Density gradient centrifugation of sperm is a common assisted-reproduction procedure in humans used to improve semen quality. The technique allows sperm separation based on their isopycnic points. Sperm with morphologic abnormalities are often more buoyant, leading to their retention above centrifuged density gradients, with structurally normal sperm passing through the gradient. Three experiments were conducted to evaluate the effects of tube size, sperm number following centrifugation, and density gradient volume (height) on stallion sperm quality and recovery rate in sperm pellets following centrifugation. In all three experiments, equine semen was initially centrifuged to increase sperm concentration. In Experiment 1, one-mL aliquots were layered over EquiPure? Bottom Layer (1-Layer) or over-tiered EquiPure? Top and Bottom Layers (2-Layer). For Experiment 2, one-mL aliquots were layered over three different heights of EquiPure? Bottom Layer in 15-mL or 50-mL conical-bottom tubes. For Experiment 3, four different aliquots containing a sperm load of 1-4x were layered over a constant volume of EquiPure? Bottom Layer in 15-mL or 50-mL conical bottom tubes. The tubes were then centrifuged. Resulting sperm pellets were evaluated for morphologic quality, DNA integrity, motility and recovery rate. Sperm-EquiPure? centrifugation yielded improvements in motility, morphology and DNA integrity parameters (P<0.05), as compared to controls. The 1-Layer method resulted in a higher recovery rate than the 2-Layer method (P<0.05). Sperm processed in the 15-mL tubes yielded higher velocity and higher recovery rates than sperm processed in the 50-mL tubes (P<0.05). Within tube type, gradient volume did not impact parameters of semen quality or recovery rate. An increase in sperm number for density gradient centrifugation resulted in a decreased recovery rate (P<0.05) when 15-mL tubes were used.

The application of silicon nanocrystals as non-toxic bio-labels and downconverters requires their uniform size distribution in order to minimize the inhomogeneous broadening of the photoluminescence peak. In this thesis, we set the basis for their size-separation via the density-gradient centrifugation method. To be more precise, we successfully apply this technique to separate 5 and 10 nm gold nanoparticles from an ensemble by using an engineered medium layer stack. In addition, we explain how atomic force microscopy is used to measure the size of the nanoparticles, with a particular attentionon the removal of unwanted solvent-related effects. As a future plan, we will implement the technique for the size-separation of silicon nanocrystals.

Submitted by Sandro Camargo (sandro.camargo@unipampa.edu.br) on 2015-03-08T18:29:37Z No. of bitstreams: 1 117110033.pdf: 882213 bytes, checksum: 12c52b9fad305c8e13e54eeab7fca3ac (MD5) / Made available in DSpace on 2015-03-08T18:29:38Z (GMT). No. of bitstreams: 1 117110033.pdf: 882213 bytes, checksum: 12c52b9fad305c8e13e54eeab7fca3ac (MD5) Previous issue date: 2013-04-26 / A centrifugação por gradiente de Percoll ® é o método mais utilizado para a seleção de sêmen bovino para a fecundação in vitro (FIV), no entanto, até o momento não existe nenhum estudo que indique uma força de centrifugação capaz de remover com sucesso o plasma seminal e outros constituintes, sem causar perdas na recuperação espermática ou danos funcionais aos espermatozoides obtidos no pellet. O presente estudo foi desenvolvido para verificar o efeito de diferentes forças de centrifugação na seleção espermática por gradientes de Percoll ® , através de avaliação de parâmetros de qualidade espermática e posterior desenvolvimento e qualidade de embriões bovinos produzidos in vitro (PIV). Parâmetros de estresse oxidativo também foram avaliados para verificar uma possível relação com os achados na avaliação de esperma. No experimento 1, amostras de sêmen de quatro touros foram homogeneizadas e submetidas a centrifugação em gradientes descontínuo de Percoll ® (30,60 e 90%) em diferentes forças: F1 (9000 X g), F2 (6500 X g), F3 (4500 X g) e F4 (2200 X g). No Experimento 2, as amostras de sêmen de cada touro foram processadas individualmente e centrifugadas em: F1 (9000 X g) e F4 (2200 X g). Todas as amostras espermáticas foram avaliadas quanto a concentração, motilidade, vigor, morfologia, espécies reativas de oxigênio (EROs) e integridade da membrana plasmática, sendo no experimento 2 avaliados ainda a peroxidação lipídica, defesas antioxidantes e desenvolvimento embrionário. Não foi observada diferença na concentração espermática nos sêmens submetidos a diferentes forças centrifugação. No Experimento 1, a percentagem de espermatozoides móveis foi superior (p <0,05) após centrifugação em F3 e F4 e a produção de EROs de F1 foi superior (p <0,05) em comparação com outras forças. No Experimento 2, quando o sêmen de cada touro foi processado individualmente, não foram observadas diferenças significativas nos parâmetros de qualidade espermática, peroxidação lipídica, defesas antioxidantes, taxa de clivagem e tempo médio da primeira clivagem entre F1 e F4. No entanto, o aumento da força de centrifugação reduziu a taxa de penetração e a fertilização normal (P<0,05). Este trabalho demonstrou pela primeira vez que a força de centrifugação de 2200 X g aumentou a penetração e a taxa de fecundação em relação à força de centrifugação habitual (9000 X g) utilizadas na separação de espermatozoides por gradientes descontínuos Percoll ® em bovinos. Estes resultados sugerem que esta força de centrifugação pode ser utilizada com sucesso na PIV de embriões bovinos, uma vez que não reduz a recuperação de espermatozoides e aumenta a taxa de fecundação. / Centrifugation by Percoll® gradient is the most widely used method in the preparation of bull sperm for the purpose of in vitro fertilization, however at the moment, no scientific study determined the best centrifugation speed to remove seminal plasma and other constituents, i.e. the speed at which the loss of sperm cells is minimized and where the spermatozoa in the pellet still remain functional. The present study was designed to examine the efficiency of different centrifugation forces in sperm separation Percoll by methods evaluating sperm quality parameters and subsequent development and quality of bovine in vitro production (IVP) embryos. Additionally, we evaluated oxidative stress parameters to verify a possible relationship with the findings in the sperm evaluation. In Experiment 1, the semen samples from each bull were pooled and submitted to centrifugation in discontinuous gradients Percoll (30,60 and 90%) at different forces: F1 (9000 X g), F2 (6500 X g), F3 (4500 X g) and F4 (2200 X g). In Experiment 2, the semen samples from each bull were done separately and submitted to: F1 (9000 X g) and F4 (2200 X g). All sperm samples were evaluated to the concentration, motility, vigor, morphology, reactive oxygen species (ROS) and integrity of the plasma membrane, and in experiment 2 also were evaluated lipid peroxidation, antioxidants assays and embryo development. No difference was observed in the concentration of sperm submitted to different centrifugation forces. In Experiment 1, the total percentage of motile sperm was increased (p < 0.05) after centrifugation in F3 and F4 and the ROS production to F1 was superior (p < 0.05) compared to other forces. In Experiment 2, when the bull semen was processed individually, no significant difference was observed in the sperm quality parameters assessment, lipid peroxidation, antioxidants assays, cleavage rate and average time of the first cleavage between F1 and F4, sires or interaction between them after Percoll. However, the increased force centrifugation reduced the rate of penetration and normal fertilization (P < 0.05). This work demonstrated for the first time that 2200 X g centrifugation force enhanced the penetration and fertilization rates in relation to usual centrifugation force (9000 X g) using sperm separation by discontinuous Percoll gradients in bovine. These findings suggest that this centrifugation force could be used with successful in the bovine IVP embryo since it does not reduce sperm recuperation and increases the fecundation rate.

Gold nanoclusters possess both theoretical and practical importance in the development of ultrasensitive biosensors based on surface-enhanced Raman spectroscopy (SERS). Manipulation of gold nanoclusters in a predictable and reproducible manner for the application of refined biochemical analysis still remains challenging. In this study, high-purity gold nanoclusters are isolated via a simple method based on density gradient centrifugation. Three distinct bands including monomers, small aggregates (2-4 nanospheres), and large aggregates (>5 nanospheres) can be separated via density gradient centrifugation. The isolated gold nanoclusters greatly enhance the Raman intensity of the trapped dye molecules such that single nanocluster detection is feasible. To develop a gold nanoparticle-based biosensor for influenza virus, effort was also made to modify recognition moieties such as aptamers to gold nanoparticles via distinct approaches. The increase of hydraulic diameter and the shift of optical absorbance spectrum indicate the success of surface modification to gold nanoparticles. / Master of Science

No description available.

No description available.

Hedgehog (Hh) proteins control animal development and tissue homeostasis. They activate gene expression by regulating processing, stability, and activation of Gli/Cubitus interruptus (Ci) transcription factors. Hh proteins are secreted and spread through tissue, despite becoming covalently linked to sterol during processing. Multiple mechanisms have been proposed to release Hh proteins in distinct forms; in Drosophila, lipoproteins facilitate long-range Hh mobilization but also contain lipids that repress the pathway. Here, we show that mammalian lipoproteins have conserved roles in Sonic Hedgehog (Shh) release and pathway repression. We demonstrate that lipoprotein-associated forms of Hh and Shh specifically block lipoprotein-mediated pathway inhibition. We also identify a second conserved release form that is not sterol-modified and can be released independently of lipoproteins (Hh-N*/Shh-N*). Lipoprotein-associated Hh/Shh and Hh-N*/Shh-N* have complementary and synergistic functions. In Drosophila wing imaginal discs, lipoprotein-associated Hh increases the amount of full-length Ci, but is insufficient for target gene activation. However, small amounts of non-sterol-modified Hh synergize with lipoprotein-associated Hh to fully activate the pathway and allow target gene expression. The existence of Hh secretion forms with distinct signaling activities suggests a novel mechanism for generating a diversity of Hh responses.

The presence of pathogens in semen can compromise the outcome of assisted reproductive treatment, together with the possibility of the female partner or offspring becoming infected. This is cause for concern, especially in South Africa with a high prevalence of HIV-1. Most of these infected individuals are in their reproductive years with the desire to have their own genetically related children. Therefore, assisted reproductive treatment with effective risk reduction procedures, such as semen processing for the elimination of these pathogens is crucial. However, during sperm preparation by standard discontinuous density gradient centrifugation, the supernatant is aspirated to allow access to the purified sperm pellet. Pathogens from the upper layers can adhere to the inside surface of the test tube and flow down to re-infect the purified sperm sample. The use of a centrifuge tube insert may prevent the re-contamination of sperm samples after discontinuous density gradient centrifugation. Furthermore, seminal pathogens can bind specifically or non-specifically to spermatozoa, rendering semen decontamination procedures ineffective. Serine proteases, such as trypsin, have been demonstrated to effectively inactivate viruses and to break pathogen-sperm bonds. However, the addition of a protease to density gradient layers during semen processing could have a negative impact on sperm parameters. This research was therefore aimed towards the determination of: i) The effect of semen processing with trypsin and trypsin inhibitor on sperm parameters. ii) The prevalence of various bacteria in semen samples from men attending the Reproductive and Endocrine Unit at Steve Biko Academic Hospital. iii) The effectiveness of semen processing by discontinuous density gradient centrifugation with a centrifuge tube insert, for the elimination of some of the most prevalent bacteria, white blood cells and in vivo derived HIV-1. Evaluation of sperm parameters after semen processing indicated that trypsin and trypsin inhibitor did not have an impact on sperm mitochondrial membrane potential, vitality, motility and zona binding potential, or acrosin activity, respectively. Seminal bacteria were highly prevalent in patients wishing to participate in the Unit’s assisted reproductive program, with 49.5% of semen samples presenting with positive bacterial cultures. Semen processing by means of discontinuous density gradient centrifugation with the tube insert, eliminated significantly more in vitro derived (spiked) bacteria and white blood cells from semen compared to processing without the insert. Furthermore, the semen decontamination procedure was effective in removing HIV-1 RNA from 100% of samples and proviral DNA from 98.1% of semen samples from HIV-1 sero-positive patients. The effectiveness of discontinuous density gradient centrifugation for the elimination of seminal pathogens could, therefore, be improved by the addition of trypsin to the upper density layer, without supplementing the bottom layer with trypsin inhibitor. Additionally, semen decontamination efficiency could also be improved by the prevention of re-contamination of processed sperm samples by the utilization of a tube insert during discontinuous density gradient centrifugation. / Thesis (PhD)--University of Pretoria, 2014. / gm2014 / Obstetrics and Gynaecology / unrestricted

Hedgehog (Hh) proteins control animal development and tissue homeostasis. They activate gene expression by regulating processing, stability, and activation of Gli/Cubitus interruptus (Ci) transcription factors. Hh proteins are secreted and spread through tissue, despite becoming covalently linked to sterol during processing. Multiple mechanisms have been proposed to release Hh proteins in distinct forms; in Drosophila, lipoproteins facilitate long-range Hh mobilization but also contain lipids that repress the pathway. Here, we show that mammalian lipoproteins have conserved roles in Sonic Hedgehog (Shh) release and pathway repression. We demonstrate that lipoprotein-associated forms of Hh and Shh specifically block lipoprotein-mediated pathway inhibition. We also identify a second conserved release form that is not sterol-modified and can be released independently of lipoproteins (Hh-N*/Shh-N*). Lipoprotein-associated Hh/Shh and Hh-N*/Shh-N* have complementary and synergistic functions. In Drosophila wing imaginal discs, lipoprotein-associated Hh increases the amount of full-length Ci, but is insufficient for target gene activation. However, small amounts of non-sterol-modified Hh synergize with lipoprotein-associated Hh to fully activate the pathway and allow target gene expression. The existence of Hh secretion forms with distinct signaling activities suggests a novel mechanism for generating a diversity of Hh responses.

La structure spécifique des nanotubes rend ce matériau très intéressant dans l’élaboration de nanohybrides. La cavité interne des nanotubes permet l’encapsulation de molécule laissant la paroi externe libre pour une fonctionnalisation. Les nanotubes de carbone sont déjà bien connus pour l’élaboration de nanosondes Raman. Les molécules de colorants encapsulé dans leurs cavité interne sont protégées de l’irradiation du laser. Les propriétés électroniques de cette structure en carbone permettent le transfert d’énergie entre le colorant et le nanotube engendrant ainsi une extinction de la fluorescence du colorant. La surface du nanotube de carbone est libre pour réaliser des fonctionnalisations permettant de modifier certaines propriétés de la nanosonde. L’élaboration de nanohybride à partir de cette structure permet les analyses de « multiplexage » en changeant simplement le colorant encapsulé dans la cavité interne du nanotube et la fonctionnalisation en surface. La structure des nanotubes de nitrure de bore (BNNTs) est très similaire à celle de leurs homologues en carbone. La cavité interne permet également l’encapsulation de colorant cependant les propriétés électroniques résultantes de cette structure ne permet pas le transfert d’énergie. Les molécules de colorant encapsulé dans les BNNTs conservent donc leurs fluorescences. Des études précédentes démontrent qu’après encapsulation, le spectre de fluorescence du colorant α-sexithiophène (6T) est élargi et décalé vers les longueurs d’ondes plus grandes, c.-à-d. vers le rouge. L’hypothèse la plus probable, quant à la raison de ce phénomène, est que la grande distribution de taille de diamètre de l’échantillon de BNNTs permet différentes agglomérations de 6T. Les nanosondes résultantes sont composées d’un mélange d’agglomération de colorant absorbant à différentes longueurs d’onde. Afin de confirmer cette hypothèse, nous allons procéder au triage en taille de diamètre des BNNTs. Pour cela, plusieurs étapes sont nécessaires, comme la fonctionnalisation de la surface des BNNTs pour les rendre dispersible dans l’eau, l’encapsulation du colorant de 6T selon un protocole déjà connus dans la littérature et enfin le test d’une méthode de triage de nanotubes en fonction de leurs diamètres et donc de leurs densités. La méthode de triage sélectionnée parmi les méthodes découvertes dans la littérature, a démontré son efficacité sur les nanotubes de carbone mais n’a cependant jamais été testée sur les BNNTs. Ce mémoire présente les premiers résultats d’une séparation de nanosondes fluorescentes en fonction de leurs tailles de diamètre. / The specific structure of nanotubes is interesting for the synthesis of nanohybrides. Molecules are encapsulated in the internal cavity of the tube while the external wall remain free for further manipulation. Carbon nanotubes are already known for synthesizing Raman nanoprobes. Dyes encapsulated inside the nanotube are protected from irradiation. The electronic properties of the carbon structure lead to energy transfer between the dyes and the nanotubes, this result by the the extinction of the dye’s fluorescence. The carbon nanotube’s surface is free for functionalisation that can add some properties to the nanoprobe. The preparation process of nanohybrides with that structures permit some analyse in « multiplexing » by easily change the dye encapsulated or the functionalisation on the surface of the nanotube. The structure of boron nitride nanotubes (BNNTs) is similar to the carbon one. The internal cavity can encapsulate dyes but the electronic properties don’t permit the energy exchange. Encapsulated dyes inside BNNTs emit some fluorescence. Previous studies show some changes in the fluorescence spectrum of α-sexithiophene (6T) after encapsulation inside BNNTs. The spectrum shows larger bands and a red shift. This caracteristic can come from a large distribution of diameter sizes in the BNNT sample. Différent diameter sizes of nanotubes results in different agglomeration of dyes inside their internal cavities, and these differents nanoprobes are absorbing at different wavelengths. To confirm this hypothesis, we will separate BNNTs into their diameter sizes. Before that some manipulation is necesary, like the functionnalisation of the nanotubes’ surfaces for a better dispersion in water, the encapsulation of 6T realized with the process already known and the experience of a new method to separate nanotubes by size. This separating method is chose from all the method of separating carbon nanotubes but has never been tested on BNNTs. This document shows the first results of separating fluorescents nanoprobes by diameter size.