Dissertations / Theses on the topic 'Plant Biochemistry'

This thesis reports the research I conducted on aspects of the pharmacology and biological activities of two natural stilbenes, Resveratrol (Rv) and Pterostilbene (Pt), major polyphenolic components of grapevines and blueberries respectively. Over the years, these two molecules have drawn attention from the scientific community thanks to their beneficial bioactivities, relevant for many areas of health care. Numerous papers describe their protective roles against the metabolic syndrome, cancer development and neurodegeneration. These striking effects nowadays are not exclusively attributed to their redox properties but also to their capacity to modulate, directly or indirectly, the activities of key proteins of interconnected cellular signalling networks. Importantly, no noxious side effects have been reported. However, in spite of their positive features, Rv and Pt are not free of shortcomings. They are subjected to an intensive phase II metabolism, largely limiting their bioavailability. Moreover, while much research has been carried out to elucidate the molecular mechanisms of action of Rv, those of Pt still largely remain to be established. During my PhD program, I addressed both these themes. I describe below: 1) the attempts to increase the bioavailability of Rv and Pt 2) my investigations on intracellular processes affected by Pt as well as the demonstration of its therapeutic potential in two different in vivo models 1) A variety of approaches has been developed to overcome the poor bioavailability of many drugs. My research group turned to the development of prodrugs for oral administration. In the case of polyphenols, a useful prodrug implies the use of protecting groups to mask the characteristic hydroxyl groups and to avoid metabolic transformations by phase II conjugative enzymes. These substituents, technically defined as pro-moieties, are attached to the backbone of the molecule through a chemical bond which can be broken in the relevant physiological environment. The choice of the pro-moiety is important for the process of absorption from the intestinal tract. The type of linkage is crucial to achieve “cargo” regeneration with appropriate kinetics. We created prodrugs for oral administration. Thus, they ought to exhibit relative stability in the gastro-intestinal tract and lability in other biological compartments (body fluids and organs). When I joined the group of Dr. Mario Zoratti, N,N-disubstituted carbamoyl derivatives of Rv bearing a PEG 350 or a sugar pro-moiety had been created. Although they increased the poor water solubility of the natural compound, these prodrugs were too stable under physiological conditions. Thus, we moved to the N-monosubstituted carbamoyl bond, expected to be less stable, for new prodrugs. Importantly, this type of chemical bond is essentially stable in acid, while it can be more easily lysed at neutral or basic pH values. A first set of compounds incorporated amino acids as pro-moieties. We hypothesized that in addition to increasing water solubility, amino acids might be recognized and transported by specific carrier systems, expressed in the enterocytes, and thus favor the intestinal absorption of the molecules. In another set of Nmonosubstituted carbamoyl derivatives polyhydroxylated groups (dihydroxypropyl or 6-deoxygalactose) were attached to all three, two or one phenolic oxygen(s) of Rv. In this case, the hope was that sugar transporters might intervene to facilitate absorption through the intestinal epithelium. All synthetized compounds displayed hydrolysis kinetics suitable for their use as prodrugs. However, absorption after oral administration to rats turned out to be rather unsatisfactory, with no evidence of an involvement of membrane carriers. In no case did galactosylated Rv derivatives reach the bloodstream of rats. The best results were obtained by administering monosubstituted aminoacid derivatives. We speculate that the extended, three-pronged structure of the fully protecting prodrugs might interfere with recognition and transport. All synthesis procedures and the characterization and pharmacokinetic evaluation of these families of compounds have been already published. The papers are included in this thesis as chapters 1 and 2. Once completed the assessment of Rv prodrugs, I started a new research project involving Pterostilbene, a more compact and possibly more effective compound than Resveratrol. This represented the main theme of my PhD program investigations. As Pt was still poorly defined from a pharmacological point of view, we first evaluated its distribution in blood and organs after oral administration (see chapter 3). Thus, we developed an appropriate method for the quantification of the molecule and its metabolites in organs after administration to rats. The resulting protocol preserves stability and allows the quantitative extraction and clarification from biological matrices of Pt itself and its metabolites, and their quantitative analysis by HPLC/UV. We used this method to determine the levels of these compounds in the blood and several other organs as a function of time after administration as a single intra-gastric dose of 88µmoles/kg of body weight. Pterostilbene levels in several organs turned out to be much higher than those measured in blood, reaching nmoles/gr (μM)-range concentrations. Moreover, Pt-4’-Sulfate was identified as the major metabolic species. Its levels were higher than those of Pt in all tissues examined except the brain. In light of this, we next aimed to prevent metabolic modifications by phase II conjugative enzymes. I exploited the experience derived from the work with the prodrugs of Rv and in collaboration with the research group of Prof. Paradisi of the Department of Chemical Sciences of the University of Padua, we developed and characterized a collection of Pt prodrugs bearing amino acids as pro-moieties, linked to the Pt 4’-OH via an Nmonosubstituted carbamate ester group. The rates of hydrolysis of all compounds fell in a range suitable for their use. Derivatives with hydrophobic aminoacid side chains were notable for the high levels of Pterostilbene they regenerated in blood after intragastric administration. We selected the most promising one and we measured its levels in rat organs, following the same methodology developed for Pterostilbene. This time, the pro-drug itself was the major specie measured in all organs considered (except the brain) but, most importantly, the levels of (regenerated) Pterostilbene were drastically increased and those of its sulfate decreased in comparison with the administration of the polyphenol as such (see chapter 4). 2) As mentioned above, the intracellular processes underlying Pt benefits are still largely undefined. Several papers suggest that the induction of autophagy may contribute importantly. I decided to investigate this aspect. Autophagy is a catabolic pathway whose dysregulation is associated with various pathological conditions. As discovered by Prof. Ballabio and co-workers it is mainly controlled by transcription factor EB (TFEB), normally inhibited by mTORC1. Thus, I evaluated the effect of Pt on the autophagy master regulator. We demonstrated that this polyphenol stimulates TFEB activity by promoting its translocation to the nucleus as well as its expression. Accordingly, Pt induced an increase of LC3B protein, an autophagy marker, and an up-regulation of TFEB lysosomal target genes. These investigations have been extended to two Pt major metabolites, Pt-4’-Sulfate and DiHydroPt, as they are the main species formed upon ingestion of the natural compound. Interestingly, while the former was ineffective, the reduced form showed an activity similar to that of the parent compound but required higher concentrations. Further studies have been carried out to clarify the upstream signalling cascade. FRET-sensor based measurements have shown that Pt is able to modestly increase the concentration of cAMP, followed by the activation of CREB. This cyclic nucleotide has been previously reported to indirectly activate AMPK, a well-recognized mTORC1 antagonist. Thus, the enhancement of this cellular axis may be responsible for the activation of TFEB. Accordingly, we observed a reduction of the activity of the mammalian target of the rapamycin. However, pharmacological interventions expected to drastically increase cAMP and AMPK activity were less effective than Pt, suggesting that this phenolic compound promotes TFEB nuclear migration by modulating more than one signalling pathway. In addition to mTORC1 inhibition, the activation of TFEB is mediated by Calcineurin. Recently, it has been demonstrated that both endogenous and exogenous ROS may promote the activity of this phosphatase. We observed in in vitro studies with cultured cells that indeed Pt increased the production of these species by mitochondria and that this phenomenon is related to TFEB migration. In light of this, it is possible to speculate that this may represent an alternative way through which this phenolic compound perturbs TFEB intracellular localization (see chapter 5). The establishment of the capability of Pt to induce autophagy in cultured cells led me to test Pt as a potential therapeutic treatment for Collagen VI (ColVI) muscular dystrophies. These disorders are mainly characterized by the presence of dysfunctional mitochondria. The concomitant deficiency of the autophagic process results in the exacerbation of the pathological conditions since the apoptotic death of myofibers occurs. As known from the literature, the use of an antisense morpholino is nowadays the best strategy to obtain zebrafish animal models with a strong phenotype of ColVI-related muscle dystrophy. Therefore, we injected into fertilized eggs a designed oligonucleotide, specifically directed against the ColVI exon 9 splicing region. This results in a frame-shift deletion of the N-terminal region of the ColVIa1 triple helical domain and therefore in a strong impairment of the organization of the muscular fibers. The data I have obtained using this model indicate that Pt treatment induces a recovery of muscular structure by more than 30%, as well as a clear recovery of motor activity.This part of the project has been carried out in collaboration with Prof. Paolo Bernardi and Dr. Marco Schiavone of the University of Padua. Although these results do not provide evidence that the amelioration of the dystrophic phenotype is due to the induction of autophagy, it is credible to suppose that the same cascade of events enhanced in vitro by Pt may be relevant also in vivo. Supporting this hypothesis, Pt provoked a significant increase of a mCherry protein, whose expression is under control of cAMP responsive elements (CRE) in a zebrafish transgenic reporter (this model has been generated by Dr. Patrizia Porazzi and Prof. Natascia Tiso of the University of Padua). Further investigations need to be performed (see chapter 6). Finally, during my graduate training I have also participated in another project still ongoing in my research laboratory. The pharmacokinetic study we performed in rats showed that Pt was particularly abundant in the brain. This observation is consistent with several papers showing a role of this phenolic compound in ameliorating performance of old rodents in behavioral tests. However, also in this context, the mechanisms accounting for these effects have been poorly characterized. My in vitro data, confirmed later in zebrafish, suggest that Pt may activate CREB following an increase of cAMP. This transcription factor has been demonstrated to play a crucial role in memory consolidation as it promotes neurogenesis in the dentate gyrus, a subarea of the hippocampus, in adult individuals. Memory, among other functions, undergoes deterioration in old age. In light of this, we set up an experimental work (see chapter 7), in collaboration with Prof. Nicoletta Berardi and the research group of Dr. Alessandro Sale, aimed to evaluate the molecular changes in the dentate gyrus and the hippocampus related to the recovery of cognitive impairments in old rats by Pt, if any. The results confirmed that after chronic administration of Pt aged animal presented a remarkable improvement in memory-related behavioral tasks. Moreover, although statistics need to be improved by adding more animals, Western blot and gene expression analyses suggest that the treatment up-regulates the activity of transcription factor CREB. An increase of mitochondrial mass has been also measured. These effects were more evident in the dentate gyrus if compared to the remaining hippocampus and strongly support the occurrence of neuronal remodeling processes. However, no differences in PSD95 levels have been observed. Further markers of synaptic plasticity will need to be taken into account in future investigations. Concluding, the studies I carried out have outlined one of the mechanisms accounting for the striking properties of the Pt, characterized its organ pharmacokinetics in the rat and allowed to boost its bioavailability. Thus, they may be helpful for the development of Pt-based therapeutic/preventive treatments.<br>Questa tesi riguarda la ricerca che ho condotto su aspetti della farmacologia e delle attività biologiche di due stilbeni, il Resveratrolo (Rv) e lo Pterostilbene (Pt), componenti polifenoliche principali rispettivamente della vite e dei mirtilli. Negli ultimi anni queste due molecole hanno attirato l’attenzione della comunità scientifica grazie alle loro attività biologiche di rilievo per molte settori della medicina. Numerosi studi descrivono le loro azioni protettive nei confronti di sindromi metaboliche, contro il cancro e la neurodegenerazione. Al giorno d’oggi, questi effetti non vengono attribuiti tanto alle loro proprietà antiossidanti quanto alla loro capacità di modulare, direttamente o indirettamente, l’attività di proteine-chiave in vie di segnalazione intracellulare tra loro interconnesse. Un punto importante è che non sono stati riportati effetti collaterali negativi del loro utilizzo. Nonostante questi aspetti positivi, Rv e Pt presentano dei punti deboli. Entrambi sono soggetti ad un intenso metabolismo di fase II che ne limita significativamente la biodisponibilità. Inoltre, mentre molto lavoro è stato condotto con il fine di elucidare i meccanismi di azione del Rv, quelli dello Pt rimangono ancora in gran parte da chiarire. Durante il mio corso di dottorato, mi sono occupata di entrambe queste problematiche. Descrivo di seguito: 1) I tentativi effettuati per aumentare la biodisponibilità del Rv e dello Pt 2) Gli studi dei processi intracellulari innescati dallo Pt e la valutazione del suo potenziale terapeutico in due modelli in vivo 1) Per far fronte al problema della scarsa biodisponibilità sono stati adottati diversi metodi. Il mio gruppo di ricerca è ricorso alla produzione di pro-farmaci. Nel caso dei polifenoli, un buon pro-farmaco implica l’utilizzo di gruppi protettori per mascherare i gruppi ossidrilici tipici di queste molecole e per evitare le modifiche metaboliche da parte degli enzimi coniugativi di fase II. Tali sostituenti, chiamati “pro-moieties”, sono legati chimicamente alla struttura stilbenica in modo reversibile nelle condizioni fisiologiche d’impiego. Essi assumono particolare importanza per l’assorbimento dei composti, mentre la tipologia del legame è cruciale affinché le molecole attive vengano rigenerate con cinetiche adeguate. Noi abbiamo voluto creare dei pro-farmaci da somministrare oralmente. Pertanto essi dovevano essere relativamente stabili nel tratto gastro-intestinale e più labili negli altri compartimenti biologici (fluidi corporei e organi). Quando ho iniziato la mia esperienza nel gruppo di ricerca del Dott. Mario Zoratti, erano stati sintetizzati dei derivati del Rv come esteri carbammici N,N-bisostituiti recanti come sostituenti (pro-moieties) PEG 350 o gruppi glicosidici. Questi profarmaci aumentavano la scarsa solubilità in acqua del composto fenolico, ma si sono rivelati troppo stabili in ambiente fisiologico per essere utilizzati come pro-drugs. Abbiamo quindi deciso di creare dei derivati carbamoilici N-monosostituiti che si prevedeva sarebbero stati meno stabili. Un aspetto importante è che questa struttura chimica è essenzialmente stabile in acido, mentre può essere idrolizzata a pH vicino alla neutralità o basico. In un primo set di composti si sono utilizzati come promoieties degli aminoacidi. Oltre ad aumentare la solubilità in acqua, ci si aspettava che gli aminoacidi potessero essere riconosciuti da specifici sistemi di trasporto espressi a livello degli enterociti favorendo l’assorbimento intestinale della molecola. In un altro gruppo di derivati carbamoilici N-monosostituiti, tutti e tre, due o uno solo degli ossigeni fenolici del Resveratrolo sono stati decorati con gruppi poliossidrilati (diidrossipropil o 6-deossigalattosil). In questo caso si sperava che i trasportatori di zuccheri potessero intervenire per facilitarne l’assorbimento attraverso l’epitelio intestinale. Come ci si attendeva, tutti i composti sintetizzati venivano idrolizzati con cinetiche compatibili con la loro utilizzazione come pro-drugs. L’assorbimento dopo somministrazione orale invece è risultato essere insoddisfacente, senza alcuna evidenza di un coinvolgimento di traslocatori di membrana. Il Rv galattosilato non ha raggiunto la circolazione sistemica in nessun caso. I risultati migliori sono stati ottenuti con i derivati amminoacidici mono-sostituiti. Ipotizziamo quindi che la struttura estesa e tripartita dei prodrugs con protezione completa interferisca con il riconoscimento ed il trasporto da parte dei sistemi di membrana. Tutte le procedure sintetiche, la caratterizzazione e la valutazione farmacocinetica di queste famiglie di composti sono già state pubblicate. Gli articoli relativi sono inclusi in questa tesi come capitoli 1 e 2. Dopo aver completato il lavoro coi pro-farmaci del Rv, ho iniziato un nuovo progetto riguardante lo Pt. Questo ha rappresentato il principale tema delle ricerche del mio programma di PhD. Poiché lo Pt era stato poco studiato da un punto di vista farmacologico, ne abbiamo dapprima valutato la distribuzione nel sangue e negli organi (capitolo 3). Abbiamo quindi messo a punto un metodo per la quantificazione di questa molecola e suoi metaboliti negli organi di ratto. Il protocollo che abbiamo sviluppato ha permesso di preservare la stabilità del composto e di ottenere un’estrazione quantitativa dello Pt e dei suoi metaboliti dalle matrici biologiche. Le analisi HPLC/UV hanno rivelato che, in seguito ad una somministrazione singola di una dose pari a 88µmoli/Kg di peso corporeo, i livelli di Pt in diversi organi erano maggiori rispetto a quelli riscontrati nel sangue, raggiungendo valori nell’ambito di varie nmoli/gr (µM). Inoltre, lo Pt-4’-solfato è stato identificato come la principale specie metabolica. I suoi livelli erano più alti rispetto a quelli dello Pt in tutti gli organi presi in considerazione tranne che nel cervello. Alla luce di questi risultati, abbiamo lavorato ulteriormente per cercare di limitare le modificazioni metaboliche da parte degli enzimi di fase II. Ho quindi sfruttato l’esperienza acquisita dal lavoro con i pro-farmaci del Rv e in collaborazione con il gruppo di ricerca della Prof. Paradisi del Dipartimento di Scienze Chimiche dell’Università di Padova, abbiamo sintetizzato e caratterizzato una serie di derivati dello Pt recanti amminoacidi come pro-moieties, collegate all’ossidrile in posizione 4’ tramite un legame carbammico mono-sostituito. Le cinetiche di idrolisi di questi composti sono risultate adatte per il loro utilizzo. I derivati con catene laterali idrofobiche si sono distinti per gli elevati livelli di Pterostilbene rigenerato nel sangue dopo somministrazione intra-gastrica ai ratti. Abbiamo pertanto selezionato il più promettente tra questi composti e ne abbiamo controllato la distribuzione negli organi, seguendo lo stesso protocollo utilizzato per lo Pt. In questo caso, il pro-farmaco per se è stato identificato come la specie predominante in tutti gli organi eccetto che nel cervello, ma soprattutto, i livelli di Pt rilasciati sono risultati notevolmente maggiori, e quelli di solfato minori, se paragonati a quelli ottenuti somministrando la molecola originaria (capitolo 4). 2) Come accennato in precedenza, i meccanismi intracellulari alla base degli effetti benefici dello Pt non sono stati ancora completamente delucidati. Alcune ricerche suggeriscono che le sue proprietà potrebbero essere attribuite all’induzione dell’autofagia. Ho quindi deciso di indagare questo aspetto (capitolo 5). L’autofagia è una via di degradazione la cui errata regolazione è associata a varie condizioni patologiche. Il Prof. Ballabio ed i suoi collaboratori hanno dimostrato che questo processo è regolato principalmente dal Fattore di Trascrizione EB (TFEB), inibito da mTORC1. Ho quindi voluto verificare gli effetti dello Pt su questo fattore di trascrizione. Ho dimostrato che questo polifenolo è in grado di stimolare l’attività di TFEB inducendo la sua migrazione nel nucleo e promuovendo la sua espressione. In accordo con quanto osservato, lo Pt ha provocato un aumento della lipidazione della proteina LC3 e dei livelli di espressione di alcuni geni lisosomiali target di TFEB. È stata valutata anche l’efficacia dei due metaboliti principali dello Pt, Pt-4’-solfato e DiidroPt, poiché essi rappresentano le specie principali ritrovate in circolo dopo somministrazione dello Pt. Mentre il primo composto è risultato essere inerte, il secondo si è mostrato attivo come lo Pt, ma a concentrazioni più alte. Ulteriori studi sono stati effettuati per esplorare le vie di segnalazione a monte di questi fenomeni. Misurazioni basate sull’utilizzo di sonde FRET hanno messo in evidenza che lo Pt incrementa la concentrazione di cAMP e attiva CREB. Come riportato in precedenti lavori, questo nucleotide ciclico può indurre indirettamente l’attivazione dell’AMPK, noto antagonista di mTORC1. La stimolazione di questo asse di segnalazione cellulare potrebbe quindi essere alla base dell’attivazione del TFEB. In accordo con questa ipotesi, abbiamo osservato una riduzione dell’attività chinasica di mTORC1. Trattamenti farmacologici volti ad aumentare le concentrazioni di cAMP o attivare l’AMPK si sono tuttavia rivelati meno efficaci dello Pt indicando che questo polifenolo induce la migrazione di TFEB al nucleo modulando diverse vie di segnalazione cellulare. La traslocazione nucleare di TFEB, oltre ad essere sotto il controllo di mTORC1, può anche essere regolata dalla Calcineurina. Di recente, è stato dimostrato che questa fosfatasi può essere attivata da ROS esogeni ed endogeni. Nel contesto di queste ricerche, ho evidenziato che lo Pt aumenta la produzione mitocondriale di queste specie e che questo fenomeno è correlato alla migrazione del TFEB. Alla luce di ciò, è possibile ipotizzare che questa rappresenti una via alternativa attraverso la quale lo Pterostilbene influenza la localizzazione subcellulare del fattore di trascrizione (capitolo 5). Lo stabilirsi dell’azione pro-autofagica dello Pt in cellule in coltura mi ha spinto a saggiare il suo potenziale terapeutico per il trattamento delle distrofie muscolari da carenza di Collagene VI. Queste malattie sono caratterizzate principalmente dall’accumulo di mitocondri non funzionali. Concomitanti difetti del processo autofagico aggravano ulteriormente le condizioni patologiche e conducono alla morte di tipo apoptotico delle miofibrille. Come indicato dalla letteratura scientifica, l’utilizzo di un morfolino antisenso è attualmente la strategia migliore per ottenere un fenotipo marcato di distrofie muscolari da carenza di Collagene VI in zebrafish. Abbiamo quindi iniettato specifici oligonucleotidi antisenso diretti contro l’esone 9 dell’mRNA codificante per il Collagene VI nelle uova fecondate di tali pesci. In questo modo abbiamo indotto una delezione “in frame” della regione N-terminale del dominio a tripla elica della molecola di Collagene VI e una profonda alterazione della struttura delle fibre muscolari. I dati che ho ottenuto indicano che il trattamento con lo Pt induce un recupero pari a più del 30% nella struttura delle fibre muscolari ed un notevole aumento dell’attività motoria. Questa parte del progetto è stata svolta in collaborazione con il Prof. Bernardi e il Dott. Marco Schiavone del Dipartimento di Scienze Biomediche dell’Università di Padova. Nonostante i nostri risultati non dimostrino direttamente che il miglioramento delle condizioni patologiche sia dovuto all’induzione dell’autofagia, è plausibile che la stessa serie di eventi che si verificano in vitro possano aver luogo anche in vivo. A supporto di questa ipotesi, abbiamo verificato che lo Pt è in grado di aumentare i livelli di una proteina mCherry la cui espressione è posta sotto il controllo di elementi influenzati dal cAMP (CRE) in una linea transgenica reporter di zebrafish (questo modello è stato generato dalla Dott.ssa Patrizia Porazzi e dalla Prof.ssa Natascia Tiso del Dipartimento di Biologia dell’Università di Padova). Tali fenomeni necessitano di essere ulteriormente approfonditi (capitolo 6). Infine, durante il mio percorso di dottorato, ho preso parte ad un altro progetto ancora in corso nel mio laboratorio di ricerca. Le analisi farmacocinetiche condotte in ratti hanno riportato che lo Pt è particolarmente abbondante nel cervello. Questa evidenza è in linea con diversi studi che dimostrano la capacità del polifenolo di migliorare le prestazioni di animali anziani in test comportamentali. I meccanismi molecolari alla base di questi effetti sono stati poco caratterizzati anche in questo contesto. Il lavoro che ho condotto in vitro, e confermato in zebrafish, dimostra che lo Pt è in grado di indurre un aumento di cAMP attivando CREB. È stato dimostrato che questo fattore di trascrizione svolge un ruolo cruciale per il consolidamento della memoria perché in grado di promuovere la neurogenesi in soggetti adulti a livello del giro dentato, una parte dell’ippocampo. La memoria, insieme ad altre funzioni cognitive, peggiora notevolmente con l’invecchiamento. Alla luce di ciò, in collaborazione con la Prof.ssa Nicoletta Berardi ed il gruppo di ricerca del Dott. Alessandro Sale (Istituto di Neuroscienze - Pisa), abbiamo messo a punto un lavoro sperimentale volto a valutare cambiamenti molecolari nel giro dentato e nell’ippocampo in seguito ad un eventuale miglioramento cognitivo in ratti anziani da parte dello Pt (capitolo 7). I risultati che abbiamo ottenuto hanno confermato che, dopo somministrazione cronica di questo composto, gli animali dimostrano un miglioramento della memoria. Inoltre, nonostante la statistica necessiti di essere ampliata aggiungendo più individui, sia analisi Western blot che RT-qPCR suggeriscono che tale trattamento ha indotto una up-regolazione di CREB. Allo stesso tempo, è stato misurato un aumento della massa mitocondriale. Questi effetti sono più evidenti a livello del giro dentato piuttosto che nella parte restante dell’ippocampo e suggeriscono fortemente il verificarsi di processi di rimodellamento neuronale. Nonostante ciò, non sono state riportate differenze nei livelli di PSD95. Per le prossime analisi saranno presi in considerazione marker diversi di plasticità sinaptica. In conclusione, le ricerche che ho condotto hanno permesso di delineare uno dei meccanismi responsabili delle proprietà dello Pt e di incrementare la sua biodisponibilità. Pertanto, essi potrebbero esse utili per lo sviluppo di futuri trattamenti terapeutici o preventivi.

This thesis is a study of the routes by which proteins are incorporated into organelles in plant cells. The mechanism of protein translocation into protein bodies, leucoplasts and glyoxysomes - three major types of organelle in castor bean endosperm cells - are investigated. While the synthesis of protein body and glyoxysome matrix proteins has previously been studied, nothing is currently known about the synthesis and incorporation of proteins destined for their membranes. This work describes the purification and characterization of the major integral membrane proteins of these organelles. Rabbit antisera raised against these proteins was used to follow their synthesis both in vivo and in vitro by a variety of techniques. The results of this investigation suggest that protein body membrane proteins are initially synthesized on and inserted into the rough endoplasmic reticulum, and are subsequently transported to protein bodies by a process of membrane flow and vesiculation. Glyoxysome membrane proteins, however, appear to be posttranslationally imported directly into the organelle from the cytoplasm. Neither class of protein appear to be synthesized as precursors nor are any co- or post-translationa1 modifications evident. Leucoplasts also import proteins from the cytoplasm, apparently by mechanisms similar to those operating in chloroplasts and mitochondria. Chloroplast precursor proteins are competent for translocation into leucoplasts and are apparently correctly processed on import by a leucoplast stromal peptidase. Other features of leucoplast and chloroplast protein import are also similar. The results presented suggest that plant cells utilize a variety of routes and mechanisms for transporting proteins. These mechanisms are compared with those employed, and more extensively studied, in animal and yeast cells.

The gum exudate from Acacia calcigera, a species recently discovered in Australia, has been shown to have a highly positive specific rotation and high molecular weight with a low rhamnose content. These results are characteristic of species within the Sec?tion Gummiferae, a predominantly African section of the genus Acacia. Analytical data for the gum exudate from a cultivar of Leucaena leucocephala from India and for gum arabic (Acacia senega!) fromAfrica were compared. The Leucaena gum had a chemical composition and properties sim ilar to gum arabic but was of higher viscosity and molecular weight; these differences could be commercially important if gum collection from Leucaena could be organised. in a series of studies in laboratory rats, gum arabic was com?pletely degraded on incorporation into a standard rat diet at levels of 2g/day/rat and 4g/day/rat. On incorporation into an elemental, low residue diet ( ?Flexical1) gum arabic was partially degraded when fed to rats at 2g/day/rat but was found to be degraded more exten?sively if fed at a reduced level (lg/day/rat). Gum arabic, mixed with faeces from rats fed the elemental diet was partially degraded by faecal bacteria. The different results obtained when gum arabic was incorporated into two different diets indicated the importance of choice of type of diet and dose level used in dietary studies. VFaecal extracts obtained from rats fed a standard diet supple?mented with gum karaya (1.2g/day/rat) were shown to be similar, but not identical, to gum karaya that had been mixed with faeces then re-extracted. A similar result was obtained when an elemental diet was used. It was not possible to conclude whether or not the gum karaya extracted from test faeces had been degraded because of the difficulties found to be associated with attempted molecular weight measurements of the impure forms of the gum extracted. Seven commercial gum tragacanth samples from Iran were found to vary in composition and in viscosity and in the ratio of their water-insoluble and water-soluble components. Their amino acid con?tents did not differ extensively. Five commercial gum tragacanth samples from Turkey showed less variation than the Iranian samples; although having lower viscosity, their amino acid compositions were sim ilar to those of the Iranian samples. A Turkish gum tragacanth sample from Astragalus microcephalus (the major source of the gum) differed extensively analytically from Turkish gum tragacanth sam?ples from Astragalus kurdicus and Astragalus gummifer (minor sources) The Test Article used in a dietary study of gum tragacanth in Man was shown to have been well-chosen, representing gum tragacanth of fair average quality.

Plant growth regulators have a vital role in plant growth and development. The cellular response to these regulators depends on the presence and the action of specific receptors. The plant growth regulators and their receptors act together in complexes which determine the final effects of the plant growth regulators. In the research reported here, emphasis has been given to the regulation of the activity of the receptors themselves. The regulation of the N-l-naphthylphthalamic acid (N~A) receptor through phosphorylation and dephosphorylation and the regulation of the auxin binding protein (ABP) through gene manipulation have been investigated. NPA, an auxin transport inhibitor, was found to bind specifically to a crude membrane preparation from sugar beet seedling leaf cell suspension cultures. The in vitro binding was optimal at pH 4.5 and 4?C. Binding parameters for NP A binding were determined by Scatchard analysis. The dissociation constant (Kd) and binding protein concentration were found to be 1.71 x 10-7 mol dm-3 and 220 pmoles g-I membrane protein respectively. It was found that the amount of specific 3H-NPA binding was significantly increased by adding Mg2+ A TP to the binding assay solution; treatment of membrane preparations with acid phosphatase, prior to the NP A binding assay, resulted in lower specific binding. A TP activation and phosphatase inactivation were culture stage dependent. Although a considerable effect could be detected when using cells from day 8 (representing the linear phase), the same treatment did not alter the binding if cells from day I (representing lag phase) or day 14 (representing the stationary phase) were used. These observations have strongly highlighted the possible involvement of a phosphorylation and dephosphorylation mechanism in vivo in the regulation of the activity of the NP A receptor. High phosphatase activity was found in the supernatant, but not in the membrane pellet, after 50,000 g centrifugation. The presence of a membrane-bound auxin receptor, ABP, was demonstrated by Scatchard analysis in sugar beet seedlings. The Kd value and the receptor concentration were found to be 2.15 x 10-6 mol dm-3 and 68 pmoles g-I membrane protein. The protein could be solubilised either with the detergent Triton X-I 00 or by acetone-washing, with a recovery of about 40%. An acetone-solubilised ABP preparation could be partially purified by DEAE-Sephacel ion exchange chromatography, NAA-linked AH-Sepharose 4B affmity chromatography or Sephadex G-200 gel filtration. The recovery after any of these chromatographic treatments was very low so that successive chromatography for further purification was unsuccessful. The low level of detectable binding after purification resulted mainly from the low abundance of ABP in the plant material. Non-radioactive labelling and detection techniques were used to show that an ABP-probe hybridized to sugar beet genomic DNA during dot blotting. The present study has indicated that receptor activity could be regulated by a phosphorylation and dephosphorylation mechanism in plants. The investigation has also suggested that the effect of plant growth regulators on plant development could be regulated through the manipulation of the expression of their receptor genes.

The endogenous Mg²⁺-ATPase activity of isolated intact turnip and mung bean mitochondria is very low relative to that of animal mitochondria, and is not stimulated by uncouplers. Results indicate that the endogenous ATPase activity is not low due to either the existence of a permeability barrier to ATP, or to a reduced content of the ATPase enzyme in turnip mitochondria. The absence of uncoupler-stimulation with mung bean mitochondria is partly due to the transport and/or permeability characteristics of the membrane. However, in both cases, an (additional) 'ATPase-inhibitory' factor is implicated. The ATPase activity of turnip mitochondria and membrane particles can be 'activated' (up to 50-fold) kinetically, and in a time-dependent manner, evidence which points to the existence of a proteinaceous ATPase-inhibitor, like that in mammalian mitochondria, which is responsible for the low ATPase activity and lack of uncoupler-stimulation in turnip mitochondria. However, alternate inhibitory ligands cannot be dismissed at present. The 'activated' ATPase activity is similar to that of the F_OF₁-ATPase with respect to inhibitor sensitivity, optimum pH, bivalent cation requirement, and sensitivity to 'activating anions'. The F₁ sector has been solubilized from turnip mitochondria by dichloromethane-extraction, and further purified to yield a preparation containing the five polypeptides (α, β, γ, δ, ε) characteristic of MF_1, BF_1 and CF_1, plus an additional sixth polypeptide (δ') which has been found in certain plant (and animal) MF₁ preparations, but whose identity has yet to be determined. The M_r values of the δ and ε polypeptides are closer to those of BF₁ and CF₁ than MF₁. The specific activity of the purified enzyme is higher than previously reported for a plant F₁-ATPase. Other notable features include apparent negative cooperativity for ATP hydrolysis, even with 'activating anions' present, reduced specificity for adenine nucleotides, high Ca²⁺-ATPase activity, and slight stimulation by the chloride anion.

The adhesion between neighbouring plant cells is established as cells are formed during cytokinesis through the middle lamella that is made principally of pectins and proteins. Pectins are secreted into the cell wall in a highly methylesterified form and subsequently de-esterified in muro by pectin methyl esterase (PME, E.C. 3.1.11). The present study reports on the biochemical characterization and immunochemical analyses of phosphate buffer/EDTA pectic extracts associated with cell-cell adhesion in suspension cultures of wild type (WT), salt tolerant (HHS) cell lines and synchronized Arabidopsis suspension cultures. Using the synchronized cultures, The PME-mediated configuration of pectins at the onset of adhesion during cytokinesis, was assessed through the analysis of the expression patterns of the PME isoforms annotated to be expressed throughout the cell cycle The wild type Arabidopsis seemed to maintain the intercellular adhesion through the gelling of the highly methylated JIM7 recognized homogalacturonans that were shown to be abundant in the primary cell walls, middle lamellae and cellular junctions, possibly due to the hydrophobic interactions between the methoxy groups. The rhamnogalacturonan-I fraction was rich in arabinan side chains reflecting the proliferative state of the cells. The increase in arabinan content was accompanied by a reduction in the galactan content 4 days after subculturing. The cell walls of salt tolerant Arabidopsis contained the JIM7 and LM7recognized epitopes along with a high degree of branching of rhamnogalacturonan-I carrying galactans and arabinans as side chains. The change in the detected epitopes is thought to play a role in the ability of the cells to withstand the high osmotic pressure and increase the in the level of adhesion between cells. The JIM5 low methylesterified HGs were less abundant in both cultures, and the absence of the 2F4 antibody recognizing the Ca2+ egg boxes could be attributed to the scarce amounts of Ca2+ present in the culturing medium The immunochemical studies of the pectin extracted from the synchronized Arabidopsis suspension cultures after washing out aphidicolin indicated that the recognition of both of JIM7 and JIM5 varied in parallel during the cell cycle, whereas, the recognition of arabinan increased during the cell division. The sequence and phylogenetic analysis of ten PME isoforms that were annotated to be expressed at one or more phases of the cell cycle of synchronized Arabidopsis thaliana suspension cultures (Menges and Murray, 2002 and 2003), revealed that only five of these genes could be PMEs. The genes At4g02330, At1g02810, At2g26440, and At2g47550 were thought to be of type II PMEs which have a pre-pro-catalytic domains and At5g47500 is a type I PME that lack the pro-region. The amino acid sequence of At4g12390 showed similarities with the N-terminal pro-peptides of plant PME and invertase inhibitors. The expression of several PME genes was studied in suspension cultures of Arabidopsis thaliana synchronised using aphidicolin. Semi-quantitative PCR experiments showed that the expression of At5g47500 transcript was always detected during M phase of the cell cycle. The rest of the genes failed to show consistent patterns of expression. Northern blots revealed that mRNA coding for At5g47500 decreases during S and G2 phases and accumulates during the M phase of the cell cycle. Our results suggest that this PME isoform is involved in the modulations of the cell walls as the cells are going through division and cytokinesis.

The plant endoplasmic reticulum is the location of storage oil and membrane lipid assembly, and for fatty acid modifying reactions (desaturation, elongation, hydroxylation). It therefore represents a source of enzymes involved in these processes. Many of these defy traditional purification strategies. In this study, ER membranes have been isolated biochemically pure and in milligram quanties from the endosperm of developing and germinating castor bean. One-dimensional SDS- PAGE, used to routinely assess sample integrity, showed resolution limitations. Two-dimensional gel electrophoresis was optimized regarding sample preparation and solubilization, and reproducible profiles confirmed its suitability as a sound basis for analysis of stage-specific ER components. In large format 2-D experiments, preparative loadings were reproducibly resolved. MALDI TOP mass spectrometry was evaluated for high throughput peptide signature generation with individual ER components. Resolution problems were again highlighted with 1-D separations, although some functional assignments were made. Subsequently analysis of selected spots from a preparative 2-D gel of germinating ER was used to establish the limitations of the procedure. Database matching of a single component at very low levels of mass error tolerance also demonstrated the power and accuracy of the technology. Membranes were subfractionated to simplify protein patterns. It is proposed that an organellar approach, including subfractionation, provides enrichment of specific subsets of cellular components. A putative plant phosphatidic acid phosphatase gene has been investigated following identification from the EST database. The aim of this research is the identification of proteins involved in storage lipid synthesis in castor bean in reactions specific to the endoplasmic reticulum.

Recently, three distinct isoforms of phospholipase D (PLD) were identified in Arabidopsis thaliana. PLD α represents the well-known form found in plants, while PLD β and γ have been only recently discovered (Pappan et al., 1997b; Qin et al., 1997). These isoforms differ in substrate selectivity and cofactors required for activity. Here, I report that PLD β and γ isoforms were active toward N-acylphosphatidylethanolamine (NAPE), but PLD α was not. The ability of PLD β and γ to hydrolyze NAPE marks a key difference from PLD α. N-acylethanolamines (NAE), the hydrolytic products of NAPE by PLD β and γ, inhibited PLD α from castor bean and cabbage. Inhibition of PLD α by NAE was dose-dependent and inversely proportional to acyl chain length and degree of unsaturation. Enzyme kinetic analysis suggested non-competitive inhibition of PLD α by NAE 14:0. In addition, a 1.2-kb tobacco (Nicotiana tabacum L.) cDNA fragment was isolated that possessed a 74% amino acid identity to Arabidopsis PLD β indicating that this isoform is expressed in tobacco cells. Collectively, these results provide evidence for NAE producing PLD activities and suggest a possible regulatory role for NAE with respect to PLD α.

Mass spectrometry has emerged as a powerful tool in the field of proteomics enabling characterization of proteins, their post translational modifications and determining protein-protein interactions. This versatile analytical technology allows both qualitative and quantitative measurements on a large number of proteins that is required to understand the complex molecular biology underlying the various cellular processes such as growth, development and response to stimuli. There are several mass spectrometry-based quantitative proteomic strategies reported in literature, all of which can be broadly classified into two groups: stable isotope-labeling approaches and label-free approaches. While the stable isotope labeling strategies are well established and applied extensively, label-free approaches are relatively new in the field of quantitative proteomics. The aim of this work was to evaluate the use of both labeling and label-free quantitative proteomic approaches with specific application to plant hormone signaling pathways. The use of stable isotope labeling for phosphorylation quantification was evaluated by the development and application of a new Phosphoprotein Acid cleavable Solid-phase Isotope-coded Reagent (PhASIR) to both model phosphopeptides and a phosphoprotein. The difficulties encountered with this approach such as complex labeling chemistries, presence of by-product, poor reproducibility and considerable loss of sample suggested that this approach was not suitable for quantitative phosphoproteomics. On the other hand, label-free quantification strategies are relatively simple, fast and can be easily applied to large-scale proteomic studies. A label-free, intensity-based quantitative strategy using a unique parallel fragmentation and a data-independent acquisition mode, termed LC/MSE was developed to carry out the functional analysis of the Arabidopsis thaliana receptor kinase BRI1 in vivo phosphorylation sites in response to brassinolide treatment. This label-free approach was also applied to carry out a comprehensive analysis of the changes in protein abundance in response to gibberellin treatment in Arabidopsis seedlings. The gel-based, LC/MSE approach termed GeLC/MSE allowed the simultaneous identification and quantification of over 200 gibberellin-responsive cytoplasmic proteins with atleast a greater than two-fold change in abundance. Overall, the LC/MSE approach proved to be a reliable method for label-free quantitative proteomics with the advantages of increased protein/proteome coverage, good analytical reproducibility and accurate quantitative results.

Biliography : leaves 162-184.<br>Resurrection plants, including Myrothamnus flabellifolius, grow in shallow soil upon rocky outcrops where they experience regular periods of water stress. Associated with this is light stress. The presence of light under water limiting conditions can result in photo-oxidation which causes damage to plant tissues. M flabellifolius is a homoichlorophyllous plant and thus retains chlorophyll during desiccation. The mechanisms whereby this plant prevents photo-oxidation damage are not known and thus one of the objectives of this study was to characterise the chloroplasts and the changes they undergo during dehydration. It was shown that chloroplasts from M flabellifolius could only be isolated using trehalose gradients (instead of sucrose gradients) and were found to have a higher buoyant density than chloroplasts isolated from another resurrection plant, Craterostigma wilmsii. The latter had the same buoyant density as those isolated from the desiccation sensitive plant Pisum sativum. The increased buoyant density in M flabellifolius was ascribed to the unusual ultrastructure of the thylakoid membranes. The latter have a staggered conformation (staircase arrangement) rather than the discrete granal and intergranal conformation found in most plants.

In plants, saturated and unsaturated N-acylethanolamines (NAEs) with acyl chains 12C to 20C are reported for their differential levels in various tissues and species. While NAEs were shown to play a vital role in mammalian neurological and physiological functions, their metabolism and functional implications in plants however, remain incomplete. Fatty acid amide hydrolase (FAAH) is one of the metabolic enzymes that breaks the amide bond in NAEs to release free fatty acid and ethanolamine. FAAH orthologs, putative PpFAAHs (Physcomitrella patens FAAH) were identified based on the sequence blast of ratFAAH, and named as PpFAAH1 to PpFAAH10. Based on the highest mRNA expression of the PpFAAH homologs upon NAE treatment, PpFAAH1 was selected for further in vitro characterization, which shares 31% sequence identity with ratFAAH. PpFAAH1 was heterologously expressed in E. coli and purified for characterization. Highest amidohydrolysis activity of PpFAAH1 was observed in vitro at pH 8.0 and temperature 37°C. Methoxy arachidonyl fluorophosphonate (MAFP), an inhibitor showed highest inhibition with 10mM concentration, however, one of the principal classes of FAAH inhibitor O-aryl carbamates (URB597) exhibited only 22% inhibition with the same concentration. Both in vivo and in vitro studies showed that unsaturated NAE substrate (NAE 20:4) is hydrolyzed faster than the saturated NAE (NAE16:0); more than 50- and 10-fold higher in vitro and in vivo assays, respectively. Amidohydrolase activity in vivo was mostly associated with microsomes compared with cytoplasmic fractions. Additionally, microsomal fraction of mature gametophytes showed higher amidohydrolase activity than of the protonemal or early gametophyte stages; however, PpFAAH expression was not significantly different between the developmental stages. Further functional characterization of NAE metabolic pathway is ongoing by generation of PpFAAH knock out (KO) and overexpressor (OE) to understand the biological implications of FAAH in growth and development of early land plants.

Feruloyl esterases (E.C. 3.1.1.73), a subclass of the carboxylic acid esterases (E.C. 3.1.1.1), are able to hydrolyse the ester bond between the hydroxycinnamic acids and sugars present in the plant cell walls, and have been classified as Types A or B based on their substrate specificity for aromatic moieties. They constitute an interesting group of enzymes that have the potential for use over a broad range of applications in the agri-food industries. In order to expand the range of available enzymes, we have examined the production of feruloyl esterases by the filamentous fungi Talaromyces slipitalus and Neurospora crassa. Neurospora crassa has been shown to produce multiple feruloyl esterase activities depending upon the time of fermentation with either sugar beet pulp or wheat bran substrates. A gene identified on the basis of its expression on sugar beet pulp has been cloned and over-expressed in Pichia pasloris. The gene encodes a single domain feruloyl esterase (NcFae-l), which represents the first report of a nonmodular Type-B enzyme and the purified recombinant protein has been shown to exhibit concentration dependent substrate inhibition. The kinetic behaviour of the non-modular enzyme is discussed in terms of the diversity in the roles of the feruloyl esterases in the mobilisation of plant cell wall materials and their respective modes of action. A novel feruloyl esterase (TsF AEC, Type-C feruloyl esterase) that exhibits broad substrate specificity in culture supernatants of Talaromyces slipitalus when grown on sugar beet pulp, has been cloned and over-expressed in Pichiapasloris. Various gene fusions have been constructed to investigate the use of alternative signal peptides by P. pastoris and to produce an authentic feruloyl esterase featuring the N-terminal sequence determined for the native enzyme. It has been demonstrated that additional amino acids at the N-terminus of the FAEC sequence do not influence the catalytic capacity of the enzyme, and that the nature of the signal sequence does not appreciably alter the yield of the secreted enzyme. NcFae-l and TsF AEC contain internal peptide sequences that correspond with the consensus motif G-X-S-X-G that contains the catalytic serine nucleophile conserved in the esterase enzyme supcrfamily. The serine residues at the centre of these peptide motifs have been independently mutated and the corresponding enzymes overexpressed in P. pastoris to identify essential serine residues as candidate nucleophiles responsible for catalysing the enzymatic reaction. Based on activity profile data and supported by the characterisation of a recombinant Type-D feruloyl esterase from N. crassa, a feruloyl esterase sub-classification is proposed and discussed in terms of the evolutionary relationships existing between carbohydrate esterases.

Plants are resilient organisms that are continually evolving and continue to withstand an adverse and dynamic world. SABP2-interacting protein (SIP)-470 is a non-specific lipid transfer protein (nsLTP) that was identified in tobacco. SIP470 was discovered during a yeast two-hybrid screening with SABP2, which is an important methyl esterase enzyme which catalyzes the conversion of immobile MeSA into active salicylic acid (SA) during pathogenic challenge. SA activation and mobility allows for immunity to be carried to other, non-infected parts of the plant. This induced responses is called systemic acquired resistance (SAR) and it is a broad spectrum defense. Like many nsLTPs, SIP470 is small and has a predicted characteristic hydrophobic cavity. nsLTPs are found in higher plants and have repeatedly demonstrated protection in biotic stress including disease resistance, and greater resistance to both bacterial and fungal pathogens in overexpressed transgenic lines. This diverse class is also significantly involved in plant adaptation to environmental changes, namely drought, salinity, and freezing, but also in osmotic stress and wounding. Furthermore, nsLTPs are involved in wax metabolism and seed development. Subcellular localization of nsLTPs varies considerably during in vitro and in recent in vivo studies. SIP470 was originally identified in tobacco plants, and therefore, it is important to study its role directly in tobacco plants. SIP470 and eGFP fusion construct has been generated to study the subcellular localization of SIP470 in tobacco cells. SIP470 localization has shown a discontinuous, punctate arrangement around the membrane periphery which is being further verified by subcellular fractionation. Transgenic tobacco lines that are silenced in SIP470 via RNAi have been generated, and these plants are being screened. Overexpressor transgenic lines of SIP470 have been generated and are under the control of an estradiol-inducible promoter. These transgenic lines will be tested for their response in basal resistance and SAR.

Aromatic and medicinal plants have been recognized since antiquity as possessing biological activities; chief amongst these are their antibacterial, antifungal and antioxidant properties. In this study, the chemical composition, the antimicrobial and in vitro antioxidant bioactivities the volatile oils extracted by hydrodistillation from aromatic and medicinal members of the plant families Geraniaceae: geranium (Pelargonium graveolens L'Herit); Lamiaceae: melissa (Melissa officinalis L.), monarda (Monarda citriodora var. citriodora Cerv. ex Lag.), oregano (Origanum vulgare ssp. hirtum (Link) Letsw.) and thyme (Thymus vulgaris L.); Myristicaceae: nutmeg (Myristica fragrans Houtt.); Myrtaceae: clove (Syzygium caryophyllus Gaertn.); Piperaceae: black pepper (Piper nigrum L.) and Umbelliferae: lovage (Levisticum officinalis L.) were investigated. The chemical percentage composition of the volatile oils extracted from black pepper, clove, geranium, lovage (from both leaf and stem material), melissa, monarda, nutmeg, oregano and thyme were analysed using gas chromatography and mass spectroscopy. The findings of these analyses confirmed that volatile oils from aromatic and medicinal members of different plant families are principally mixtures of mono- and sesqui- terpenoids compounds. Furthermore, oil samples extracted from species of the same plant family, samples sourced from different parts of the same plant and a commercial and authenticated volatile oil described as from the same species may exhibit different compositions, i.e. the percentage composition and variation in individual phytochemicals. A series of experiments were carried out in an attempt at assessing the antibacterial properties of black pepper, clove, geranium, melissa, nutmeg, oregano and thyme volatile oils and their main components. A collection of 25 test microorganisms [9 Gram-positive and 16 Gram-negative strains] including human, plant and veterinary pathogens and food spoilage organisms. All the volatile oils demonstrated some degree of antiseptic activity with the oils of oregano and thyme being particularly active. The phenyipropanoid eugenol and the phenolic constituents carvacrol and thymol were found to be strongly antiseptic. The antifungal activity of black pepper, clove, melissa, oregano and thyme volatile oils against the agriculturally important Aspergillus species Aspergillus flavus (Link) Fries and Aspergillus niger van Tieghen, and the Fusarium species Fusarium culmorum W.G. Smith was investigated. All the volatile oil samples demonstrated antifungal properties with variable degrees of efficacy across the concentration levels used in this study, with the volatile oils of oregano and thyme being particularly inhibitory. The in vitro antioxidant properties of the volatile oils of black pepper, clove, nutmeg, oregano and thyme and their major components were evaluated by using a method routinely used in this laboratory, a simplified plate diffusion technique for determining lipid antioxidant activity using linoleic acid/β-carotene. To investigate further the potential antioxidant activity of these plant extracts and their main components and characterise their underpinning mechanism of action, an attempt to develop and optimize current antioxidant screening techniques was carried out. These methods included a thiobarbituric reactive species assay, a conjugated diene assay and a free-radical trapping assay. Finally, a series of feeding trials were carried out to assess whether volatile oils feed at various concentrations prior to and during pregnancy can beneficially affect the compositional levels of major fatty acids in a maternal and foetal/neonatal model extracted from cholesteryl ester, triacylglyceride, free fatty acid and phosphoglyceride lipid fractions in an organ dependent manner. Particular focus centered upon the saturated fatty acids lauric, palmitic and stearic acids; the essential fatty acids[LA](18:2n-6) and α-linoleic [LA] (18:2n-6) and α-linolenic [ALA (18:3n-3) acids and the long chain polyunsaturated fatty acid metabolic derivatives, e.g. arachidonic acid [ARA] (20:4n-6), docosahexaenoic [DHA] (22:6n-3) and eicosapentnoic acid [EPA] (20:5n-3). The volatile oil of oregano was administered orally to female rats at 167mg Kgˉ¹; 334 mg Kgˉ¹ and 843 mg Kgˉ¹ concentration levels immediately prior to and during pregnancy to determine whether they affected the fatty acids composition in a variety of major maternal and neonatal organs. In addition to the aforementioned experiment, further feeding trails were carried out using the volatile oils of clove and nutmeg at a 5Oµg gˉ¹ concentration level.

The Gram-positive, strictly anaerobic bacterium, <I>R. flavefaciens</I>, plays an important role in the degradation of plant cell wall polysaccharides in the rumen. There is a paucity of information available, however, regarding the multiplicity and organisation of <I>R. flavefaciens </I>cellulolytic and xylanolytic enzyme systems. A technique involving PCR amplification of DNA with primers designed from conserved sequences, followed by hybridisation of the PCR products to chromosomal DNA, has led to an estimate of xylanase gene multiplicity in <I>R. flavefaciens</I>. The xylanase-specific primers were also useful in the isolation and sequencing of a partial xylanase gene, <I>xynC</I>. Although <I>R. flavefaciens</I> 17 appears to produce a cellulose-binding enzyme-complex, none of the individual enzymes examined was found to bind cellulose in isolation. However, a 210 kDa protein which is present in the complex was shown to bind cellulose after isolation from a renatured SDS-gel. In order to look for genetic evidence for a cellulose-binding mechanism, sequencing of the <I>R. flavefaciens</I> 17 endoglucanase gene, <I>endA</I>, was completed from PCR products. The carboxy-terminus of the predicted <I>endA</I> product consists of a domain which is similar to dockerins found in <I>Clostridium thermocellum </I>polysaccharidases. Homologous domains are also found in the <I>R. flavefaciens </I>xylanases, XynB and XynD. As the <I>C. thermocellum</I> dockerin domains mediate binding to the 210 kDa scaffolding protein in the cellulosome complex, it is likely that the <I>R. flavefaciens</I> domains play a similar role in assembly of a cellulosome-like complex (Lamed and Bayer, 1994). A gene which maps approximately 1.5 kb downstream from <I>endA</I> on the <I>R. flavefaciens</I> 17 chromosome was sequenced and found to be homologous to <I>nifS</I> genes from nitrogen-fixing bacteria (Zheng <I>et al, </I>1993). The <I>R. flavefaciens</I> NifS product catalyses the production of sulphur from cysteine, and is suspected to partake in the assembly of iron-sulphur clusters. The precise role of NifS is not yet known, but may be related to the degradation of crystalline cellulose.

The bioassay guided fractionation of two Latin American plants, structure elucidation of pure isolates, and LC/MS studies of six plant extracts is presented here along with the structure determination of two compounds using X-ray diffraction. The bioassay guided fractionation of the antibacterial and antitubercular CH2Cl2-MeOH extracts of the Argentinean plant Caiophora coronata Hook. et Arn. (Loasaceae) and the Chilean plant Myrcianthes coquimbensis (Barn.) Landrum et Grifo (Myrtaceae) respectively led to the isolation and complete structure elucidation of nine compounds from the active fractions. Three of these isolates were determined to be new. Namely, a new triterpene, 1beta,3beta-dihydroxyurs-12-en-27-oic acid, a new iridoid, 1alpha-methoxy-6alpha,10-dihydroxyisoepiiridomyrmecin (caiophoraenin) from C. coronata and a new monoterpene (1S,3 S,4R)-1-methyl-4-(1-methylethenyl)-1,3-cyclohexanediol from M. coqumibensis. All chemical structures were established unequivocally by physical and spectroscopic techniques including-melting point, optical rotation, 1D and 2D NMR, HR-FABMS, and FT-IR. Absolute configuration of the new monoterpene was established by Mosher's esterification. The antibacterial activity of all isolates from C. coronata were determined against methicillin-sensitive (MSSA) and -resistant (MRSA) strains of Staphylococcus aureus, Bacillus subtilis (BS), vancomycin-resistant Enterococcus faecium (VREF), Escherichia coli (EC), E. coli imp (ECimp), and Candida albicans (CA). 1beta,3beta-Dihydroxyurs-12-en-27-oic acid was found active against BS, MSSA, MRSA, VREF, and ECimp with MIC values of 2, 4, 4, 4 and 16 mug/ml respectively, whereas, other isolates were essentially inactive. The antitubercular activity of all isolates from M. coquimbensis was evaluated against M. tuberculosis using the microplate alamar blue assay. Oleanolic acid was determined to be the active principle of the extract with an MIC value of 29.66 mug/mL whereas other isolates were regarded as inactive (MIC > 128 mug/mL). Chemical investigations by LC/MS of species closely related to C. coronata and M. coquimbensis were also conducted. Structure solutions by single crystal X-ray crystallography, of an iridoid (4R,5R,7S,8S,9 S)-(-)-7-hydroxy-8-hydroxymethyl-4-methylperhydrocyclopenta[ c]pyran-1-one, and a fernane (3R,5S,9 R,10S,13S,14S,17 R,18R,21R)-(-)-fern-7-ene-3alpha-ol, isolated from the antitubercular methanolic extracts of Valeriana laxiflora DC (Valerianaceae) and Sebastiania brasiliensis Spreng. (Euphorbiaceae) respectively is also presented. The absolute configuration of these compounds was also determined.

Thesis (PhD)--Stellenbosch University, 2013.<br>ENGLISH ABSTRACT: The present study employed a two-phased approach to investigate the possible mechanisms involved in the chemopreventive properties of rooibos (Aspalathus linearis) and different honeybush species (Cyclopia spp.) in vitro. In the first phase, the effect of unfermented methanol and aqueous herbal tea extracts against the growth parameters (cell viability, proliferation and apoptosis) of normal (CRL 7761); premalignant (HaCaT); and malignant (CRL 7762) skin cells was evaluated and compared to green tea extracts. The predictive potential of polyphenol content (total polyphenol and flavanol/proanthocyanidins) and antioxidant properties (ABTS; ORAC; FRAP and LPO) in the biological activity of extracts in cells was also assessed. Of the herbal teas, the methanol extract of rooibos was the most active and it inhibited the growth of skin cells presumably by inducing mitochondrial dysfunction via membrane depolarisation. At lower concentrations, this activity was associated with inhibition of cell proliferation that was selective for cancer cells whilst higher concentrations induced apoptosis that was more prominent in premalignant cells. The strong antioxidant properties of the extracts implicated the role of pro-oxidative polyphenol/iron interactions involving monomeric flavonoids and polymeric proanthocyanidins in the cytotoxic effects of rooibos. The strong relationship between total polyphenolic and flavanol/proanthocyanidins content, antioxidant properties and reduction of cell viability indicated that these parameters (polyphenols and antioxidant properties) can serve as predictive tools for the cytotoxic effects of rooibos in vitro. The aqueous extracts of honeybush species, although weaker, displayed similar effects to rooibos extracts in cells with C. genistoides being the most effective at selectively inhibiting the proliferation of cancer cells whilst the pro-apoptotic activity of C. subternata and C. intermedia was more prominent in premalignant cells. The underlying mechanisms are also likely to result from pro-oxidative mechanisms resulting from polyphenol/iron interactions that mainly involve polymeric flavanol-like proanthocyanidin compounds in honeybush. In contrast, the methanol extracts exhibited weaker cytotoxic effects and protected cancer cells from going into apoptosis. The cytoprotective effects of honeybush species are possibly mediated by the major monomeric compounds such as mangiferin and hesperidin through antioxidant mechanisms that result in reduction of oxidative stress. Due to the possible dual role of the monomeric and polymeric compounds in the honeybush extracts, the total polyphenolic content of these herbal teas may not be a good indicator of biological activity in vitro. However, as aqueous extracts displayed high flavanol/proanthocyanidins content and exceptional activity in the ABTS assay, these parameters may be considered as indicators of cytotoxicity. On the other hand, methanol extracts, particularly from the xanthone-rich species (C. genistoides and C. longifolia) which exhibited the weakest cytotoxic effects, were more active in the ORAC thus this assay may be a useful predictor for cytoprotective activity. In the second phase, an in vitro UVB/HaCaT model which used IL-1α as a biomarker for early inflammation was developed and validated with known anti-inflammatory compounds, dexamethasone and ibuprofen. It was used to determine the specific mechanisms involved in the modulatory effects of the herbal tea extracts against inflammation. Rooibos extracts and the aqueous extract of honeybush enhanced the cytotoxic effects of UVB in the model and exhibited indirect anti-inflammatory effects as they removed icIL-1α containing cells via apoptosis. In contrast, methanol extracts of honeybush exacerbated icIL-1α by protecting UVB stimulated cells from undergoing apoptosis. In conclusion, methanol extract of rooibos and aqueous extracts of honeybush species may be useful in protecting the skin after UVB exposure. These herbal tea extracts may block initiation and delay the promotion stage during skin carcinogenesis by removing premalignant cells via apoptosis and preventing onset of inflammation. In contrast, due to their cytoprotective effects, methanol extracts of honeybush may be more effective at preventing oxidative stress in skin before UVB exposure. Future studies should focus on the effects of extracts and polyphenolic fractions on the oxidative status of the cells and development of biomarkers of chemoprevention that can be utilised in vivo and in human skin.<br>AFRIKAANSE OPSOMMING: In hierdie studie word moontlike velkankerwerende eienskappe van rooibos (Aspalathus linearis) en ‘n aantal heuningbos (Cyclopia spp.) spesies deur twee afsonderlike benaderings bestudeer. Die eerste benadering ondersoek die effek van die kruietee op groeiparameters van velselle [lewensvatbaarheid, groei en dood van normale selle (CRL 7761), vroeë kankerselle (HaCaT) en kankerselle (CRL 7762)]. Tydens eksperimente is die moontlikheid om polifenoolinhoud (totale polifenole, en flavanol/proantosianidiene verhouding) en antioksidant-eienskappe te gebruik om die biologiese funksies van die ekstrakte in die selle te voorspel, geevalueer. Die metanolekstrak van rooibos het die groei van selle die effektiefste gestop, moontlik deur depolarisasie van die mitokondriale membraan. By lae konsentrasies van die ekstrak is die groei van kankerselle selektief gestop, terwyl vroeë kankerselle die sensitiefste by hoër konsentrasies was. Die hoë antioksidant-aktiwiteit van die rooibosekstrak kan moontlik ‘n rol speel in die indusering van sitotoksiese effekte in die selle en kan toegeskryf word aan die pro-antioksidant aktiwiteit van die polifenole weens hul interaksie met yster. ‘n Spesifieke funksie word vir die monomeriese flavonoïede en die polimeriese proantosianidiene geïmpliseer. Die sterk verwantskap tussen die totale polifenoolinhoud, flavanol/proantosianidien inhoud en antioksidant aktiwiteit met die verlaging in selgroei, maak hul relevante parameters van die voorspellingsmodel. Die waterekstrakte van heuningbos induseer ook soortgelyke maar swakker effekte met die induksie van kankersel dood, met C. genistoides die selektiefste en C. subternata en C. intermedia die aktiefste spesies wat die groei van die vroeë kanker selle inhibeer. Die onderliggende meganismes betrokke blyk ook aan ‘n pro-oksidant effek toe geskryf te wees, waartydens spesifieke polifenool/yster interaksies betrokke is. In teenstelling met rooibos, beskerm die metanolekstrak van heuningbos kankerselle teen seldood, wat moontlik verband hou met die antioksidant-eienskappe van die hoof monomeriese polifenole, mangiferien/isomangiferien en hesperidien. Vanweë die dubbele rol van die monomeriese polifenole en polimeriese verbindings in heuninghbosekstrakte is die totale polifenol inhoud nie ‘n goeie indikator van die biologiese aktiwiteit in vitro nie. Daarenteen is die flavanol/proantosianien inhoud en die hoë aktiwiteit in die ABTS antioksidanttoets goeie indikators om seldood te voorspel. In teenstelling hiermee het die metanolekstrakte van die xantoon-ryke spesies (C. genistoides en C. longifolia) ‘n baie lae effek op seldood, maar ‘n hoë aktiwitiet in die ORAC toets getoon, wat ‘n goeie rigtingwyser is om die beskermende effek in selle te voorspel. Met die tweede benadering is die anti-inflammatoriese eienskappe en die onderliggende meganismes van die kruietee ondersoek in ‘n UVB/HaCaT selmodel. Intrasellulêre interleukin 1α (IL-1α) is as merker gebruik en die model is geëvalueer deur bekende anti-inflammatoriese verbindings soos dexamethasone en ibuprofin te gebruik. Die metanolekstrak van rooibos en die waterekstrak van heuningbos het die toksiese effek van UVB in die model verhoog deur selle met verhoogde vlakke,van icIL-1α te verwyder deur middel van die induksie van seldood. Die metanolekstrak beskerm die selle teen die oksidatiewe skade wat deur UVB geïnduseer word en verwyder nie selle met hoë IL-1α vlakke nie. Ter opsomming blyk dit dat die metanolekstrak van rooibos en die waterekstrak van heuningbos moontlik gebuik kan word om die vel te beskerm teen die induksie van icIL-1α en sodoende die inisiëring van kanker te blokkeer en ook die promosie van kanker te vertraag. Die beskermende effek van die metanolekstrak kan moontlik aangewend word om die oksidatiewe skade wat deur UVB veroorsaak word teen te werk deur dit aan te wend voordat blootstelling plaasvind. Toekomstige studies behoort verdere karakterisering van die polifenoolsamestelling van die ekstrakte in te sluit en hul effek op die oksidatiewe status en anti-inflammoriese effekte van selle te bepaal ten einde sekere merkers te identifiseer vir vel studies in vivo.