Dissertations / Theses on the topic 'Plant stem cells'

Plants are sessile organisms that cannot escape environmental hazards, which induce DNA damage and cause mutations. Plants are also subject to DNA damage caused by endogenous processes such as transposon movement. Plant stem cell populations in particular must be protected from genotoxicity, as they are the origin of all organs, together with the germline. In accordance with this premise, plant stem cells were found to be hypersensitive to Double Strand Breaks (DSBs), leading to their specific killing via the ATAXIA TELANGIECTASIA MUTATED (ATM) and SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1) genes. However, the components of the pathway leading to programmed cell death (PCD) in response to DSBs in plant stem cells are still unknown, and the in vivo DNA damaging agents relevant to this mechanism have not been characterised, providing the starting point of this thesis. Here, a candidate gene approach and a forward genetics screen in the root stem cells did not yield new factors of the pathway leading to PCD in root stem cells. However, a specific ecotype showed an absence of DSBs-induced PCD, revealing natural variation in stem cell responses to DSBs. In relation to responses to endogenous DNA damage in plant stem cells, I identified several chromatin‐silencing mutants showing spontaneous PCD in the root meristem, and studied the link between transposon silencing and the ATM/SOG1 pathway. Finally, by characterising responses to Cre‐catalysed recombination in the shoot meristem, I uncovered an unexpected link between the DNA damage response pathway and chromatin silencing, This silencing was dependent on ATM/SOG1, linked to the production of 24-nt siRNA, and required the RNA polymerase IV and ARGONAUTE 6. My work links DNA damage responses to chromatin silencing in Arabidopsis stem cells.

La present tesi doctoral descriu la configuració del complex proteic de BRAVO en el nínxol de cèl·lules mare d'Arabidopsis. També demostra que BRAVO-WOX5-BES1 formen part d'una xarxa organitzadora que comprèn heterodímeros de BRAVO-WOX5, i que junts mantenen la quiescencia en els nínxols de cèl·lules mare de l'arrel. I tot això està controlat pels BRs. Aquestes dades proporcionen nous coneixements sobre l'organització de la divisió del centre quiescente en les arrels vegetals. Es va investigar la composició del complex proteic BRAVO de l'arrel primària d'Arabidopsis in vivo mitjançant tècniques de InmunoPrecipitación i cromatografia líquida/masses. Però a causa de la baixa expressió nativa de BRAVO, ens vam veure obligats a realitzar un examen exhaustiu dels possibles interactores de BRAVO en llevat, per a aconseguir augmentar la sensibilitat del nostre enfocament. Les anàlisis van revelar que BRAVO, un membre de la família R2R3 MYB, interactua amb una proteïna de la superfamília homeobox. Aquest treball demostra que BRAVO, a més d'interaccionar amb WOX5, també ho fa amb BES1 i TPL. El resultat va ser confirmat mitjançant tècniques moleculars in vitro per assajos de Y2H i també in vivo utilitzant FRET-FLIM i BiFC en fulles de Nicotiana benthamiana. Les nostres dades proporcionen evidències d'interacció directa de BRAVO amb WOX5, i al mateix temps, proposem que tots dos podrien formar part del complex transcripcional BES1/TPL en el SCN, i tot això regulat mitjançant BRs. Vam mostrar que la interacció de BES1-TPL és essencial per a la divisió del QC en el nínxol de cèl·lules mare de l'arrel. L'augment dels nivells de BR indueix la divisió del QC mitjançant un mecanisme controlat amb precisió per BRAVO. Els resultats estableixen que TPL regula la divisió de cèl·lules del QC a través de la supressió mediada per BES1 de BRAVO, i en resposta als BRs. A més, mitjançant una anàlisi genètic i matemàtic revelem que la interacció de BRAVO i WOX5 és essencial per a determinar el destí de les cèl·lules mare. De particular interès és el fet que BRAVO i WOX5 es reforcen mútuament en el nínxol de cèl·lules mare d'arrel. Això és sorprenent, ja que els nivells de WOX5 estan regulats per BRs de manera oposada que ho són en BRAVO. Un anàlisi exhaustiu del patró d'expressió de tots dos gens en els mutants KO simples i dobles, secunda que BRAVO és necessari per a mantenir els nivells normals de WOX5 en el QC. A més, les nostres dades són coherents amb el fet que WOX5 pot induir l'expressió de BRAVO, però només en el seu domini natiu. D'acord amb la nostra hipòtesi, el nostre model matemàtic prediu que WOX5 es reprimeix transcripcionalmente i al mateix temps activa l'expressió de BRAVO, tenint en compte la formació de heterodímeros i del complex. En aquest escenari, les interaccions del model indiquen que BRAVO no pot activar l'expressió WOX5 fora del seu domini, d'acord amb els resultats. Vam mostrar una xarxa reguladora de les nostres interaccions predita pel model matemàtic. A més, en augmentar la concentració de BR observem una major correlació entre les concentracions de proteïnes BRAVO i WOX5 quan un d'ells està absent, però no quan falten tots dos. Aquest mecanisme podria ser un mecanisme de compensació. I finalment, en l'últim capítol, aprofundim en l'evolució de BRAVO i de WOX5 amb l'objectiu de comprendre l'organització primitiva i la funció del nostre SCN actual en l'arrel.
La presente tesis doctoral describe la configuración del complejo proteico de BRAVO en el nicho de células madre de Arabidopsis. También demuestra que BRAVO-WOX5-BES1 forman parte de una red organizadora que comprende heterodímeros de BRAVO-WOX5, y que juntos mantienen la quiescencia en los nichos de células madre de la raíz. Y todo esto está controlado por los BRs. Estos datos proporcionan nuevos conocimientos sobre la organización de la división del centro quiescente en las raíces vegetales. Se investigó la composición del complejo proteico BRAVO de la raíz primaria de Arabidopsis in vivo mediante técnicas de Inmuno-precipitación y cromatografía líquida/masas. Pero debido a la baja expresión nativa de BRAVO, nos vimos obligados a realizar un examen exhaustivo de los posibles interactores de BRAVO en levadura, para conseguir aumentar la sensibilidad de nuestro enfoque. Los análisis revelaron que BRAVO, un miembro de la familia R2R3 MYB, interactúa con una proteína de la superfamilia homeobox. Este trabajo demuestra que BRAVO, además de interaccionar con WOX5, también lo hace con BES1 y TPL. El resultado fue confirmado mediante técnicas moleculares in vitro por ensayos de Y2H y también in vivo utilizando FRET-FLIM y BiFC en hojas de Nicotiana benthamiana. Nuestros datos proporcionan evidencias de interacción directa de BRAVO con WOX5, y al mismo tiempo, proponemos que ambos podrían formar parte del complejo transcripcional BES1/TPL en el SCN, y todo esto regulado mediante BRs. Mostramos que la interacción de BES1-TPL es esencial para la división del QC en el nicho de células madre de la raíz. El aumento de los niveles de BR induce la división del QC mediante un mecanismo controlado con precisión por BRAVO. Los resultados establecen que TPL regula la división de células del QC a través de la supresión mediada por BES1 de BRAVO, y en respuesta a los BRs. Además, mediante un análisis genético y matemático revelamos que la interacción de BRAVO y WOX5 es esencial para determinar el destino de las células madre. De particular interés es el hecho de que BRAVO y WOX5 se refuerzan mutuamente en el nicho de células madre de raíz. Esto es sorprendente, ya que los niveles de WOX5 están regulados por BRs de forma opuesta que lo son en BRAVO. Un análisis exhaustivo del patrón de expresión de ambos genes en los mutantes KO simples y dobles, apoya que BRAVO es necesario para mantener los niveles normales de WOX5 en el QC. Además, nuestros datos son coherentes con el hecho de que WOX5 puede inducir la expresión de BRAVO, pero sólo en su dominio nativo. De acuerdo con nuestra hipótesis, nuestro modelo matemático predice que WOX5 se reprime transcripcionalmente y a su vez activa la expresión BRAVO, teniendo en cuenta la formación de heterodímeros y del complejo. En este escenario, las interacciones del modelo indican que BRAVO no puede activar la expresión WOX5 fuera de su dominio, de acuerdo con los resultados. Mostramos una red reguladora de nuestras interacciones predicha por el modelo matemático. Además, al aumentar la concentración de BR observamos una mayor correlación entre las concentraciones de proteínas BRAVO y WOX5 cuando uno de ellos está ausente. Pero no cuando faltan ambos. Este mecanismo podría ser un mecanismo de compensación. Y finalmente, en el último capítulo, profundizamos en la evolución de BRAVO y de WOX5 con el objetivo de comprender la organización primitiva y la función de nuestro SCN actual en la raíz.
The present PhD thesis dissertation describes the configuration of BRAVO protein complex in Arabidopsis stem cell niche, while demonstrates that BRAVO-WOX5-BES1 are part of a main regulator network that comprises BRAVO-WOX5 heterodimers, and together contribute to cell specific regulation of BR-controlled quiescence in root stem cell niches. The current data provide new insights into the QC division organization in plant roots. It was investigated the composition of BRAVO protein complex from Arabidopsis primary root in vivo by IP and LC-MS/MS techniques. Giving the low expression of native BRAVO, we conducted an exhaustive screening for BRAVO interactors in Yeast to increase the sensitivity of our approach. The analyses revealed that BRAVO, a member of the R2R3 MYB family, interact with a homeobox superfamily protein. The work further demonstrate that BRAVO interacts with WOX5, BES1 and TPL. This result was confirmed by molecular techniques in vitro by Y2H assays and in vivo using FRET-FLIM and BiFC in Nicotiana benthamiana leaves. Our data provides evidences of BRAVO directly interaction with WOX5, and at the same time both could be part of the BES1/TPL transcriptional complex at the SCN trough the BR signalling cascade. We display that the interaction of BES1-TPL is essential for the QC division in root SCN. Increasing BR levels induce QC division through a fine mechanism which is accurately controlled by BRAVO. The results establish that TPL regulates QC cell division through BES1-mediated suppression of BRAVO, and in response to BRs, the last step seems to be the promotion of the QC division. By a genetical and a mathematical analysis, we revealed that BRAVO and WOX5 interaction is essential for stem cell fate. Of particular interest is the fact that BRAVO and WOX5 reinforce each other at the root stem cell niche. This was surprising, since WOX5 levels are oppositely regulated by BRs than in BRAVO. The exhaustive analysis of the expression pattern of both genes in all the simple and double KO mutants, support that BRAVO is required to maintain normal WOX5 levels in the QC. In addition our data are coherent with the fact that WOX5 can induce BRAVO expression but only in the BRAVO native domain. Consistent with our hypothesis, a mathematical model predicted that WOX5 transcriptionally represses itself and activates BRAVO expression, taking in account the heterodimers and complex formation. In this scenario, the model interactions indicate that BRAVO is unable to activate WOX5 expression outside of its domain, in agreement with the results of the BRAVO overexpression line. We show a regulatory network of our interactions predicted by the mathematical model. We added the protein fold changes predicted by this model when changing the BR concentrations due to the BR signalling cascade in different situations and we observed better correlation of BRAVO and WOX5 protein concentrations when one of them are absent. But not when both are out. The exact reasons for these differences are not clear. This mechanism could be a compensation mechanism. And finally, in the last chapter, we delve into evolution with the aim to comprehend the primitive organisation and function of our present root SCN.

SHOOT MERISTEMLESS (STM) encodes a transcription factor in Arabidopsis essential for ensuring correct stem cell fate. STM is known to impinge on a number of key regulatory processes such as cytokinin synthesis and the cell cycle, and interacts with other core regulatory genes such as CUP-SHAPED COTYLEDON1 (CUC1). In this study inducible STM over-expression and RNAi-mediated downregulation over a time course experiment have been used to identify the genes which form STM's gene regulatory network (GRN). These results reveal for the first time how STM over-expression and knockout phenotypes are mediated and identified the temporal order of transcriptomic changes following STM over-expression. A Bayesian network approach further refined the GRN - identifying conditional dependencies among regulated TFs and core signalling components from an independent dataset (>2,000 experiments). Predictions of direct targets from the network have been tested, demonstrating a high degree of accuracy. Interplay between STM and CUC1 is a biologically interesting sub-module of the STM GRN, with unusual dynamics. Via gene expression and microscopy experiments it has been shown that STM positively regulates the CUC1-targetting microRNA miR164c. Mathematical modelling approaches show that this is consistent with a model in which the boundary is the site of highest STM mRNA production via CUC1, and STM movement with miR164c upregulation produces the observed spatial distributions of both proteins. These relationships recast the boundary zone as a particularly dynamic region of the shoot apical meristem (SAM) and significantly develop our understanding of STM developmental context.

As more patients with large body surface area burns are surviving and requiring reconstructive surgery, there is a necessity for advances in the provision of bioengineered alternatives to autologous skin cover. The aims of this Thesis are to identify feasible source tissues of Endothelial Colony Forming Cells and Mesenchymal Stem/Stromal Cells for microvascular network formation in vitro with three-dimensional dermal substitute scaffolds. The working hypothesis is that pre-vascularised dermal scaffolds will result in better quality scarring when used with split thickness skin grafts. Human umbilical cord blood, peripheral blood and adipose tissue were collected and processed with ethical approval and informed consent. Samples were cultured to form endothelial outgrowth colonies and confluent Mesenchymal Stem/Stromal Cells, which were characterised using flow cytometry and expanded in vitro. Mesenchymal Stem/Stromal Cell multipotency was confirmed with tri-lineage mesenchymal differentiation. Primary cells were tested in a two-dimensional tubule formation co-culture assay and differences assessed using a proangiogenic antibody array. Tubule formation was tested in four different acellular dermal substitute scaffolds; Integra® Dermal Regeneration Template, Matriderm®, Neuskin-F® and De-cellularised Human Cadaveric Dermis. Umbilical cord blood was the most reliable source of Endothelial Colony Forming Cells, the yield of which could be predicted from placental weight. Microvasculature dissected free from adipose tissue was a reliable source of Mesenchymal Stem/Stromal Cells which supported significantly more tubule formation than Mesenchymal Stem/Stromal Cells from whole adipose tissue. Microvasculature Mesenchymal Stem/Stromal Cells secreted significantly higher levels of the proangiogenic hormone leptin, and addition of exogenous leptin to the tubule formation assay resulted in significantly increased tubule formation. Microvasculature was cultured in all four of the scaffolds tested, but depth of penetration was limited to 100µm. The artificial oxygen carrier perfluorocarbon was shown to increase two-dimensional tubule formation and may be useful in further three-dimensional scaffolds studies to improve microvascular penetration.

The stringent requirement for hemopoietic growth factors (HGF) in the induction of hemopoiesis in vitro has raised questions as to their possible role(s) in leukemogenesis. Several recent clinical studies have shown aberrant cell growth factor gene activation in patient derived leukemic cells. Assessment of growth factor activity is often based on in vitro bioactivity assays of conditioned media or body fluids. The specificity of this type of endpoint is, however, open to question due to the overlap in biological activities of many HGFs. In assessing the role of growth factor gene expression in a murine myeloid leukemia model I have used a sensitive RNA detection procedure coupled with a vector-probe system that enables the synthesis of uniformly labelled radioactive DNA probes to detect unambiguously the expression of particular growth factor genes. The Abelson murine leukemia virus (A-MuLV) derived myeloid transformants used in this study had previously been shown to produce a multi-lineage colony stimulating activity (CSA). While these A-MuLV transformants were shown to produce GM-CSF, it seemed likely that the multi-lineage CSA was due to another factor. In addition to confirming the expression of GM-CSF mRNA, I was able to show that the cells of all four A-MuLV transformed lines tested also expressed interleukin-3 mRNA. This finding was strongly corroborated by bio-activity data obtained using the CM from the A-MuLV myeloid transformants. Additional preliminary analysis by bioactivity assays have also shown the possible presence of interleukin-6 (IL-6) and a recently described pre-B cell factor suggesting perhaps a common mechanism underlying the activation of these various growth factor genes.
Medicine, Faculty of
Medical Genetics, Department of
Graduate

Aquesta tesi doctoral descriu diversos avenços conceptuals per a la comprensió molecular de la senyalització de brassinosteroides en el nínxol de cèl·lules mare de l’arrel d’Arabidopsis thaliana. Els brassinoesteroides són les hormones esteroides de les plantes que juguen un paper important en el creixement i desenvolupament vegetal. En l’arrel primària d’Arabidopsis, els brassinoesteroides estan involucrats en el desenvolupament del meristem i manteniment de les cèl·lules mare. Les cèl·lules mare són les cèl·lules més indiferenciades, aquestes es van dividint i diferenciant per generar els diferents tipus cel·lulars de l’arrel. Aquests processos estan estretament controlats per factors interns i externs. El baix nombre de cada població de cèl·lules mare fa complicat el seu estudi de manera individual, per tant, el desenvolupament de mètodes amb resolució per estudiar tipus cel·lulars i fins i tot cèl·lules individuals representa una oportunitat única per investigar aquesta població cel·lular tan escassa. En aquesta tesi doctoral, utilitzem una estratègia multidisciplinària, que inclou genètica, anàlisi transcriptòmica i models matemàtics, per identificar les característiques moleculars de les cèl·lules mare de l’arrel enfocant-nos en el paper dels brassinoesteroides en aquestes cèl·lules. Defectes en els processos de creixement i desenvolupament vegetal generalment són deguts a defectes en el creixement de l’arrel principal. Com que la quantificació precisa de la longitud de l’arrel requereix molt de temps, en aquesta tesi doctoral es descriu el desenvolupament de l’eina MyROOT per a la mesura d’arrels d’Arabidopsis d’una manera semiautomàtica (Capítol 2). A més a més, els resultats presentats en aquesta tesi revelen quin és el paper dels brassinoesteroides en el nínxol de cèl·lules mare. Mitjançant una estratègia de biologia de sistemes s’ha estudiat el paper del factor de transcripció BRAVO, regulat per brassinoesteroides, juntament amb WOX5 en el creixement i desenvolupament de l’arrel (Capítol 3). Igualment, una aproximació específica per tipus cel·lulars ha revelat la resposta transcripcional mediada per BRAVO al centre quiescent i en les cèl·lules mare vasculars adjacents (Capítol 4). Finalment, l’ús de seqüenciació massiva de l’RNA (RNAseq) amb resolució cel·lular ha estat implementat per generar el primer atles transcriptòmic del nínxol de cèl·lules mare de l’arrel. Aquesta aproximació ha permès identificar les característiques moleculars de les cèl·lules mare i la presència de diferents poblacions d’aquestes en el domini d’expressió de BRAVO (Capítol 5). Aquesta tesi doctoral avança en el coneixement de les cèl·lules mare de les plantes i posa de manifest la necessitat d’estratègies multidisciplinàries per descobrir processos fonamentals de el desenvolupament vegetal.
Esta tesis doctoral reporta avances conceptuales en la respuesta molecular mediada por la ruta de señalización de los brasinoesteroides en el nicho de células madre de Arabidopsis thaliana. Los brasinoesteroides son las hormonas esteroideas de las plantas y juegan un papel importante en el crecimiento y desarrollo vegetal. En la raíz primaria de Arabidopsis, los brasinoesteroides están involucrados en el desarrollo del meristemo y mantenimiento de las células madre. En el nicho de células madre, las células madre son las células más indiferenciadas que se van dividiendo y diferenciando para generar los distintos tipos celulares de la raíz. Estos procesos están estrechamente controlados por factores internos y externos. El bajo número de cada población de células madre hace complicado su estudio individualmente, por lo tanto, el desarrollo de métodos con resolución para estudiar tipos celulares e incluso células individualmente representa una oportunidad única para investigar esta población celular tan escasa. En esta tesis doctoral, utilizamos una estrategia multidisciplinar, que incluye genética, análisis transcriptómicos y modelos matemáticos, para identificar las características moleculares de las células madre de la raíz y enfocándonos al papel de los brasinoesteroides en esas células. Defectos en procesos de crecimiento y desarrollo vegetal se reflejan generalmente en defectos en el crecimiento de la raíz principal. Como la cuantificación precisa de la longitud de la raíz requiere mucho tiempo, en esta tesis doctoral se describe el desarrollo de la herramienta MyROOT para la medida de raíces de Arabidopsis de una forma semiautomática (Capítulo 2). Además, los resultados presentados en esta tesis revelan el papel de los brasinoesteroides en el nicho de células madre. Una estrategia de biología de sistemas revela el papel del factor de transcripción BRAVO, regulado por brasinoesteroides, junto con WOX5 en el crecimiento y desarrollo de la raíz (Capítulo 3). Igualmente, una aproximación específica para tipos celulares revela la respuesta transcripcional mediada por BRAVO en el centro quiescente y en las células madre vasculares adyacentes (Capítulo 4). Por último, el uso de RNAseqs con resolución celular ha sido implementado para generar el que creemos es el primer atlas transcriptómico del nicho de células madre de la raíz. Esta aproximación ha permitido identificar las características moleculares de las células madre y la presencia de diferentes poblaciones de estas células en el dominio de expresión de BRAVO (Capítulo 5). Esta tesis doctoral avanza en el conocimiento de las células madre de las plantas y pone de manifiesto la necesidad de estrategias multidisciplinares para descubrir procesos fundamentales del desarrollo vegetal.
This PhD thesis dissertation reports a number conceptual advances for the molecular understanding of brassinosteroid signaling in the root stem cell niche of Arabidopsis thaliana. Brassisnosteroids are the plant steroid hormones that play important roles in plant growth and development. In the Arabidopsis primary root, brassinosteroids are involved in meristem development and stem cell maintenance. At the root stem cell niche, stem cells are the more undifferentiated cells that divide and differentiate to give rise to the distinct cell types of the root. These processes are tightly controlled by internal and external factors. The low number each stem cell population makes it difficult to study them individually, therefore, the advent of cell-type and single-cell specific approaches represents a unique opportunity to investigate this rare cell population. In this PhD thesis, we used an interdisciplinary approach, including genetics, transcriptomics analysis and mathematical modelling, to identify the molecular signatures of the root stem cells with a focus on the role of brassinosteroid hormones in those cells. Defects in growth and development processes is often reflected in abnormal primary root growth. As the accurate quantification of plant primary root length is time consuming, in this PhD dissertation, we describe the development of MyROOT software for the semi-automatic measurement of Arabidopsis primary roots (Chapter 2). In addition, the results presented in this thesis uncover the role of brassinosteroids in the stem cell niche. A systems biology approach revealed a role of the brassinosteroid-mediated BRAVO transcription factor together with WOX5 in overall root growth and development (Chapter 3). Moreover, cell-type specific transcriptomic analysis uncover the transcriptional response mediated by BRAVO in the QC and adjacent vascular stem cells (Chapter 4). Finally, the use of single-cell RNAseq has been implemented to generate to our knowledge the first transcriptomic atlas of the root stem cell niche. This approach allowed to characterize the molecular signatures of the stem cells and to find novel stem cell populations within the BRAVO expression domain (Chapter 5). Overall, the present PhD thesis advances in the understanding of stem cells in plants and expose the necessity of multidisciplinary approaches to uncover fundamental biological questions in plant development.
Universitat Autònoma de Barcelona. Programa de Doctorat en Biologia i Biotecnologia Vegetal

The development of safe and efficient gene delivery systems in pluripotent human embryonic stem cells (hESc) is essential to realising their full potential for basic and clinical research. The purpose of this study was to develop an efficient, non-integrating gene expression system in pluripotent hESc using human artificial chromosomes (HAC). Similar to endogenous chromosomes, HAC are capable of gene expression, replication and segregation during cell division. Unlike retroviral-mediated gene delivery vectors, HAC do not integrate into the host genome and can encompass large genomic regions for the delivery of multiple genes. Despite the advantages HAC offer, their use has been limited due to laborious cloning procedures and poor transfection efficiencies, and thus only studied in immortalised and tumour-derived human cell lines. In this study, the high transduction efficiency of herpes simplex virus type-1 (HSV-1) amplicons was utilised to overcome the described difficulties and delivered HAC vectors into pluripotent hESc. Analysis of stable hESc clones showed that de novo gene-expressing HAC were present at high frequencies ranging from 10-70% of metaphases analysed, without integrating into the genome. The established HAC contained an active centromere, and were stably maintained without integration or loss in the absence of selection for 90 days. Stable HAC-containing hESc clones retained their pluripotency as demonstrated by neuronal differentiation, in vitro germ layer and teratoma formation assays. HAC gene expression persisted, with some variation, post-differentiation in the various deriving cell types. This is the first report of successful de novo HAC formation in hESc for gene expression studies. These findings show potential for delivering high-capacity genomic constructs safely and efficiently into pluripotent cells for the purpose of genetic manipulation and ultimately patient-specific somatic gene therapy.

Haematopoietic stem cells (HSC) are strictly regulated by intrinsic regulators and extrinsic signals from the microenvironment. Nov (CCN3), a matricellular protein of the CCN family, has been reported as a suppressor gene in solid tumours and chronic myeloid leukaemia (CML). Recent study identified Nov as a positive regulator in human cord blood CD34+ stem cells. However, the functions of Nov in haematopoiesis and adult HSC remain largely unknown.

Improving the repopulation potential of human umbilical cord blood (UCB) haemopoietic stem cells (HSCs) remains a paramount goal in HSC transplantation (HSCT) therapy. This implies enhancing the homing and engraftment potential of UCB-CD34+CD133+ cells to the bone marrow (BM). Although an array of molecules continues to be identified as ‘key’ homing molecules, the molecular mechanisms controlling HSC homing are still not fully understood. The regulatory implications of hypoxia in the BM, with the concomitant stabilisation of hypoxia inducible transcription factor-1α (HIF-1α), are becoming more apparent, yet at the commencement of this thesis no study had explored whether hypoxia induced signalling can be adopted to regulate the homing and engraftment of transplanted HSCs. The aim of this DPhil project was thus to investigate whether hypoxic conditions as detected in the BM influence the adhesion of UBC-CD133+ cells to osteoblasts, BM stromal cells and BM endothelial cells-60 (BMEC-60), as well as their transmigration towards chemokine SDF-1α across BMEC-60. Increasing the exposure of UCB-CD133+ cells to 1.5% O2 doubled the percentage of transmigrating cells (p<0.05), and while hypoxia stimulated UCB-CD133+ cells preferentially adhered to IL-1β stimulated BMEC-60, their adhesion to non-stimulated (BMEC-60) was significantly improved (p<0.001). To help unravel the underlying molecular mechanisms, we attempted to examine the potential involvement of hypoxia regulated scaffolding protein HEF-1/NEDD9/Cas-L (HEF-1) in the increased percentage of migrating UCB-CD133+ cells after hypoxia pre-conditioning. The role of HEF-1 in HSCs is unexplored, and its multifunctional contribution in a variety of processes including cell migration, attachment and invasion make HEF-1 a prime candidate as a contributing homing molecule. After identifying a suitable short-hairpin RNA (shRNA) sequence to knockdown HEF-1, generating lentiviral (LV)-particles in house and optimising transduction protocols, HEF-1 knockdown was achieved in haemopoietic model cell lines KG-1 and KG-1A (KG-1/KG-1A–HEF1). Significantly decreased KG-1A–HEF1 cell adhesion to non-stimulated BMEC-60 was detected. Together, these studies provide a promising platform to further explore the role of HEF-1 in hypoxia induced UCB-CD133+ cell transmigration towards the key homing molecule SDF-1α.

Carbonic anhydrase IX (CA9) is strongly induced by hypoxia and its overexpression is associated with poor therapeutic outcome in cancer. The function of CAIX is to catalyze the reversible hydration of CO2 to bicarbonate and a proton. This helps hypoxic tumours to maintain a more neutral intracellular pH (pH_i) promoting survival, but produces a more acidic extracellular (pH_e) which promotes invasion and metastasis. Recent evidence has expanded on the role of hypoxia and CAIX by relating them to stem cell niches. In this study, taking advantage of the transmembrane location of CAIX, we show for the first time, a direct marked heterogeneity in response to hypoxia within each tumour cell population studied, associated with major biological differences. Based on CAIX expression pattern under hypoxic conditions, we identify, isolate and characterize two distinct populations of tumour cells, one that express CAIX and the other that does not. Interestingly, we discover that the CAIX positive population is enriched with cells expressing cancer stem cell markers. These include ALDHA1, IGF1, LIN28 and genes involved in epithelial-mesenchymal transition (EMT) and multi-drug resistance (i.e. WNT2, TWIST1, and ABCC2). Accordingly, CAIX+ve cells show higher self-renewal capacity and form tumours significantly faster compared to the CAIX-ve population. Importantly, functional suppression of CAIX in vitro and in vivo, in two breast cancer cell lines resulted in the downregulation of breast cancer stem cell signatures, suggesting that CAIX is not just a marker of stemness but also a regulator of stemness. The molecular mechanism underlying the differential expression of CAIX in the two populations is not HIF-1α-dependent, but instead driven by hypoxia-induced reorganization of chromatin structure. In line with this, we provide experimental evidence showing that the genomic locus encoding CA9 has a more “open” and transcriptionally active chromatin structure in CAIX+ve cells, and a condense and transcriptionally silent chromatin structure in the CAIX-ve cells. Given that HIF induces the transcription of CA9 by binding to hypoxia response elements (HREs) in its promoter we show a significant reduction in binding of HIF to the CA9 promoter of the negative population. We suggest that the reduce HIF binding is a result of the compact chromatin structure of CA9 promoter of the negative cells. Analysis of the transcriptome of the positive and negative populations suggests a symbiotic relationship between these two subpopulations and their environment, likely required to promote tumour growth. This is based on the following observations: Firstly, we identified that CAIX-ve cells express high levels of cytokines and based on this, we suggest that the cytokines secreted by CAIX-ve cells may transmit paracrine signals that regulate the CAIX+ve cells, thus providing a wider hypoxia tolerant microenvironment to protect the stem cell population. Secondly, we identified a metabolic heterogeneity between the CAIX+ve and CAIX-ve cells. The CAIX+ve cells show an upregulation of genes implicated in oxidative phosphorylation, TCA cycle and fatty acid synthesis. Whereas in CAIX-ve cells there is an upregulation of genes implicated in autophagy and mitophagy. Given the above together with the upregulation of oxidative phosphorylation and TCA cycle in the CAIX+ve cells, we proposed the existence of a metabolic symbiosis between the CAIX+ve and CAIX-ve cells. We postulate that the catabolic process such as autophagy and mitophagy in the CAIX-ve cells may results in the overproduction of high-energy metabolites such as lactate, glutamine and ketone bodies which in turns they are been utilized by CAIX+ve cells to fuel mitochondria respiration. Finally, we also demonstrated that in the CAIX+ve cells mTORC1 signaling is upregulated, and contributes to the regulation of CAIX expression. Given the role of mTORC1 in stem cell maintenance and EMT as well as the interdependence of mTORC1 and CAIX expression in the CAIX+ve cells we suggest that mTORC1 signaling may be the critical factor by which CAIX regulates stemness. Interestingly, the subpopulations show a differential sensitivity to HDAC inhibitors, NaBu and SAHA as treatment of MCF7 breast cancer cell line and HCT116 colon cancer cell line leads to elimination of the CAIX+ve population. This is not driven by the downregulation of HIF-1α, the major transcriptional regulator of CAIX. In contrast, we demonstrate that SAHA causes downregulation mTORC1. This suggests that SAHA-induced downregulation of CAIX expression could be due to its effect on mTORC1 pathway. Of wider significance, our findings show that tumours are not homogenous in their response to hypoxia, and distinct signal transduction networks regulate different populations of cells within the tumour. This highlights the need for the utilization of biomarkers, which reveal distinct functional hypoxia profiles of human cancers, and permit the stratification of tumours. Furthermore, the identification of epigenetic regulation of the histones in response to hypoxia for highly selective gene regulation, provides a connection between the epigenetic mechanisms under environmental stress and cancer progression, and is model for development of novel epigenetic cancer therapeutic drugs.

A major therapeutic challenge in musculoskeletal regenerative medicine is how to effectively replenish bone tissue lost due to pathological conditions such as fracture, osteoporosis, or rheumatoid arthritis. Mesenchymal stem cells are currently investigated for applications in bone-tissue engineering and human bone marrow-derived mesenchymal stem cells (hMSCs) could be a promising source for generation of tissue-engineered bone. However, the therapeutic potential of MSCs has not been fully exploited due to a lack of knowledge regarding the identity, nature, and differentiation of hMSCs. Epigenetic mechanisms regulating the chromatin structure as well as specific gene transcription are crucial in determination of stem cell differentiation. With the aim to systematically identify epigenetic factors that modulate MSC differentiation, the work in this thesis encompasses an approach to identify epigenetic mechanisms underlying, initiating, and promoting osteoblast differentiation, and the investigation of individual epigenetic modulators. Various osteogenic inducers were validated for differentiation of MSCs and an assay allowing assessment of differentiation outcome was developed. This assay was subsequently employed in knockdown experiments with lentiviral short hairpin RNAs and inhibitor screens with small molecules targeting putative druggable epigenetic modulator classes. This approach identified around 100 epigenetic modulator candidates involved in osteoblast differentiation, of these candidates approximately 2/3 downregulated and 1/3 upregulated alkaline phosphatase (ALP) activity. Serving as a proof-of-concept, orthogonal validation experiments employing locked nucleic acid (LNA) knockdown were performed to validate a subset of candidates. Two identified target genes were selected for further investigation. Bromodomain-containing protein 4 (BRD4) was identified as one component of epigenetic regulation; its inhibition led to a decrease in ALP expression, downregulation of key osteoblast transcription factors Runx2 and Osterix, as well as impaired bone matrix formation. Knockdown of lysine (K)-specific demethylase 1A (KDM1A/LSD1) upregulated ALP activity and treatment with a small molecule inhibitor targeting KDM1A led to an increase in ALP, RUNX2, and bone sialoprotein expression. Intriguingly, in a transgenic mouse model overexpressing Kdm1a a decrease in bone volume and bone mineral density was observed, thus supporting the hypothesis that KDM1A is a central regulator of osteoblast differentiation.

Modelling and simulation are essential to modern research in cell biology. This thesis follows a journey starting from the construction of new stochastic methods for discrete biochemical systems to using them to simulate a population of interacting haematopoietic stem cell lineages. The first part of this thesis is on discrete stochastic methods. We develop two new methods, the stochastic extrapolation framework and the Stochastic Bulirsch-Stoer methods. These are based on the Richardson extrapolation technique, which is widely used in ordinary differential equation solvers. We believed that it would also be useful in the stochastic regime, and this turned out to be true. The stochastic extrapolation framework is a scheme that admits any stochastic method with a fixed stepsize and known global error expansion. It can improve the weak order of the moments of these methods by cancelling the leading terms in the global error. Using numerical simulations, we demonstrate that this is the case up to second order, and postulate that this also follows for higher order. Our simulations show that extrapolation can greatly improve the accuracy of a numerical method. The Stochastic Bulirsch-Stoer method is another highly accurate stochastic solver. Furthermore, using numerical simulations we find that it is able to better retain its high accuracy for larger timesteps than competing methods, meaning it remains accurate even when simulation time is speeded up. This is a useful property for simulating the complex systems that researchers are often interested in today. The second part of the thesis is concerned with modelling a haematopoietic stem cell system, which consists of many interacting niche lineages. We use a vectorised tau-leap method to examine the differences between a deterministic and a stochastic model of the system, and investigate how coupling niche lineages affects the dynamics of the system at the homeostatic state as well as after a perturbation. We find that larger coupling allows the system to find the optimal steady state blood cell levels. In addition, when the perturbation is applied randomly to the entire system, larger coupling also results in smaller post-perturbation cell fluctuations compared to non-coupled cells. In brief, this thesis contains four main sets of contributions: two new high-accuracy discrete stochastic methods that have been numerically tested, an improvement that can be used with any leaping method that introduces vectorisation as well as how to use a common stepsize adapting scheme, and an investigation of the effects of coupling lineages in a heterogeneous population of haematopoietic stem cell niche lineages.

Thesis (Ph. D.)--University of Sydney, 2008.
Includes graphs and tables. Includes list of publications: p. iv. Title from title screen (viewed May 5, 2008). Thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Chemical and Biomolecular Engineering. Includes bibliographical references. Also available in print form.

A mist reactor was used to study plant growth and development under various environmental conditions towards the production of healthy plantlets ready for soil transplant in one step from inoculation. In addition, a 3D type of cultivation via surface attachment of explants to vertically hanging strips inside the mist reactor was also investigated to maximize productivity with minimal footprint. Using carrot as the model species, pre-embryogenic cell suspensions were successfully spray-inoculated onto hanging poly-L-lysine (PLL)-coated nylon mesh to which they then attached and remained for several weeks while they developed into rooted plantlets. To study single step micropropagation from shoot explants to fully acclimatized plantlets, Artemisia annua was used as the model species. Nodal cuttings of A. annua were inoculated onto PLL-coated mesh strips by briefly immersing the strips in the suspension of nodal cuttings. Investigation of medium, phytohormones, CO2, ventilation level and humidity ensued resulting in selection of a preferred final process that reduced physiological aberrations like hyperhydricity and was time efficient. The nodal cuttings that attached to the strips were first misted with half strength shooting medium for 7 days to develop new shoots. Then the new shoots were misted with the rooting medium supplemented with NAA for 12 days to develop roots. Rooted plantlets were acclimatized in the same rooting medium for 9 days to acquire fully functional stomata prior to planting into soil. Taken together this study suggested that fully developed plantlets ready for planting into soil could be obtained in a single step in a bioreactor from embryogenic cells or from nodal explants.

Detailed thermodynamic, kinetic, geometric, and cost models are developed, implemented, and validated for the synthesis/design and operational analysis of hybrid solid oxide fuel cell (SOFC) – gas turbine (GT) – steam turbine (ST) systems ranging in size from 1.5 MWe to 10 MWe. The fuel cell model used in this thesis is based on a tubular Siemens-Westinghouse-type SOFC, which is integrated with a gas turbine and a heat recovery steam generator (HRSG) integrated in turn with a steam turbine cycle. The SOFC/GT subsystem is based on previous work done by Francesco Calise during his doctoral research (Calise, 2005). In that work, a HRSG is not used. Instead, the gas turbine exhaust is used by a number of heat exchangers to preheat the air and fuel entering the fuel cell and to provide energy for district heating. The current work considers instead the possible benefits of using the exhaust gases in an HRSG in order to produce steam which drives a steam turbine for additional power output. Four different steam turbine cycles are considered in this M.S. thesis work: a single-pressure, a dual-pressure, a triple-pressure, and a triple-pressure with reheat. The models have been developed to function both at design (full load) and off-design (partial load) conditions. In addition, different solid oxide fuel cell sizes are examined to assure a proper selection of SOFC size based on efficiency or cost. The thermoeconomic analysis includes cost functions developed specifically for the different system and component sizes (capacities) analyzed. A parametric study is used to determine the most viable system/component syntheses/designs based on maximizing total system efficiency or minimizing total system life cycle cost.
Master of Science

M. Tech. (Biotechnology, Faculty of Applied and Computer Science), Vaal University of Technology
Tulbaghia violacea Harv. (wild garlic) has been used in traditional medicine in Southern Africa for the treatment of various ailments. Despite the widespread use and popularity of this medicinal plant as a herbal medicine, there is contradictory evidence regarding the safety and toxicity of the plant. The phytochemical profiling of the plant has also been neglected in research. The determination of chemical constituents present in plant material as well as the potential toxicity found in plants are preliminary steps necessary for the discovery and development of novel therapeutic agents with improved efficacy. The aim of this study was to evaluate the cytotoxic and genotoxic potential of crude extracts from the leaves, stems and roots of T. violacea. This was performed in vitro using aqueous and ethanol extracts of the leaves, stems and roots. The aim of the study was achieved by three major objectives; (1) to identify the active phytocompounds present in the leaves, stems and roots, (2) to assess the cytotoxicity using the MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) cell proliferation assay, and (3) to evaluate the genotoxic potential of the leaf, stem and root water extracts using the Allium cepa assay. A total of 14 phytochemicals were each extracted separately with distilled water and 70% ethanol by maceration from the leaves, stem and roots of T. violacea. The results of the qualitative phytochemical analysis showed that pharmacologically active compounds such as tannins, terpenoids, flavonoids, saponins, proteins, steroids, cardiac glycosides, phenols and coumarins were present in some organs of T. violacea. However, phlobatannins, leucoanthocyanins, alkaloids, carbohydrates and anthocyanins were absent in all plant parts. Overall, the leaves of the plant contained more active compounds than those present in the stems and roots when both water and 70% ethanol were used as the extractants. The quantitative phytochemical analysis for the Total Flavonoids Content (TFC) and Total Phenolic Contents (TPC) was also assessed. The water (0.027 mg/g) and 70% ethanol (0.053 mg/g) were most effective in extracting flavonoids from the leaves while the least amounts were obtained from the stems and roots. This observation was similar to the TFC were the water extracts of the leaves were the most effective in extracting phenols followed by the stems and roots. The MTT assay was conducted using two cell lines RAW 264.7 and C2C12. The experiment was conducted in triplicates for the leaf, stem and root extracts (water and ethanol) of T. violacea. The experimental design employed a 23 factorial design where three independent variables (concentration, incubation time and type of extracts) were selected using two levels for each variable (high (+) and low (-)). The results illustrated that both the water and ethanol vi extracts only showed a significant reduction in the number of viable cells at the concentration higher than 250 μg/ml treatment for both RAW 264.7 and C2C12 cells. The ethanol extracts from the leaves, stems and roots were found to be toxic towards the RAW 264.7 cells even at lower concentrations at both 24 and 48 h incubation periods (% cell viability < 50%). The water extracts were non-toxic to RAW 264.7 cells except for the water stem extract which showed toxicity after 48 h incubation (IC50 = 9.475 (4.061 to 23.39)). For the C2C12 cells, the lowest potent toxic concentration was 250 μg/ml for the ethanol extract of the stem after 48 h incubation. Overall, the T. violacea plant extracts were non-toxic as percentage cell viability greater than 50% was noted for both extraction solvents in all the plant parts of T. violacea. No cytotoxic activity was observed in all T. violacea plant parts with the C2C12 cell line (IC50 > 30 μg/ml). For the Allium cepa assay, only the water crude extracts of the leaves, stems and roots of T. violacea were used. A similar trend of potent genotoxic activity in the water stem extracts compared to the leaf and root extracts at the concentration ranges studied. Similar to the MTT assay, it is clear from the study that at higher concentrations, the water crude extracts from the leaves, stems and roots of T. violacea is toxic. From this study, it can be concluded that the extraction of compounds using water is more efficient than using ethanol. Overall, the T. violacea leaf extracts extracted the most phytocompounds and showed the highest percentage of viable cells as well as desirable IC50 values. However, preparation of herbal remedies using T. violacea plant extracts should be done with caution due to their possible genotoxic and cytotoxic potential at higher concentrations. This study raises a need to further conduct in vivo cytogenetic studies to ascertain the possible toxic effects of T. violacea crude extracts.

The present PhD thesis work reports the molecular and genetic dissection for the role of plant steroid hormones Brassinosteroids (BRs) in root growth and development of Arabidopsis thaliana (Arabidopsis). A genetic, physiological and cellular analysis of existing BR mutants, together with the discovery of a novel pathway for the control of BR mediated stem cell divisions. The results presented here provide experimental evidence for a role of BRs in stem cell homeostasis in the primary root of Arabidopsis. Specifically, BRs promote columella stem cell differentiation and the division of a set of low mitotic cells called quiescent center (QC) that maintain the surrounding stem cells. Using a microgenomic approach, a novel BR signaling component, BRAVO (Brassinosteroids at Vascular and Organizing Centre) has been identified that is specifically expressed in the stem cells. BRAVO encodes a R2-R3 MYB transcription factor (MYB56) that acts as a negative regulator of BRmediated QC divisions. This study uncovers a fine example of negative regulation model; BRAVO is directly repressed and interacts with BES1 creating a molecular switch that controls QC divisions. The work carried out during this PhD thesis shed light to a new function of brassinosteroids in the regulation of stem cells. BRAVO provides plasticity to the stem cells to response to DNA damage, and at the same time robustness to ensure QC function upon damage. The control of plant stem cell homeostasis is pivotal to understand plant adaptation to environmental changing conditions and provides a new mechanism to understand plants life span.
Aquesta tesi doctoral té com a objectiu principal investigar els efectes de les hormones vegetals esteroides, Brassinosteroids (BRs), durant el desenvolupament de lʼarrel primària dʼArabidopsis thaliana (Arabidopsis). Per tal dʼassolir aquest objectiu hem realitzat una caracterització genètica, fisiològica i anàlisi cel•lular de mutants de BRs. Així mateix, sʼha descobert una nova ruta de senyalització que controla les divisions de les cèl•lules mare mediades per BRs, Els nostres resultats experimentals mostren com els BRs controlen la homeòstasi de les cèl•lules mare de lʼarrel. En concret, els BRs promouen la diferenciació de les cèl•lules mare de la columel•la i la divisió dʼun grup de cèl•lules mitòticament inactives que actuen en el manteniment de les cèl•lules mare, el centre quiescent (QC). Mitjançant un abordatge microgenòmic hem identificat un nou element de la ruta de senyalització dels BRs específic de les cèl•lules mare, BRAVO (Brassinosteroids at Vascular and Organizing Centre). BRAVO és un factor de transcripció R2R3 de la família MYB (MYB56), que actua com a regulador negatiu de les divisions de QC. Els nostres resultats mostren un model de regulació negativa, on BES1 reprimeix directament i interacciona amb BRAVO, creant un interruptor molecular que controla les divisions del QC. El treball realitzat durant aquesta tesis doctoral permet proposar una nova funció dels BRs en el control de les cèl•lules mare. BRAVO dóna plasticitat a les cèl•lules mare per a poder respondre al dany sobre lADN, així com robustesa per evitar-lo. El control de la homeòstasi de les cèl•lules mare en plantes és vital per entendre lʼadaptació dʼaquests organismes sèssils i la longevitat que presenten algunes espècies.

Tissue engineers – in order to develop therapies for the treatment of complex neurological injuries and diseases – attempt to recreate elaborate developmental mechanisms in vitro. Neuronal precursor cells are excellent candidates for the study of developmental operations such as cell adhesion, differentiation, and axonal pathfinding. Hyaluronan (HA) is a common polysaccharide that is found extensively throughout the neuronal extracellular matrix (ECM), and can be functionalised and crosslinked to form stable hydrogels that support growing neuronal cells. Hyaluronan hydrogels can be modified chemically and mechanically to mimic the ECM of the developing brain, awarding control over mechanisms such as differentiation and axonal pathfinding. This thesis is concerned with the functionalisation and characterisation of HA hydrogels, ultimately in order to simulate vital properties of the developing brain. Here we show that HA hydrogels can be finely tuned mechanically (by modulating stiffness and viscosity), and chemically, by the conjugation of peptides that mimic the neural cell adhesion molecule (NCAM). NCAM mimics and novel mimics of sialylated NCAM significantly influence the differentiation of NSPCs in 2D and 3D. HA hydrogels successfully support long term culture of neural cells in 3D, and encourage the formation and extension of neurites of several cell types including human, mouse and rat neuronal precursor and stem cells. These results demonstrate for the first time that novel NCAM mimicking peptides can be conjugated to well defined hydrogel matrices that influence intricate developmental behaviours in 3D. Understanding how neural cells form functional networks is essential for the development of clinical approaches that attempt to address the injuries and diseases that affect these systems.

Today, many organisations are adopting offices that have an open design with or without flexible seating. While advocates of open-plan offices propose that these office types lead to cost savings and aid inter and intra-team communication, opponents argue that these office types are associated with decreased performance and worsened health among employees. This thesis investigates how the type of office (cell offices, shared room offices, small open-plan offices, medium-sized open plan offices, large open-plan offices and flex offices) influences employee health and performance, and whether this is different for different personalities and jobs with different concentration demands. Data were gathered by means of surveys and cognitive tests from five organisations with different office types. In Study I (N=1241), the aim was to investigate the main effect of office type on indicators of health and performance and the interaction effect of office type with the need to concentrate in order to carry out work tasks. Office type alone was associated with distraction and cognitive stress in such a way that cell offices were associated with fewest problems, followed by flex offices, while open-plan offices were associated with the most problems. While employees in open-plan offices and employees in flex offices reported more problems as the need for concentration increased, employees in cell offices reported the same level of problems regardless of the need of concentration. Study II (N=527) investigated how performance on a memory test was affected during normal working conditions as compared to a quiet baseline. There was a negative dose-response relationship between the size of the open-plan office environment and the drop in word recall during the normal working condition. However, Study II also showed that individuals working in cell offices had as high a drop in performance during normal working conditions as did those working in large open-plan office environments. Study III (N=1133–1171) focused on the interaction effect between office type and individual differences in personality. The personality trait agreeableness interacted with office type on the outcome variables distraction and job satisfaction. Specifically, Study III may indicate that as offices get more open and flexible, agreeable people will report more problems. In conclusion, the studies in the present thesis have implications for practice and suggest that office type impacts on employee health and performance, while concentration demands of the job and agreeableness moderate the effects. Although employees report higher level of distraction in open-plan office environments, when performance on a demanding task is measured, cell offices are not as favourable during normal working conditions as self-reported data usually indicate. Organisations should also be aware that, among open-plan offices, small open-plan offices are associated with fewer problems.
Kontorslandskap med eller utan fasta arbetsstationer förekommer idag i många organisationer. Förespråkare för kontorslandskap hänvisar till kostnadsbesparing samt förbättrade förutsättningar för kommunikation, medan motståndare hävdar att kontorslandskap leder till försämrad prestation och hälsa bland medarbetarna. Denna avhandling undersöker om kontorstyp påverkar de anställdas hälsa och prestation, samt om effekten av kontorstyp varierar beroende på de anställdas personlighet och typ av arbetsuppgifter. Data i form av enkätsvar och prestation på kognitiva tester samlades in från fem organisationer med olika typer av kontorslösningar (cellkontor, delade kontorsrum, små kontorslandskap, mellanstora kontorslandskap, stora kontorslandskap och flexkontor). I studie I (N = 1241) var syftet att undersöka huvudeffekten av kontorstyp på indikatorer för hälsa och prestation samt om effekten är beroende av koncentrationskraven i arbetet. Kontorstyp visade samband med distraktion och kognitiv stress på så sätt att medarbetare i cellkontor uppgav minst problem, följt av de i flexkontor, medan kontorslandskap var förknippade med mer problem. Vidare rapporterade anställda som hade arbetsuppgifter som krävde koncentration mer problem i kontorslandskap och flexkontor, medan anställda i cellkontor, oavsett arbetets krav, rapporterade lika mycket problem. I studie II (N = 527) undersöktes hur prestation på ett minnestest påverkades under normala arbetsförhållanden jämfört med en tyst referensmätning i olika kontorstyper. Det fanns ett negativt dos-responssamband mellan storleken på kontorslandskapet och hur många procent sämre medarbetare presterade under normala arbetsförhållanden. Men Studie II visade också att personer som arbetar i cellkontor hade ett lika högt bortfall i prestation under normala arbetsförhållanden som de som arbetade i stora kontorslandskap. I studie III (N = 1133-1171) låg fokus på interaktionseffekten mellan kontorstyp och personlighet. Vänlighet var den enda personlighetsvariabeln som interagerade med kontorstyp på utfallsvariablerna distraktion och arbetstillfredsställelse. Mer specifikt visade Studie III att när kontoret blir mer öppet och flexibelt, så rapporterar människor som skattar sig högt på personlighetsvariabeln vänlighet fler problem. Resultaten i avhandlingen kan få flera praktiska implikationer då den visar att kontorstyp påverkar medarbetarnas hälsa och prestation, medan koncentrationskrävande arbetsuppgifter och vänlighet modererar effekterna. Vidare visar avhandlingen att även om anställda rapporterar mindre distraktion i cellkontor jämfört med i kontorslandskap, behöver inte cellkontor vara lika gynnsamma som självskattade mått visar när prestationen mäts med objektiva mått under normala arbetsförutsättningar, i det här fallet ett minnestest. Slutligen bör organisationer även vara medvetna om att avhandlingen visar en viss tendens att små kontorslandskap är förknippade med mindre problem än stora.

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.

Vegetative propagation allows the amplification of selected genotypes for research, breeding, and commercial planting. However, efficient in vitro regeneration and genetic transformation remains a major obstacle to research and commercial application in many plant species. Our aims are to improve knowledge of gene regulatory circuits important to meristem organization, and to identify genes that might be useful for improving the efficiency of in vitro regeneration. In this thesis, we have approached these goals in two ways. First, we analyzed gene expression during poplar (Populus) regeneration using an AffymetrixGeneChip® array representing over 56,000 poplar transcripts. We have produced a catalog of regulated genes that can be used to inform studies of gene function and biotechnology. Second, we developed a GUS reporter system for monitoring meristem initiation using promoters of poplar homologs to the meristem-active regulatory genes WUSCHEL (WUS) and SHOOTMERISTEMLESS (STM). This provides plant materials whose developmental state can be assayed with improved speed and sensitivity. For the microarray study, we hybridized cDNAs derived from tissues of a female hybrid poplar clone (INRA 717-1 B4, Populus tremula x P. alba) at five sequential time points during organogenesis. Samples were taken from stems prior to callus induction, at 3 days and 5 days after callus induction, and at 3 and 8 days after the start of shoot induction. Approximately 15% of the monitored genes were significantly up-or down-regulated based on both Extraction and Analysis of Differentially Expressed Gene Expression (EDGE) and Linear Models for Microarray Data (LIMMA, FDR<0.01). Of these, over 3,000 genes had a 5-fold or greater change in expression. We found a very strong and rapid change in gene expression at the first time point after callus induction, prior to detectable morphological changes. Subsequent changes in gene expression at later regeneration stages were more than an order of magnitude smaller. A total of 588 transcription factors that were distributed in 45 gene families were differentially regulated. Genes that showed strong differential expression encoded proteins active in auxin and cytokinin signaling, cell division, and plastid development. When compared with data on in vitro callogenesis from root explants in Arabidopsis, 25% (1,260) of up-regulated and 22% (748) of down- regulated genes were in common with the genes that we found regulated in poplar during callus induction. When ~3kb of the 5' flanking regions of close homologs were used to drive expression of the GUSPlus gene, 50 to 60% of the transgenic events showed expression in apical and axillary meristems. However, expression was also common in other organs, including in leaf veins (40% and 46% of WUS and STM transgenic events, respectively) and hydathodes (56% of WUS transgenic events). Histochemical GUS staining of explants during callogenesis and shoot regeneration using in vitro stems as explants showed that expression was detectable prior to visible shoot development, starting 3 to 15 days after explants were placed onto callus inducing medium. Based on microarray gene expression data, a paralog of poplar WUS was detectably up-regulated during shoot initiation, but the other paralog was not. Surprisingly, both paralogs of poplar STM were down-regulated 3- to 6-fold during early callus initiation, a possible consequence of its stronger expression in the secondary meristem (cambium) than in shoot tissues. We identified 15 to 35 copies of cytokinin response regulator binding motifs (ARR1AT) and one copy of the auxin response element (AuxRE) in both promoters. Several of the WUS and STM transgenic events produced should be useful for monitoring the timing and location of meristem development during natural and in vitro shoot regeneration.
Graduation date: 2008

Dissertação de Mestrado em Biotecnologia Vegetal, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra
O tamarilho (Cyphomandra betacea (Cav.) Sendt sin. Solanum betaceum), pertencente à família das solanáceas, é uma árvore que produz frutos comestíveis com elevado valor nutricional. Alguns estudos têm mostrado o interesse desta espécie para a compreensão de aspectos particulares da morfogénese in vitro, particularmente a embriogénese somática (ES). A ES tem demostrado ser uma importante ferramenta na biotecnologia com um grande potencial para a rápida propagação de clones em larga-escala. Além disso, os procedimentos de transformação genética e criopreservação de muitas espécies vegetais, baseiam-se em protocolos eficazes de ES. Uma das formas de induzir ES no tamarilho consiste um processo em duas fases, no qual células embriogénicas são inicialmente induzidas num meio de cultura suplementado com uma auxina (fase de indução) e depois após a sua transferência para um meio de cultura desprovido de auxinas, desenvolvem-se embriões somáticos (fase de desenvolvimento). Vários tipos de explantes, com origem em material adulto ou previamente estabelecido in vitro, e várias condições de indução, têm sido testados para optimizar a resposta do tamarilho à indução de ES. Para superar o reduzido potencial de ES nos tecidos adultos, neste trabalho seguiu-se uma abordagem indirecta, na qual os rebentos de uma árvore adulta foram, primeiramente, estabelecidos in vitro, e uma abordagem directa, na qual se induziu ES em secções do caule de ramos jovens de tamarilho. De modo a optimizar o protocolo de indução de ES no tamarilho, precedeu-se à caracterização da resposta da indução de ES sob vários tipos de stresse, assim como face à indução de ES em diferentes genótipos, de plantas diploides e tetraploides, que foram previamente caracterizados citológica e morfologicamente. Os resultados demonstram que factores como o tipo de auxina, a concentração de sacarose, a presença de ácido ascórbico e o tempo de manutenção dos meios de cultura interferem na resposta à indução de ES. Na indução de ES em tamarilho, células embriogénicas e células não embriogénicas surgem lado a lado nos mesmos explantes, o que se trata de uma condição ideal para avaliar as alterações moleculares e bioquímicas observadas nos diferentes tipos de calos. No trabalho realizado estabeleceu-se, pela primeira vez, um protocolo para a multiplicação de massas de células embriogénicas através do uso de suspensões celulares. O teste de várias massas de células e vários volumes de meio de cultura permitiram analisar a cinética de crescimento das células e optimizar a razão x massa de células / volume a utilizar em ensaios futuros. Além disso, analisaram-se os perfis proteicos das secreções dos dois tipos diferentes de células, sendo as secreções do tecido não embriogénico as que apresentam mais diversidade e quantidade de proteínas. Este tipo de análise pode ser utilizada para compreender a embriogénese numa perspectiva mais integradora. Uma das muitas aplicações do calo embriogénico obtido por indução de ES é a transformação genética. Trabalhos anteriores para outras espécies têm demostrado que as taxas mais elevadas de sucesso na transformação genética têm sido obtidas com este tipo de explante. Neste trabalho procurou-se estabelecer um protocolo de transformação genéticas de células embriogénicas, utilizando três estirpes diferentes de Agrobacterium tumefaciens possuindo o plasmídeo p35SGUSINT. A quantificação da massa de células resistentes à canamicina e os resultados da coloração do ensaio GUS indicaram a estirpe C58C1,como a mais eficiente na transformação de células embriogénicas do tamarilho, sendo, no entanto, necessária uma análise mais detalhada em trabalhos futuros. A informação recolhida neste trabalho, nomeadamente com a análise comparativa da resposta de diferentes genótipos e com o desenvolvimento de um protocolo para a cultura de suspensões celulares, poderá contribuir para responder a alguns dos passos restritivos da ES e da transformação genética no tamarilho, para a qual os conhecimentos fundamentais sobre as plantas modelo tradicionais têm sido insuficientes. Encontrar as proteínas directamente envolvidas na aquisição de competência embriogénica poderá ajudar a compreender os mecanismos reguladores deste processo. Para além disso, o desenvolvimento de protocolos de transformação genética optimizados constitui um importante recurso, não apenas para o melhoramento da espécie, mas também como uma ferramenta da genómica funcional para a identificação e caracterização das vias moleculares envolvidas no processo de ES.
Cyphomandra betacea (Cav.) Sendt (tamarillo) (syn. Solanum betaceum) is a small solanaceous tree which produces edible high nutritional fruits. Several lines of research have shown the interest of this species to understand particular aspects of in vitro morphogenesis, in particular somatic embryogenesis (SE). SE is an important biotechnological tool with great potential for rapid large-scale clone propagation. In addition, genetic transformation and cryopreservation procedures in many plant species rely on efficient SE protocols. One of the pathways to induce SE in tamarillo is a two-step process in which embryogenic tissues and non-embryogenic callus are first produced (induction phase) in an auxin-rich medium and then developed into embryos, following the transfer to an auxin-free medium (development phase). Several explants, with origin from an adult tree or from plants previously established in vitro have been tested in optimization assays of the SE induction protocol in tamarillo. To overcome the lack of potential of adult tissues for SE, an indirect approach was attempted, in which shoots from an adult tree were first established in vitro, and a direct approach, in which SE was intended to be induced from juvenile plant material (intermodal stem segments). To improve the induction protocol of SE in tamarillo several stress conditions were tested for different genotypes of diploid and tetraploid plants. These genotypes were previously characterized cytological and morphologically. The results have showed that different factors, such as the auxin kind, the sucrose concentration, the presence of ascorbic acid on the medium and the medium´s maintenance time were crucial for the SE induction. In tamarillo SE, embryogenic and non-embryogenic cells arise side by side from the same cultured explants, which is an ideal condition to evaluate molecular and biochemical changes occurring in the different types of calli. In this work, a protocol for the embryogenic cells multiplication through cell suspension culture was established for the first time. Several weights of cells and several culture media volumes were tested in order to evaluate the kinetic growth of the cell suspension and optimize the ratio weight/volume for future approaches. Furthermore, protein profiles were obtained from the secretions produced by embryogenic and non-embyogenic cells after the culture period. The profiles analysis showed that the non-embryogenic cells were the ones producing a higher protein diversity and quantity. This type of analysis can be extended to understand embryogenesis in a more integrated perspective. xii One of the many applications for embryogenic tissue obtained by SE induction is its use for genetic transformation. Previous work with other species have reported the highest success levels achieved with this type of explant. In this work, the establishment of an efficient protocol for Agrobacterium-mediated transformation of embrygenic cells, using three different strains carrying the p35SGUSINT plasmid, was for the first time attempted. The quantification of kanamycin resistance cells and the results obtained for the Gus staining assay indicated that C58C1 was the most efficient strain, nevertheless more detailed analysis are needed for future assays. The information gathered in this work, with the comparative analysis of the responsiveness of different genotypes, under several culture conditions, and the development of a cell suspension culture protocol, can contribute to answer to some of the limiting steps of SE in tamarillo, to which fundamental knowledge from the classical model plants has been insufficient. Finding proteins directly involved in the acquisition of embryogenic competence may help to understand the regulatory mechanisms of this process. Furthermore, the development of optimized transformation protocols is critical, not only for the species improvement, but also for functional genomics approaches that would allow to better understand the molecular pathways involved in SE.