Diverse biological activities of polyketide natural products are often associated with specific structural motifs, biosynthetically introduced after construction of the polyketide core. Therefore, investigation of such "post-polykektide synthase (PKS)" modifications is important, and the accumulated knowledge on these processes can be applied for combinatorial biosynthesis to generate new polyketide derivatives with enhanced biological activities. In addition to the practical value, a lot of unprecedented chemical mechanisms can be found in the enzymes involved therein, which will significantly advance our understanding of enzyme catalysis. The works described in this dissertation focus on elucidating a number of post-PKS modifications involved in the biosynthesis of an insecticidal polyketide, spinosyn A, in Saccharopolyspora spinosa. First, three methyltransferases, SpnH, SpnI, and SpnK, responsible for the modification of the rhamnose moiety, have been investigated to verify their functions and to study how they are coordinated to achieve the desired level of methylation of rhamnose. In vitro assays using purified enzymes not only established that SpnH, SpnI, and SpnK are the respective rhamnose 4ʹ-, 2ʹ-, and 3ʹ-O-methyltransferase, but also validated their roles in the permethylation process of spinosyn A. Investigation of the order of the methylation events revealed that only one route catalyzed by SpnI, SpnK, and SpnH in sequence is productive for the permethylation of the rhamnose moiety, which is likely achieved by the proper control of the expression levels of the methyltransferase genes involved in vivo. The key structural feature of spinosyn A is the presence of the unique tetracyclic architecture likely derived from the monocyclic PKS product. To elucidate this "cross-bridging" process, which had been hypothesized to involve four enzymes, SpnF, SpnJ, SpnL, and SpnM, the presumed polyketide substrate was chemically synthesized using Julia-Kocienski olefination, Stille cross-coupling, and Yamaguchi macrolactonization as key reactions. Incubation of the synthesized substrate with SpnJ produced a new product where the 15-OH group of the substrate is oxidized to the ketone. Next, it was demonstrated that incubation of this ketone intermediate with SpnM produces a tricyclic compound, via a transient monocyclic intermediate with high degree of unsaturation. Whereas it was initially thought that SpnM catalyzes both dehydration and [4+2] cycloaddition in sequence, detailed kinetic analysis revealed that SpnM is only responsible for the dehydration step, and the [4+2] cycloaddition step is indeed catalyzed by SpnF. Finally, successful conversion of the tricyclic intermediate to the tetracyclic core was demonstrated using SpnL. Proposed chemical mechanisms of SpnF and SpnL, Diels-Alder and Rauhut-Currier reactions, respectively, are interesting because enzymes capable of catalyzing these reactions have yet to be characterized in vitro. This work not only establishes the biosynthetic pathway for constructing the spinosyn tetracyclic core, but also epitomizes the significance of the post-PKS modification as a rich source of new enzyme catalysis. / text

Small molecules are important not only for treating human diseases but also for studying disease-related biological processes. This dissertation focuses on the effects of small molecules on pancreatic ductal adenocarcinoma cells. Here, I describe the discovery of two small-molecule tool compounds and their applications for interrogating the biological processes related to two distinct diseases in the human pancreas. First, BRD4770 was identified as a histone methyltransferase inhibitor through a target-based biochemical approach, and was used as a probe to study the function of methyltransferases in cancer cells. Second, BRD7552 was discovered as an inducer of Pdx1 using a cell-based phenotypic screening approach, and was used to induce the expression of Pdx1, a master regulatory transcription factor required for \(\beta\)-cell transdifferentiation. This compound is particularly interesting for the study of type-1 diabetes (T1D). The histone methyltransferase G9a catalyzes methylation of lysine 9 on histone H3, a modification linked to aberrant silencing of tumor-suppressor genes. The second chapter describes the collaborative effort leading to the identification of BRD4770 as a probe to study the function of G9a in human pancreatic cancer cells. BRD4770 induces cellular senescence and inhibits both anchorage-dependent and -independent proliferation in PANC-1 cell line, presumably mediated through ATM-pathway activation. Chapter three describes the study of a natural product gossypol, which significantly enhances the BRD4770 cytotoxicity in p53-mutant cells through autophagic cell death. The up-regulation of BNIP3 might be responsible for the synergistic cell death, suggesting that G9a inhibition may help overcome drug resistance in certain cancer cells. Ectopic overexpression of Pdx1, Ngn3, and MafA can reprogram pancreatic exocrine cells to insulin-producing cells in mice, which sheds light on a new avenue for treating T1D. The fourth chapter focuses on a gene expression-based assay using quantitative real-time PCR technique to screen >60,000 compounds for induction of one or more of these three transcription factors. A novel compound BRD7552 which up-regulated Pdx1 mRNA and protein levels in PANC-1 cells was identified. BRD7552 induces changes of the epigenetic markers within the Pdx1 promoter region consistent with transcriptional activation. Furthermore, BRD7552 partially complements Pdx1 in cell culture, enhancing the expression of insulin induced by the introduction of the three genes in PANC-1 cells. In summary, the central theme of my dissertation is to identify novel bioactive small molecules using different screening approaches, as well as to explore their effects in pancreatic ductal cells. / Chemistry and Chemical Biology

The type III secretion system (T3SS) of pathogenic Yersinia pseudotuberculosis is involved in virulence. The syringe-like secretion system spans both bacterial membranes and is responsible for the ability of Yersinia to transfer toxic proteins (Yop proteins) into the eukaryotic target cell. The T3SS is believed to have evolved from the flagellum and regulation of the T3SS is a complex event that involves a series of regulatory proteins, whereby two are YscP and YscU. In a regulatory model, called the substrate specificity switch, both proteins act together to ensure proper T3SS structure and function by regulating a stop in YscF needle protein export with a shift to Yop effector secretion. YscU undergoes autoproteolysis at a conserved motif consisting of amino acids Asparagine-Proline-Threonine-Histidine (NPTH). Processing generates a C-terminal 10 kDa peptide, YscUCC. Processing is crucial for proper T3SS regulation and function both in vitro and in vivo. Full-length YscU does not support Yop secretion and after cleavage, YscUCC remains attached to the rest of YscU and acts as a negative block on T3S. Relief of this negative block is suggested to occur through displacement of YscUCC from the rest of YscU. Thorough control of many different cellular processes is brought by the heat shock proteins (HSPs) DnaK and DnaJ. Due to their multiple regulatory functions, mutations in the hsp-genes lead to pleiotropic effects. DnaK and DnaJ are essential for proper flagellum driven motion of bacteria, but more so; they ensure proper Yersinia T3SS function in vivo. Furthermore, DnaJ interacts with YscU and may be directly involved in T3SS regulation. Virulence of Yersinia is regulated on many levels. A previously identified virulence associated protein, VagH, is now characterized as an S-adenosyl-methionine dependent methyltransferase. The targets of the methylation activity of VagH are release factors 1 and 2 (RF1 and RF2), that are important for translation termination. The enzymatic activity of VagH is important for Yop secretion and a vagH mutant up-regulates a T3SS negative regulatory protein, YopD. Furthermore, a vagH mutant is avirulent in a mouse infection model, but is not affected in macrophage intracellular survival. The importance of VagH in vivo makes it a possible target for novel antimicrobial therapy.

Epigenetic modification of DNA plays an important role in gene regulation. During gametogenesis and early embryogenesis epigenetic states are reset to ensure embryonic-specific gene expression patterns after fertilization. However, certain genomic regions may resist epigenetic reprogramming. This may result in transgenerational epigenetic inheritance. Earlier, a grandparental origin dependent (GPO) transmission ratio distortion (TRD) of alleles in the distal region of mouse chromosome 12 had been found (Croteau et al ., 2002). The distorted region overlaps with the imprinted region of chromosome 12. The mechanism underlying this TRD is unknown, and we hypothesized that it was due to failure to reset imprints in the imprinted region in a proportion of germ cells. Such an imprint resetting failure would represent a particular case of transgenerational epigenetic inheritance. DNA (Cytosine-5) methyltransferase 1 (DNMT1) plays a key role in the maintenance of epigenetic states in mammalian genomes. To test the role of DNA methylation and DNMT1 in the genesis of TRD and its relationship to epigenetic inheritance we investigated the effect of Dnmt1 loss-of-function mutations using two mouse models: GPO (grandparental origin dependent)-TRD (transmission ratio distortion) and epigenetic inheritance at the agouti locus. Here, we report that Dnmt1 mutations have a modifying parental effect on the transmission of grandparental chromosome 12 alleles. However, the same Dnmt1 mutation did not affect the agouti coat color inheritance patterns in mice that inherited the Avy (agouti viable yellow) mutant allele from the father. Our results suggest that Dnmt1 is a trans-acting modifier of allelic transmission and support the role of epigenetic states in the genesis of TRD.

Investigation of RNA and DNA function often requires sequence-specific incorporation of various reporter and affinity probes. This can be achieved using AdoMet-dependent methyltransferases (MTases) as they can be active with synthetic AdoMet analogues equipped with transferable chains larger than the methyl group. These chains usually carry reactive groups that can be further chemically appended with required reporters. For this, azide-alkyne 1,3-cycloaddition (AAC), also called “click”, reaction is particularly attractive. This work shows that the HhaI cytosine-5 DNA MTase (variant Q82A/Y254S/N204A) catalyzes efficient sequence-specific transfer of hex-2-ynyl side chains containing terminal alkyne or azide groups from synthetic cofactor analogues to DNA. Both the enzymatic transfer and subsequent “click” coupling of a fluorophore can be performed even in cell lysates. For RNA labeling, the activity of an archaeal RNA 2‘-O-MTase C/D ribonucleoprotein complex (RNP) with synthetic cofactors was investigated. It was shown that synthetically reprogrammed guide RNA sequences can be used to direct the C/D RNP-dependent transfer of a prop-2-ynyl group to predetermined nucleotides in substrate RNAs. Followed by AAC this can be used for programmable sequence-specific labeling of a variety of RNA substrates in vitro. These new possibilities for specific labeling of nucleic acids can be adopted in biochemistry, biomedical, nanotechnology, etc. research. / Tiriant DNR ir RNR, neretai svarbu prijungti įvairius reporterinius ar giminingumo žymenis griežtai apibrėžtose (sekos) vietose – t.y. specifiškai. Tam galima pasitelkti fermentus metiltransferazes (MTazes). Natūraliai jos naudoja kofaktorių AdoMet, tačiau gali būti aktyvios ir su sintetiniais jo analogais, turinčiais ilgesnes nei metil- pernešamas grandines. Jei šios grandinės turi galines funkcines grupes, prie jų vėliau cheminių reakcijų pagalba galima prijungti norimus žymenis. Tam itin patogi azidų-alkinų cikloprijungimo (AAC), dar vadinama „click“, reakcija. Šiame darbe parodyta, kad DNR citozino-5 MTazė HhaI (variantas Q82A/Y254S/N204A) efektyviai katalizuoja sekai specifinę heks-2-inil- grandinių, turinčių galines alkinil- arba azido- grupes, pernašą nuo sintetinių kofaktorių ant DNR. Naudojant šią MTazės-kofaktorių sistemą bei AAC, visą specifinio DNR žymėjimo procesą galima atlikti netgi ląstelių lizate. RNR žymėjimui ištirtas archėjų RNR 2‘-O-MTazės C/D ribonukleoproteininio komplekso aktyvumas su sintetiniais kofaktoriais. Parodyta galimybė sintetiškai keičiant kreipiančiąją RNR, prop-2-inilgrupės pernašą nukreipti į norimas įvairių substratinių RNR sekos vietas ir po to AAC reakcijos pagalba prijungti fluoroforą. Taigi, sukurtas naujas molekulinis įrankis, leidžiantis be suvaržymų pasirinkti norimą pažymėti RNR seką. Šios naujos specifinio nukleorūgščių žymėjimo galimybės gali būti pritaikytos biochemijos, biomedicinos, nanotechnologijų ir kitose tyrimų srityse... [toliau žr. visą tekstą]

Tiriant DNR ir RNR, neretai svarbu prijungti įvairius reporterinius ar giminingumo žymenis griežtai apibrėžtose (sekos) vietose – t.y. specifiškai. Tam galima pasitelkti fermentus metiltransferazes (MTazes). Natūraliai jos naudoja kofaktorių AdoMet, tačiau gali būti aktyvios ir su sintetiniais jo analogais, turinčiais ilgesnes nei metil- pernešamas grandines. Jei šios grandinės turi galines funkcines grupes, prie jų vėliau cheminių reakcijų pagalba galima prijungti norimus žymenis. Tam itin patogi azidų-alkinų cikloprijungimo (AAC), dar vadinama „click“, reakcija. Šiame darbe parodyta, kad DNR citozino-5 MTazė HhaI (variantas Q82A/Y254S/N204A) efektyviai katalizuoja sekai specifinę heks-2-inil- grandinių, turinčių galines alkinil- arba azido- grupes, pernašą nuo sintetinių kofaktorių ant DNR. Naudojant šią MTazės-kofaktorių sistemą bei AAC, visą specifinio DNR žymėjimo procesą galima atlikti netgi ląstelių lizate. RNR žymėjimui ištirtas archėjų RNR 2‘-O-MTazės C/D ribonukleoproteininio komplekso aktyvumas su sintetiniais kofaktoriais. Parodyta galimybė sintetiškai keičiant kreipiančiąją RNR, prop-2-inilgrupės pernašą nukreipti į norimas įvairių substratinių RNR sekos vietas ir po to AAC reakcijos pagalba prijungti fluoroforą. Taigi, sukurtas naujas molekulinis įrankis, leidžiantis be suvaržymų pasirinkti norimą pažymėti RNR seką. Šios naujos specifinio nukleorūgščių žymėjimo galimybės gali būti pritaikytos biochemijos, biomedicinos, nanotechnologijų ir kitose tyrimų srityse... [toliau žr. visą tekstą] / Investigation of RNA and DNA function often requires sequence-specific incorporation of various reporter and affinity probes. This can be achieved using AdoMet-dependent methyltransferases (MTases) as they can be active with synthetic AdoMet analogues equipped with transferable chains larger than the methyl group. These chains usually carry reactive groups that can be further chemically appended with required reporters. For this, azide-alkyne 1,3-cycloaddition (AAC), also called “click”, reaction is particularly attractive. This work shows that the HhaI cytosine-5 DNA MTase (variant Q82A/Y254S/N204A) catalyzes efficient sequence-specific transfer of hex-2-ynyl side chains containing terminal alkyne or azide groups from synthetic cofactor analogues to DNA. Both the enzymatic transfer and subsequent “click” coupling of a fluorophore can be performed even in cell lysates. For RNA labeling, the activity of an archaeal RNA 2‘-O-MTase C/D ribonucleoprotein complex (RNP) with synthetic cofactors was investigated. It was shown that synthetically reprogrammed guide RNA sequences can be used to direct the C/D RNP-dependent transfer of a prop-2-ynyl group to predetermined nucleotides in substrate RNAs. Followed by AAC this can be used for programmable sequence-specific labeling of a variety of RNA substrates in vitro. These new possibilities for specific labeling of nucleic acids can be adopted in biochemistry, biomedical, nanotechnology, etc. research.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Soybean [Glycine max (L.) Merr.] is an important crop worldwide. It has been widely consumed for protein, oil and other soy products. To develop soybean cultivars with greater resistance against pests and improved meal quality, it is important to elucidate the molecular bases of these traits. This dissertation aims to investigate the biochemical and biological functions of soybean genes from four gene families, which are hypothesized to be associated with soybean defense against pests and soybean meal quality. There are three specific objectives in this dissertation. The first one is to determine the function of components in the salicylic acid (SA) signaling pathway in soybean resistance against soybean cyst nematode (Heterodera glycines, SCN). The second one is to determine whether insect herbivory induce the emission of volatiles from soybean, and if so, how these volatiles are biosynthesized. The third objective is to identify and characterize soybean mannanase genes that can be used for the improvement of soybean meal quality. The soybean genome has been fully sequenced, which provides opportunities for cross-species comparison of gene families of interest and identification of candidate genes in soybean. The cloned cDNAs of putative genes were expressed in Escherichia coli to produce recombinant enzymes. Through biochemical assays, these proteins were proved to be soybean salicylic acid methyltransferase (GmSAMT1), methyl salicylate esterase (GmSABP2-1), α[alpha]-farnesene synthase (GmTPS1) and E-β[beta]-caryophyllene synthase (GmTPS2), and endo-β[beta]-mannanase (GmMAN1). Through a transgenic hairy root system harboring overexpression of GmSAMT1 and GmSABP2-1, both of these two genes were evaluated for their biological function related to resistance against SCN. The results showed that the over-expression of GmSAMT1 and GmSABP2-1 in the susceptible soybean background lead to enhanced resistance against SCN. Among four putative soybean mannanase genes, one gene was cloned and characterized. GmMAN1 showed the endo-β[beta]-mannanase hydrolyse activity and can hydrolyze cell walls isolated from soybean seeds. In summary, using comparative and functional genomics, a number of genes involved in soybean defense and meal quality were isolated and characterized. This study provides novel knowledge and molecular tools for the genetic improvement of soybean for enhanced resistance and improved meal quality.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Made available in DSpace on 2014-06-11T19:35:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-26Bitstream added on 2014-06-13T18:46:32Z : No. of bitstreams: 1 perecin_f_dr_jabo.pdf: 591625 bytes, checksum: f3212bddc59577b0aa73b9d7ef9f1d91 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A clonagem por transferência de núcleo é freqüentemente associada a resultados insatisfatórios devido à reprogramação nuclear anormal da célula somática doadora de núcleo e à expressão gênica alterada. O primeiro objetivo deste trabalho foi estudar a freqüência dos RNAs mensageiros das DNA metiltransferases (DNMT) 1, 3A e 3B, e do gene de expressão constitutiva gliceraldeído 3-fosfato desidrogenase (GAPDH) em blastocistos bovinos isolados produzidos in vivo e in vitro por transferência nuclear (TN) de célula somática, ativação partenogenética e fertilização in vitro (FIV). O segundo objetivo foi avaliar a expressão das DNMTs e dos genes imprinted IGF2, IGF2R e H19 em membranas cório-alantóide e fetos bovinos produzidos in vivo e in vitro por TN, ativação partenogenética e FIV e recuperados entre os dias 33 e 36 de gestação. Houve decréscimo (P<0,05) na freqüência do GAPDH nos blastocistos TN e partenogenéticos quando comparados aos embriões fertilizados, e também diferença entre blastocistos TN produzidos com diferentes protocolos de sincronização celular (células em G0 ou G1 do ciclo celular). Com relação às DNMTs, não foram identificados transcritos da DNMT1 nos blastocistos do grupo TN-G0; ocorreu diminuição na freqüência dos transcritos da DNMT3B nos embriões TN quando comparados aos partenotos. Não se observou diferença na freqüência relativa das DNA metiltransferases em membranas cório-alantóide e fetos. Com relação aos genes imprinted, o grupo partenogenético apresentou menor nível de expressão de IGF2 em relação aos os demais grupos; baixos níveis de expressão de IGF2 e IGF2R foram observados, respectivamente, em amostras de feto e de cório-alantóide derivadas de animais clonados por TN, quando comparadas aos grupos fertilizados in vivo e in vitro. / Cloning by nuclear transfer is often associated with poor results due to abnormal nuclear reprogramming of somatic cell donor and altered gene expression. The first objective of this study was to evaluate the frequency of DNA methyltranferases (DNMT) 1, 3A and 3B, and the housekeeping glyceraldehyde 3- phosphate dehydrogenase (GAPDH) mRNAs in single bovine blastocysts produced in vivo or in vitro by somatic cell nuclear transfer (SCNT), parthenogenetic activation and in vitro fertilization (IVF). The second objective was to evaluate the expression of DNMTs and imprinted genes IGF2, IGF2R and H19 in chorio-alantois membrane of bovine fetuses produced in vivo or in vitro by SCNT, parthenogenetic activation and IVF, and recovered between days 33 and 36 of gestation. There was strong GAPDH downregulation (P<0.05) in parthenogenetic and cloned by SCNT blastocysts when compared to fertilized ones, and also differences between cloned blastocysts produced with different cell synchronization (G0 or G1) protocol. Regarding DNMTs expression, we did not identify DNMT1 transcrips in SCNT-G0 derived blastocysts, and observed DNMT3B downregulation in SCNT-derived embryos when compared to parthenotes. No differences in DNA methyltransferase relative frequency were seen in chorio-alantois membrane and fetuses. Regarding imprinted genes expression, downregulation of IGF2 in the parthenogenetic group was observed in comparision to all other groups, and also, downregulation of IGF2 and IGF2R in the cloned-derived fetuses and chorio-alantois samples, respectively, were observed comparing to in vivo and in vitro fertilized groups.