This master's thesis was written on 148 leaves (A4 format, font Times New Roman, font size 14, 1.5 line spacing), excluding attachments. Number of tables-33 (excluding annexes). The number of figures – 53 (excluding applications). Master's thesis consists of an introduction, three parts, conclusion, bibliography, applications. The theoretical part presents the basic concepts, basic approaches, models, methods, analysis of talent management as a system and process, the conceptual model of assessing the potential of employees of the HIPO category, including the model of assessing the potential of employees of "ECOPSI". In the practical part describes the General characteristics of the organization under study, the analysis and evaluation of staff in the "kindergarten combined type", the identification of employees of the category HIPO based on the model "ECOPSI". The 3rd Chapter offers recommendations for the implementation of technology work with HIPO through the development of individual career development plans for employees and individual development plans for HIPO staff. In conclusion, the results were summed up in accordance with the tasks. / Данная магистерская диссертация выполнена на 148 листах (формат А4, шрифт Times New Roman, кегль 14, интерлиньяж 1.5) без учета приложений. Количество таблиц – 33 (без учета приложений). Количество рисунков – 53 (без учета приложений). Магистерская диссертация состоит из введения, трех частей, заключения, библиографического списка, приложений. В теоретической части представлены основные понятия, основные подходы, модели, методы, проведен анализ талант-менеджмента как системы и процесса, концептуальной модели оценки потенциала сотрудников катеории hipo, включая модель оценки потенциала сотрудников «ЭКОПСИ». В практической части описывается общая характеристика исследуемой организации, проведены анализ и оценка персонала в «детском саду комбинированного вида», выявление сотрудников категории hipo на основе модели «ЭКОПСИ». В 3-й главе предложены рекомендации по внедрению техноогий работы с hipo путем разработки индивидуальных карьерных планов развития сотрудников и индивидуальных планов развития для hipo персонала. В заключении подведены итоги в соответствии с поставленными задачами.

Τhe purpose of this study is to investigate and analyze the influence of the possible errors eventually occurring in a 3D-US based HDR Brachytherapy of prostate cancer on the quality of dose delivery. The influence of modulation restriction tool on the plan quality and sensitivity is also investigated. Materials: Twelve clinical implants for HDR Brachytherapy of prostate cancer have been selected out of the clinical routine. The range of the prostate volumes was 26-101 cm3. Due to the fact that the implanted needles are fixed on the template, the most probable error should be a systematic shift of the implanted catheters on the cranial-caudal direction caused by the movement of the patient relative to the template. The planning was done using HIPO which is implemented in the real time intraoperative planning system Oncentra Prostate (OcP). HIPO offers a unique modulation restriction option that limits the free modulation of dwell times. Firstly the reference plans, where no catheter shift has been simulated, the clinical with MR >0 and the theoretical with MR=0, for all 12 implants have been compared. Then for each of the 12 clinical implants, 10 systematic shifts of the implanted catheters in the range of [-5, +5] mm in step of 1mm were simulated. The influence of this systematic shift on DVH-, COIN, EI and radiobiological parameters of PTV and OARs is calculated and recorded. The analysis of the observed changes has been done firstly by addressing the quality of the implant. For this purpose the range of shift was estimated that the resulted 3D dose distributions keep fulfilling the clinical dosimetric protocol. Secondly, the focus was placed to the stability of the dose distribution. Here the range for the shift has been estimated which enables that the dosimetric, conformity and radiobiological parameters of the implant remain within ±5% or ±10% of the originally planned values. Results: The use of modulation restriction (MR>0) results in plans with more conformal dose distribution (COIN, EI) but slightly lower D90 and V100 , gEUD, EUD2,v and EUD2,s values. The quality analysis demonstrate that for the DVH based parameters values of prostate a maximal shift of ±1.0 mm can be tolerated, although in case of using the modulation restriction the sensitivity from the influence of the systematic shift is greater. Similar were the results for the DVH parameters for urethra, rectum and bladder. For the stability analysis in order to keep the dosimetric parameters within ±5% of the originally planned value for the prostate and OARs, a maximum shift of around ±0.5 mm can be tolerated and for the ±10% criterion this is -1.0/+0.5 mm. The same behavior applies for the radiobiological parameters. The analysis based on COIN considering only the target and also the OARs have shown a maximum shift range of ±1.5 mm. For the EI analysis this range is ±0.0 mm. For ±10% criterion this is ±2.5 mm and ±0.5 mm respectively. Conclusion: Our study has demonstrated that high modulated, high conformal Brachytherapy dose distributions for prostate HDR implants are sensitive to systematic catheter shift. The consequence of shift changes is not clear. We can generally speak about a required geometrical stability of the implant as high as ±1.0mm. Modulation restriction without improving this reduces significantly the total dwell time keeping the plan quality and increasing conformity (COIN, EI). / Ο σκοπός αυτής της μελέτης είναι να ερευνήσουμε και να αναλύσουμε την επιρροή που μπορεί να έχουν τα πιθανά λάθη που συμβαίνουν στην Υψηλού Ρυθμού Δόσης (HDR) Βραχυθεραπεία του καρκίνου του προστάτη, η οποία βασίζεται σε τρισδιάστατες εικόνες (3D) υπερήχου, στη ποιότητα εναπόθεσης δόσης. Επίσης διερευνάται η επίδραση του Modulation Restriction (MR) στην ποιότητα και ευαισθησία του πλάνου θεραπείας. Υλικά και Μέθοδοι: Επιλέχθηκαν 12 κλινικά εμφυτεύματα για την HDR Βραχυθεραπεία του καρκίνου του προστάτη από την κλινική ρουτίνα μας. Το εύρος του όγκου του προστάτη είναι 26-101 cm3. Επειδή οι βελόνες που εμφυτεύθηκαν στον προστάτη είναι σταθεροποιημένες πάνω στο template, το πιο πιθανό λάθος που μπορεί να συμβεί είναι η συστηματική μετατόπιση των εμφυτευμένων καθετήρων σε cranial – caudal (κρανιακή – ουραία ) διεύθυνση η οποία έχει προκληθεί από την κίνηση του ασθενούς σε σχέση με το template. Το πλάνο θεραπείας έγινε χρησιμοποιώντας την επιλογή HIPO του προγράμματος real time intraoperative planning system Oncentra Prostate (OcP). Το HIPO προσφέρει την δυνατότητα επιλογής του Modulation Restriction (MR) το οποίο περιορίζει την ελεύθερη διαμόρφωση των χρόνων παραμονής της πηγής στους καθετήρες. Στα αρχικά μας πλάνα θεραπείας (reference plans) δεν έχει γίνει προσομοίωση μετακίνησης του καθετήρα. Συγκρίνουμε τα κλινικά μας πλάνα (MR>0) και τα θεωρητικά μας (MR=0) και για τα 12 εμφυτεύματα. Στην συνέχεια για κάθε ένα από τα 12 εμφυτεύματα γίνεται η προσομοίωση 10 συστηματικών μετακινήσεων των εμφυτευμένων καθετήρων με εύρος [-5,+5]mm και με βήμα 1mm. Υπολογίζεται και καταγράφεται η επίδραση της συστηματικής μετακίνησης στα ιστογράμματα δόσης - όγκου (DVH), δείκτη συμμορφίας (conformity index- COIN), External Index (EI) και στις ραδιοβιολογικές παραμέτρους για τον όγκο στόχου (PTV) και των ευαίσθητων σε κίνδυνο οργάνων (OARs). Αρχικά η ανάλυση των παρατηρούμενων αλλαγών έχει γίνει σύμφωνα με την ποιότητα του εμφυτεύματος (quality analysis). Για αυτό τον λόγο το εύρος της μετακίνησης έχει υπολογιστεί έτσι ώστε τα αποτελέσματα από τις 3D κατανομές δόσεις να πληρούν το κλινικό δοσιμετρικό μας πρωτόκολλο. Στην συνέχεια εστιάσαμε στην σταθερότητα της κατανομής της δόσης (stability analysis). Σε αυτή την περίπτωση το εύρος μετακίνησης των καθετήρων έχει υπολογιστεί έτσι ώστε οι τιμές των DVH, COIN και ραδιοβιολογικών παραμέτρων των εμφυτευμάτων να παραμένουν μέσα στο ±5% ή στο ±10% των αρχικών πλάνων (reference). Αποτελέσματα: Χρησιμοποιώντας την επιλογή του Modulation Restriction (MR>0) προκύπτουν πλάνα με πιο ομοιόμορφη κατανομή της δόσης (COIN, EI) αλλά με ελαφρώς μικρότερες τιμές των D90, V100, gEUD, EUD2,v και EUD2,s. H “quality analysis” έδειξε ότι για τις δοσιμετρικές παραμέτρους του προστάτη η μέγιστη μετατόπιση που μπορούμε να έχουμε είναι ±1mm. Χρησιμοποιώντας την επιλογή του MR η μετατόπιση αυτή γίνεται ακόμα πιο ευαίσθητη. Παρόμοια ήταν τα αποτελέσματα μας για τις δοσιμετρικές παραμέτρους των OARs (ουρήθρα, κύστη και ορθό). Σύμφωνα με την “stability analysis” η μέγιστη μετατόπιση που μας επιτρέπεται έτσι ώστε να διατηρήσουμε τις τιμές των δοσιμετρικών παραμέτρων του προστάτη και των OARs μέσα στο ±5% της τιμής του αρχικού μας πλάνου είναι ±0.5mm ενώ για το ±10% το όριο αυτό είναι -1.0/+0.5 mm. Την ίδια συμπεριφορά παρατηρούμε και για τις ραδιοβιολογικές παραμέτρους. Η ανάλυση που βασίζεται στο COIN, συμπεριλαμβάνοντας αρχικά μόνο τον στόχο μας και στην συνέχεια και τα OARs έδειξε ότι η μέγιστη μετακίνηση μας έχει εύρος ±1.5mm . Για την ανάλυση που βασίζεται στο EI αυτό το εύρος είναι ±0.0 mm . Για το ±10% τα όρια είναι ±2.5mm και 0.5mm αντίστοιχα. Συμπεράσματα: Η μελέτη μας έδειξε ότι οι υψηλά διαμορφωμένες και οι υψηλά ομοιόμορφες κατανομές δόσης των εμφυτευμάτων της HDR βραχυθεραπείας του προστάτη είναι ευαίσθητες στις συστηματικές μετακινήσεις των καθετήρων. Οι συνέπειες από τις αλλαγές αυτών των μετακινήσεων δεν είναι ξεκάθαρες. Μπορούμε γενικά να μιλήσουμε για μια απαιτούμενη γεωμετρική σταθερότητα του εμφυτεύματος τόσο υψηλή όσο ±1.0mm. Η δυνατότητα επιλογής του MR χωρίς να βελτιώνει αυτό, μειώνει σημαντικά τον ολικό χρόνο παραμονής της πηγής στους καθετήρες διατηρώντας την ποιότητα του πλάνου θεραπείας και αυξάνοντας την ομοιομορφία στην κατανομή της δόσης (COIN, EI).

A acidez do solo é um dos principais fatores limitantes à produção vegetal. A toxicidade por alumínio, que ocorre apenas a pH baixo, tem sido extensamente investigada, enquanto o estresse causado pelo pH baixo tem recebido pouca atenção. Os estudos nesta área quase sempre presumem efeitos aditivos, e portanto independentes, da toxicidade por Al3+ e H+. Este provavelmente não é o caso, sendo que o pH baixo pode ser um fator de predisposição das células ao Al3+. As evidências indicam que o pH baixo causa desarranjos na parede de células em crescimento, gerando estresse que pode comprometer a sua funcionalidade e integridade. É provável que a susceptibilidade a este estresse deve ser dependente da pressão de turgor. Por sua vez, o metabolismo oxidativo e a geração de espécies reativas de oxigênio (ROS) na parede celular podem modular a sua extensibilidade por romper ou criar ligações dentro ou entre cadeias de polissacarídeo. Há grande interesse em se conhecer se, à semelhança do que ocorre em leveduras, as células vegetais possuem um sistema de percepção e resposta a estresse da parede. Os pêlos radiculares em crescimento são sensíveis a pH baixo e estresse hipo-osmótico e constituem um bom modelo experimental para estes estudos. Os objetivos deste trabalho foram: a) Otimizar um sistema experimental para o estudo de pêlos radiculares de tomateiro (Solanum lycopersicum L. cv Micro-Tom); b) Avaliar as respostas dos pêlos radiculares ao estresse por pH baixo e hipo-osmolaridade; c) Examinar o papel da modulação oxidativa da parede celular nestas respostas; e d) Avaliar a resposta de diferentes mutantes hormonais de Micro-Tom a estes fatores de estresse. Os principais parâmetros avaliados foram a taxa de alongamento (µm.min-1) e a freqüência de rompimento dos pêlos. Tanto o estresse por pH baixo quanto choques hipo-osmóticos resultaram em taxas de alongamento significativamente diminuídos e o rompimento de pêlos radiculares, mas os efeitos dos tratamentos hipo-osmóticos foram mais marcantes. Uma curva de resposta frente à osmolaridade da solução externa revelou que a taxa de alongamento aumentou com a diminuição da osmolaridade até alcançar um limiar em que houve redução drástica da taxa de alongamento e começou-se a observar o rompimento de pêlos. Também se observou uma interação entre hipo-osmolaridade e pH baixo. O emprego do inibidor difenileno iodônio não forneceu evidências do envolvimento de NADPH oxidases da membrana plasmática na resposta de pêlos radiculares a choque hipo-osmótico ou pH baixo. Já no caso do inibidor ácido salicilhidroxâmico, encontrou-se evidências do envolvimento de peroxidases da parede. Nos mutantes hormonais dgt (pouco sensível a auxina) e epi (super produtor de etileno), mas não em not (deficiente em ácido abscísico), os pêlos radiculares apresentaram uma melhor resposta de ajustamento a choque hipo-osmótico do que Micro-Tom, reduzindo o alongamento e o rompimento dos pêlos. Este trabalho fornece fortes evidências de que os pêlos radiculares possuem um mecanismo de percepção e resposta a estresse da parede visando à manutenção de sua integridade e que apresentam bom potencial como sistema modelo nesta linha de pesquisa / Soil acidity is a major factor limiting plant growth worldwide. Aluminum toxicity, which occurs only at low pH, has been extensively studied, whereas low pH stress has received much less attention. Studies on Al3+ and H+ toxicity make the underlying assumption that the effects of these stress factors are additive, and, therefore independent of each other. However, this is most likely not the case and low pH may be a factor which increases susceptibility to further injury by Al3+. There is evidence that low pH causes disruption in cell wall structure of growing cells, which might jeopardize cell wall functionality and integrity. It is likely that turgor pressure plays an important role in cell wall stress caused by low pH. The apoplastic metabolism of reactive oxygen species (ROS) can modulate cell wall extensibility by making or breaking bonds within and between cell wall polysaccharides. A major question is whether, similarly to yeast, plant cells have a cell wall integrity signaling and response system. Growing root hairs are sensitive to low pH and hypo-osmotic stress and are potentially good experimental systems for such investigations. The objectives of this study were: a) Optimize an experimental system to examine tomato (Solanum lycopersicum L. cv Micro-Tom) root hairs; b) Examine the response of root hairs to low pH and hypo-osmotic stress; c) Examine the role of oxidative modulation of the cell wall in these responses; and d) Evaluate the response of different hormonal mutants of Micro-Tom to these stress factors. Root hair elongation rates (µm.min-1) and the frequency of cell bursting were the major experimental parameters which were evaluated. Both low pH and, more markedly, hypo-osmotic stress caused significant reductions in elongation rates and the bursting of root hair tips. In a response curve to varying osmolarities of the external medium, root hair elongation rates increased with decreasing osmolarities until a threshold was reached and elongation rates decreased drastically and the bursting of root hairs began to be observed. Interactions between low pH and hypo-osmolarity were observed. The use of the inhibitor diphenylene iodonium (DPI) did not provide evidence for the involvement of plasma membrane NADPH in the response of root hairs to low pH and hypo-osmotic shock. However, a role for cell wall peroxidases was provided by use of the inhibitor salicylhydroxamic acid (SHAM). Root hairs of the hormonal mutants dgt (low sensitivity to auxin) and epi (ethylene super producer), but not not (deficient in abscisic acid), displayed a more effective response to hypo-osmotic shock than Micro-Tom, by decreasing elongation rates and cell bursting to a greater degree. This study provides strong evidence to suggest that root hairs have a cell wall integrity response system and that root hairs are potentially good cell model systems for such research

A acidez do solo é um dos principais fatores limitantes à produção vegetal. A toxicidade por alumínio, que ocorre apenas a pH baixo, tem sido extensamente investigada, enquanto o estresse causado pelo pH baixo tem recebido pouca atenção. Os estudos nesta área quase sempre presumem efeitos aditivos, e portanto independentes, da toxicidade por Al3+ e H+. Este provavelmente não é o caso, sendo que o pH baixo pode ser um fator de predisposição das células ao Al3+. As evidências indicam que o pH baixo causa desarranjos na parede de células em crescimento, gerando estresse que pode comprometer a sua funcionalidade e integridade. É provável que a susceptibilidade a este estresse deve ser dependente da pressão de turgor. Por sua vez, o metabolismo oxidativo e a geração de espécies reativas de oxigênio (ROS) na parede celular podem modular a sua extensibilidade por romper ou criar ligações dentro ou entre cadeias de polissacarídeo. Há grande interesse em se conhecer se, à semelhança do que ocorre em leveduras, as células vegetais possuem um sistema de percepção e resposta a estresse da parede. Os pêlos radiculares em crescimento são sensíveis a pH baixo e estresse hipo-osmótico e constituem um bom modelo experimental para estes estudos. Os objetivos deste trabalho foram: a) Otimizar um sistema experimental para o estudo de pêlos radiculares de tomateiro (Solanum lycopersicum L. cv Micro-Tom); b) Avaliar as respostas dos pêlos radiculares ao estresse por pH baixo e hipo-osmolaridade; c) Examinar o papel da modulação oxidativa da parede celular nestas respostas; e d) Avaliar a resposta de diferentes mutantes hormonais de Micro-Tom a estes fatores de estresse. Os principais parâmetros avaliados foram a taxa de alongamento (µm.min-1) e a freqüência de rompimento dos pêlos. Tanto o estresse por pH baixo quanto choques hipo-osmóticos resultaram em taxas de alongamento significativamente diminuídos e o rompimento de pêlos radiculares, mas os efeitos dos tratamentos hipo-osmóticos foram mais marcantes. Uma curva de resposta frente à osmolaridade da solução externa revelou que a taxa de alongamento aumentou com a diminuição da osmolaridade até alcançar um limiar em que houve redução drástica da taxa de alongamento e começou-se a observar o rompimento de pêlos. Também se observou uma interação entre hipo-osmolaridade e pH baixo. O emprego do inibidor difenileno iodônio não forneceu evidências do envolvimento de NADPH oxidases da membrana plasmática na resposta de pêlos radiculares a choque hipo-osmótico ou pH baixo. Já no caso do inibidor ácido salicilhidroxâmico, encontrou-se evidências do envolvimento de peroxidases da parede. Nos mutantes hormonais dgt (pouco sensível a auxina) e epi (super produtor de etileno), mas não em not (deficiente em ácido abscísico), os pêlos radiculares apresentaram uma melhor resposta de ajustamento a choque hipo-osmótico do que Micro-Tom, reduzindo o alongamento e o rompimento dos pêlos. Este trabalho fornece fortes evidências de que os pêlos radiculares possuem um mecanismo de percepção e resposta a estresse da parede visando à manutenção de sua integridade e que apresentam bom potencial como sistema modelo nesta linha de pesquisa / Soil acidity is a major factor limiting plant growth worldwide. Aluminum toxicity, which occurs only at low pH, has been extensively studied, whereas low pH stress has received much less attention. Studies on Al3+ and H+ toxicity make the underlying assumption that the effects of these stress factors are additive, and, therefore independent of each other. However, this is most likely not the case and low pH may be a factor which increases susceptibility to further injury by Al3+. There is evidence that low pH causes disruption in cell wall structure of growing cells, which might jeopardize cell wall functionality and integrity. It is likely that turgor pressure plays an important role in cell wall stress caused by low pH. The apoplastic metabolism of reactive oxygen species (ROS) can modulate cell wall extensibility by making or breaking bonds within and between cell wall polysaccharides. A major question is whether, similarly to yeast, plant cells have a cell wall integrity signaling and response system. Growing root hairs are sensitive to low pH and hypo-osmotic stress and are potentially good experimental systems for such investigations. The objectives of this study were: a) Optimize an experimental system to examine tomato (Solanum lycopersicum L. cv Micro-Tom) root hairs; b) Examine the response of root hairs to low pH and hypo-osmotic stress; c) Examine the role of oxidative modulation of the cell wall in these responses; and d) Evaluate the response of different hormonal mutants of Micro-Tom to these stress factors. Root hair elongation rates (µm.min-1) and the frequency of cell bursting were the major experimental parameters which were evaluated. Both low pH and, more markedly, hypo-osmotic stress caused significant reductions in elongation rates and the bursting of root hair tips. In a response curve to varying osmolarities of the external medium, root hair elongation rates increased with decreasing osmolarities until a threshold was reached and elongation rates decreased drastically and the bursting of root hairs began to be observed. Interactions between low pH and hypo-osmolarity were observed. The use of the inhibitor diphenylene iodonium (DPI) did not provide evidence for the involvement of plasma membrane NADPH in the response of root hairs to low pH and hypo-osmotic shock. However, a role for cell wall peroxidases was provided by use of the inhibitor salicylhydroxamic acid (SHAM). Root hairs of the hormonal mutants dgt (low sensitivity to auxin) and epi (ethylene super producer), but not not (deficient in abscisic acid), displayed a more effective response to hypo-osmotic shock than Micro-Tom, by decreasing elongation rates and cell bursting to a greater degree. This study provides strong evidence to suggest that root hairs have a cell wall integrity response system and that root hairs are potentially good cell model systems for such research

A acidez do solo é um dos principais fatores limitantes à produção vegetal. Apesar da toxicidade por alumínio ter sido extensamente investigada, pouca atenção tem sido dada ao estresse causado pelo baixo pH em si. Existem diferenças marcantes entre células quanto à sensibilidade ao pH baixo que dependem do seu estado de crescimento e desenvolvimento celular e que devem ser exploradas para se entender o que determina a sensibilidade e tolerância a pH baixo. Em alguns casos, a suscetibilidade a pH baixo está relacionada a desarranjos na parede de células em crescimento, chegando a causar o rompimento da célula, como já foi demonstrado em pêlos radiculares em expansão. Por outro lado, o metabolismo oxidativo e a geração de espécies reativas de oxigênio (ROS) na parede podem influenciar neste processo por romper ou criar ligações dentro ou entre cadeias de polissacarídeos, modulando assim a extensibilidade da parede celular. Em células de tabaco (Nicotiana tabacum) cv. BY-2, há um aumento acentuado na sensibilidade ao pH baixo no final da fase lag da cultura, que ocorrre entre 12 e 24 h de cultivo. Os objetivos deste trabalho foram: a) Investigar se a mudança na sensibilidade pH baixo ocorre durante a retomada do ciclo celular e determinar, com o uso de inibidores do ciclo celular, o período do ciclo em que isto ocorre; b) verificar se o aumento da sensibilidade a pH baixo está relacionado com a expansão celular ou com alterações no potencial osmótico da célula; c) examinar o efeito da aplicação de H2O2 ou ascorbato sobre a resposta de células sensíveis a pH baixo; d) testar a hipótese de que a sensibilidade a pH baixo pode ser revertida por meio de um choque hipo-osmótico prévio; e) avaliar o possível papel da modulação oxidativa da parede celular na reversão de sensibilidade das células a pH baixo expostas ao choque hipo-osmótico. A retomada do ciclo celular é necessária para que ocorra a alteração de sensibilidade a pH baixo, pois a remoção de auxina (2,4-D) ou a adição de bloqueadores de canais de K+ impediu ou atrasou, respectivamente, a alteração na sensibilidade a pH baixo. O uso de inibidores do ciclo celular demonstrou que as células de BY-2 se tornam mais sensíveis a pH baixo durante o final da fase G1 mas antes do ponto de checagem da transição G1/S do ciclo celular. A aplicação de H2O2, diminuiu a suscetibilidade das células a pH baixo, ao contrário da aplicação de ascorbato. Foi demonstrado que a aplicação prévia de tratamento hipo-osmótico por 60 min reverteu a sensibilidade de células a pH baixo. A aplicação de inibidores de NAPDH oxidase da membrana plasmática e de peroxidases revelou a participação destas enzimas na reversão de sensibilidade das células a pH baixo, indicando a possibilidade de geração de ROS e de modulação oxidativa da parede. Embora já tenha sido descrito que ocorre uma explosão oxidativa com choque hipo-osmótico, ainda não havia sido demonstrado a conseqüência disto. Este trabalho fornece indícios de que uma explosão oxidativa poderia modificar a parede tornando-a mais resistente e a célula menos suscetível a pH baixo / Soil acidity is a major factor limiting plant growth worldwide. Although aluminum toxicity, which occurs only at low pH, has been extensively studied, little attention has been given to stress caused by low pH. There are marked differences in the sensitivity of cells to low pH which are contingent on the growth and developmental stage of the cells. These differences should be explored to further the understanding of the factors governing sensitivity and tolerance to low pH. In at least some cases, the susceptibility of cells to low pH is related to derangements in the wall of growing cells, which can cause ruptures or bursting of the cells, as has been clearly demonstrated in expanding root hairs. On the other hand, the oxidative metabolism and generation of reactive oxygen species (ROS) can modulate cell wall extensibility by breaking or making bonds within and between cell wall polymers. In tobacco (Nicotiana tabacum) cv. BY-2 cells, there is a sharp increase in sensitivity to low pH at the end of the lag phase of the cell culture, which occurs between 12 and 24 h of subculture. The objectives of this study were: a) determine if the changes in sensitivity to low pH occurred during the restart of the cell cycle and, by employing cell cycle inhibitors, at which points of the cycle does this occur; b) examine if the changes in sensitivity to low pH are related to cell expansion or changes in osmotic potential of the cell; c) examine how the application of H2O2 or ascorbate affects the response of cells to low pH; d) test the hypothesis that sensitivity of cells to low pH can be reverted by the previous application of a hypo-osmotic shock; e) evaluate the possible role of oxidative modulation of the cell wall in hypo-osmotic-induced reversal of the sensitivity of cells to low pH. The restart of the cell cycle was shown to be necessary for the change in sensitivity to low pH occur, since the absence of auxin (2,4-D) or the addition of K+ channel blockers prevented or delayed this change, respectively. The use of cell cycle inhibitors demonstrated that BY-2 cells become sensitive to low pH at the end of G1 but before the G1/S transition restriction point of the cell cycle. Exogenous H2O2, but not ascorbate, reduced the effect of low pH on sensitive cells. Sensitive cells submitted to 60 min hypo-osmotic treatment became insensitive to low pH. This reversal of sensitivity depended on the activity of plasma membrane NADPH oxidase and peroxidase, as evidenced by the use of DPI and SHAM, inhibitors of these enzymes, respectively. This suggests that ROS is generated and that oxidative modifications of the cell wall occur. Although hypo-osmotic treatments have been shown to generate an oxidative burst, its purpose or implication has not yet been shown. This study provides evidence that an oxidative burst might modify and strengthen the cell wall, making cells less susceptible to low pH

A acidez do solo é um dos principais fatores limitantes à produção vegetal. Apesar da toxicidade por alumínio ter sido extensamente investigada, pouca atenção tem sido dada ao estresse causado pelo baixo pH em si. Existem diferenças marcantes entre células quanto à sensibilidade ao pH baixo que dependem do seu estado de crescimento e desenvolvimento celular e que devem ser exploradas para se entender o que determina a sensibilidade e tolerância a pH baixo. Em alguns casos, a suscetibilidade a pH baixo está relacionada a desarranjos na parede de células em crescimento, chegando a causar o rompimento da célula, como já foi demonstrado em pêlos radiculares em expansão. Por outro lado, o metabolismo oxidativo e a geração de espécies reativas de oxigênio (ROS) na parede podem influenciar neste processo por romper ou criar ligações dentro ou entre cadeias de polissacarídeos, modulando assim a extensibilidade da parede celular. Em células de tabaco (Nicotiana tabacum) cv. BY-2, há um aumento acentuado na sensibilidade ao pH baixo no final da fase lag da cultura, que ocorrre entre 12 e 24 h de cultivo. Os objetivos deste trabalho foram: a) Investigar se a mudança na sensibilidade pH baixo ocorre durante a retomada do ciclo celular e determinar, com o uso de inibidores do ciclo celular, o período do ciclo em que isto ocorre; b) verificar se o aumento da sensibilidade a pH baixo está relacionado com a expansão celular ou com alterações no potencial osmótico da célula; c) examinar o efeito da aplicação de H2O2 ou ascorbato sobre a resposta de células sensíveis a pH baixo; d) testar a hipótese de que a sensibilidade a pH baixo pode ser revertida por meio de um choque hipo-osmótico prévio; e) avaliar o possível papel da modulação oxidativa da parede celular na reversão de sensibilidade das células a pH baixo expostas ao choque hipo-osmótico. A retomada do ciclo celular é necessária para que ocorra a alteração de sensibilidade a pH baixo, pois a remoção de auxina (2,4-D) ou a adição de bloqueadores de canais de K+ impediu ou atrasou, respectivamente, a alteração na sensibilidade a pH baixo. O uso de inibidores do ciclo celular demonstrou que as células de BY-2 se tornam mais sensíveis a pH baixo durante o final da fase G1 mas antes do ponto de checagem da transição G1/S do ciclo celular. A aplicação de H2O2, diminuiu a suscetibilidade das células a pH baixo, ao contrário da aplicação de ascorbato. Foi demonstrado que a aplicação prévia de tratamento hipo-osmótico por 60 min reverteu a sensibilidade de células a pH baixo. A aplicação de inibidores de NAPDH oxidase da membrana plasmática e de peroxidases revelou a participação destas enzimas na reversão de sensibilidade das células a pH baixo, indicando a possibilidade de geração de ROS e de modulação oxidativa da parede. Embora já tenha sido descrito que ocorre uma explosão oxidativa com choque hipo-osmótico, ainda não havia sido demonstrado a conseqüência disto. Este trabalho fornece indícios de que uma explosão oxidativa poderia modificar a parede tornando-a mais resistente e a célula menos suscetível a pH baixo / Soil acidity is a major factor limiting plant growth worldwide. Although aluminum toxicity, which occurs only at low pH, has been extensively studied, little attention has been given to stress caused by low pH. There are marked differences in the sensitivity of cells to low pH which are contingent on the growth and developmental stage of the cells. These differences should be explored to further the understanding of the factors governing sensitivity and tolerance to low pH. In at least some cases, the susceptibility of cells to low pH is related to derangements in the wall of growing cells, which can cause ruptures or bursting of the cells, as has been clearly demonstrated in expanding root hairs. On the other hand, the oxidative metabolism and generation of reactive oxygen species (ROS) can modulate cell wall extensibility by breaking or making bonds within and between cell wall polymers. In tobacco (Nicotiana tabacum) cv. BY-2 cells, there is a sharp increase in sensitivity to low pH at the end of the lag phase of the cell culture, which occurs between 12 and 24 h of subculture. The objectives of this study were: a) determine if the changes in sensitivity to low pH occurred during the restart of the cell cycle and, by employing cell cycle inhibitors, at which points of the cycle does this occur; b) examine if the changes in sensitivity to low pH are related to cell expansion or changes in osmotic potential of the cell; c) examine how the application of H2O2 or ascorbate affects the response of cells to low pH; d) test the hypothesis that sensitivity of cells to low pH can be reverted by the previous application of a hypo-osmotic shock; e) evaluate the possible role of oxidative modulation of the cell wall in hypo-osmotic-induced reversal of the sensitivity of cells to low pH. The restart of the cell cycle was shown to be necessary for the change in sensitivity to low pH occur, since the absence of auxin (2,4-D) or the addition of K+ channel blockers prevented or delayed this change, respectively. The use of cell cycle inhibitors demonstrated that BY-2 cells become sensitive to low pH at the end of G1 but before the G1/S transition restriction point of the cell cycle. Exogenous H2O2, but not ascorbate, reduced the effect of low pH on sensitive cells. Sensitive cells submitted to 60 min hypo-osmotic treatment became insensitive to low pH. This reversal of sensitivity depended on the activity of plasma membrane NADPH oxidase and peroxidase, as evidenced by the use of DPI and SHAM, inhibitors of these enzymes, respectively. This suggests that ROS is generated and that oxidative modifications of the cell wall occur. Although hypo-osmotic treatments have been shown to generate an oxidative burst, its purpose or implication has not yet been shown. This study provides evidence that an oxidative burst might modify and strengthen the cell wall, making cells less susceptible to low pH