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A thesis submitted by Melinda Jane Mayer, B.Sc.(Bristol) in accordance with the requirements of the University of Durham for the degree of Doctor of Philosophy. Department of Biological Sciences, August 1993.Two cDNA libraries were constructed from desiccating pea cotyledons. Differential screening of the libraries with cDNA from an earlier developmental stage (physiological maturity) demonstrated that the abundant message population during dehydration shows some noticeable differences to the message populations present before desiccation. Clones hybridising to a polyubiquitin probe were isolated from a cDNA library. These clones were identified as messages for the two types of ubiquitin extension proteins (with 52 and 79 residue tails), already characterised in other species as being involved in ribosome biogenesis. The pea ubiquitin extension tail amino acid sequences showed considerable homology to tails from other plants, animals, yeast and protozoa, including a nuclear localisation site and a putative zinc-binding nucleic acid binding domain, the positions of which are conserved within the tail sequences. Sequencing of a second polyubiquitin cDNA from pea leaf demonstrated that pea contains a ubiquitin multigene family of at least four members. The expression of several genes associated with plant response to stress and two abundant seed messages (Leg A and J) was examined in developing and dehydrating cotyledons and axes. This confirmed conspicuous variations in the message levels of the genes examined as the cotyledons aged, with different members of the ubiquitin and legumin multigene families showing differential expression with age. It was also demonstrated that the expression pattern of certain messages in the cotyledons was different to that in the axes and other seed tissues. This was confirmed by an analysis of total and albumin protein fractions in cotyledons and axes. The effect on specific message and protein levels of premature desiccation treatments indicated that the temporal expression of several seed genes is related to the state of hydration of the seed, artificial desiccation leading to premature maturation. Seed storage protein message and protein levels were especially increased by premature desiccation. Legumin seed storage protein messages were also shown to be responsive to exogenous ABA applied to immature cotyledons during the seed filling stage. However, the other stress-related messages examined in pea (ubiquitin and a pea putative metallothionein) were not responsive to exogenous ABA at this developmental stage.

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Severe dehydration is lethal for most biological species, however there are a number of organisms which have evolved mechanisms to avoid damage during dehydration. One of these mechanisms is the accumulation of small solutes (e.g. sugars), which have been shown to preserve membranes by inhibiting deleterious phase changes at low hydration. Specifically, sugars reduce the gel to fluid phase transition temperatures of model lipid/water mixtures. However, there is debate about the precise mechanism, the resolution of which hinges on the location of the sugars. An experimental investigation into the effects of small solutes on the phase behaviour of phospholipid membranes is presented in order help identify the mechanisms by which solutes facilitate desiccation tolerance. Differential Scanning Calorimetry (DSC) was used to determine the first comprehensive phase diagram for the synthetic phospholipid DPPC over a wide range of hydration and solute molar ratios between 0.1 and 1.0 sugars per lipid. Over the same range of hydrations and solute concentrations Small Angle X-Ray Scattering (SAXS) was used to measure the structural parameters of the membrane bilayers necessary to determine both the phase of the membrane lipids and the location of the solutes. SAXS was also used to conduct the first comprehensive study of the effect of solutes on the kinetics of the fluid - gel transition of DPPC over a range of both hydration and solute concentration. Finally, contrast variation Small Angle Neutron Scattering (SANS) was used to quantitatively determine the location of the solutes. Data from these complimentary techniques are presented which show a monotonic relationship between both transition temperature and repeat spacing with respect to solute concentration. This relationship exists between solute:lipid molar ratios between 0.1 to approximately 0.5, after which higher concentrations of solute are shown to have no further effect on either the bilayer repeat spacing or transition temperature. It is proposed that the exclusion of small solutes into sugar/water micro-phases external to the bilayer can account for this behaviour. A theoretical model previously used to describe membrane phase behaviour at low hydrations is modified to account for the presence of solutes between membrane bilayers. This model is shown to be in quantitative agreement with the experimental data up until approximately 0.5 sucrose molecules per lipid, the point of solute exclusion. Once exclusion is taken into account, the model is quantitative over the whole range of sugar concentrations.

Tardigrades are microscopic aquatic invertebrates that are known for their ability to survive extreme conditions. Different species of tardigrades tolerate extreme conditions to a varying degree. It has been suggested that limnic tardigrades would have a lower tolerance to desiccation compared to limno-terrestrial tardigrades. In this study limno-terrestrial species Ramazzottius oberhaeuseri and the limnic species Hypsibius dujardini is compared in regard to their tolerance to desiccation and freezing. The results show that there indeed is a difference in the tolerance and that Ramazzottius oberhaeuseri show better tolerance.

Η Κορινθιακή Σταφίδα, ποικιλία της αμπέλου και προϊόν μεγάλης διατροφικής αξίας, άρχισε να καλλιεργείται στη χώρα μας πριν από επτά αιώνες περίπου. Η καλλιέργειά της εντοπίζεται αρχικά στη Βόρεια, Βορειοδυτική και Δυτική Πελοπόννησο, καθώς και σε νησιά του Ιονίου αργότερα, περιοχές στις οποίες καλλιεργείται μέχρι σήμερα. Η παραγωγή και το εμπόριο της σταφίδας, αποτελούσαν τον κορμό της οικονομίας της χώρας μας, στο δεύτερο μισό του 19ου αιώνα. Για την περιοχή της Αιγιαλείας, η Κορινθιακή Σταφίδα αποτελεί ακόμη και σήμερα το βασικό αγροτικό προϊόν της, με αποτέλεσμα να καθορίζει την οικονομική ζωή της περιοχής, αλλά και να συμβάλλει στη διαμόρφωση των κοινωνικών και πολιτιστικών χαρακτηριστικών της. Η ξήρανση της σταφίδας, δηλαδή η μετατροπή του σταφυλιού σε ξηρό καρπό, γίνεται σήμερα με Άμεση Ηλιακή Ξήρανση, δηλαδή με άμεση έκθεση του προϊόντος στην ηλιακή ακτινοβολία. Διαδικασία επίπονη για τους παραγωγούς και χωρίς ιδιαίτερο ποιοτικό αποτέλεσμα για το προϊόν. Η Έμμεση Ηλιακή Ξήρανση, που προτείνεται από την παρούσα εργασία, διαφέρει από την Άμεση Ηλιακή Ξήρανση, σε δύο βασικά σημεία: Το προϊόν, * δεν εκτίθεται άμεσα στην ηλιακή ακτινοβολία και * δεν απλώνεται απροστάτευτο σε μεγάλη επιφάνεια στο έδαφος. Συγκεκριμένα: Με τη βοήθεια ενός ηλιακού συλλέκτη αέρος, συλλέγεται η ηλιακή ενέργεια, μετατρέπεται σε θερμότητα και στη συνέχεια, με τη βοήθεια του φυσικού ή εξαναγκασμένου ρεύματος αέρα που δημιουργείται, χρησιμοποιείται για την αποξήρανση της σταφίδας, που βρίσκεται προστατευμένη σε κλειστό χώρο. Υπολογίστηκε το ποσό της απαιτούμενης για την ξήρανση ενέργειας, η δυνατότητα δέσμευσης αυτής από το συλλέκτη, καθώς και τα χαρακτηριστικά του συλλέκτη και κατασκευάστηκε αρχικά ένα μικρό, πειραματικό μοντέλο ξηραντηρίου, με το οποίο έγιναν οι βασικές αρχικές διαπιστώσεις. Στην συνέχεια, κατασκευάστηκε ένα μεγαλύτερο με παραγωγική ικανότητα ξηραντήριο, με στόχο να μελετηθεί η διαδικασία ξήρανσης σε πραγματική κλίμακα. Για πέντε συνεχόμενα χρόνια μετρήθηκαν οι αποδόσεις του συλλέκτη και η αποτελεσματικότητα της διάταξης στη διαδικασία αποξήρανσης του προϊόντος, ενώ κάθε χρόνο με βάσει τις παρατηρήσεις της προηγούμενης χρονιάς, γίνονταν μεταβολές στον τρόπο λειτουργίας του ξηραντηρίου, αλλά και στη γεωμετρία του. Μελετήθηκε η λειτουργία του συλλέκτη και του χώρου ξήρανσης με εξαναγκασμένη κυκλοφορία, καθώς και με φυσική κυκλοφορία και έγινε σύγκριση των αποτελεσμάτων σε κάθε περίπτωση. Το κύριο συμπέρασμα είναι ότι, οι υψηλότερες θερμοκρασίες (κατά 10 οΚ), που ανεπτύχθησαν στην περίπτωση της φυσικής κυκλοφορίας αέρα, χωρίς δηλαδή χρήση ανεμιστήρα, οδηγούν σε ταχύτερη ξήρανση, παρά τη μικρότερη παροχή αέρα. Το αποτέλεσμα αναφορικά με την ποιότητα του αποξηραμένου προϊόντος, είναι άριστο, δεδομένου ότι είναι απαλλαγμένο από τα προβλήματα της έκθεσής του στο έδαφος (μικροβιακό και χημικό φορτίο, καταστροφές, κ.τ.λ.), στο ήλιο (χρώμα, συστατικά) και η ξήρανση του γίνεται με λιγότερη εργασία. Το κόστος της εγκατάστασης είναι αρχικά μεγαλύτερο από αυτό της άμεσης ηλιακής ξήρανσης, αλλά ο χρόνος ζωής αυτής, σε συνδυασμό με το πολύ μικρότερο κόστος εργασίας ξήρανσης, καθιστούν την Έμμεση Ηλιακή Ξήρανση σε έναν ορίζοντα 10 ετών, πολύ φθηνότερη. / -

This study examined some of the environmental, physiological, and genetic factors or mechanisms which contribute to L. monocytogenes’ desiccation survival under food processing conditions. Desiccation experiments were carried out on stainless steel coupons stored at 43% RH, 15°C. The level of initial contamination had no impact (p>0.05), whereas the presence of a mature biofilm, prior osmoadaptation, and the presence of salt (5%) and lard (20-60%) on the SS coupons significantly (p<0.05) increased the bacterium’s desiccation survival. An Lm568 transposon mutant library was constructed to screen for novel genes involved in desiccation survival. Fifteen tolerant and 16 sensitive desiccation mutants were sequenced. Interrupted genes involved in motility and FA membrane modification were the most common in tolerant mutants whereas energy and membrane transport related genes were the most recognized in sensitive mutants. Lastly, a spontaneous desiccation resistant Lm568 variant was isolated, emphasizing the importance of understanding desiccation tolerance for food safety.

Resumo: O presente trabalho foi realizado no Departamento de Botânica, Instituto de Biociências - UNESP, Campus de Botucatu, e teve como objetivo avaliar a tolerância à dessecação de sementes de araticum-de-terra-fria (Annona emarginata (Schldtl.) H. Rainer), os açúcares envolvidos no processo e o efeito de GA4+7 N- (fenilmetil)-aminopurina na germinação das sementes submetidas aos níveis de secagem e além de avaliar o comportamento fisiológico das sementes após armazenadas no fruto (repouso pós-colheita). Foram instalados dois experimentos em delineamento inteiramente casualizado, com quatro repetições de 25 sementes por parcela. No primeiro As sementes foram secas em estufa de circulação forçada de ar (50°C) obtendo-se níveis de água de: 31% testemunha, 19%, 12% e 5%. Após secagem, as sementes foram embebidas em GA4+7 N-(fenilmetil)-aminopurina (0, 250, 500, 750 e 1000 mg L-1) por 60 horas. Já O segundo experimento foi divido em duas etapas. Na primeira os frutos maduros foram armazenados a 5°C pelo período de 0, 7, 14, 21 e 28 dias. Após armazenamento as sementes foram colocadas para embeber em água destilada e em solução de GA4+7 N-(fenilmetil)-aminopurina (800 mg L-1 ) por 60 horas. Na segunda etapa, as sementes foram armazenadas em sacos de papel tipo Kraft, envoltas por um saco de polietileno e acondicionadas em câmaras de germinação nas temperaturas de 5°C, 15°C e 25°C, onde permaneceram armazenadas por 30, 60, 90, 120 e 150 dias sendo o tempo 0 a testemunha. Após armazenamento as sementes foram colocadas para embeber em água destilada e em solução de GA4+7 N-(fenilmetil)- aminopurina (800 mg L-1 ) por 60 horas. As variáveis analisadas foram: condutividade elétrica, porcentagem, tempo médio, índice de velocidade, freqüência e sincronização da germinação, porcentagem de sementes dormentes, porcentagem de plântulas normais, comprimento ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The present study was carried out at the Department of Botany, Institute of Biosciences - UNESP, Botucatu Campus, São Paulo State, Brazil, aimed to assess the tolerance to desiccation of "araticum-de-terra-fria" (Annona emarginata (Schldtl.) H. Rainer) seeds, the sugars involved in this process, the effect of GA4+7 + N- (phenylmethyl)-1H-purine-6-amine on the germination of seeds subjected to different desiccation levels, and the physiological behavior of seeds stored in the fruit (postharvest resting period) and after fruit extraction. Two experiments were carried out in completely randomized design, including 4 replicates of 25 seeds per plot. In the first one, seeds were drought in forced aeration oven (50°C) and the following water levels were obtained: 31% control, 19%, 12% and 5%. After desiccation, seeds were immersed in GA4+7 + N-(phenylmethyl)-1H-purine-6-amine (0, 250, 500, 750 and 1000 mg L-1) for 60h. The second experiment was divided into two steps: in the first one, mature fruits were stored at 5°C for 0, 7, 14, 21 and 28 days. Then, the fruits were manually pulped and the seeds were allowed to imbibe in distilled water and in GA4+7 + N- (phenylmethyl)-1H-purine-6-amine solution at the concentration of 800 mg L-1 for 60h. In the second step, the seeds were involved with polyethylene bags, placed inside Krafttype paper bags and stored in a germination chamber at 5°C, 15°C and 25°C for 30, 60, 90, 120 and 150 days; time 0 was adopted as control. Following storage, seeds were allowed to imbibe in distilled water and in GA4+7 + N-(phenylmethyl)-1H-purine-6- amine solution at the concentration of 800 mg L-1 for 60h. The evaluated variables were electrical conductivity; germination percentage, mean time, speed index, frequency and synchronization; percentage of dormant seeds; quantitative analysis of total and reducing soluble sugars; and sugar profile by using ... (Complete abstract click electronic access below) / Orientador: Gisela Ferreira / Coorientador: Claudio José Barbedo / Banca: José Marcio Faria / Banca: João Nakagawa / Mestre