Human umbilical cord blood (HUCB) cells consist of a heterogeneous population of cells, rich in hematopoietic stem and progenitor cells. These cells have been used in the treatment of various nonmalignant and malignant hematopoietic diseases. With in the last few years HUCB cells have been used in pre-clinical animal models of brain and spinal cord injuries, in which functional recovery has been shown. The properties of cord blood cells that could be important in cell transplantation (repair or replacement) of CNS injury or disease are currently being evaluated. The major focus of this study was to determine whether HUCB cells would migrate to ischemic tissue extracts. In addition, factors that may be inducing the cells to migrate were examined by identifying the cytokines or chemokines present in the ischemic tissue extracts. The secondary focus was to establish whether cultured HCUB cells are releasing cytokines and chemokines (in vitro) in response to their environment. The results of these studies showed that HUCB cells migrate to ischemic tissue in a time dependent manner. In which there is a 48 to 72 hour window of opportunity for the delivery of HUCB cells to the ischemic brain. In addition, the cord blood cells were shown to release cytokines that may be aiding in the behavioral recovery seen in the transplantation studies. The results from this study are promising in that the current 3-hour therapeutic window for the treatment of stroke victims, using approved anticoagulant treatment, may be extended with the use of cord blood cell therapy with the peak at 48 hours.

Neutrophils playa key role in the systemic inflammatory response which may lead to serious tissue injury and multiple organ dysfunction. In this setting, activated neutrophils, largely in response to tumour necrosis factor-alpha (TNF-α), secrete reactive oxidants, granule proteases and bioactive lipids, as well as pro-inflammatory cytokines, emphasising the importance of these cells as targets for anti-inflammatory therapies. There are, however, only a few currently available agents that directly modulate neutrophil pro-inflammatory responses in clinical practice, with corticosteroids being relatively ineffective against these cells. Although, the anti-inflammatory potential of cAMP-elevating agents has been recognised, the exact molecular/biochemical mechanisms which underlie the anti-inflammatory actions of epinephrine and related β-agonists with neutrophils, have not been established. Epinephrine treatment of neutrophils resulted in increased intracellular cAMP and dose-related inhibition of both superoxide production and elastase release, which was potentiated by the type 4 phosphodiesterase inhibitor, rolipram, further supporting a cAMP-mediated effect. Although epinephrine did not affect the release of Ca2+ from neutrophil intracellular stores, the rate of clearance of cytosolic Ca2+ was accelerated by this agent. In the setting of decreased efflux and a reduction in store-operated influx of Ca2+, these effects of epinephrine are compatible with enhancement of the cAMP-dependent Ca2+ sequestering/resequestering endo-membrane Ca2+-ATPase. Epinephrine therefore down-regulates the pro-inflammatory activation of neutrophils by cAMP-mediated enhancement of the clearance of cytosolic Ca2+. Comparison of the effects of 4 selective (fenoterol, formoterol, salbutamol and salmeterol) and 3 non-selective (epinephrine, norepinephrine and isoproterenol) β-adrenoreceptor agonists, on the pro-inflammatory activities of human neutrophils, demonstrated that the agents tested clearly differ with respect to anti-inflammatory potential. Epinephrine, isoproterenol, fenoterol and formoterol significantly increased intracellular concentrations of cAMP in neutrophils, an activity which was paralleled by inhibition of the production of reactive oxidants and release of elastase from FMLP-activated cells. Salbutamol and salmeterol on the other hand, did not cause significant suppression of the pro-inflammatory activities of these cells. The effect of norepinephrine was intermediate between these two groups. The inhibitory effects of βagonists are mediated via β2-adrenergic receptors on the neutrophil membrane. The relationship between activation of NAOPH oxidase, alterations in membrane potential and triggering of Ca2+ fluxes in human phagocytes has been investigated using neutrophils from 4 subjects with chronic granulomatous disease (CGO). Activation of CGO neutrophils was accompanied by a prolonged increase in cytosolic Ca2+, occurring in the setting of trivial membrane depolarisation and accelerated influx of Ca2+. This was associated with hyperactivity of the cells with excessive elastase release, which was attenuated by the type 4 phosphodiesterase inhibitor, rolipram. These findings support the involvement of NAOPH oxidase in regulating membrane potential and Ca2+ influx in activated neutrophils, and may explain the disordered inflammatory responses, and granuloma formation, which are characteristic of CGO. Store-operated influx of Ca2+ into activated neutrophils is stringently regulated, presumably to prevent hyperactivation of the cells. The major contributors to this physiologic, anti-inflammatory process are NAOPH oxidase which, by its membrane depolarising actions excludes extracellular Ca2+, and the plasma membrane and endomembrane Ca2+-ATPases, which mediate clearance of store-derived cation. Subsequent influx of the cation, through store-operated Ca2+ channels is controlled by the relatively slow, restraining, membrane repolarising action of the Na+/Ca2+ exchanger, enabling efficient diversion of incoming cation into stores. / Thesis (DPhil (Immunology))--University of Pretoria, 2005. / Immunology / unrestricted

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Chez les plantes à fleurs, l’ovaire est l’organe reproducteur femelle et il interagit de façon importante avec les gamètes mâles durant la croissance, le guidage, la réception et la rupture du tube pollinique ainsi que la fusion des gamètes. Le processus débute lorsque de nombreux gènes de l’ovule sont activés à longue distance lors de la réception du pollen sur le stigmate. Afin d’explorer les signaux provenant de l’ovule ayant un impact important sur les interactions pollen–pistil, particulièrement les molécules sécrétées impliquées dans la signalisation espècespécifique, l’expression génique des ovules sous forme d’ARNm ainsi et la sécrétion protéique ont été étudiées chez Solanum chacoense, une espèce diploïde de pomme de terre sauvage. S. chacoense a subi beaucoup d’hybridation interspécifique avec d’autres espèces sympathiques de solanacées, facilitant ainsi grandement l’étude des interactions pollen–ovule de façon espècespécifique ainsi que leur évolution. Dans ce projet, des ovules provenant de trois conditions différentes ont été comparés: des ovules matures de type sauvage, des ovules légèrement immatures, récoltés deux jours avant l’anthèse et des ovules provenant du mutant frk1 pour lesquels le sac embryonnaire est absent. Un séquençage d’ARN à haut débit a d’abord été effectué sur les ovules de type sauvage de S. chacoense afin de générer un assemblage de référence comprenant 33852 séquences codantes. D’autres séquençages ont été effectués sur les trois conditions d’ovules et sur les feuilles afin de faire une analyse d’expression différentielle des gènes. En comparaison avec les ovules de type sauvage, 818 gènes sont réprimés dans les ovules du mutant frk1. Un sous-groupe de 284 gènes, étaient également sous-exprimés dans les ovules légèrement immatures, suggérant un rôle spécifique dans les stades tardifs de la maturation du sac embryonnaire (stade de développent FG6 à FG7) ainsi que du guidage du tube pollinique, puisque ni les ovules du mutant frk1 ni ceux légèrement immatures ne sont capables d’attirer les tubes polliniques lors d’essais de croissance semi in vivo. De plus, 21% de ces gènes sont des peptides riches en cystéines (CRPs). En utilisant un transcriptome assemblé de novo provenant de deux proches parents de S. chacoense, S. gandarillasii et S. tarijense, une analyse d’orthologie a été effectuée sur ces CRPs, révélant une grande variabilité et une évolution rapide chez les solanacées. De nouveaux motifs de cystéine uniques à cette famille ont également été découverts. En comparant avec des études similaires chez Arabidopsis, le sac embryonnaire de S. chacoense montre un transcriptome fortement divergent, particulièrement en en ce qui a trait à la catégorisation fonctionnelle des gènes et de la similarité entre les gènes orthologues. De plus,même si la glycosylation n’est pas requise lors du guidage mycropylaire du tube pollinique chez Arabidopsis, Torenia ou le maïs, des extraits d’ovules glycosylés de S. chacoense sont capables d’augmenter la capacité de guidage de 18%. Cette étude est donc la première à montrer une corrélation entre glycosylation et le guidage du tube pollinique par l’ovule. En complément à l’approche transcriptomique, une approche protéomique portant sur les protéine sécrétées par l’ovule (le secrétome) a été utilisée afin d’identifier des protéines impliquées dans l’interaction entre ovule et tube pollinique. Des exsudats d’ovules matures (capables d’attirer le tube pollinique) et d’ovules immatures (incapables d’attirer le tube pollinique) ont été récoltés en utilisant une nouvelle méthode d’extraction par gravité permettant de réduire efficacement les contaminants cytosoliques à moins de 1% de l’échantillon. Un total de 305 protéines sécrétées par les ovules (OSPs) ont été identifiées par spectrométrie de masse, parmi lesquelles 58% étaient spécifiques aux ovules lorsque comparées avec des données de protéines sécrétées par des tissus végétatifs. De plus, la sécrétion de 128 OSPs est augmentée dans les ovules matures par rapport aux ovules immatures. Ces 128 protéines sont donc considérées en tant que candidates potentiellement impliquées dans la maturation tardive de l’ovule et dans le guidage du tube pollinique. Cette étude a également montré que la maturation du sac embryonnaire du stade FG6 au stade FG7 influence le niveau de sécrétion de 44% du sécrétome total de l’ovule. De façon surprenante, la grande majorité (83%) de ces protéines n’est pas régulée au niveau de l’ARN, soulignant ainsi l’importance de cette approche dans l’étude du guidage du tube pollinique comme complément essentiel aux études transcriptomiques. Parmi tous les signaux sécrétés par l’ovule et reliés au guidage, obtenus à partir des approches transcriptomiques et protéomiques décrites ci-haut, nous avons spécifiquement évalué l’implication des CRPs dans le guidage du tube pollinique par l’ovule chez S. chacoense, vu l’implication de ce type de protéine dans les interactions pollen-pistil et le guidage du tube pollinique chez d’autres espèces. Au total, 28 CRPs étaient présentes dans les ovules capables d’attirer le tube pollinique tout en étant absentes dans les ovules incapables de l’attirer, et ce, soit au niveau de l’ARNm et/ou au niveau du sécrétome. De celles-ci, 17 CRPs ont été exprimées dans un système bactérien et purifiées en quantité suffisante pour tester le guidage. Alors que des exsudats d’ovules ont été utilisés avec succès pour attirer par chimiotactisme le tube pollinique, les candidats exprimés dans les bactéries n’ont quant à eux pas été capables d’attirer les tubes polliniques. Comme l’utilisation de systèmes d’expression hétérologue eucaryote peut permettre un meilleur repliement et une plus grande activité des protéines, les candidats restants seront de nouveau exprimés, cette fois dans un système de levure ainsi que dans un système végétal pour produire les peptides sécrétés. Ceux-ci seront ensuite utilisés lors d’essais fonctionnels pour évaluer leur capacité à guider les tubes polliniques et ainsi isoler les attractants chimiques responsable du guidage du tube pollinique chez les solanacées comme S. chacoense. / In flowering plants, the ovary is the female reproductive organ that interacts extensively with the male gametophyte during pollen tube (PT) growth, guidance, reception, discharge and gamete fusion. The process begins when numerous ovule-expressed genes are activated when pollen lands on the stigma. To explore the ovular signals that have a great impact on successful pollen–pistil interactions, especially the secreted molecules that mediate species-specific signalling events, ovule mRNA expression and protein secretion profiles were studied in Solanum chacoense, a wild diploid potato species. Solanum chacoense has undergone extensive interspecific hybridization with sympatric solanaceous species that greatly facilitates the study of species-specific pollen–ovule interactions and evolution. In this project, three ovule conditions were studied: wild-type mature ovules, slightly immature ovules at two days before anthesis (2DBA), and frk1 mutant ovules that lack an embryo sac (ES). RNA-seq was performed on S. chacoense ovules to provide a scaffold assembly comprising 33852 CDS-containing sequences, then to provide read counts for differential gene expression analyses on three ovule conditions as well as on leaf. Compared to wild-type ovules, 818 genes were downregulated in frk1 ovules. A subset of 284 genes was concurrently under-expressed in 2DBA ovules, suggestive of their specific involvement in late stages of ES maturation (female gametophyte (FG), FG6 to FG7 developmental stage), as well as in PT guidance processes, as neither frk1 nor 2DBA ovules attract semi in vivo-grown PTs. Of these 284, 21% encoded cysteine-rich peptides (CRPs). Using de novo assembled ovule transcriptomes of two close relatives, S. gandarillasii and S. tarijense, an orthology survey was conducted on these CRPs, revealing their highly polymorphic nature among species and rapid evolution. Interestingly, novel cysteine motifs unique to this family were also uncovered. As compared to parallel studies in Arabidopsis, S. chacoense was found to possess a highly divergent ES transcriptome, in terms of both functional categories and individual ortholog similarities. Although glycosylation is not required for micropylar guidance cues to attract PTs in Arabidopsis, Torenia or maize, glycosylated ovule extracts from S. chacoense showed enhanced PT guidance competency by 18%. This is the first time a positive regulation between glycosylation and ovular PT guidance has been observed. As a complement to the transcriptomic approach, a proteomic approach using secreted proteins from the ovule (secretome) was employed to identify proteins involved in pollen–pistil interactions. Ovule exudates were collected from mature ovules (PT attracting) and immature ovules at 2DBA (PT nonattracting), using a novel tissue free-gravity extraction method (tf-GEM), which efficiently reduced the cytosolic contamination to less than 1%. Through mass spectrometry analyses, a total of 305 ovule-secreted proteins (OSPs) were identified, of which 58% were considered ovule-specific when compared to secretome studies conducted in other plant tissues. The secretion of 128 OSPs was upregulated in mature ovules vs. immature ovules. These OSPs were considered as candidate proteins involved in late ovule maturation and PT guidance. This study demonstrated that the ES maturation from FG6 to FG7 stages influenced the secretion status of 44% of ovule secretome. Surprisingly, the majority (83%) of these proteins were not regulated at the RNA level, vindicating this novel approach in the study of PT guidance as a robust complement to transcriptomic studies. Among all identified guidance-related ovular signals from the transcriptomic and proteomic approaches described above, we focused on the evaluation of the involvement of CRPs in ovular PT guidance of S. chacoense, due to the implication of various CRPs in pollen–pistil interactions and, especially, in PT guidance. A total of 28 CRPs were present in PT attracting ovules while being low or absent in nonattracting ovules, at the mRNA and/or protein secretion levels. Of these, 17 CRPs were expressed in bacteria and purified in sufficient amount for PT guidance assays. However, while ovule exudates were shown to induce PT chemotropism in the bead assay, refolded candidates did not show guidance competency. Since the use of eukaryotic protein expression systems might lead to better refolding and higher protein activity, the remaining candidates will be expressed in both yeast and plant-based expression systems and tested for their ability to attract PTs in a semi in-vivo assay, in order to lead us toward the isolation of PT guidance chemoattractants in solanaceous species like S. chacoense.