exchange that suggests DMCl may be capable of DNA strand exchange. We also developed a system to determine whether haploid cells of Trichomonas vaginal is are capable of cytoplasmic fusion through the use of fluorescent proteins. Specifically, this study focuses on a line of Green Fluorescent Protein-expressing cells.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第19972号 / 薬科博第63号 / 新制||薬科||7(附属図書館) / 33068 / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 橋田 充, 教授 佐治 英郎, 教授 髙倉 喜信 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24110号 / 農博第2515号 / 新制||農||1093(附属図書館) / 学位論文||R4||N5401(農学部図書室) / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 井上 和生, 教授 谷 史人, 准教授 後藤 剛 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Brucella abortus is a facultative intracellular bacterial pathogen, which causes abortion in cattle and undulant fever in human. B. abortus strain RB51 (Strain RB51) is the official vaccine for bovine brucellosis in the USA. B. abortus strain RB51 can be used as a vector for the over-expression of its own (homologous) as well as heterologous protective antigens. The immune system can detect these heterologous antigens and produce a response. Expressing a protein in different bacterial compartments has been shown to affect its accessibility to the immune system and the way the antigen is processed by antigen presenting cells. In order to determine if the immune response is affected by the localization of the antigen, green fluorescent protein (GFP) was expressed at three different locations in B. abortus strain RB51, outer-membrane (OM), periplasmic space (PS) and in the cytoplasmic region (CR) of B. abortus strain RB51. This localization was obtained by transforming strain RB51 with plasmids pBBg18sGFP and pBBgSsGFP, in which the 18 kDa Brucella lipoprotein and the Brucella Cu/Zn SOD protein signal sequences were added to the GFP sequence to cause OM and PS expression respectively. No signal sequences were added to the plasmid pBBgGFP for CR only expression. Expression and localization of GFP in the different compartments in recombinant B. abortus strain RB51 were confirmed by electron microscopy and antibody absorption experiments. Groups of 5 female BALB/c mice each were injected and boosted with three recombinant strains and appropriate controls. Mice were bled and their anti-GFP antibody production was assessed. None of the immunized mice produced specific antibodies against GFP, probably due to the low expression of the heterologous antigen observed in this study by strain RB51 observed in this study. It will be necessary to produce new recombinants which are able to express higher amounts of GFP to answer if localization of heterologous antigen within the recombinant RB51 affects the level of a specific immune response. / Master of Science

A new synthetic methodology for the combinatorial preparation of C-terminus-modified Green and Red Fluorescent Protein chromophores is described. This method involves the modification of the previously reported [2+3] cycloaddition reaction scheme to incorporate new R2 groups in the imidate used in the final step. This is achieved through two primary routes: (a) the imidation of nitriles using hydrochloric acid gas and (b) the O-alkylation of amides using a variant of Meerwein's Salt to provide conjugated imidates. The preparation of fluorescent microcrystals and nanofibers from Green Fluorescent Protein chromophore derivatives via the reprecipitation method is also demonstrated. The properties of these microcrystals and nanofibers, especially in relation to the powder obtained from organic solvents, are also explored. Additionally, it is demonstrated that the size and shape of the microcrystals and nanofibers can be modulated with varying experimental conditions for RP. A new class of AIE-active GFP chromophores is reported. These chromophores contain a benzoxazole group on the phenyl ring and varying lengths of alkyl chains on the imidazolidinone nitrogen. These benzoxazole-based chromophores exhibit unique properties in the solid state not previously observed for GFP chromophore derivatives, namely, a broadening of the excitation spectrum and red-shifting of the emission, likely caused by excimer formation. The crystal structure also reveals a unique "hot-dog" stacking motif. Additionally, some projects which require further work are discussed at the end of the thesis. These include a stress-responsive GFP-based polymer and DNA-binding fluorophores.

No description available.

Ce travail de thèse porte sur l’étude de la réplication chez les archées, qui constituent le troisième domaine du vivant avec les bactéries et les eucaryotes. L’organisme modèle que nous avons utilisé est l'archée halophile Haloferax volcanii car les outils génétiques disponibles permettent d’exprimer des protéines fusionnées à la Protéine Fluorescente Verte (GFP) dans cet organisme mésophile et aérobe et ainsi de localiser les protéines d’intérêt dans des cellules vivantes. Nous nous sommes ainsi intéressés à la localisation cellulaire de quatre protéines de la réplication qui ont été fusionnées à la GFP et exprimées sous contrôle de leur propre promoteur : (i) la protéine ‘Flap Endonuclease 1’ (FEN1), qui intervient dans la maturation des fragments d’Okazaki, (ii) la protéine ‘Origin Recognition Complex’ (ORC1) impliquée dans la reconnaissance des origines de réplication, (iii) la protéine ‘Proliferating Cellular Nuclear Antigen’ (PCNA), anneau de processivité des ADN polymérases réplicatives, et (iv) la protéine de fixation à l’ADN simple-brin ‘Replication Protein A’ (RPA2) essentielle à la réplication chez H. volcanii. Seule la protéine PCNA n’a pu être exprimée en fusion avec la GFP, suggérant que la protéine fusion n’est pas fonctionnelle. GFP::Orc1 et GFP::Fen1 ont été exprimées dans la cellule mais ne présentent pas de localisation spécifique reflétant un rôle de ces protéines dans la réplication de l’ADN. En revanche des foyers de fluorescence de la protéine fusion GFP::Rpa2 ont été observés, dont le nombre augmente significativement dans des cellules exposées à l’aphidicoline, drogue inhibant la synthèse de l’ADN et induisant ainsi un stress réplicatif. Cependant une localisation différente de la protéine GFP::Rpa2 a été observée lorsque les cellules sont exposés à la phléomycine, qui induit notamment des cassures double-brin de l‘ADN. Dans ces cellules, GFP::Rpa2 forme un foyer de fluorescence massif qui colocalise avec l’ADN compacté dans la grande majorité des cellules observées. Nos résultats suggèrent donc que la localisation spécifique observée pour GFP::Rpa2 reflète son rôle dans la réparation de l’ADN et/ou le redémarrage des fourche de réplication arrêtées. / The aim of this thesis project was to improve our understanding of DNA replication in archaea, the third domain of life with bacteria and eukarya. The model organism chosen for these studies is the halophilic archaea Haloferax volcanii, a mesophilic aerobe for which genetics tools allow studying in living cells the localization of proteins fused to the Green Fluorescent protein (GFP). Four proteins involved in DNA replication were fused to the GFP and expressed under the control of their own promoter: (i) the ‘Flap Endonuclease 1’ (FEN1), involved in Okazaki fragments maturation, (ii) the ‘Origin Recognition Complex’ (ORC1), involved in DNA replication origin recognition, (iii) the ‘Proliferating Cellular Nuclear Antigen’ (PCNA), processivity factor of replicative DNA polymerases, and (iv) the ‘Replication Protein A’ (RPA2), single-stranded DNA binding protein essential for DNA replication in H. volcanii. Only the PCNA fusion to the GFP was not successful, suggesting that the GFP hinders essential roles of PCNA in DNA replication. Fen1 and Orc1 were successfully fused to the GFP and expressed in living cells, but specific localization in cells related to growth phase, reflecting different replication dynamics, were not observed. In contrast, we could observed fluorescent foci formed by the fully functional GFP::Rpa2 protein that actively responded to DNA damage in H. volcanii cells. The number of these fluorescent foci per cell was constant during cell growth but it significantly increased in cells exposed to aphidicoline, which inhibits DNA synthesis during replication. When cells were treated with phleomycine, a DNA damaging agent mainly causing double-strand breaks, formation of a massive fluorescent focus coinciding with DNA compaction was observed. Our results suggest that the specific cellular localization of GFP::Rpa2 observed reflects Rpa2 roles in DNA repair and/or DNA replication fork restart.

Indiana University-Purdue University Indianapolis (IUPUI) / The discovery and isolation of naturally occurring fluorescent proteins, FPs, have provided much needed tools for molecular and cellular level studies. Specifically the cloning of green fluorescent protein, GFP, revolutionized the field of biotechnology and biochemical research. Recently, a red fluorescent protein, DsRed, isolated from the Discosoma coral has further expanded the pallet of available fluorescent tools. DsRed shares only 23 % amino acid sequence homology with GFP, however the X-ray crystal structures of the two proteins are nearly identical. DsRed has been subjected to a number of mutagenesis studies, which have been found to offer improved physical and spectral characteristics. One such mutant, DsRed-Monomer, with a total of 45 amino acid substitutions in native DsRed, has shown improved fluorescence characteristics without the toxic oligomerization seen for the native protein. In our laboratory, we have demonstrated that DsRed proteins have a unique and selective copper-binding affinity, which results in fluorescence quenching. This copper-binding property was utilized in the purification of DsRed proteins using copper-bound affinity columns. The work presented here has explored the mechanism of copper-binding by DsRed-Monomer using binding studies, molecular biology, and other biochemical techniques. Another focus of this thesis work was to demonstrate the applications of DsRed-Monomer in biochemical studies based on the copper-binding affinity and fluorescence properties of the protein. To achieve this, we have focused on genetic fusions of DsRed-Monomer with peptides and proteins. The work with these fusions have demonstrated the feasibility of using DsRed-Monomer as a dual functional tag, as both an affinity tag and as a label in the development of a fluorescence assay to detect a ligand of interest. Further, a complex between DsRed-Monomer-bait peptide/protein fusion and an interacting protein has been isolated taking advantage of the copper-binding affinity of DsRed-Monomer. We have also demonstrated the use of non-natural amino acid analogues, incorporated into the fluorophore of DsRed-Monomer, as a tool for varying the spectral properties of the protein. These mutations demonstrated not only shifted fluorescence emission compared to the native protein, but also improved extinction coefficients and quantum yields.

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Lactobacillus plantarum 285, isolated from sorghum beer, produces bacteriocin 285, which displays activity against several food spoilage organisms. For future application of bacteriocin 285 in the food industry, it was important to characterize the peptide and identify the genes encoding its production. The effect of bacteriocin 285 on sensitive cells was determined through the use of an indicator (sensitive) organism, Lactobacillus sakei DSM 20017. The indicator strain was genetically modified to express GFP (green fluorescent protein), with the aim of quantifying the antibacterial activity of bacteriocin 285 as a function of GFP fluorescence. Bacteriocin 285 proved to be identical to plantaricin 423 produced by L. plantarum 423. Plantaricin 423 is a class lIa bacteriocin and displays antimicrobial activity towards a broad spectrum of bacteria, including several food spoilage organisms. The sensitivity of L. sakei DSM 20017 towards antibacterial peptides produced by Lactobacillus curvatus DF38, L. plantarum 285, Lactobacillus casei LHS and Lactobacillus salivarius 241 is not limited to the growth stage of the organism. Cells remained sensitive to all four of these bacteriocins, from lag phase to late exponential growth. To inhibit growth of up to 90% of the cells of L. sakei DSM 20017, 1 AU/ml bacteriocin 285 (7 ng/ml) of partially purified bacteriocin 285 was required. However, to kill all viable cells of L. sakei DSM 20017, 16 AU/ml (110 ng/ml) of partially purified bacteriocin 285 was required. The gfpuv gene, encoding GFPuv, was cloned downstream of the Idh promoter and successfully expressed in L. sakei DSM 20017. However, GFPuv fluorescence could not be used as a direct method to quantify the antimicrobial activity of bacteriocin 285, since cells of strain DSM 20017 remained fluorescent for prolonged periods after treatment with lethal concentrations of the bacteriocin. The non-viability of the cells was confirmed with epifluorescence microscopy and a L1VE/DEAD® Baclight™ Bacterial Viability Probe. Cells that were stained with the viability probe indicated that the majority of untreated L. sakei DSM 20017 cells were viable. However, treatment of strain DSM 20017 with 16 AU/ml bacteriocin 285 rendered all visible cells non-viable. / AFRIKAANSE OPSOMMING: Lactobacillus plantarum 285 wat uit sorgumbier geïsoleer is, produseer bakteriosien 285. Die bakteriosien toon aktiwiteit teen verskeie organismes wat voedselbederi veroorsaak. Vir toekomstige aanwending van bakteriosien 285 in die voedselindustrie was dit belangrik om die peptied te karakteriseer en die gene wat vir die produksie daarvan kodeer, te identifiseer. Die effek van bakteriosien 285 op sensitiewe selle is bepaal deur die gebruik van 'n indikator (sensitiewe)-organisme, Lactobacillus sakei DSM 20017. Die indikator-organisme is geneties verander om die GFP (groen fluoreserende proteïen) uit te druk. Die doel was om die antibakteriese aktiwiteit van bakteriosien 285 te kwantifiseer as 'n funksie van GFP fluorisensie. Bakteriosien 285 is identies aan plantarisien 423 wat deur L. plantarum 423 produseer word. Plantarisien 423 is 'n klas Iia bakteriosien en vertoon antimikrobiese aktiwiteit teenoor 'n wye verskeidenheid bakterieë, insluitende verskeie organismes wat voedsel bederf. Die sensitiwiteit van L. sakei DSM 20017 teenoor antibakteriese peptiede wat deur Lactobacillus cutveius DF38, L. plantarum 285, Lactobacillus casei LHS en Lactobacillus salivarius 241 geproduseer word, word nie beïnvloed deur die groeifase van die organisme nie. Selle het sensitief gebly teenoor al vier die bakteriosiene van sloer- tot laat eksponensiële groei. Om groei van tot 90% van L. sakei DSM 20017 selle te inhibeer, word 1 AU/ml (7 ng/ml) gedeeltelik gesuiwerde bakteriosien 285 benodig. Om alle lewensvatbare L. sakei DSM 20017 selle te dood, word 16 AU/ml (110 ng/ml) gedeeltelik gesuiwerde bakteriosien 285 benodig. Die gfpuv-geen, wat GFPuv kodeer is stroomaf van die Idh-promoter gekloneer en suksesvol in L. sakei DSM 20017 uitgedruk. GFPuv fluoresensie kon nie as direkte metode gebruik word om die antimikrobiese aktiwiteit van bakteriosien 285 te bepaal nie, aangesien die selle van L. sakei DSM 20017 fluoreserend gebly het lank na behandeling met dodelike konsentrasies van die bakteriosien. Die lewensvatbaarheid van die selle is bevestig deur epifluoresensiemikroskopie en 'n LlVE/DEAD® Bac/ight™ bakteriese lewensvatbaarheidspeiler. Selle van L. sakei DSM 20017 wat deur die peiler gekleur is, het gewys dat die meeste selle wat nie deur bakteriosien 285 behandel was nie, lewensvatbaar was. Behandeling van L. sakei DSM 20017 met 16 AU/ml bakteriosien 285 het al die sigbare selle gedood.

Stem cells have great potential value for treating a number of diseases and conditions, including diabetes, Parkinson's, and spinal cord injuries. Applying stem cells for therapeutic purposes will require an in-depth understanding of their biology, not only of the genes they express, but also the functions of the proteins encoded by the genes. The goal of the project presented in this thesis was to develop a method for high-throughput analyses of protein localization in mouse stem cells. Localization information can provide insight into the functions and biological roles of proteins. <br /><br /> One means of studying protein localization involves creating proteins with a green fluorescent protein (GFP) reporter gene and analyzing their localization using fluorescence microscopy. The research outlined in this thesis focused on developing a system to create a large number of GFP-tagged proteins by constructing a cDNA?GFP fusion library. This involved exploring methods for optimizing cDNA synthesis, designing a retroviral vector (pBES23) for the expression of cDNA?GFP fusions in mouse stem cells, and constructing a cDNA?GFP fusion library in this vector using R1 mouse embryonic stem cell mRNA. The library constructed was not successfully delivered to target cells for GFP-tagged protein expression; it was therefore not possible to characterize protein localization in mouse stem cells. Suggestions are given as to how the methods used in this thesis might be optimized further.