Redox regulation and homeostasis are critically important in the regulation of cell function; however, there are significant challenges in quantitatively measuring and monitoring intracellular redox potentials. The work in this thesis details a novel approach to intracellular redox monitoring. The approach is based on the use of nanosensors, which comprise molecules capable of sensing the local redox potential, assembled on gold nanoshells. Since the Raman spectra of the sensor molecules change depending on their oxidation state, and since the nanoshells allow a large enhancement of the Raman scattering, intracellular potential can be calculated by simple optical measurements. A full description of the design, fabrication and characterisation (spectroscopic and electrochemical) of the nanosensors is provided within. The ability to deliver nanosensors into cells in a controllable fashion was confirmed using electron microscopy. Results from a range of assays are also presented which reveal that introduction of nanosensors does not result in any cytotoxicity. Sensor utility in monitoring redox potentials as cells responded to physiological and superphysiological oxidative and reductive stimuli was investigated. Importantly, the capability of the nanosensors in monitoring intracellular potentials in a reversible, non-invasive manner, and over a previously unattainable potential range, is demonstrated.

Thesis (MCur)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Motivation. Homeostasis is a dynamic and balanced process that must be maintained in order to for health to be sustained (Scales & Pilsworth, 2008:50-57). In critically illness, homeostasis is disrupted and along with inadequate tissue perfusion potentially leads to multiple organ failure (Elliot, Aitken & Chaboyer, 2007:437). The fluid balance of a patient is essential for preserving homeostasis and to maintain optimal tissue perfusion, thus monitoring fluid balance plays an important role in the managing a critically ill patient. Current literature and best nursing practice emphasise the importance of accurate and correct fluid balance monitoring in critically ill patients including recording fluid intake and output on a purpose designed fluid balance sheet. Research has shown that the patient’s outcome after critical illness is influenced by the fluid balance management including fluid balance monitoring (Vincent, Sakr, Sprung, Ranieri, Reinhart, Gerlach, Moreno, Carlet, Le Gall & Payen, 2006:344-353), while several studies have questioned accuracy of fluid balance calculation in various acute care settings (Johnson & Monkhouse, 2009:291; Smith, Fraser, Plowright, Dennington, Seymour, Oliver & MacLellan, 2008:28-29). In an informal audit performed in a local critical care unit, seven out of ten fluid balances were incorrectly calculated. Clinical experience of nurses’ inattention to fluid balance monitoring, together with the informal audit data, reveals that fluid balance monitoring is generally not performed correctly or accurately by nurses working in critical care units. The aim of the study was to describe the perspectives and practices of registered nurses in critical care units with regard to fluid balance monitoring. Methods. A quantitative approach in the form of an audit was applied to establish the current practice of fluid balance monitoring. A survey was conducted among registered nurses to gain insight into their perspectives and knowledge of fluid balance monitoring. The sample for the audit was drawn from fluid balance records, which met the study inclusion criteria. The survey was conducted with a sample of participants from registered nurses in critical care units from a particular hospital group, in compliance with the inclusion criteria. The researcher collected the data using a purpose designed audit tool and questionnaire. Results. The audit revealed that 90 % of the sampled fluid balance records were inaccurate (tolerated deviation 0-10ml) and 79% were inaccurate if a deviation of 50ml would be tolerated. Furthermore the inaccuracy in calculation was larger in patients whoreceived diuretics. The questionnaire data revealed that registered nurses considered fluid balance monitoring as an important part of patient nursing care and were aware that inaccuracy can pose a risk to the patient. The nurses feel responsible for performing fluid balance monitoring. In addition the nurses gave recommendations for the practice. Discussion. The results of this study are similar to other studies done internationally. The nurses are aware of the importance of the fluid balance, and recognise the inaccuracies. With our limited resources, both financial and in terms of nursing staff, the solutions have to be very basic and practical. Key words: fluid balance, critical care, accuracy and auditing, best practice / AFRIKAANSE OPSOMMING: Motivering. Homeostase is ’n dinamiese en gebalanseerde proses wat onderhou moet word vir gesondheid om handhaaf te word (Scales & Pilsworth, 2008:50-57). Onder toestande van kritieke siekte, word homeostase onderbreek en kan dit saam met onvoldoende weefselperfusie moontlik tot veelvuldige orgaanmislukking lei (Elliot, Aitken & Chaboyer, 2007:437). Die vloeistofbalans van ’n pasiënt is van die uiterste belang vir die preservering van homeostase en om optimale weefselperfusie te onderhou, en dus speel die monitering van vloeistofbalans ’n belangrike rol in die bestuur van die pasiënt wat kritiek siek is. Die huidige literatuur en beste verpleegkundige praktyk beklemtoon die belangrikheid van akkurate en korrekte vloeistofbalansmonitering in pasiënte wat kritiek siek is, insluitend die aantekening van vloeistofinname en -afskeiding op ’n vorm wat vir die doel pasgemaak is. Navorsing het getoon dat die pasiënt se uitkoms ná kritiese siekte deur vloeistofbalansbestuur, insluitend vloeistofbalansmonitering, beïnvloed word (Vincent, Sakr, Sprung, Ranieri, Reinhart, Gerlach, Moreno, Carlet, Le Gall & Payen, 2006:344-353), terwyl verskeie studies die akkuraatheid van die vloeistofbalansberekening in ’n verskeidenheid kritiekesorgeenhede bevraagteken het (Johnson & Monkhouse, 2009:291; Smith, Fraser, Plowright, Dennington, Seymour, Oliver & MacLellan, 2008:28-29). In ’n informele oudit wat in ’n plaaslike kritiekesorgeenheid uitgevoer is, is daar gevind dat sewe uit tien vloeistofbalanse verkeerdelik bereken is. Kliniese ervaring van verpleërs se agtelosigheid met betrekking tot vloeistofbalansmonitering, tesame met die data vanuit die informele oudit, wys dat vloeistofbalansmonitering oor die algemeen nie korrek of akkuraat deur verpleërs in die kritiekesorgeenheid uitgevoer word nie. Die doelwit van hierdie studie was om die perspektiewe en praktyke van geregistreerde verpleërs in kritiekesorgeenhede met betrekking tot vloeistofbalansmonitering te beskryf. Metodes. ’n Kwantitatiewe benadering in die vorm van ’n oudit is gebruik om die huidige praktyk van vloeistofbalansmonitering te bepaal. ’n Opname is onder geregistreerde verpleërs gedoen om insig te bekom oor hulle perspektiewe oor en kennis van vloeistofbalansmonitering. Die steekproef vir die oudit is geneem uit vloeistofbalansrekords wat aan die studiekriteria voldoen het. Die opname is gedoen onder ’n steekproef van geregistreerde verpleërs in ’n kritiekesorgeenheid van ’n spesifieke hospitaalgroep, in ooreenstemming met die insluitingskriteria. Die navorser het die data met ’n pasgemaakte ouditinstrument en vraelys versamel. Resultate. Die oudit het gewys dat 90% van die vloeistofbalansrekords in die steekproef onakkuraat was (toleransie verskil 0-50ml) en 79% was onakkuraat als een verskil van 50 ml was tolereer. Verder was die onakkuraatheid in die berekenings groter in pasiënte wat urineermiddels ontvang het. Die data vanaf die vraelys het gewys dat geregistreerde verpleërs vloeistofbalansmonitering as ’n belangrike deel van die verpleging van ’n pasiënt beskou en daarvan bewus is dat onakkuraatheid ’n risiko vir die pasiënt kan inhou. Die verpleërs voel daarvoor verantwoordelik om die vloeistofbalansmonitering uit te voer. Hulle het ook aanbevelings vir die praktyk gemaak. Bespreking. Die resultate van hierdie studie is baie soortgelyk aan dié van ander internasionale studies. Die verpleërs is bewus van die belangrikheid van die vloeistofbalans en is bewus van die onakkuraathede. Met ons beperkte hulpbronne, beide finansieel en in terme van verpleegpersoneel, is dit noodsaaklik dat die oplossings baie basies en prakties is. Sleutelwoorde: vloeistofbalans, kritieke sorg, akkuraatheid en ouditering, beste praktyk

Thesis (PhD (Genetics))--University of Stellenbosch, 2010. / Dissertation presented for the degree of Doctor of Philosophy Stellenbosch University. / ENGLISH ABSTRACT: Phosphorus (P) is essential for the survival of all living organisms and forms part of several key biological molecules and processes. The basic biological function of all cells depends on the availability of P as structural element in phospholipids and nucleic acids. P plays a central role in the energy metabolism of the cell by activating metabolic intermediates of carbohydrate metabolism and by acting as an energy currency in the form of adenosine tri-phosphate (ATP). ATP is produced during photosynthesis from the energy derived from sunlight, probably the most important biological process on earth. The balance of P supply and demand is of critical importance here. Plants assimilate P in the form of orthophosphate (Pi) via its roots and utilises complex mechanisms to redistribute and balance the Pi concentrations throughout the plant. These processes are collectively known as phosphate homeostasis and in this study we utilised molecular techniques to study some key aspects of this complex network of mechanisms in the plant Arabidopsis thaliana. When the role of the PHT1;5 Pi transporter was investigated in photosynthesis under Pi limitation a new mechanism utilised by plants to supply Pi for the production of ATP in the chloroplast was discovered. During periods of adequate Pi supply plants make use of the triose phosphate / phosphate translocator (TPT) to exchange Pi for phosphorylated carbon intermediates. This transporter does, however, not function at the low Pi concentrations present during Pi limitation and the plant therefore express an alternative transporter i.e. PHT1;5. Together with this transporter several genes were identified that was expressed to allow the export of carbon intermediates from the chloroplast via starch turnover. Amongst these, several alternative isoforms of the enzymes responsible for starch turnover are expressed during Pi limiting conditions. It is therefore suggested that the products of starch degradation, e.g. glucose and maltose are the potential candidates for carbon export from chloroplasts under Pi limiting conditions. In an attempt to perturb the Pi concentrations in the Arabidopsis vacuole we expressed the three genes of a newly discovered polyphosphate (PolyP) polymerase from the yeast Sacharomyces cerevisiae in Arabidopsis. This enzyme complex accumulates PolyP in the yeast vacuole and since the plant vacuole is playing a key role in buffering Pi concentrations we anticipated some observable effects that could lead to the elucidation of the mechanisms involved. Production of PolyP was conclusively shown in plant callus, but it was only at very low concentrations with no detectable perturbing effect and undetectable in whole plants. With the aim to apply this technology to the PolyP and PHT1;5 lines developed in the other parts of this study, newly developed fluorescent indicator protein nanosensors (FLIPPi) were evaluated as a method for detecting and monitoring in vivo Pi concentrations in multicellular plant organs. This technique is capable of detecting changes in metabolite concentrations in real-time and it was applied to the roots of Arabidopsis seedlings subjected to Pi limitation. We specifically looked at changes in the cytosol, but our results revealed no detectable changes occurring in the Pi concentrations in this compartment. This was interpreted to indicate lower levels of Pi in this compartment as was previously expected. / AFRIKAANSE OPSOMMING: Fosfaat (P) is essensieël vir die oorlewing van alle organismes en maak deel uit van etlike kern biologiese prosesse en molekules. Die basiese biologiese funksionering van alle selle hang direk af van die beskikbaarheid van P as strukturele element van fosfolipiede en nuklëinsure. Fosfaat speel 'n sentrale rol in die energie metabolisme van 'n sel deur metaboliese intermediante te aktiveer en deur op te tree as die geld eenheid van sellulere energie in die vorm van adenosien tri-fosfaat (ATP). ATP word gegenereer gedurende fotosintese vanaf die energie wat van sonlig vasgevang word, dit is waarskeinlik die belangrikste biologiese proses op aarde. Dit is van kritiese belang dat die fosfaat vraag en aanbod hier fyn gebalanseer word. Plante assimileer P in die vorm van ortofosfaat (Pi) deur hulle wortels en maak gebruik van komplekse meganismes om Pi deur die plant te versprei en konsentrasies te balanseer. Hierdie prosesse staan gesamentlik bekend as fosfaat homeostase en in die huidige studie het ons gebruik gemaak van molekulêre tegnieke om 'n paar belangrike aspekte van hierdie komplekse netwerk van prosesse in die plant Arabidopsis thaliana te bestudeer. Toe die rol van die PHT1;5 Pi transporter in fotosintese onder toestande van Pi tekort bestudeer is, is 'n nuwe meganisme ontdek waarmee plante Pi verskaf aan chloroplaste vir die proses van fotosintese onder toestande van Pi tekort. Gedurende periodes wat die plant genoegsame Pi tot sy beskikking het, word van die triose fosfaat / fosfaat uitruiler (TPT) gebruik gemaak om Pi uit te ruil vir gefosforileerde koolstof metaboliete. Hierdie transporter kan egter nie onder die lae Pi konsentrasies wat voorkom in die sitoplasma onder Pi tekort toestande funksioneer nie, en gevolglik moet die plant van 'n alternatiewe transporter naamlik PHT1;5 uitdruk. Verskeie ander gene is ook geidentifiseer wat saam met hierdie transporter onder toestande van Pi tekort uitgedruk word en die plant toelaat om koolstof tussengangers uit die chloroplaste uit te vervoer via die proses van stysel produksie en afbraak. Onderandere is verskeie alternatiewe isoforme van die gene wat verandwoordelik is vir stysel produksie en afbraak identifiseer wat uitgedruk word onder toestande van Pi tekort. In 'n poging om die Pi konsentrasies in die Arabidopsis vakuool te versteur is drie gene van die nuut ontdekte polifosfaat (PolyP) polimerase kompleks van die gis Sacharomyces cerevisiae in Arabidopsis uitgedruk. Hierdie ensiem kompleks is verandwoordelik vir die akkumulasie van PolyP in die gis vakuool en siende die plant vakuool 'n kern rol speel in die buffering van Pi konsentrasies in die plant, het ons sekere waarneembare gevolge verwag wat kon lei tot die ontrafeling van die meganismes hierby betrokke. Die produksie van PolyP in plant kallus is duidelik gedemonstreer, maar dit was slegs teen baie lae konsentrasies met geen waarneembare versteuringseffek nie, en kon glad nie in heel plante waargeneem word nie. Met die oog daarop om hierdie tegnologie toe te pas op die bestudering van die PolyP en PHT1;5 lyne wat in die ander dele van hierdie studie ontwikkel is, is 'n nuut ontwikkelde fluoresente indikator protein nanosensor (FLIPPi) tegnologie evalueer as 'n metode om Pi konsentrasies in vivo in multisellulere plant organe waar te neem en te monitor. Hierdie tegniek is in staat daartoe om veranderinge in Pi konsentrasies in selle direk te monitor en is gevolglik op die wortels van Arabidopsis saailinge onder Pi tekort toestande toegepas. Daar is spesifiek na veranderinge in die sitosol gekyk, maar ons resultate kon geen waarneembare veranderinge in Pi konsentrasies in hierdie kompartement uitwys nie. Hierdie resultaat beteken waarkeinlik dat die Pi konsentrasies in hierdie kompartement waarskeinlik baie laer is as wat voorheen verwag is.

KIT receptor (CD117) is a receptor tyrosine kinase crucial for homeostasis of mast cells (MCs) in tissues and recruitment to sites of inflammation and tumors in response to its ligand Stem cell factor (SCF). Gain of function mutations in KIT (e.g. D816V) are frequently observed in systemic mastocytosis and other cancer types. Src Homology 2 domain containing phosphatase-2 (SHP2 or PTPN11) is a protein tyrosine phosphatase that promotes cell proliferation, survival and motility in multiple pathways and cell types. To study SHP2 function in MCs, we generated novel MC-specific Shp2 knock-out (KO) mice (MC-shp2 KO). These mice had reduced numbers of MCs in skin and peritoneum, and defective contact hypersensitivity responses compared to control mice, consistent with SHP2 promoting MC homeostasis. Using an inducible SHP2 KO bone marrow-derived MC (BMMC) culture model, we found that SHP2 KO cells were prone to apoptosis and had no MC repopulating activity in vivo. Mechanistically, SHP2 enhanced ERK activation and downregulation of pro-apoptotic protein Bim. SHP2 KO BMMCs also had defects in chemotaxis towards SCF, due to impaired activation of a Lyn/Vav/Rac pathway in SHP2 KO BMMCs. This correlated with defects in cell spreading, and F-actin polymerization in response to SCF. Treatment of BMMCs with a SHP2 inhibitor (II-B08) also led to reduced chemotaxis, consistent with SHP2 phosphatase activity being required for KIT-induced chemotaxis. Lastly, we tested whether SHP2 regulates oncogenic KIT signaling using a P815 mouse mastocytoma model. Stable silencing of SHP2 in P815 cells led to reduced cell growth and survival in vitro, and less aggressive systemic mastocytosis development in syngeneic mice. Overall, these studies identify SHP2 as a key node in SCF/KIT and oncogenic KIT pathways, and as a potential therapeutic target in several human diseases. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2013-06-25 12:03:57.818

Calcium is a highly versatile intracellular signal molecule that can regulate numerous different cellular functions. In filamentous fungi there is evidence for it being involved in regulating various processes, including spore germination, hyphal tip growth, hyphal branching and conidiation. During colony initiation in the filamentous fungus Neurospora crassa, conidia form germ tubes which are involved in colony establishment, and conidial anastomosis tubes (CATs) which are involved in generating fused networks of conidial germlings. The primary research aim of this thesis was to analyze the role of Ca2+-signalling and homeostasis during colony initiation in N. crassa. Removal of Ca2+ from the growth medium showed that external Ca2+ was necessary for CAT fusion and, more specifically, was required for CAT chemoattraction. Two L-type Ca2+ channel blockers (verapamil and diltiazem) with different modes of action were found to inhibit both conidial germination and CAT fusion in wild type strains and CAT fusion was shown to be more sensitive to these two drugs. These channel blockers were additionally found to inhibit Ca2+ uptake by conidial germlings of the wild type expressing the aequorin Ca2+ reporter. However, the channel blockers also, unexpectedly, raised the cytosolic free Ca2+ ([Ca2+]c) resting level in these germlings suggesting that they did not just inhibit L-type Ca2+ activity. The morphological phenotypes (conidial germination, hyphal extension rate, conidiation and hyphal branching) of 22 mutants defective in different components of their Ca2+-signalling and homeostasis machinery were characterized in order to identify their possible roles of Ca2+ during colony initiation and development. The ∆cch-1 mutant lacking the CCH-1 L-type Ca2+ channel gene exhibited a reduction in CAT fusion. CAT fusion was decreased even further in a double mutant (∆cch-1∆mid-1) suggesting that that the CCH-1 and MID-1 proteins operate in combination during this process. Increased extracellular Ca2+ partially restored the phenotypes of the ∆cch-1, ∆mid-1 smco-1 and ∆cch-1∆mid-1 mutants which is consistent with CCH-1 and MID-1 being involved in Ca2+ uptake from the external medium. Calcium signatures following mechanical perturbation were successfully measured in populations of conidial germlings using aequorin expressed in the wild type and in deletion mutants (∆cch-1, ∆yvc-1, ∆fig-1) lacking different Ca2+ channels. The removal of external Ca2+ completely abolished the [Ca2+]c increase in response to mechanical perturbation and CCH-1 was found to partly contribute to this increase in [Ca2+]c. Various Ca2+-sensitive dyes (Oregon green 488, Fluo-4 and Calcium Green-1) were also tested to determine if they can be used to image [Ca2+]c at the single cell and subcellular levels. Only Fluo-4 allowed the measurement of [Ca2+]c in individual cells but the changes in dye fluorescence in response to changes in [Ca2+]c were too small to be useful for imaging [Ca2+]c dynamics at the subcellular level. The other two dyes underwent rapid compartmentalization in organelles when loaded into germlings. The plant antifungal proteins (defensins), MsDef1, MtDef4 and PAF were all found to disrupt Ca2+ signaling/homeostasis in conidial germlings of N. crassa. They all inhibited the [Ca2+]c increase and raised the resting level of [Ca2+]c in response to mechanical perturbation. Analysis of an aequorin expressing mutant that was defective in glucosylceramide synthase (∆gcs) showed that the effects of MsDef1 (but not MtDef4) on [Ca2+]c were mediated by the sphingolipid glucosylceramide. All of the defensins tested were found to exhibit different potencies with regard to their inhibitory effects on conidial germination and CAT fusion.

Ingeniero Civil en Biotecnología / A lo largo del tiempo, los organismos vivos han desarrollado la capacidad de utilizar y acumular metales de transición. Esta capacidad resulta de suma importancia al considerar que estos elementos, entre los cuales se encuentra el cobre, están asociados a distintos procesos y rutas metabólicas. A pesar de esto, pueden llegar a ser tóxicos para la célula sobrepasando ciertas concentraciones. En el presente trabajo se proponen mecanismos cinéticos de los componentes de la homeostasis del cobre en Escherichia coli para estudiar la resistencia de éste elemento en bacterias. Estos mecanismos se relacionan mediante un modelo cuantitativo de flujos a partir de EDOs, donde se identificó y caracterizó cada componente del sistema. La cinética de tres proteínas (CopA, CueO y CusCBA) es estudiada en base a sus actividades reportadas para la obtención de los parámetros cinéticos, los cuales son ajustados por los mecanismos propuestos mediante la técnica de análisis numérico de mínimos cuadrados. Para desarrollar el modelo se realizaron algunos supuestos, como considerar las concentraciones de ATP, H+ y O2 en exceso para las reacciones, y mantener la cantidad de proteínas constante. Como resultado principal, se obtiene que la cantidad de cobre en el citoplasma y periplasma varía entre 2 a 12 átomos (0.003 a 0.02 [uM]) y 55 a 570 átomos (0.09 a 0.95 [uM]) respectivamente; otorgando a este segundo compartimiento 48 veces más capacidad de almacenamiento de cobre libre. Además, las escalas de tiempo de respuesta de las simulaciones, están en el orden de los 30 minutos, acercándose al tiempo de respuesta que se espera para mantener el supuesto de proteínas constante. Por otra parte, el resultado del modelo es más sensible a perturbaciones de los parámetros cinéticos de CueO y del volumen extracelular; mientras que se observan variaciones menores del 1% al perturbar los parámetros del sistema transportador CusCBA-CusF. El modelo se validó mediante ensayos de comparación entre distintos casos de mutaciones de tipo knockdown y los valores de las MICs asociadas a ellos, pudiendo recuperar los valores vinculando las MICs reportadas con las MICs obtenidas mediante una relación. En los casos de silenciamiento genético de CopA, se observa la importancia de insertar un transportador putativo que transporte cobre desde el periplasma hacia el citoplasma. A través del modelo, es posible estudiar el comportamiento de los elementos principales de la homeostasis de cobre en bacteria, validando la metodología utilizada. A partir de esto, se puede tener aproximaciones de la dinámica del sistema de homeostasis de cobre y dada la flexibilidad del modelo, extrapolarlo a otros organismos para evaluar concentraciones críticas de este elemento en distintos casos como polímeros biocidas o enfermedades relacionadas con la absorción del cobre.

De part ses propriétés physico-chimiques, le fer est un cofacteur de choix pour de nombreuses enzymes, impliquées dans de multiples processus biologiques, comme la photosynthèse ou la respiration. Cependant, sa capacité à gagner/perdre des électrons le rend très réactif, et potentiellement toxique. Son homéostasie doit donc être finement régulée. Chez A. thaliana, le gène de ferritine AtFER1 est régulé transcriptionnellement par le fer, et son expression est régulée par le rythme circadien. Au début de ce travail, aucun facteur de transcription impliqué dans cette régulation n'était identifié. Des cribles simple hybride en levure ont été réalisés, permettant l'identification de deux facteurs de transcription régulant le gène AtFER1: AtPHR1 et AtPIF7.PHR1 (Phosphate starvation Response1) et son homologue PHL1 (PHr1 Like 1) sont des facteurs de transcription impliqués dans la réponse à la carence en phosphate. Ils régulent directement le gène AtFER1, en se fixant sur l'élément 2 du promoteur d'AtFER1. Cette régulation ne fait pas intervenir l'IDRS et est indépendante du statut en fer des plantes. Par ailleurs, l'homéostasie du fer est affectée dans le double mutant phr1 phl1. Ces résultats montrent l'existence d'un lien moléculaire direct entre les homéostasies du fer et du phosphate.PIF7 (Phytochrome Interacting Factor 7) est un facteur de transcription de type bHLH impliqué dans la régulation circadienne des gènes DREB. Il se fixe probablement sur la G-box présente dans l'élément 5 du promoteur d'AtFer1. Dans un mutant perte de fonction pour le gène PIF7, l'amplitude des oscillations de l'expression du gène AtFer1 en cycles jour/nuit est augmentée. Un résultat similaire est obtenu lorsque l'élément 5 est muté dans des lignées trasngéniques exprimant le gène rapporteur LUC sous le contrôle du promoteur d'AtFer1. Ces résultats montrent que PIF7 est un répresseur de l'expression d'AtFer1. / Due to its redox properties, iron is a major cofactor for numerous proteins involved in many biological processes such as photosynthesis or respiration. Nevertheless, its ability to easily gain or lose electrons makes it highly reactive with oxygen and potentially toxic. Iron homeostasis has to be tightly regulated. In A. thaliana, AtFER1 ferritin gene is regulated at the transcriptional level by iron, and its expression is regulated by the circadian clock. Before this work, no transcription factor involved AtFer1 regulation has been identified. A yeast one hybrid was performed and allowed us to identify PHR1 and PIF7 as transcription factors involved in AtFER1 regulation. PHR1 (Phosphate starvation Response1) and its homolog PHL1 (PHr1 Like 1) are transcription factors involved in phosphate starvation response. They directly regulate AtFER1 expression and bind to the element 2 present in AtFER1 promoter. This regulation does not involve the IDRS sequence and is independent of iron status of plants. Moreover, iron homeostasis is affected in phr1phl1 double mutant. These results highlight a direct molecular link between iron and phosphate homeostasis.PIF7 (Phytochrome Interacting Factor 7) is a bHLH transcription factor involved in the circadian regulation of DREB genes. PIF7 probably interacts with the G-box found in element 5 in AtFER1 promoter region. In a pif7 knock-out mutant, amplitude of AtFer1 oscillations during light dark cycles are increased. Such a result was also obtained with transgenic lines expressing LUC reporter gene under the control of AtFER1 promoter region harboring a mutation in element 5. These results show that PIF7 is a repressor of AtFer1 expression.

Orientador: Renato de Mello Prado / Abstract: Global warming is predicted to increase the intensity and duration of extreme weather events, such as droughts, heat waves, and floods, especially in tropical regions. Climate change affect growth of forage species. However, information regarding the effects of global climate change on the nutritional performance of tropical pastures is lacking, especially under field conditions. We, thus, conducted two field experiment with Panicum maximum and Stylosanthes capitata using a temperature free-air controlled enhancement system and evaluated the effects of two temperature conditions, ambient temperature and moderate warming (2°C above ambient canopy temperature), and two levels of water availability, irrigated and non-irrigated, on nutrients accumulation, nutrient use efficiency (NUE), the stoichiometric patterns of C:N:P and leaf biomass production. Both experiments was conducted using a randomized complete block design in a factorial arrangement. Our findings revealed in plants of P. maximum (C4- grass) that the N and P leaf concentration greatly decreased under water-stressed, which increased the C:N and C:P ratios, while warming increased the N:P ratio. Leaf biomass production was impaired by up to 16% under water stress and ambient temperature conditions, but the biomass production was improved by 20% under warming and irrigated conditions. Our results also showed that homeostatic instability under rainfed conditions resulted in decreased leaf biomass production, and it was ... (Complete abstract click electronic access below) / Resumo: Prevê-se que o aquecimento global aumente a intensidade e a duração dos eventos climáticos extremos, como secas, ondas de calor e inundações, especialmente nas regiões tropicais. Mudanças climáticas afetam o crescimento de espécies forrageiras. No entanto, faltam informações sobre os efeitos das mudanças climáticas globais no desempenho nutricional de pastagens tropicais, especialmente em condições de campo. Nós, assim, conduzimos dois experimento em campos com as forrageiras Panicum maximum e Stylosanthes capitata utilizando um sistema de temperatura controlada do aquecimento do ar (T-Face) e avaliou-se os efeitos de duas condições de temperatura, (temperatura ambiente) e aquecimento moderado (2°C acima da temperatura ambiente) e dois níveis de disponibilidade hídrica, (irrigada e não irrigados), no acúmulo de nutrientes, eficiência de uso de nutrientes (NUE), nos padrões estequiométricos de C:N:P e na produção de biomassa foliar. Ambos experimentos foram conduzidos utilizando um delineamento de blocos completos casualizados em arranjo fatorial. Nossos resultados revelaram que em plantas de P. maximum (pastagem C4) a concentração foliar de N e P diminuiu sob estresse hídrico, o que aumentou as relações C:N e C:P, enquanto o aquecimento aumentou a relação N:P. A produção de biomassa foliar foi prejudicada em até 16% sob condições de estresse hídrico e temperatura ambiente, mas a produção de biomassa foi melhorada em 20% sob condições de aquecimento e irrigação. Nossos resulta... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

Chan Chung Sing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 93-101). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.ix / List of Abbreviations --- p.xvii / Chapter CHATPER ONE --- INTRODUCTION / Chapter 1.1 --- Glucose Homeostasis --- p.1 / Chapter 1.1.1 --- Function --- p.1 / Chapter 1.1.2 --- Origins and regulation of glucose --- p.2 / Chapter 1.1.3 --- Glucoregulatory factors --- p.4 / Chapter 1.1.4 --- Insulin --- p.6 / Chapter 1.1.4.1 --- Function of Insulin --- p.7 / Chapter 1.1.4.2 --- Discovery and Production of Insulin --- p.7 / Chapter 1.1.4.3 --- Insulin Signaling Pathway --- p.8 / Chapter 1.1.4.3.1 --- Insulin Receptor --- p.8 / Chapter 1.1.4.3.2 --- MAPK Pathway --- p.9 / Chapter 1.1.4.3.3 --- Phosphatidylinositol 3-kinase (PI3-K) Pathway --- p.10 / Chapter 1.1.5 --- Glucose Transporters --- p.11 / Chapter 1.1.6 --- Role of skeletal muscle in glucose homeostasis --- p.13 / Chapter 1.1.7 --- Insulin Resistance --- p.14 / Chapter 1.1.8 --- Glucose abnormality and its complications --- p.16 / Chapter 1.2 --- Actin --- p.19 / Chapter 1.2.1 --- Function of Actin --- p.20 / Chapter 1.2.2 --- Actin Accessory Protein --- p.22 / Chapter 1.2.3 --- Actin Polymerization --- p.23 / Chapter 1.3 --- "Interaction between Insulin, GLUT4 and Actin in Glucose Homeostasis" --- p.24 / Chapter 1.3.1 --- Insulin-Induced Actin Remodeling --- p.25 / Chapter 1.3.2 --- Actin Remodeling and Insulin-Induced GLUT4 Translocation --- p.26 / Chapter 1.3.3 --- Involvement of Insulin Signaling Molecules in Actin Remodeling --- p.27 / Chapter 1.3.4 --- Actin Remodeling and Insulin Resistance --- p.30 / Chapter 1.4 --- Hypothesis and Objective --- p.30 / Chapter 1.4.1 --- Rationale --- p.30 / Chapter 1.4.2 --- Hypothesis --- p.31 / Chapter 1.4.3 --- Objective --- p.31 / Chapter CHAPTER TWO --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.33 / Chapter 2.2 --- Cell Culture --- p.36 / Chapter 2.2.1 --- Cell Culture --- p.36 / Chapter 2.2.2 --- Reagents Preparation and Incubation --- p.39 / Chapter 2.3 --- 2-Deoxyglucose Uptake --- p.39 / Chapter 2.4 --- Immunofluorescence Microscopy --- p.41 / Chapter 2.4.1 --- Permeabilized cell staining --- p.41 / Chapter 2.4.2 --- Membrane-intact cell staining --- p.43 / Chapter 2.4.3 --- The analysis of actin remodeling reduction --- p.44 / Chapter 2.5 --- Live Image Microscopy --- p.44 / Chapter 2.6 --- Transmission Electron Microscope Study --- p.44 / Chapter 2.7 --- Statistical Analysis --- p.46 / Chapter CHAPTER THREE --- RESULTS / Chapter 3.1 --- Cell Growth --- p.48 / Chapter 3.2 --- Acute Effect of Insulin on L6 myotubes --- p.48 / Chapter 3.2.1 --- Immunofluorescence Microscopy --- p.49 / Chapter 3.2.1.1 --- The time profile of insulin on actin cytoskeletonin permeabilized L6 myotubes --- p.49 / Chapter 3.2.1.2 --- The concentration effect of insulin on actin cytoskeletonin permeabilized L6 myotubes --- p.50 / Chapter 3.2.1.3 --- Relationship between actin cytoskeleton and GLUT4mycin permeabilized L6 myotubes --- p.51 / Chapter 3.2.1.4 --- Translocation of GLUT4myc in membrane-intact L6 myotubes --- p.51 / Chapter 3.2.1.5 --- "Effect of methyl-β-cyclodextrins, MeOH or EtOHin permeabilized and membrane-intact L6 myotubes" --- p.52 / Chapter 3.2.2 --- 2-Deoxyglucose Uptake --- p.52 / Chapter 3.2.2.1 --- "Effects of insulin, methyl-β-cyclodextrins, MeOH and EtOH in L6 myotubes" --- p.52 / Chapter 3.2.3 --- TEM Study --- p.53 / Chapter 3.2.3.1 --- Effects of insulin on actin cytoskeleton and GLUT4myc in L6 myotubes --- p.53 / Chapter 3.3 --- Effect of high glucose and high insulin incubation in L6 myotubes --- p.54 / Chapter 3.3.1 --- Immunofluorescence Microscopy --- p.54 / Chapter 3.3.1.1 --- High insulin and high glucose preincubation in permeabilized L6 myotubes --- p.55 / Chapter 3.3.1.2 --- Effect of high insulin and high glucose incubationin membrane-intact L6 myotubes --- p.55 / Chapter 3.3.2 --- 2-Deoxyglucose Uptake --- p.56 / Chapter 3.3.2.1 --- Effect of high insulin and high glucose incubation in L6 myotubes --- p.56 / Chapter 3.3.3 --- TEM Study --- p.57 / Chapter 3.3.3.1 --- Effect of high insulin and high glucose incubation in L6 myotubes --- p.57 / Chapter 3.4 --- Effect of FFA incubation in L6 myotubes --- p.58 / Chapter 3.4.1 --- Immunofluorescence Microscopy --- p.58 / Chapter 3.4.1.1 --- FFA preincubation in permeabilized L6 myotubes --- p.58 / Chapter 3.4.1.2 --- FFA incubation in membrane-intact L6 myotubes --- p.59 / Chapter 3.4.2 --- 2-Deoxyglucose Uptake --- p.59 / Chapter 3.4.2.1 --- FFA incubation in L6 myotubes (24 hours) --- p.60 / Chapter 3.4.3 --- TEM Study --- p.62 / Chapter 3.4.3.1 --- FFA incubation in L6 myotubes --- p.62 / Chapter 3.5 --- Effect of CHO incubation in L6 myotubes --- p.62 / Chapter 3.5.1 --- Immunofluorescence Microscopy --- p.62 / Chapter 3.5.1.1 --- CHO preincubation in permeabilized L6 myotubes --- p.63 / Chapter 3.5.1.2 --- CHO incubation in membrane-intact L6 myotubes --- p.63 / Chapter 3.5.2 --- 2-Deoxyglucose Uptake --- p.64 / Chapter 3.5.2.1 --- CHO incubation in L6 myotubes (24 hours) --- p.64 / Chapter 3.5.3 --- TEM Study --- p.65 / Chapter 3.5.3.1 --- CHO incubation in L6 myotubes --- p.65 / Chapter 3.6 --- Overall changes in glucose uptake after preincubation experiment --- p.65 / Chapter CHAPTER FOUR --- DISCUSSION / Chapter 4.1 --- Effect of insulin on L6 myotubes --- p.69 / Chapter 4.2 --- "Effect of methyl-β-cyclodextrins, MeOH and EtOH on L6 myotube" --- p.75 / Chapter 4.3 --- Effect of pretreatment of cells in conditions of insulin resistance --- p.76 / Chapter 4.3.1 --- Effect of high glucose and high insulin preincubation on L6 myotubes --- p.76 / Chapter 4.3.2 --- Effect of FFA preincubation on L6 myotubes --- p.78 / Chapter 4.3.3 --- Effect of CHO preincubation on L6 myotubes --- p.82 / Chapter 4.3.4 --- Effect of cell preincubation in conditions of insulin resistance on L6 myotubes (TEM) --- p.83 / Chapter 4.4 --- Summary of the effects of cell preincubation in conditions of insulin resistance --- p.84 / Chapter 4.5 --- Possible mechanisms involved in insulin resistance induction --- p.86 / Chapter 4.5.1 --- Possible changes in GLUT expression and activities --- p.87 / Chapter 4.5.2 --- Possible changes in insulin signaling propagation --- p.88 / Chapter 4.5.3 --- Altered functioning of various actin accessory proteins --- p.89 / Chapter 4.6 --- Limitation of the study --- p.90 / Chapter 4.7 --- Conclusion --- p.90 / Chapter 4.8 --- Future study --- p.91 / REFERENCES --- p.93 / TABLES

Actin cytoskeletal regulation is of critical importance for a number of diverse cellular functions, including cell motility, endocytosis, cell division and transcription. Tight regulation of actin is critical for many aspects of cancer biology and in particular invasion and metastasis. ADF/cofilins are among the most important actin regulatory proteins. Mammals have three highly conserved members, ADF, CFL1 and CFL2, which regulate actin dynamics by severing and depolymerizing actin filaments. Despite a huge literature on the roles of ADF/cofilins in actin treadmilling and cell migration in vitro and in cancer cell behavior during invasion, very little is known about their collective roles in tissue homeostasis. By employing genetic knock-outs of ADF, in conjunction with conditional depletion of CFL1 using a Cre-LoxP system under the control of the keratin 14 promoter, we were able to study the effects of ADF/CFL1 loss in vivo in the mouse epidermis. Furthermore, by generating ADF-null squamous cell carcinoma (SCC) cell lines and by transiently downregulating CFL1 with RNAi, we were able to investigate further the cellular responses after ADF/CFL1 depletion in vitro. Co-depletion of ADF and CFL1 from the mouse epidermis triggered loss of tissue homeostasis characterized by abnormal thickening of the tissue, actin filament accumulation and nuclear deformation. Loss of ADF/CFL1 in cultured malignant keratinocytes also led to aberrant cell morphology accompanied by unrestrained accumulation of actin stress fibers tethered to enlarged focal adhesions. Enhanced SRF/MAL-mediated transcription fuels this uncontrolled actin polymerization which is also mediated by Arp3. Furthermore, these actin filaments are decorated with phospho-myosin light chain, which indicates their contractile nature. As a consequence, the increased intracellular acto-myosin tension results in nuclear deformation, which is promoted by the deregulated actin filaments tethered to the nuclear envelope via the linker of nucleoskeleton and cytoskeleton (LINC) complex. Overall, we describe new conceptual insight into the cellular functions of ADF/cofilins. We show that their activities are essential for the dynamic regulation of contractile actin filaments that, if left unchecked, lead to loss of cellular homeostasis and cell death promoted by loss of nuclear integrity. Additionally, the critical roles of nuclear actin and actin-associated proteins have recently started being appreciated. Thus, for the first time we set out to investigate new functions of cofilins in the nucleus using proteomics, and identify new cofilin binding partners that implicate them in novel cellular pathways, expanding our knowledge on these small actin-binding proteins.