Thesis (MSc) Stellenbosch University, 2014 / ENGLISH ABSTRACT: In this study, the effect of the amino acid residues at positions 40 and 407 on the catalytic activity of porcine CYP17A1 was investigated. Porcine cofactor CYB5 was cloned from porcine liver tissue and its effect on the catalytic activity of porcine CYP17A1 was determined. The influence of rat, human and angora CYB5 on the lyase activity of porcine CYP17A1 was subsequently determined and compared to the influence of porcine CYB5. Wt porcine CYP17A1, which has residues Val40 and His407, catalysed the conversion of prog efficiently with ~50% prog converted to 17OHprog (~40%) and A4 (~10%) after 3 hr. After 24 hr, negligible levels prog remained with ~71% 17OHprog and ~25% A4 being produced. Low levels of 16OHprog were formed (~9%). The Leu105Ala mutation reduced wt 17α-hydroxylase activity, with 70% prog remaining after 24 hr while 16OHprog (~10%) levels remained unchanged. Porcine CYP17A1 with residues Leu40 and His407, exhibited similar catalytic activity towards prog as did wt porcine CYP17A1 (Val40 and His407 residues), while porcine CYP17A1 with residues Leu40 and Leu407 increased the formation of A4 2-fold to 54% at 24 hr and porcine CYP17A1 with residues Val40 and Leu407 resulted in the highest formation of A4 (90%). Wt porcine CYP17A1, while having converted 95% of the prog substrate, produces only ~16% A4 after 24 hr. In the presence of porcine CYB5, however, the lyase activity was stimulated with 85% of prog being converted to A4 and only 13% 17OHprog remaining. The lyase activity was also stimulated by CYB5 from other species, resulting in an increase in A4 production of 60.6%, 24% and 11.6% by rat, angora and human CYB5, respectively. The degree of lyase stimulation correlated to the percentage identity of the CYB5 amino acid sequences to porcine CYB5. While the Val and Leu residues at position 40 do not appear to influence the lyase activity of porcine CYP17A1 as prominently as the residue at position 407, it is the charged residue at 407 that plays a significant role in the production of A4, decreasing A4 production irrespective of the Val and the Leu residues at position 40. It would, furthermore, appear that the stimulation of lyase activity of CYP17A1 is the greatest when assaying this activity in the presence of CYB5 of the same species as was detected when co-expressing porcine CYP17A1 and porcine CYB5. / AFRIKAANSE OPSOMMING: In hierdie studie is die invloed van die aminosuurresidue by posisies 40 en 407 op die katalitiese aktiwiteit van vark CYP17A1 ondersoek. Vark CYB5 is geklooneer vanuit vark lewer weefsel en die effek van hierdie kofaktor op die katalitiese aktiwiteit van vark CYP17A1 is bepaal. Die invloed van rot, mens en angora CYB5 op die liase aktiwiteit van vark CYP17A1 is daarna bepaal en vergelyk met die invloed van vark CYB5. Vark CYP17A1-VH, (kodeer Val40 en His407), kataliseer die omskakeling van prog doeltreffend met ~50 % prog wat omgeskakel word na 17OHprog (~40%) en A4 (~10%) na 3 uur. Na 24 uur, is feitlik alle prog omgeskakel, met ~71% 17OHprog en ~25% A4 geproduseer. Lae vlakke 16OHprog is ook gevorm (~9%). Die Leu105Ala mutasie verminder 17α- hidroksilase aktiwiteit, met 70% prog wat na 24 uur nie omgesit is nie, terwyl 16OHprog (~10%) vlakke onveranderd gebly het. Vark CYP17A1-LH (kodeer Leu40 en His407), en CYP17A1-VH het diselfde katalitiese aktiwiteit teenoor prog getoon, terwyl vark CYP17A1-LL (kodeer Leu40 en Leu407) die vorming van A4 2-voudig verhoog het tot 54% na 24 uur. Vark CYP17A1-VL (kodeer Val40 en Leu407) se katalitiese aktiwiteit het gelei tot die hoogste vorming van A4 (90%). Alhoewel CYP17A1-VH, 95% van die prog substraat omgeskakel het is slegs ~16% A4 geproduseer na 24 uur. In die teenwoordigheid van vark CYB5 is die liase aktiwiteit egter gestimuleer, en is 85% van die prog substraat omgeskakel na A4 met slegs 13% 17OHprog teenwoordig na 24 uur. Die liase aktiwiteit is ook gestimuleer deur CYB5 van ander spesies, wat lei tot 'n toename in A4 produksie van 60,6% , 24% en 11,6% deur rot, angora en menslike CYB5, onderskeidelik. Daar is gevind dat daar’n sterk korrelasie is tussen die stimulering van die liase aktiwitieit en die persentasie aminosuur volgorde identiteit van CYB5 afkomstig vanaf die verskillende spesies. Terwyl die Val en die Leu aminosuurresidu op posisie 40 wel die liase aktiwitiet tot ‘n mate beȉnvloed, blyk dit uit die data dat die potitief gelaaide residue by 407 'n belangrike rol speel in die produksie van A4, en A4 produksie verlaag ongeag van die Val en die Leu residu by posisie 40. Dit wil ook verdermeer voorkom asof die stimulering van die liase aktiwiteit van CYP17A1 die hoogste is wanneer die ensiem gekataliseerde reaksie deurgevoer word in die teenwoordigheid van CYB5 en CYP17A1 afkomstig vanaf dieselfde spesies.

La protéine murine AKR1B7 appartient à la famille des aldoses réductases. Par son expression limitée à un petit nombre de tissus et son contrôle hormonal, elle constitue un modèle de choix pour l'étude des fonctions physiologiques de cette famille enzymatique. D'une part, ces travaux de thèse ont permis de démontrer ex vivo le rôle de l'activité prostaglandine F synthase des aldoses réductases murines et humaines dans la glande surrénale. Nous avons mis en évidence une nouvelle boucle de régulation négative des fonctions endocrines surrénaliennes qui utilise la prostaglandine F2alpha comme signal paracrine et/ou autocrine entre le cortex et la medulla. D'autre part, nous avons développé la première lignée de souris transgéniques exprimant spécifiquement la recombinase Cre dans la cortico-surrénale pour réaliser l'invalidation conditionnelle de gènes dans ce tissu. Cette lignée constitue un outil très puissant pour la création de modèles murins de pathologies tumorales surrénaliennes

Thesis (MSc. (Biochemistry)) -- University of Limpopo, 2013 / Hydroxynitrile lyases (HNLs) are enzymes that catalyse enantioselective cleavage of the substrate in a reaction and are also used as important industrial biocatalysts for the synthesis of chiral cyanohydrins. The aim of the study was to screen indigenous South African plants for potential hydroxynitrile lyase activity, purify and biochemically characterise the active hydroxynitrile lyase(s) from the selected plants. Several indigenous plants were randomly collected, identified and screened for HNL activity. The plant parts (leaves, seeds or fruits) were processed using established experimental protocols in order to obtain the crude enzyme extracts. The enzymatic conversion of benzaldehyde and potassium cyanide to mandelonitrile was optimised and consequently used for the screening of HNL activity. Enzyme activity was detected in the crude enzyme extracts of Kalanchoe spp and Senecio spp and these were then designated as Ks and Sb, respectively. Ammonium sulphate fractionation, DEAE Toyopearl 650M and Concanavalin A chromatography techniques were then used in the purification process of the active crude enzyme extracts. Subsequently, two purified active fractions were isolated from each plant species with molecular masses estimated at 64.64 kDa and 64.06 kDa for the KsHNL enzymes and 70.60 kDa and 74.04 kDa for SbHNL enzymes. The optimum temperature and pH of all the isolated enzymes were determined as 50°C and pH 5, respectively. The experimental Km and Vmax values of the enzymes were respectively determined to be 0.33 and 0.73 mM and 1.238 and 1.948 μM/min for KsHNL; while that for SbHNL enzymes were 5.86 and 0.22 mM and 9.741 and 1.905 μM/min. The effect of additives and metal ions (viz., DTT, DEP, mercury chloride, magnesium chloride and zinc chloride) was determined. The experimental data obtained alluded to the notion that both KsHNL and SbHNL enzymes may contain the cysteine and serine residues next to their active sites and that a histidine residue may be involved in the catalytic activities of both the isolated KsHNL enzymes and one of the SbHNL enzymes. All the isolated enzymes from the two plant species did not seem to contain an FAD group. These findings compared favourably to the theoretical type II HNLs, although with a slight difference in that they displayed high molecular weights. Kalanchoe spp and Senecio spp are the two indigenous South African plants that were found to contain active HNLs. The isolated HNLs from the two plants have a potential to be xv purified to homogeneity, cloned and overexpressed into robust recombinant enzymes that can be used for large scale industrial applications.

Diss. med. dent. Zürich. / Literaturverz.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Partial embargo. Vita. Bibliography: 128-131.

This study set out to express, purify and characterize twelve ammonia lyase enzymes for potential application as a supplement to a treatment of an inborn error of metabolism disease. The DNA sequence for two wild-type ammonia lyases, three modified ammonia lyases and seven resurrected ancestral ammonia lyases had been synthesized and cloned in vectors. These were transformed into Escherichia coli, expressed, purified using immobilized metal affinity chromatography and size exclusion chromatography and characterized. Ten of the enzymes were successfully expressed and purified. All enzymes had a higher turnover number with substrate 1 than with substrate 2. The wild-types showed the highest catalytic turnover and one of them displayed substrate cooperativity. The modified enzymes were inactive. Some ancestral enzymes were active and had decreasing kcat with age. A promising ancestral enzymes was found that showed a kcat of 2,85 s-1 with substrate 1 and 1,82 s-1 with substrate 2. The ancestral enzymes had a lower Km with substrate 2 compared to substrate 1, while one of the wild-types had a higher Km with substrate 2 than with substrate 1, indicating that the substrate affinity has switched. The ancestral enzymes had increased thermostability compared to the wild-types which increased with age. Ranging from a +7C increase in melting temperature with the youngest ancestral enzyme to +10,7C with the oldest tested enzyme, comparing with one of the wild-types. The promising ancestral enzyme displayed a higher stability than the wild-types during long term incubation in 37_C and 25_C, since it did not become prone to aggregation,it did not show visible degradation on SDS-PAGE and it retained the highest activity following incubation. It was also demonstrated that neither wild-types nor the promising ancestral enzyme were stable in a simulated gut environment. The promising ancestral enzyme and one of the wild-types degraded substrate 1 and 2 in serum. Using the resurrection of ancestral sequences a promising enzyme has been produced and characterized, displaying properties that are desired in therapeutic enzymes. The enzyme did not aggregate or become prone to aggregation over time, it was thermostable, it was active in serum and had acceptable catalytic properties. For therapeutic application of the ancestral enzyme, immunogenicty should be analyzed in silico and in vitro followed by further investigation in vivo. / Målet med denna studie var att uttrycka, rena och karaktärisera tolv ammonia lyase enzymer, för potentiell användning som komplement till en behandling utav en sjukdom, som tillhör sjukdomsgruppen medfödda ämnesomsättningsrubbningar. DNA sekvensen för två vild-typammonia lyaser, tre modifierade ammonia lyaser och sju återuppväckta ammonia lyaser hade blivit syntetiserade och klonade i vektorer. E.coli celler blev transformerade med vektorerna, vilka uttryckte enzymerna, som renades med hjälp av immobilized metal affinity chromatography och gelfiltrering och karaktäriserades. Tio utav enzymerna kunde uttryckas och renas. Alla enzymer hade högre katalytisk omsättning av substrat 1 än substrat 2. Vildtyperna hade högst kcat med båda substrat och en utav dem uppvisade substratsammarbete. De modifierade enzymerna var inaktiva. Några av de återuppväckta ammonia lyaserna var aktiva och kcat minskade med ålder. Ett av de återuppväckta enzymerna var lovande och hade ett kcat värde av 2,85 s-1 med substrat 1 och 1,82 s-1 med substrat 2. De återuppväckta enzymerna hade ett lägre Km värde för substrat 2 än substrat 1, jämfört med en utav vildtyperna som hade ett högre Km värde för substrat 2 än substrat 1, vilket indikerar ett skifte i substrataffinitet. De återuppväckta enzymerna var mer termostabilia än vild-typerna och termostabiliteten ökar med ålder. Ökningen i smälttemperatur låg i spannet av +7C för de yngsta återuppväckta enzymerna till + 10,7C för det äldsta testade återuppväckta enzymet, vid jämförelse med en utav vild-typerna. Det lovande återuppväckta enzymet demonstrerade även en högre stabilitet än vild-typerna under långtidsinkubering, eftersom den inte blev benägen att aggregera, den uppvisade ingen nedbrytning på SDS-PAGE och den behöll högst aktivitet efter inkubering. Det bevisades även att varken vild-typerna eller det lovande återuppväckta enzymet var stabila i en simulerad magsäcksmiljö. Både det lovande återuppväckta enzymet och en av vild-typerna bröt ner substrat 1 och 2 i serum. Genom att återuppväcka sekvenser kunde ett lovande enzym produceras och karaktäriseras, vilket uppvisade egenskaper som är eftertraktade i terapeutiska enzymer. Enzymet aggregerade ej, det blev inte benäget att aggregera över tid, det var termostabilt, det var aktivt i serum och hade acceptabla katalytiska egenskaper. För terapeutisk applikation av det återuppväckta enzymet, borde analys av dess immunogenicitet utföras in silico och in vitro följt av vidare undersökning in vivo.

Dehydroquinate synthase (DHQS) catalyses the five-step transformation of the seven carbon sugar 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) to the carbacycle dehydroquinate (DHQ). Multiple studies have described in detail the mechanism of most of the steps carried out by DHQS with the exception of the final cyclisation step. In this study, (3S)-3-fluoro-DAH7P and (3R)-3-fluoro-DAH7P (fluorinated analogues of DAH7P) were produced and assayed across three phylogenetically distinct sources of DHQS in order to determine the role of the enzyme during the cyclisation step of the reaction. Incubation of (3S)-3-fluoro-DAH7P with DHQS from Escherichia coli, Pyrococcus furiosus, and Kiwifruit resulted in the production of different ratios of (6S)-6-fluoro-DHQ and 1-epi-(6S)-6-fluoro-DHQ for each enzyme. In addition, enzyme catalysis showed a slowing of reaction rates when (3S)-3-fluoro-DAH7P was used, suggesting that the fluorine at C-3 is stabilising the enol pyranose. An increase in the stabilisation of the fluoro-enol pyranose would allow release of this substrate intermediate from the enzyme to compete with the on-going on-enzyme reaction. The differences in the ratio of products formed suggest that the cyclisation occurs in part on the enzyme and that the epimeric product arises only by an abortive reaction pathway where the (3S)-3-fluoro-enol pyranose is prematurely released and allowed to cyclise free in solution. Once in solution, the (3S)-3-fluoro-enol pyranose could undergo a conformational change in the ring leading to the formation of the epimeric product. Furthermore, it is suspected that the position of fluorine influences the likely transition-state in carbacycle formation leading to the production of the epimeric product. This research has illuminated the role of the enzyme in guiding the correct stereochemistry of the product and illustrates the important molecular interplay between the enzyme and substrate.

No / Methionine dependence is unique to cancer cells and defined as the inability to grow in a methionine-deprived environment even if supplemented with the metabolic precursor homocysteine. Cobalamin-dependent methionine synthase (MS) catalyses the formation of methionine and tetrahydrofolate from homocysteine and methyltetrahydrofolate, thus linking the methionine and folate pathways. The apparent altered methionine metabolism in methionine-dependent cancer cells suggests a role for MS, although results to date are conflicting. We have analysed key metabolites of the MS-associated transmethylation, transsulphuration and folate pathways of the methionine-dependent MAC15A tumour model as a function of tumour progression over a 10-day period. MS activity increased 2-fold from day I to day 10. Cysteine, homocysteine, S-adenosylmethionine and S-adenosylhomocysteine levels in tumour cytosolic fractions decreased as a function of tumour progression. Plasma cysteine levels also decreased, whilst the distribution of folates in erythrocytes was altered, with a maximum increase in methyltetrahydrofolate observed by day 5. The increasing MS activity and decreasing cysteine levels suggest an increasing methionine requirement by the tumour, whilst the induction of enzyme activity indicates that MS is not defective in the methionine-dependent MAC15A tumour. The decrease in tumour S-adenosylmethionine and S-adenosylhomocysteine levels suggests that methionine is required for some function other than cellular methylation, e.g., incorporation into protein. Overall, the results support a theory of methionine conservation in response to tumour growth, where the methionine-dependent MAC15A tumour has a higher than normal methionine requirement.

L’une des particularités fondamentales caractérisant les cellules végétales des cellules animales est la présence de la paroi cellulaire entourant le protoplaste. La paroi cellulaire joue un rôle primordial dans (1) la protection du protoplaste, (2) est impliquée dans les mécanismes de filtration et (3) est le lieu de maintes réactions biochimiques nécessaires à la régulation du métabolisme et des propriétés mécaniques de la cellule. Les propriétés locales d’élasticité, d’extensibilité, de plasticité et de dureté des composants pariétaux déterminent la géométrie et la forme des cellules lors des processus de différentiation et de morphogenèse. Le but de ma thèse est de comprendre les rôles que jouent les différents composants pariétaux dans le modelage de la géométrie et le contrôle de la croissance des cellules végétales. Pour atteindre cet objectif, le modèle cellulaire sur lequel je me suis basé est le tube pollinique ou gamétophyte mâle. Le tube pollinique est une protubérance cellulaire qui se forme à partir du grain de pollen à la suite de son contact avec le stigmate. Sa fonction est la livraison des cellules spermatiques à l’ovaire pour effectuer la double fécondation. Le tube pollinique est une cellule à croissance apicale, caractérisée par la simple composition de sa paroi et par sa vitesse de croissance qui est la plus rapide du règne végétal. Ces propriétés uniques font du tube pollinique le modèle idéal pour l’étude des effets à courts termes du stress sur la croissance et le métabolisme cellulaire ainsi que sur les propriétés mécaniques de la paroi. La paroi du tube pollinique est composée de trois composantes polysaccharidiques : pectines, cellulose et callose et d’une multitude de protéines. Pour comprendre les effets que jouent ces différents composants dans la régulation de la croissance du tube pollinique, j’ai étudié les effets de mutations, de traitements enzymatiques, de l’hyper-gravité et de la gravité omni-directionnelle sur la paroi du tube pollinique. En utilisant des méthodes de modélisation mathématiques combinées à de la biologie moléculaire et de la microscopie à fluorescence et électronique à haute résolution, j’ai montré que (1) la régulation de la chimie des pectines est primordiale pour le contrôle du taux de croissance et de la forme du tube et que (2) la cellulose détermine le diamètre du tube pollinique en partie sub-apicale. De plus, j’ai examiné le rôle d’un groupe d’enzymes digestives de pectines exprimées durant le développement du tube pollinique : les pectate lyases. J’ai montré que ces enzymes sont requises lors de l’initiation de la germination du pollen. J’ai notamment directement prouvé que les pectate lyases sont sécrétées par le tube pollinique dans le but de faciliter sa pénétration au travers du style. / One of the most important features characterizing plant cells and differentiating them from animal cells is the cell wall that surrounds them. The cell wall plays a critical role in providing protection to the protoplast; it acts as a filtering mechanism and is the location of many biochemical reactions implicated in the regulation of the cell metabolism and the mechanical properties of the cell. The local stiffness, extensibility, plasticity and elasticity of the different cell wall components determine the shape and geometry of the cell during differentiation and morphogenesis. The goal of my thesis is to understand the role played by the different cell wall components in shaping the plant cell and controlling its growth behaviour. To achieve this goal, I studied the pollen tube, or male gametophyte, as a cellular model system. The pollen tube is a cellular protuberance formed by the pollen grain upon its contact with the stigma. Its main purpose is to deliver the sperm cells to the female gametophyte to ensure double fertilization. The pollen tube is a tip-growing cell characterized by its simple cell wall composition and by the fact that it is the fastest growing cell of the plant kingdom. This makes it the ideal model to study the effects of drugs, mutations or stresses on cellular growth behaviour, metabolism and cell wall mechanics. The pollen tube cell wall consists mainly of proteins and three major polysaccharidic components: pectins, cellulose and callose. To understand the role played by these components in regulating pollen tube growth, I investigated the effects of mutations, enzymatic treatments, hyper-gravity and omni-directional gravity on the pollen tube cell wall. Using mathematical modeling combined with molecular biology and high-resolution electron and fluorescent microscopy I was able to show that the regulation of pectin chemistry is required for the regulation of the growth rate and pollen tube shape and that cellulose is crucial for determining the pollen tube diameter in the sup-apical region. Moreover, I investigated the role of the pectate lyases, a group of pectin digesting enzymes expressed during pollen tube development, and I showed that this enzyme activity is required for the initiation of pollen germination. More importantly, I directly showed for the first time that the pollen tube secretes cell wall loosening enzymes to facilitate its penetration through the style.

L’une des particularités fondamentales caractérisant les cellules végétales des cellules animales est la présence de la paroi cellulaire entourant le protoplaste. La paroi cellulaire joue un rôle primordial dans (1) la protection du protoplaste, (2) est impliquée dans les mécanismes de filtration et (3) est le lieu de maintes réactions biochimiques nécessaires à la régulation du métabolisme et des propriétés mécaniques de la cellule. Les propriétés locales d’élasticité, d’extensibilité, de plasticité et de dureté des composants pariétaux déterminent la géométrie et la forme des cellules lors des processus de différentiation et de morphogenèse. Le but de ma thèse est de comprendre les rôles que jouent les différents composants pariétaux dans le modelage de la géométrie et le contrôle de la croissance des cellules végétales. Pour atteindre cet objectif, le modèle cellulaire sur lequel je me suis basé est le tube pollinique ou gamétophyte mâle. Le tube pollinique est une protubérance cellulaire qui se forme à partir du grain de pollen à la suite de son contact avec le stigmate. Sa fonction est la livraison des cellules spermatiques à l’ovaire pour effectuer la double fécondation. Le tube pollinique est une cellule à croissance apicale, caractérisée par la simple composition de sa paroi et par sa vitesse de croissance qui est la plus rapide du règne végétal. Ces propriétés uniques font du tube pollinique le modèle idéal pour l’étude des effets à courts termes du stress sur la croissance et le métabolisme cellulaire ainsi que sur les propriétés mécaniques de la paroi. La paroi du tube pollinique est composée de trois composantes polysaccharidiques : pectines, cellulose et callose et d’une multitude de protéines. Pour comprendre les effets que jouent ces différents composants dans la régulation de la croissance du tube pollinique, j’ai étudié les effets de mutations, de traitements enzymatiques, de l’hyper-gravité et de la gravité omni-directionnelle sur la paroi du tube pollinique. En utilisant des méthodes de modélisation mathématiques combinées à de la biologie moléculaire et de la microscopie à fluorescence et électronique à haute résolution, j’ai montré que (1) la régulation de la chimie des pectines est primordiale pour le contrôle du taux de croissance et de la forme du tube et que (2) la cellulose détermine le diamètre du tube pollinique en partie sub-apicale. De plus, j’ai examiné le rôle d’un groupe d’enzymes digestives de pectines exprimées durant le développement du tube pollinique : les pectate lyases. J’ai montré que ces enzymes sont requises lors de l’initiation de la germination du pollen. J’ai notamment directement prouvé que les pectate lyases sont sécrétées par le tube pollinique dans le but de faciliter sa pénétration au travers du style. / One of the most important features characterizing plant cells and differentiating them from animal cells is the cell wall that surrounds them. The cell wall plays a critical role in providing protection to the protoplast; it acts as a filtering mechanism and is the location of many biochemical reactions implicated in the regulation of the cell metabolism and the mechanical properties of the cell. The local stiffness, extensibility, plasticity and elasticity of the different cell wall components determine the shape and geometry of the cell during differentiation and morphogenesis. The goal of my thesis is to understand the role played by the different cell wall components in shaping the plant cell and controlling its growth behaviour. To achieve this goal, I studied the pollen tube, or male gametophyte, as a cellular model system. The pollen tube is a cellular protuberance formed by the pollen grain upon its contact with the stigma. Its main purpose is to deliver the sperm cells to the female gametophyte to ensure double fertilization. The pollen tube is a tip-growing cell characterized by its simple cell wall composition and by the fact that it is the fastest growing cell of the plant kingdom. This makes it the ideal model to study the effects of drugs, mutations or stresses on cellular growth behaviour, metabolism and cell wall mechanics. The pollen tube cell wall consists mainly of proteins and three major polysaccharidic components: pectins, cellulose and callose. To understand the role played by these components in regulating pollen tube growth, I investigated the effects of mutations, enzymatic treatments, hyper-gravity and omni-directional gravity on the pollen tube cell wall. Using mathematical modeling combined with molecular biology and high-resolution electron and fluorescent microscopy I was able to show that the regulation of pectin chemistry is required for the regulation of the growth rate and pollen tube shape and that cellulose is crucial for determining the pollen tube diameter in the sup-apical region. Moreover, I investigated the role of the pectate lyases, a group of pectin digesting enzymes expressed during pollen tube development, and I showed that this enzyme activity is required for the initiation of pollen germination. More importantly, I directly showed for the first time that the pollen tube secretes cell wall loosening enzymes to facilitate its penetration through the style.