One strategy to repair an injured or degenerated retina is to stimulate the replacement of damaged or dead neurons with cells derived from endogenous stem- or progenitor cells. A successful strategy requires knowledge about how the proliferation and differentiation of the endogenous cells are regulated. In particular, this knowledge will be important in the establishment of protocols that produce sufficient numbers of specific neurons. The main aim of this thesis was to find and characterise factors regulating the proliferation and differentiation of retinal progenitor cells (RPCs) and hence, contribute to the knowledge of how to use progenitor cells for retinal repair.    The major result in this thesis is that GABA contributes to and maintains RPC proliferation. Inhibition of GABAA receptors decreases the proliferation of non-pigmented ciliary epithelial (NPE) cells and RPCs in the intact retina. We propose that this effect is mediated through changes in the membrane potential and voltage-gated calcium channels, which in turn regulate components of the cell cycle. Furthermore, we show that one of the endogenous RPC sources, the Müller cells, consists of two subpopulations based on Pax2 expression. This is interesting because Pax2 may suppress the neurogenic potential, characterised by de-differentiation and proliferation, in Müller cells. Finally, we show that over-expression of FoxN4 induces differentiation-associated transcription factors in the developing chick retina. However, FoxN4 over-expression did not trigger differentiation of NPE cells. These results indicate that the intrinsic properties of the RPCs are determinant for FoxN4-induced differentiation. The results presented in this thesis advance our understanding of how specific cells may be generated from different sources of RPCs. Our results show that the different sources are highly diverse in their potential to proliferate and produce neurons. GABA, Pax2 and FoxN4 may be factors to consider when designing strategies for retinal repair. However, the results indicate that the specific responses to these factors are highly associated with the specific properties of the progenitor cells. / <p>Doctor of Philosophy <strong>(Faculty of Medicine)</strong></p>

Heart valve interstitial cells (VICs) undergo activation and proliferation in repair and disease, but the mechanisms are not fully understood. We hypothesize that the establishment of N-cadherin/β-catenin cell-cell contacts may decrease VIC activation, and that Wnt3a/β-catenin signaling may increase VIC proliferation. VIC cultures of different densities are stained for α-SMA, cofilin, TGF-β, pSmad2/3, N-cadherin and β-catenin, and probed for phospho-β-catenin by Western blot. Low density VIC cultures are treated with exogenous Wnt3a and measured for cell number, proliferation, apoptosis, α-SMA, β-catenin, and β-catenin-mediated transcription. β-Catenin siRNA knockdown is used to assess β-catenin specificity. Increased staining of α-SMA, cofilin, TGF-β, pSmad2/3, nuclear β-catenin, and increased phospho-β-catenin are associated with few cell-cell contacts. Wnt3a increased VIC cell number, proliferation, nuclear β-catenin and β-catenin-mediated transcription without affecting activation and apoptosis, and proliferation is abolished by β-catenin siRNA. Thus, N-cadherin/β-catenin cell-cell contacts may inhibit VIC activation and Wnt3a/β-catenin signaling may increase VIC proliferation.

Heart valve interstitial cells (VICs) undergo activation and proliferation in repair and disease, but the mechanisms are not fully understood. We hypothesize that the establishment of N-cadherin/β-catenin cell-cell contacts may decrease VIC activation, and that Wnt3a/β-catenin signaling may increase VIC proliferation. VIC cultures of different densities are stained for α-SMA, cofilin, TGF-β, pSmad2/3, N-cadherin and β-catenin, and probed for phospho-β-catenin by Western blot. Low density VIC cultures are treated with exogenous Wnt3a and measured for cell number, proliferation, apoptosis, α-SMA, β-catenin, and β-catenin-mediated transcription. β-Catenin siRNA knockdown is used to assess β-catenin specificity. Increased staining of α-SMA, cofilin, TGF-β, pSmad2/3, nuclear β-catenin, and increased phospho-β-catenin are associated with few cell-cell contacts. Wnt3a increased VIC cell number, proliferation, nuclear β-catenin and β-catenin-mediated transcription without affecting activation and apoptosis, and proliferation is abolished by β-catenin siRNA. Thus, N-cadherin/β-catenin cell-cell contacts may inhibit VIC activation and Wnt3a/β-catenin signaling may increase VIC proliferation.

Hoxa2 null embryos display a high incidence of cleft secondary palate which has previously been described as secondary to altered tongue development. The experiments described in this thesis demonstrate that expression of Hoxa2 does occur within the developing palate, with the highest levels appearing in the early stages of palatogenesis (E12.5 and E13.5). Increased cell proliferation was observed throughout the palate in the absence of Hoxa2, without a detectable difference in apoptosis or the ability of the shelves to fuse. In addition, the palate shelves of the null embryos failed to elevate above the tongue, suggesting a mechanism by which the increased cell proliferation results in cleft palate.<p> Numerous downstream targets of Hoxa2 were also identified in the palate (Msx1, Bmp4, Barx1, Ptx1, Six2, Lef1 and Tbx1). In all cases, Hoxa2 appears to act as a transcriptional repressor. Increases in palatal Msx1, Bmp4 and Barx1 expression have all been previously described to lead to increases in cell proliferation. Hoxa2, Ptx1, Lef1 and Tbx1 may be involved in a novel pathway that regulates proliferation in the palate. In addition, three novel gene targets were identified in the palate, Six2, Fgf8 and Htra3.<p> Together these data show that there is a direct role for Hoxa2 in regulating palate development, apparently through regulating the expression of downstream genes involved in maintaining normal cell proliferation rates.

Phosphorous in its inorganic form, orthophosphate (Pi), is found in every compartment of the plant cell and serves as a substrate, product or effector for a wide range of metabolic processes. Several Pi transporters exist in plants and these help regulate Pi homeostasis within different cellular compartments. The PHT4 family of organellar Pi transporters consists of six members in the model plant Arabidopsis thaliana, and five of these are localized to plastids. I used gene expression analyses and reverse genetics to demonstrate functional specialization for the PHT4 family members with a focus on PHT4;1 and PHT4;2. The PHT4;1 Pi transporter is localized to chloroplast thylakoid membranes and it is expressed in a circadian manner. Plants that lack a functional copy of the PHT4;1 gene have reduced rosette size and altered responses to photooxidative stress. The PHT4;2 transporter is localized to heterotrophic plastids in roots and other sink organs and pht4;2 mutants exhibit decreased starch accumulation, which is consistent with a defect in Pi export, and increased rosette size, which is caused by increased cell proliferation. These results confirm that PHT4;1 and PHT4;2 have specialized functions and that plastidic Pi homeostasis influences broad aspects of plant metabolism, including abiotic stress response and control of lateral organ growth.

La política exterior y de seguridad de la Unión Europea (UE) varía sustancialmente dependiendo de las circunstancias específicas de cada caso. Esto es particularmente evidente en el ámbito de la no proliferación de armas de destrucción masiva (ADM). Por ejemplo, en el caso de la crisis nuclear iraní la UE se muestra un actor propio con un papel bastante coherente y enérgico, mientras que durante la disputa con Irak del 2003, la UE se porta más bien como una organización internacional profundamente dividida e incapaz de realizar acciones independientes. En la presente tesis se asume que las principales variables independientes que pueden explicar este fenómeno no son los 'intereses nacionales' sino las ideas en forma de creencias normativas y causales que sustentan la construcción de intereses, la elección de los instrumentos y, en última instancia, la política exterior colectiva. Por lo tanto, la cuestión central de esta investigación es: ¿Cómo afectan las ideas a la política exterior colectiva, en particular de la UE en el ámbito de la no proliferación?En la primera parte de la tesis, se desarrolla el marco teórico para comprender mejor la relación entre las ideas y los diferentes grados de acción colectiva por grupos de estados en materia de 'alta política.' Basado en un modelo concreto sobre el papel de las ideas en la cooperación internacional, se examina cómo funcionan estas ideas en el caso específico de la política exterior y de seguridad europea. En este sentido, se identifican cuatro grupos dominantes de ideas ('complejos de ideas') que influyen en la política europea común: 'Europa nacional,' 'Europa integracionista,' 'Europa cosmopolita' y 'Europa multilateral.' En estos complejos de ideas son particularmente importantes las creencias causales y normativas sobre seguridad, el uso de los medios y relaciones estatales. El argumento fundamental es que un número limitado de complejos de ideas hace probable el consenso en una política relativamente fuerte. Esto es particularmente cierto si se toma en consideración la maleabilidad de las ideas y el alto grado de institucionalización de grupos de estados como la UE. La competencia entre los complejos de ideas, sin embargo, deja un margen considerable para el desacuerdo. Por tanto, los complejos de ideas pueden explicar la fuerte variación de la política de la UE entre los diferentes campos de actividad.La segunda parte de la tesis analiza empíricamente la política europea en el ámbito de la no proliferación de ADM. Se han elegido tres casos de estudio: (a) una comparación entre las políticas de la UE durante la crisis nuclear iraní y la invasión estadounidense de Iraq; (b) los esfuerzos desiguales de la UE contra la proliferación en el sur y este de la vecindad europea, y (c) las políticas de la UE hacia las instituciones internacionales de no proliferación a la luz del concepto de 'multilateralismo eficaz.' El objetivo es demostrar cómo las ideas influyen en la práctica la política exterior desigual de la UE bajo situaciones diferentes. Del análisis de los estudios de caso se pueden extraer tres conclusiones principales: en primer lugar, el consenso en la UE para la acción colectiva sólo es posible si ciertos límites relativos a la percepción de seguridad, la utilización de medios y las relaciones con otros estados no se cruzan; en segundo lugar, la necesidad de un equilibrio entre los complejos de ideas opuestos explican la política frecuentemente moderada de la UE ('equilibrio de ideas'); y, por último, las ideas como 'multilateralismo eficaz' se pueden utilizar de manera limitada como un foco para fomentar la cohesión, coherencia y legitimidad de la UE en los asuntos internacionales. / The foreign and security policy outputs of the European Union (EU) vary substantially depending on the issue at stake. This has been particularly clear in the field of non-proliferation of Weapons of Mass Destruction (WMD). For example, in the case of the Iranian nuclear crisis, the EU shows the characteristics of a fairly coherent and forceful actor in its own right, whereas during the 2003 Iraq standoff the EU is merely a deeply divided international organization incapable of independent action. The dissertation argues that the principal independent variables that can explain this phenomenon are not 'national interests' but ideas in the form of normative and causal beliefs, which underpin the construction of interests, the choice of instruments and, ultimately, collective foreign policy outputs. Hence, the central research question is: How do ideas affect collective foreign policy output, in particular by the EU in the field of non-proliferation?In the first part, the dissertation develops a theoretical framework to understand better the relation between ideas and the different degrees of collective action by groups of states in matters of 'high politics.' Based on a concrete model outlining the role of ideas in international cooperation, it continues examining theoretically how ideas work in the specific case of the European foreign and security policy. In this regard, it identifies four dominant sets of ideas ('idea complexes') that influence common European policy output: 'national Europe,' 'integrationist Europe,' 'cosmopolitan Europe' and 'multilateral Europe.' In these idea complexes, causal and normative beliefs about security, the use of means and state relations are particularly important. The key argument is that the limited number of relatively malleable foreign policy idea complexes makes consensus for relatively forceful policy output likely, in particular taking into consideration the high degree of institutionalization of groups of states such as the EU. The competition between idea complexes leaves, however, substantial room for disagreement. Therefore, idea complexes can explain the EU's strong output variation between different fields of activity.The second part of the dissertation analyzes empirically the EU's policy in the field of non-proliferation of WMD. Three specific case studies have been chosen: (a) a comparison between the EU policies during the Iranian nuclear crisis and the US led invasion of Iraq; (b) the EU's uneven non-proliferation efforts in the Southern and Eastern neighbourhood; and (c) EU policies towards international non-proliferation institutions in light of the concept of 'effective multilateralism.' The aim is to demonstrate how ideas influence in practice the uneven EU foreign policy output in different situations. Three major conclusions can be drawn from the analysis of the case studies: First, consensus in the EU on collective action is only possible, if certain limits regarding security perception, use of means and relations with other states are not crossed; secondly, the need for striking a balance between competing idea complexes explains the frequently moderate policy output by the EU ('ideational balancing'); and, finally, ideas such as 'effective multilateralism' can be used to a limited extent as focal points to foster cohesion, coherence and legitimacy of the EU in international affairs.

Eukaryotic translation initiation factor 5A (eIF-5A) is the only known cellular protein that contains the post-translationally derived amino acid, hypusine. Initially, eIF-5A was named as a translation initiation factor because of its capability to stimulate the formation of methionyl-puromycin, which mimics the first peptide bond formation during protein synthesis, under <em>in vitro</em> conditions. Subsequently, however, this proposed function of eIF-5A has been questioned because a similar effect on translation was not observed <em>in situ</em>. Moreover, eIF-5A appears not to be required for general protein synthesis. Rather, there is evidence that it facilitates the translation of specific subsets of mRNAs required for cell proliferation as well as apoptosis. <br /><br /> There are two isoforms of eIF-5A in the human genome which have designated eIF-5A1 and eIF-5A2. The objective of the present study was to gain an increased understanding of the roles of eIF-5A1 and eIF-5A2 during apoptosis and cell proliferation using human cell lines as a model system. Apoptosis was induced by treating the cells with Actinomycin D or sodium nitroprusside (SNP), which initiate programmed cell death by different mechanisms. It was observed for both normal and cancer cells that eIF-5A1 protein is up-regulated during apoptosis induced by Actinomycin D or SNP, whereas eIF-5A1 mRNA is constitutively expressed and does not change in abundance during this treatment. The up regulation of eIF-5A1 protein levels in the absence of a corresponding up-regulation in eIF-5A1 mRNA suggests that eIF-5A1 may be post-transcriptionally regulated. Moreover, eIF-5A1 protein up-regulation was stronger in normal cells than in cancer cells. By contrast, eIF-5A2 protein was below detection levels during apoptosis in both normal and cancer cells, although the corresponding transcript was detectable by semi-quantitative RT-PCR. This is attributable to inefficient translation of eIF-5A2 mRNA. <br /><br /> The effects of eIF-5A1 and eIF-5A2 on cell proliferation were examined by modulating the levels of serum in cultures of UACC-1598 cells, which are ovarian cancer cells that express high levels of both isoforms of eIF-5A. Serum starvation, which induces cell cycle arrest and ensuing apoptosis, followed by the re-addition of serum had no effect on the transcript levels of either eIF-5A1 or eIF-5A2. However, eIF-5A1 and eIF-5A2 proteins were both up-regulated within 24 hours of the initiation of serum starvation, and this coincided temporally with the onset of apoptosis as measured by TUNEL and a subsequent decline in viable cells. <br /><br /> The data indicate that eIF-5A1 and eIF-5A2 are both post-transcriptionally regulated and that they have functionally redundant roles in apoptosis.

Hoxa2 null embryos display a high incidence of cleft secondary palate which has previously been described as secondary to altered tongue development. The experiments described in this thesis demonstrate that expression of Hoxa2 does occur within the developing palate, with the highest levels appearing in the early stages of palatogenesis (E12.5 and E13.5). Increased cell proliferation was observed throughout the palate in the absence of Hoxa2, without a detectable difference in apoptosis or the ability of the shelves to fuse. In addition, the palate shelves of the null embryos failed to elevate above the tongue, suggesting a mechanism by which the increased cell proliferation results in cleft palate.<p> Numerous downstream targets of Hoxa2 were also identified in the palate (Msx1, Bmp4, Barx1, Ptx1, Six2, Lef1 and Tbx1). In all cases, Hoxa2 appears to act as a transcriptional repressor. Increases in palatal Msx1, Bmp4 and Barx1 expression have all been previously described to lead to increases in cell proliferation. Hoxa2, Ptx1, Lef1 and Tbx1 may be involved in a novel pathway that regulates proliferation in the palate. In addition, three novel gene targets were identified in the palate, Six2, Fgf8 and Htra3.<p> Together these data show that there is a direct role for Hoxa2 in regulating palate development, apparently through regulating the expression of downstream genes involved in maintaining normal cell proliferation rates.

The unique gene, pleiotrophin (ptn) was identified in the expressed sequence taq (EST) derived from the developing tilapia brain in our lab. The cDNA full length of ptn was cloned. ptn play a role in the differentiation of nerve cell. In this study, bioinformatics were searched for EE723939.1 (ptn), which is a gene with 1026 bp of cDNA sequence, open reading frame(ORF) is 483bp, and deduced as 160 amino acids. The protein of PTN was expressed in the prokaryotic system, BL21(E.coli), and purified with Ni-NTA affinity chromatography. In the present studies, ptn, cloned from tilapia, Oreochromis mossambicus. The influence of ptn on the proliferation of Neuro-2a cell was also investigated.The ORF of ptn was cloned, and the pEGFP-ptn plasmid was constructed. The distribution of ptn in the pEGFP-ptn transfected Neuro-2a cell was identified by fluorescence and laser confocal microscopy.

Understanding the influence of environment and correlation/relationships among traits is necessary in selection for crop quality improvement. Therefore, correlations among antioxidant activity (AOA), total phenolics (TP), phenolic composition, and specific gravity (SPG) in four potato (Solanum tuberosum, L.) cultivars (Atlantic, Red La Soda, Russet Norkotah, and Yukon Gold) grown in nine states (California, Idaho, Michigan, Minnesota, New Jersey, North Carolina, Oregon, Texas, and Wisconsin) for three years, and in 15 advanced selections grown in Texas were investigated. Cultivars within and between locations were significantly different in AOA, TP, and SPG. Significant effects of cultivar, year, location and their interactions on AOA, TP, and SPG were observed. There were significant positive correlations among the four cultivars between AOA and TP, and negative correlations between AOA and SPG, and between TP and SPG. However, correlations between AOA and SPG, and between TP and SPG, in the advanced selections were not significant. Some tuber-bearing wild potato species were higher in AOA and TP than the commercial cultivars; therefore, they could be used as parental material in breeding for high AOA and TP. However, use of wild species that might be higher in total glycoalkaloids (TGA) than cultivars could result in progenies with high TGA if the traits are positively correlated. To elucidate the relationships among AOA, TP and TGA, accessions of Solanum jamesii and S. microdontum from the US Potato Genebank were screened for these traits and their correlations determined. Also, anti-proliferative and cytotoxic effects of 15 S. jamesii tuber extracts (5 and 10 μg/ml) on human prostate (LNCaP) and colon (HT-29) cancer cells was determined in vitro. Alpha-solanine and α-chaconine were found in both species, while tomatine and dehydrotomatine were quantified in some S. microdontum accessions. Both species were higher in all above traits than the Atlantic, Red La Soda, and Yukon Gold cultivars. More than 90% of S. jamesii accessions had TGA levels < 20 mg/100g fresh weight, while only two accessions of S. microdontum, P1 500041 and PI 473171, exhibited TGA < 20 mg/100g. Neither AOA nor TP was significantly correlated with TGA in both species. Also, individual phenolics were not correlated with TGA. Solanum jamesii accessions significantly reduced proliferation of HT-29 (5 and 10μg/ml) and LNCaP (10μg/ml) cells and were not cytotoxic compared to the control (DMSO). Therefore, since AOA and TP were not found to be correlated with TGA, using wild accessions in breeding for increased health promoting compounds would not necessarily increase glycoalkaloids in newly developed potato cultivars.