Thesis (MSc)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: Sugarcane, a complex polyploid, may theoretically contain up to 12 alleles for a particular gene at a single locus. The number of alleles and the extent of their variation is of particular importance due to the potential for the exploitation of genetic variation through breeding. Also, allelic variation has implications for the manipulation of gene function via genetic engineering. Pyrophosphate dependent phosphofructokinase (PFP) is considered a key regulatory enzyme involved in sucrose biosynthesis, which may provide a target for genetic manipulation to increase sucrose yields in sugarcane. The enzyme is composed of a regulatory (alpha) and catalytic (beta) protein subunit encoded by the PFP-a and PFP-p genes respectively. The PFP-p gene, which has been shown to be a single locus gene in other plant species, was used in this study as a model for allelic variation in sugarcane. Two main areas of investigation involved genomic and expression analyses to further characterise the gene. Polymerase Chain Reaction (PCR) using specific primers previously designed from conserved regions of the PFP-p gene from different plant sources, were used to amplify across exons 10 to 12 of the sugarcane PFP-p gene. Two PCR products, designated PFP-81/881250bp and PFP-81/881100bp respectively, were obtained from commercial cultivars N19 and N21. Numerous clones of the fragments were obtained and sequenced. International database searches confirmed that both amplicons were identifiable as PFP-p. Comparative sequence analysis indicated that the PFP-81/881250bP and PFP-81/881100bp fragments were poorly homologous to each other, with higher regions of homology residing in the putative exon regions (77-78%) compared to the intron regions (34- 56%). Although minor sequence variation was detected within the amplicon populations, it was evident that two major variants of the PFP-p gene are present in sugarcane. Southern hybridisation analysis revealed a simple banding pattern for PFP-p. Also, there are DNA polymorph isms for the genomic regions corresponding to the PFP-81/881250bP and PFP-81/881100bPfragments. Previous evidence indicates that both variants are also present in the ancestral sugarcane germplasm and maintain the same level of heterozygosity. The presence of both gene forms in the ancestral and commercial germplasm prompts speculation that the two variants may not segregate. This theory, together with the simple Southern hybridisation pattern obtained for PFP-p, leads to the hypothesis that the two gene forms are at separate loci in the sugarcane genome, which may be closely linked on the same chromosome. The expression of the variants was investigated during different stages of sucrose accumulation in the sugarcane culm using a Reverse Transcription (RT)- peR approach. A single, identical transcription product was isolated from these and other selected tissues of the plant. In addition, the same transcript was obtained from the ancestral species representatives of modern sugarcane, Saccharum officinarum and Saccharum spontaneum. Sequence comparison of the transcribed product and the derived exon regions of the two variants implies that the PFP-p gene represented by the PFP-B 1/B81250bpvariant is being expressed in sugarcane while the gene form characterised by the PFP-B1/B81100bp amplicon is silent. Northern hybridisation analysis indicates that PFP-p is differentially expressed at different stages of sucrose accumulation. PFP-p expression is higher in the immature culm tissue of sugarcane and low in the mature culm, which suggests that PFP-p is highly regulated during maturation. It is hypothesised that the PFP-p gene underwent duplication and that one gene form was subject to accumulative mutations evolving into a pseudogene. On the basis of present results, it can be suggested that future genetic manipulation of PFP-p should involve the gene variant characterised by the PFP-B 1/B81250bp fragment. / AFRIKAANSE OPSOMMING: Suikerriet, 'n komplekse poliploïede organisme, kan teoreties tot 12 allele vir 'n spesifieke geen by 'n enkele lokus bevat. Die aantal allele en hul mate van variasie is veral van belang, weens die potensiaal vir die benutting van hierdie genetiese variasie deur teëling. Alleelvariasie het ook implikasies vir die manipulering van geenfunksie via genetiese inginieurswese. Pirofosfaat-afhanklike fosfofruktokinase (PFP) is 'n belangrike regulatoriese ensiem vir sukrose biosintese en kan geteiken word vir genetiese manipulasie met die oog op verhoogde sukroseproduksie in suikerriet. Die ensiem bestaan uit 'n regulatoriese (alfa) en katalitiese (beta) proteïen subeenheid wat deur die PFP-a en PFP-~ gene respektiewelik gekodeer word. Die PFP-~ geen, wat in ander plantspesies as 'n enkellokusgeen aangedui is, is in hierdie studie gebruik as 'n model vir alleelvariasie in suikerriet. Die twee hoofroetes van ondersoek wat gevolg is, behels genoom- en uitdrukkingsanalises om die geen verder te karakteriseer. Eksons 10 tot 12 van die suikerriet PFP-~ geen is geamplifiseer met behulp van die Polimerase Ketting Reaksie (PKR), bemiddel deur die gebruik van spesifieke voorvoerders wat voorheen ontwerp was vanaf gekonserveerde areas van die PFP-~ geen uit verskillende plantbronne. Twee PKR produkte, genoem PFP-B1/B81250bPen PFP-B1/B81100bPrespektiewelik, is deur die kommersiële kultivars N19 en N21 opgelewer. Verskeie fragmentklone is gekonstrueer en hul DNA basisvolgorde is bepaal. Soektogte in internasionale databasisse het bevestig dat beide amplikons PFP-~ was. Vergelykende DNA basisvolgorde analise het aangedui dat PFP-B1/B81250bP en PFP-B1/B81100bP fragmente swak homologie toon, terwyl 'n hoër mate van homologie in die oënskynlike ekson areas (77-78%), vergeleke met die intronareas (34-56%) gevind is. Alhoewel klein basisvolgordeverskille opgemerk is binne die amplikonpopulasies, was dit duidelik dat twee hoofvariante van die PFP-~ geen in suikerriet teenwoordig is. Southern hibridisasie analisie het 'n eenvoudige band patroon vir PFP-~ onthul. Daar is ook DNA polimorfismes vir die genoomstreke wat met die PFP-B1/B81250bPen PFP-B1/B81100bPfragmente ooreenstem. Vorige bewyse het aangetoon dat beide variante ook in die voorvaderlike suikerriet kiemplasma voorkom en dat dieselfde vlak van heterosigositeit gehandhaaf word. Die voorkoms van beide geenvorme in die voorvaderlike asook kommersiële kiemplasmas stel voor dat hierdie twee variante miskien nie segregeer nie. Hierdie teorie, tesame met die eenvoudige Southern hibridisasiepatroon vir PFP-p, lei tot die hipotese dat die twee geen vorme by verskillende lokusse in die suikerrietgenoom voorkom, en dat hierdie lokusse nou gekoppel mag wees op dieselfde chromosoom. Die uitdrukking van die variante is ondersoek gedurende verskillende stadia van sukrose akkumulasie in die suikerrietstingel deur van Trutranskripsie (TT)-PKR gebruik te maak. 'n Enkele, identiese transkripsie produk is hieruit en uit ander geselekteerde plantweefsels geïsoleer. Dieselfde transkrip is ook verkry vanaf die voorouerlike spesieverteenwoordigers van hedendaagse suikerriet, Saccharum officinarum en Saccharum spontaneum. 'n DNA basisvolgordevergelyking tussen die getranskribeerde produk en die ekson-areas van die twee variante impliseer dat die PFP-p geen verteenwoordig deur die PFP-B1/B81250bPvariant uitgedruk word in suikerriet, terwyl die geen verteenwoordig deur die PFP-B1/B81100bpamplikon stil is. Northern hibridisasie analise toon aan dat PFP-p verskillend uitgedruk word gedurende verskillende stadia van sukrose akkumulasie. PFP-p uitdrukking is hoër in die onvolwasse stamweefsel van suikerriet en laag in die volwasse stam, wat voorstel dat PFP-p hoogs gereguleer word gedurende maturasie. Daar word gehipotetiseer dat die PFP-p geen duplikasie ondergaan het en dat een geenvorm onderworpe was aan akkumulerende mutasies wat deur evolusie tot 'n pseudogeen gelei het. Dit word voorgestel, gebaseer op die huidige resultate, dat toekomstige genetiese manipulasie van die PFP-p geen, die geenvariant gekarakteriseer deur die PFP- B1/B81250bPfragment moet behels.

This thesis examines the interconnection between the in vitro and in vivo phosphorylation of rabbit muscle phosphofructokinase. The first goal of the project was to show whether a novel site of rabbit muscle phosphofructokinase that is subject to in vitro phosphorylation, serine 376, may also become phosphorylated in vivo. Evidence obtained through iron chelate chromatography, amino acid analysis, gas phase sequencing, ammonium sulfate reversed phase high pressure liquid chromatography (HPLC), and matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) spectroscopy of cyanogen bromide digests of the enzyme purified from epinephrine-stimulated rabbit hearts demonstrate the in vivo phosphorylation of serine 376. Parallel experiments with phosphofructokinase isolated from unstimulated rabbit hearts show no detectable phosphorylation of serine 376. The second part of the thesis examines the effects of alterations in experimental conditions on the in vitro phosphorylation of rabbit muscle phosphofructokinase that is catalyzed by the cAMP-dependent protein kinase (cAMP-dPK). Significant phosphorylation of serine 376 takes place in the presence of specific proteins, calmodulin-calcium and troponin C-calcium, that are known to stabilize the catalytically inactive phosphofructokinase dimer. Conditions that stabilize the catalytic activity of phosphofructokinase generally inhibit the in vitro phosphorylation reaction. / Graduation date: 1999

The preimplantation mouse embryo undergoes many metabolic changes as development proceeds. One major change is the switch from a pyruvate based metabolism, to a glucose based metabolism. The phosphofructokinase enzyme is the regulatory enzyme of glycolysis and is thought to be a major contributor in controlling the block to glycolysis in early preimplantation mouse embryos. This study was undertaken to construct a system that would allow detection of RNA for the highly glycolytically active subunit (muscletype) of the phosphofructokinase (PFK) enzyme. A muscle specific mutant PFK plasmid was generated to provide mutant internal control RNA. Using this internal control, initial reverse transcriptionpolymerase chain reaction data collected from early embryo stages suggest that the muscle type PFK subunit RNA is not expressed in the preimplantation mouse at the 1-cell or blastocyst stages. This result suggests that PFK activity detected at the later morula and blastocyst stages must be from either a different PFK subunit or a novel embryonic form of PFK. / Department of Biology

The regulatory enzyme 6-phosphofructo-l-kinase (PFK) controls the key, rate-limiting step in glycolysis. There are 3 known mammalian isoforms termed PFK-muscle (PFK-A), PFK-liver (PFK-B), and PFK-brain (PFK-C) that randomly aggregate to form active homo- and heterotetrameric isozymes with their respective frequencies and kinetic properties contingent upon the presence and concentration of individual subunits. This study utilized RT-PCR and densitometry analyses to characterize the expression patterns of the mRNA for each isoform during mouse preimplantation development. PFK-B is increasingly expressed across these stages with a significant increase in PFK-B transcript between 8-cell (0.425 ± 0.158) and morula (0.579 ± 0.197) stages (p < 0.0005). Neither PFK-A nor PFK-C mRNA was detected at any of the preimplantation stages tested. The statistically significant increase in PFK-B corresponded with the known juncture of the switch from the oxidation of maternally supplied pyruvate to a predominant glycolyticmetabolism. Such timing suggested the direct involvement of elevated PFK-B transcription with an increase in glycolysis. / Department of Biology

The studies described in this dissertation examine the effects of F-2,6-P2 and AMP or phosphorylation on the kinetic mechanism of d-PFK. The effect of varied pH on the activation by F-2,6-P2 is also described.

Thesis advisor: Eranthie Weerapana / The 1,3,5-triazine is a privileged scaffold in that it is planar and has three-fold symmetry which allows for controlled modification around the ring structure with various substituents. In this thesis, we report on two modular inhibitor libraries that center around a 1,3,5-triazine core scaffolding system, which have been shown to target protein disulfide isomerase A1 (PDIA1), glutaredoxin-3 (GLRX3), and 6-phosphofructo-1-kinase (PFKP). Protein disulfide isomerase A1 (PDIA1) is a thiol-disulfide oxidoreductase localized in the lumen of the endoplasmic reticulum (ER), and is an important folding catalyst and chaperone for proteins in the secretory pathway. PDIA1 contains two active-site domains (a and a’), each containing a Cys-Gly-His-Cys (CGHC) active-site motif. Here, we synthesize a targeted library o second-generation triazine-based inhibitors to optimize the potency and selectivity of our lead compound, RB-11-ca. Characterization of this targeted library afforded an optimized PDIA1 inhibitor, KSC-34, which covalently modifies C53 in the a site of PDIA1 and demonstrates time-dependent inhibition of the reductase activity of PDIA1 in vitro with a kinact/KI = 9.66 x 103 M-1s-1. Interestingly, KSC-34 treatment demonstrated that a-site inhibition led to decreased secretion of amyloidogenic antibody light chain, thus illustrating that site-selective inhibitors like KSC-34 provide useful tools for delineating the pathological role and therapeutic potential of PDIA1. In 2014, our lab first reported on RB7, a dichlorotriazine-based electrophilic small molecule which displayed extremely high reactivity and selectivity toward lysine residues in the proteome. Herein, we further on this study by investigating the unique reactivity of RB7 through the synthesis of a second-generation small molecule electrophile library and investigating proteome-wide reactivity in vitro and in situ. This library afforded KSC-46, an RB-7 analogue with p-chlorothiophenol tuning element, which provided optimal proteome reactivity to use as a scaffold for the generation of a targeted library. To take advantage of the tuned reactivity of KSC-46, a second-generation targeted library was generated to target react residues in the proteome. This library yielded two molecules, KSC-56 and KSC-65, which were identified to target glutaredoxin-3 (GLRX3) and 6-phosphofructo-1-kinase (PFKP), respectively. GLRX3 is a cytosolic, monothiol iron-sulfur cluster chaperon protein which relies on two nucleophilic cysteine residues to bind and transfer iron clusters. PFKP is known to catalyze the first irreversible step in glycolysis and regulates the flux of glucose metabolism in the cell, which makes PFKP an attract therapeutic target. KSC-56 was further characterized to bind to Cys261 in the C-terminal glutaredoxin domain of GLRX3. / Thesis (PhD) — Boston College, 2018. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Thesis (Ph. D.)--University of Missouri--Columbia, 2004. / Typescript. Vita. Includes bibliographical references (leaves 166-179). Also issued on the Internet.