Clomiphene citrate (CC) is the leading treatment for women with anovulatory infertility. The precise mechanism of action of the drug on the hypothalamic-pituitary-gonadal (HPG) axis has yet to be determined. Neurons expressing kisspeptin, neurokinin B (NKB) and dynorphin A (Dyn), collectively called KNDy neurons, in the arcuate nucleus (ARC) of the hypothalamus have been shown to play an integral role in the estradiol (E2) feedback pathways of the reproductive system in mammals. KNDy neurons are found in the ARC and the anteroventral periventricular nucleus (AVPV) in humans but have been predominantly reported to not express NKB and Dyn in rodents. The axons of these neurons project to the medial eminence (ME) in the region where the gonadotropin-releasing hormone (GnRH) terminals and fibres are located. It was hypothesised that CC upregulates the gene expression of kisspeptin and neurokinin B while down-regulating the gene expression of dynorphin A which results in a leutenizing hormone surge and an increase in oestradiol which causes ovulation. This was a randomized experiment which included 18 female Sprague-Dawley rats in which the aim was to analyse the expression of kisspeptin, NKB and Dyn in the ARC and the AVPV as well as blood plasma levels of oestradiol and leutinizing hormone (LH) in female rats after CC administration. Six of the rats constituted the control group that received a vehicle solution. The second group of 6 rats received the intervention in the form of CC and the third group of six rats received CC as well as p234-penetratin, a kisspeptin antagonist (KpA). The mRNA expression of the KNDy genes were analysed using real-time quantitative polymerase chain reaction (qPCR) and the plasma levels of E2 and LH were analysed by enzyme-linked immunosorbant assays (ELISA). ELISA results show that the E2 concentration in the group that received CC plus KpA was found to be marginally lower than that of the control group but there was no significant difference between the E2 concentrations of the control group and the group that received only CC. The LH concentration in the group that received CC plus KpA was significantly higher than both other groups but again, there was no significant difference between the LH concentration control group and the group that only received CC. The qPCR showed that in the AVPV, the kisspeptin expression of the CC group and the CC plus KpA groups are marginally higher than that of the control group. Conversely, the Dyn expression of the CC group and the CC plus KpA groups are marginally lower than that of the control group in the AVPV. There were no significant differences in NKB expression across the three groups. In the ARC, there were no significant differences in kisspeptin or Dyn expression across the groups. The NKB expression of the CC group was marginally lower than that of the control and there was no significant difference between the CC plus KpA group and the control group. In summary, CC appears to have a marginal effect on the kisspeptin and Dyn mRNA via the positive feedback systems in the rat AVPV as well as a significant decrease of NKB mRNA via the negative feedback systems in the ARC. To increase the validity of similar future studies, higher sample sizes, different drug administration doses, possibly more precise surgical techniques and more accurate age determination methods or ovariectomised rats could be used. / Dissertation (MSc)--University of Pretoria, 2016. / Physiology / MSc / Unrestricted

OBJECTIVE: To evaluate the effectiveness of offering double intrauterine insemination (IUI) to clients in our fertility program. STUDY DESIGN: In this prospective, nonrandomized study, 595 couples with ovulatory dysfunction, endometriosis, male factor, unexplained, tubal factor and combined diagnoses utilizing clomiphene citrate-hCG (CC-hCG), CC-gonadotropin-hCG (CC-Gn-hCG), Gn-hCG, lupron-Gn-hCG (L-Gn-hCG) or luteinizing hormone (LH) surge monitoring of natural cycles were offered single or double IUI in a total of 1,276 cycles. Single IUIs were performed at 36 hours following hCG or the day following LH surge; double IUIs were performed 18 and 36 hours following hCG or the day of and day following LH surge. Single versus double IUI clinical pregnancy outcomes were compared between ovarian stimulation protocols and diagnostic categories. RESULTS: One hundred ten clinical pregnancies occurred for 508 couples in 999 single IUI cycles (fecundity, 11.0%); 45 clinical pregnancies for 174 couples occurred in 277 double IUI cycles (16.2%, p < 0.004). The single IUI group was younger than the double IUI group (32.8 vs 33.7, p < 0.004). Differences for fecundity were noted regarding diagnostic categories between single and double IUI groups (ovulation dysfunction, 12.9% vs 19.5%, p < 0.048, and male factor, 7.9% vs 17.5%, p < 0.030) and ovulation protocols (CC-Gn-hCG, 13.0% vs 21.3%, p < 0.031, and L-Gn-hCG, 4.2% vs 25.0%, p < 0.002). CONCLUSION: Double IUI is superior to single IUI overall, especially when comparing Gn-containing ovarian stimulation protocols or within the ovulatory dysfunction and malefactor diagnostic categories.

no / Two regioisomeric citrate-functionalized ciprofloxacin conjugates have been synthesized and their antimicrobial activities against a panel of clinically-relevant bacteria have been determined. Cellular uptake mechanisms were investigated using wild-type and ompF deletion strains of Escherichia coli K-12.

The favorable interaction between proteins and nanoparticles has sparked potential applications of nanotechnology in medicine, and the unique electronic and chemical properties of nanoparticles also provide novel strategies for protein-related therapeutics. The formation of the biocorona has attracted substantial interest over the past decades. For instance, as a potential drug delivery mechanism, protein-coated nanoparticles can improve biocompatibility and increase targeting ability. However, the mechanistic details of protein-nanoparticle interactions remain poorly understood. For example, it is currently impossible to predict the orientation and structure of proteins on the nanoparticle surface, as well as the fate of the biocorona in vivo. Since the composition of the biocorona determines the biological response, identifying and stabilizing the biocorona seems critical for the further development of applications in biological system. In this study, we investigated the physicochemical properties of protein interactions with gold nanoparticles (AuNPs). Firstly, we developed an NMR-based approach for measuring the stoichiometry of protein adsorption to AuNPs, which can be generally applied to globular proteins of different size. Quantitative analysis enabled us to create a protein binding model that involves an initial association, structure reorientation and irreversible adsorption. Secondly, we measured the protein hydrogen-deuterium exchange rates and found that they were unperturbed in the presence of AuNPs, suggesting that proteins retain their globular structure upon adsorption. Finally, we investigated the electrostatic contribution to binding, and we identified a dynamically changing surface in which the factors of net charge, binding affinity and protein size play distinct roles at different phases.

No description available.

The discovery that negatively charged aggregates of C60 fullerene are stable in aqueous environments has elicited concerns regarding the potential environmental and health effects of these aggregates. Although many previous studies have used aggregates synthesized using intermediate organic solvents, this work primarily employed an aggregate production method that more closely emulates the fate of C60 upon accidental release into the environment — extended mixing in water. The aggregates formed via this method (aqu/nC60) differ from those produced using the more common solvent exchange methods. The aqu/nC60 aggregates are heterogeneous in size (20 nm and larger) and shape (angular to round), negatively charged, and crystalline in structure — exhibiting a face centered cubic (fcc) system. Solution characteristics such as aqu/nC60 aggregate size and concentration were found to be dependant upon preparation variables such as stirring time, initial C60 concentration, and initial particle size. Additional experiments indicate that aggregate charge, structure, and stability are highly dependant upon the identity of co-solutes (NaCl, CaCl2, sodium citrate) and their concentrations. Citrate concentrations greater than 0.5 mM resulted in the formation of very small (< 20 nm) spherical aqu/nC60 particles. At moderate citrate concentrations (~ 1 mM) a more negative surface charge was observed, which may be an indication of increased nC60 stability. In contrast, high concentrations of monovalent and divalent electrolytes result in aggregation and sedimentation of nC60 out of solution. Our research describes the effect that solution composition has on aggregate formation and stability, and suggests that C60 fate and transport will be a function of solution composition. / Master of Science

L’étude proposée s’inscrit dans un contexte de contamination croissante de la pédosphère et fait le choix d’appréhender cette problématique via une approche centrée sur la rhizospère et sur les mécanismes gouvernant son évolution. Ces travaux s'intéressent à la relation entre l’uranium – un métal naturel ubiquiste non-essentiel – et le citrate – un acide organique de faible poids moléculaire exsudé par certaines espèces végétales poussant sur un milieu pauvre en phosphore. Parmi ces espèces particulières le lupin blanc (Lupinus albus) fait office de plante modèle. Un sol naturellement riche en uranium prélevé à l’affleurement d’une veine de pechblende a été étudié. L’objectif du travail a été de savoir si l’activité racinaire est suffisante pour provoquer la remobilisation de l’uranium dans la solution du sol et favoriser son transfert. Le système sol/plante a été étudié sur rhizotest et de manière découplée en hydroponie et en réacteurs fermés. Il a été démontré que la présence d’uranium dans un milieu contrôlé (hydroponie) stimule l’exsudation de citrate chez les lupins blancs qui ne sont pas carencés en phosphore et que cette exsudation de citrate favorise la solubilisation de l’uranium dans la solution du sol en réacteurs fermés. Ces résultats ont été confirmés par les observations faites sur rhizotest même si les valeurs obtenues dans ce cas étaient plus nuancées. Ceci justifie les approches utilisées et met en évidence leur complémentarité. Les résultats obtenus ont également permis d’apporter des informations concernant la toxicité de l’uranium, son internalisation dans les cellules racinaires ainsi que sur son transfert des racines aux parties aériennes. / The present study is related to the context of pedosphere contamination increasing and chose to consider this issue through a focus on rhizosphere and mechanisms that govern its evolution. This work is oriented on the relation between uranium – a non-essential ubiquitous metal – and citrate – a low molecular weight organic acid exudated by specific species when grown on a phosphorus-deficient environment. Among these particular species white lupine (Lupinus albus) is considered as a model species. Furthermore, a natural soil containing huge amounts of uranium has been collected at the outcrop of a pitchblende vein and used during some experiments. The main aim of this PhD thesis was to determine if the root activity of white lupine is sufficient to cause the remobilization of uranium into soil solution and so to promote the transfer of this contaminant to the plant. The soil/plant system has been studied with rhizotests and by combining datas obtained with hydroponics experiments and batches tests. It has been demonstrated that the presence of uranium in a controlled growth medium (hydropony) stimulates citrate exudation in P-sufficient white lupine. More, this citrate exudation enhances uranium solubilization in soil solution in batches. These results have been confirmed by observations on rhizotest even if the values in this case were more nuanced. This justifies the employed approaches and highlights their coherence and complementarity. The results also allowed to provide additional information concerning uranium toxicity, its internalization in root cells as well as its transfer from roots to aerial parts.

L’objectif de ce travail est de concevoir, développer, mettre en œuvre et optimiser une technologie continue permettant une bonne maîtrise des réactions de précipitation, processus chimique rapide donnant naissance à une phase solide. Ce précipiteur est un réacteur multifonctionnel à lit fluidisé qui n’a pas connu le même essor que les cristallisoirs à lit fluidisé. Deux approches expérimentales ont été adoptées : (1) l’étude des phénomènes de mélange des réactifs et (2) l’étude de l’influence des conditions opératoires sur la précipitation d’un produit modèle. Le produit modèle choisi pour cette étude est la calcite, le polymorphe le plus stable du carbonate de calcium qui possède trois polymorphes : la vaterite, l’aragonite et la calcite. Cette dernière est obtenue via la réaction de précipitation entre les solutions de chlorure de calcium et de carbonate de sodium à une température de 20° C et en présence d’un agent complexant (citrate de sodium) pour orienter la forme cristalline recherchée. Une étude cinétique a été menée pour la détermination des cinétiques de nucléation et de croissance cristalline de la calcite en milieu citrate. Le réacteur à lit fluidisé, de volume de 10 L, constitué de deux zones : cylindrique de fluidisation et de classification des particules et cylindro-conique de décantation, a été conçu au Laboratoire des Sciences du Génie Chimique. L’étude du mélange global, réalisée à l’aide de la réaction de décoloration acide-base et l’étude hydrodynamique, réalisée à l’aide de suspensions de microbilles, ont permis de développer et d’optimiser ce réacteur multifonctionnel. La faisabilité de la précipitation de la calcite en lit fluidisé a été ensuite vérifiée. Les expériences effectuées en présence d’une charge solide conséquente ont permis de diminuer la sursaturation et de favoriser la croissance cristalline. Le précipité obtenu présente une distribution de taille relativement étroite et la forme des particules obtenues est sensiblement sphérique. Enfin, des essais de modélisation du réacteur à lit fluidisé ont été entrepris pour réaliser des simulations à l’aide du code de calcul commercial FLUENT / The objective of this work is to conceive, develop, implement and to optimize a continuous technology allowing a good control of the precipitation reactions, fast chemical process, giving birth to a solid phase. This precipitor is a multipurpose engine with fluidized bed which did not make the same great strides like fluidized bed crystallizers. Two experimental approaches were adopted : (1) the study of the mixing phenomena of the reagents and (2) the study of the influence of the operating conditions on the precipitation of a model product. The model product selected for this study is the calcite, the polymorphic most stable phase of calcium carbonate which has three polymorphs: vaterite, aragonite and calcite. The latter is obtained by the precipitation reaction between the calcium chloride and sodium carbonate solutions at a temperature of 20° C and a complexing agent (sodium citrate) to have the required crystalline form. A kinetic study was undertaken for the determination of the nucleation and crystalline growth kinetics of calcite in citrate medium. The fluidized bed reactor, having a volume of 10 L, consisted of two zones: cylindrical for fluidization and classification of the particles and cylindro-conical for decantation, was conceived at the Chemical Engineering Science Laboratory (LSGC). The study of mixing phenomena, performed using the decoloration acid-base reaction and the hydrodynamic study, carried out using suspensions of glass microballs, made it possible to develop and optimize this multipurpose reactor. The feasibility of the precipitation of calcite in a fluidized bed was then checked. The experiments carried out in the presence of an important solid content made it possible to decrease supersaturation and to support the crystalline growth. The precipitate obtained presents a relatively narrow size distribution and the particle shape is appreciably spherical. Lastly, the modelling tests of the fluidized bed reactor were undertaken to carry out simulations using the FLUENT commercial computer code

Au cours de cette étude, nous avons approfondi nos connaissances concernant l’effet des conditions de culture sur la quantité et la qualité d’une protéine recombinante produite par des cellules CHO. En particulier, nous avons étudié l’influence de 3 composés (citrate de fer, pluronic F-68 et éthanolamine) présents dans le milieu BDM mais absent du milieu RPMI avec sérum (FCS-RPMI) sur la croissance des cellules CHO, la production de l’interféron-gamma humain recombinant (IFN-γ) ainsi que sa qualité. L’ajout de pluronic F-68 (0,1%) et de citrate de fer (500 µM) dans le milieu RPMI sans sérum a permis d’obtenir une croissance cellulaire comparable à celle obtenue avec le milieu FCS-RPMI. Par ailleurs, dans ces conditions de culture, la production de l’IFN-g est également augmentée. L’ajout de citrate de fer dans le milieu FCS-RPMI permet non seulement d’améliorer la croissance des cellules CHO mais également la production de l’IFN-γ. Avec le milieu FCS-RPMI, la macrohétérogénéité de la glycosylation de l’IFN-γ change au cours du procédé discontinu, cette dernière est maintenue constante uniquement lorsque du citrate de fer est ajouté à ce même milieu de culture. En outre, des activités gélatinase et caséinase appartenant aux familles des métalloprotéases et des protéases à sérine ont été mises en évidence au cours des cultures de cellules CHO. Quel que soit le milieu utilisé (RPMI, BDM avec ou sans sérum), l’ajout de citrate de fer permet de maîtriser et d’éviter la protéolyse de l’IFN-γ. Enfin, la relation entre le degré de glycosylation macroscopique de l’IFN- γ et son activité biologique (immunomodulatrice) in-vitro a été établie / In this study, we characterized the effect of culture conditions on the quantity and the quality of a recombinant protein, IFN-γ, produced by CHO cells. In particular, we studied the effect of 3 components (iron citrate, pluronic F-68 and ethanolamine) that are present in the BDM medium, but completely lacking in RPMI serum medium (FCS-RPMI) on CHO cell growth, as well as the production and quality of recombinant IFN-γ.The addition of Pluronic F-68 (0.1%) and iron citrate (500 µM) in RPMI without serum resulted in growth kinetic performances similar to those observed in FCS-RPMI. Furthermore, in these culture conditions, IFN- γ production was improved. Addition of iron citrate in FCS-RPMI improved cell growth, as well as IFN-γ production. Whereas the glycosylation pattern of recombinant IFN-γ produced by CHO cells was not constant when the culture was performed in FCS-RPMI, the glycosylation pattern of IFN-γ remained constant when iron citrate was added in the medium. In addition, gelatinase and caseinase enzymatic activities in CHO batch cultures were detected, due most likely to enzymes of the metalloproteases and serine protease families. Despite the type of medium used (RPMI, BDM with or without serum), addition of iron citrate minimized IFN-g proteolysis. Finally, the relationship between the macroglycosylation pattern of IFN-g and its in-vitro biological (immunomodulatory) activity was demonstrated

Le récepteur hépatique aux lipoprotéines LSR est impliqué dans la clairance des lipoprotéines riches en triglycérides telles que les résidus de chylomicrons pendant la phase postprandiale. La réduction de l’expression du LSR chez la souris (LSR+/-) est associée à une dyslipidémie et une lipémie postprandiale élevée. Afin de mieux comprendre la régulation de la distribution des lipides alimentaires, nous avons cherché quels étaient les facteurs pouvant affecter le niveau protéique de LSR. La leptine, hormone sécrétée par le tissu adipeux et connue pour son action d’hormone de satiété au niveau du système nerveux central, a été démontrée dans cette thèse comme modulant l’expression de LSR par la régulation de la transcription du gène lsr. La leptine est impliquée dans la régulation de la lipogénèse à travers SREBP-1. Grâce à l’utilisation d’un extrait de Garcinia cambogia contenant un inhibiteur de l’ATP citrate lyase, nous avons démontré une interaction importante entre les enzymes lipogéniques, l’expression de LSR et d’autres récepteurs lipoprotéiques, afin de maintenir un équilibre entre la synthèse de lipides endogènes et l’apport alimentaire de lipides exogènes. Soumises à un régime hyperlipidique, les souris sauvages montrent une diminution de l’expression des enzymes lipogéniques hépatiques, aggravée chez les souris LSR+/-. Ces résultats indiquent qu’il existe un mécanisme de maintien de l’équilibre entre la lipogénèse (synthèse endogène de lipides), la lipolyse (utilisation lipidique comme substrat énergétique) et le stockage de lipides à travers une forte interaction entre les enzymes lipogéniques et LSR. / The hepatic lipoprotein receptor LSR is involved in the clearance of triglyceride-rich lipoproteins including chylomicrons remnants during the post-prandial phase. Reduced LSR protein expression in mice (LSR+/-) is associated with dyslipidemia and increased postprandial lipemia; these mice exhibit increased weight gain with aging or when placed under a high-fat diet. In order to better understand the regulation of the distribution of dietary lipids, we looked for factors that could regulate LSR protein levels. Leptin is a hormone secreted by the adipose tissue that is a centrally-acting satiety factor, and was demonstrated to modulate LSR mRNA and protein expression through the modulation of transcription of the gene lsr. Leptin has been reported be involved in the control of lipogenesis through SREBP-1c. Using Garcinia cambogia extract containing an inhibitor of ATP citrate lyase, we demonstrated that there is an important link between lipogenic enzymes and LSR protein levels and with other lipoprotein receptors that provides the means to maintain a balance between endogenous lipid synthesis and dietary intake of exogenous lipids. When exogenous lipid intake is increased in the form of a high-fat diet, mice exhibited a decrease in hepatic lipogenic enzymes expression, but a deficiency of LSR led to increased lipid content in the peripheral tissues. These results suggest the presence of mechanisms for the maintenance for the balance between lipogenesis (de novo endogenous lipid synthesis), lipolysis (lipids used as energy substrate), and lipid storage through an important link between lipogenic enzymes and LSR.