Submitted by Claudinei Pracidelli (cpracide@ipen.br) on 2015-04-10T14:26:55Z No. of bitstreams: 0 / Made available in DSpace on 2015-04-10T14:26:55Z (GMT). No. of bitstreams: 0 / Neste trabalho, vidros contendo um resíduo industrial denominado lama vermelha, foram desenvolvidos e caracterizados. Foi utilizada a quantidade mínima de 60% em massa de lama vermelha na produção dos vidros para promover a utilização desse resíduo. De acordo com os resultados de difração de raios X obtidos, observa-se que é possível produzir materiais a partir da lama vermelha com fases amorfas consideráveis, embora fases cristalinas referentes ao Fe originário do resíduo estejam presentes. O material denominado 60L40S, o qual possui 60% em massa de lama vermelha na sua composição nominal, apresentou as melhores propriedades dentre as composições estudadas, porém apresentou também alta temperatura de fusão. Ajustes na composição desse material foram realizados buscando diminuir essa temperatura. Os resultados mostram que os ajustes foram satisfatórios no desempenho de diminuir a temperatura de fusão, porém perdas na propriedade química desse material foram observadas. Elementos comumente encontrados na composição química de rejeitos nucleares foram adicionados aos vidros produzidos neste trabalho visando estudar os efeitos dessa adição nas propriedades químicas e térmicas desses materiais. Foi observado que é possível adicionar até 15% em massa de elementos simuladores aos materiais produzidos e essa adição promove a diminuição da temperatura de fusão. Acima de 15% em massa os elementos adicionados se precipitam na estrutura do material. Foi observado que, embora haja perdas na durabilidade química do material 60L40S após a adição dos elementos simuladores, esse material, quando em contato com água, mantém os elementos simuladores confinados em sua estrutura. Esse resultado é promissor, pois sugere que o material 60L40S é capaz de imobilizar em sua estrutura elementos provenientes de rejeitos nucleares. / Tese (Doutorado em Tecnologia Nuclear) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Este estudo objetivou avaliar os efeitos da melatonina sobre a qualidade e as taxas de blastocistos bovinos PIV. A melatonina é um poderoso eliminador de radicais livres e tem efeitos benéficos na PIV de algumas espécies. Foram feitas sessões de PIV (n=12), sendo aspirados os folículos (2 a 7 mm) de ovários obtidos de abatedouro. Complexos cumulus oócito (COCs) que apresentavam cumulus compacto e oócito com citoplasma homogêneo foram selecionados e randomicamente distribuídos ente os grupos controle (GC) e tratado com Melatonina (GM). Submetidos a MIV por 22h em 400&micro;l de TCM199(Dia-1). Para a FIV COCs foram transferidos para 400&micro;l de meio de fertilização, inseminado com sêmen descongelado previamente submetido ao gradiente descontínuo de Percoll&reg;, e incubados por 18h (dia0). As células do cumulus dos presumíveis zigotos foram removidas com hialuronidase e pipetagem sucessiva, sendo então cultivados (CIV) em 400&micro;l de KSOM- BSA (Dia 1). No Dia 3, foi avaliada a taxa de clivagem em estereomicroscópio e adicionados 400&micro;l KSOM-20% SFB. No Dia 5, 200&micro;l de meio foram removidos e 400&micro;l KSOM-10% SFB adicionado. No dia 7 foi avaliado o estágio de desenvolvimento dos embriões, e as porcentagens calculadas em relação ao total de presumíveis zigotos. Todos os procedimentos foram realizados em placa de 4 poços (Nunc Multidishes&reg;) sem cobertura de óleo mineral Na MIV e no CIV, os grupos receberam ou 0ng/ml ou 50ng/ml de melatonina, GC e GM, respectivamente. No Dia 7, de 4 sessões de PIV, todos os blastocistos expandidos foram corados com Iodeto de Propídeo e Hoechst 33342. Os demais blastocistos expandidos, das outras sessões, foram vitrificados, assim como todos os presentes no Dia 8. Uma amostra deste meio foi retirada para avaliação da concentração de espécies reativas ao ácido tiobarbitúrico (TBARS). Posteriormente os embriões foram descongelados e cultivados para a avaliação do efeito da melatonina sobre os embriões no cultivo pós-descongelação. A análise estatística foi feita usando SAS system for Windows 9.2&reg;. Dados paramétricos foram submetidos ao teste T de Student e os não paramétricos ao Wilcoxon, diferenças foram consideradas significativas quando p<0,05. Blastocistos expandidos do GM apresentaram maior número de células, sendo que estas apresentaram porcentagem menor de ruptura de membrana que os do GC. A melatonina não influenciou as taxas de clivagem, de blastocistos no Dia 7 nem a taxa de blastocistos passíveis de vitrificação. Não foi observado efeito da melatonina sobre a tolerância dos embriões à vitrificação nem no período de cultivo após o descongelamento. Inesperadamente, a melatonina aumentou a concentração de TBARS no meio. A melatonina melhorou a qualidade embrionária, contudo suas propriedades antioxidantes não foram evidenciadas. / This aimed to evaluate melatonin effect over bovine embryo quality and rates on in vitro embryo production procedures (IVP). Melatonin is an effective free radical scavenger and beneficial effects of melatonin have been demonstrated in IVP for some species. Embryo production sessions (12) were performed; follicles (2 to 7 mm) were aspirated from slaughterhouse-derived ovaries. Cumulusoocyte complexes (COCs) who had a compact cumulus and oocyte with homogeneous cytoplasm were selected and randomly allocated (35 to 55 per group) on either control Group (CG) or Melatonin Treatment Group (MG). COCs underwent in vitro maturation (IVM) for 22 hours in 400&micro;l of TCM199 (Day -1). For in vitro fertilization (IVF) COCs were moved to fertilization medium 400&micro;l and were inseminated with previously submitted to discontinuous PercollTM gradient, frozenthawed semen, incubated for 18 hours (Day 0). After IVF presumptive zygotes were striped from remaining cumulus cells by incubation with hyaluronidase and gentile pipetting, then moved to 400&micro;l of KSOM- BSA (Day 1). On Day 3 embryos were inspected under a stereomicroscope to evaluate cleavage rate, and 400&micro;l KSOM-20% FCS was added. On Day 5 200&micro;l of medium were removed and 400&micro;l KSOM-10% FCS was added. On Day 7 embryos were evaluated regarding their developmental stage, percentages (%) were calculated over the total of presumptive zygotes, expanded blastocysts were vitrified. All procedures were performed on a Nunc MultidishesTM 4 well dish without mineral oil overlay. During IVM and IVC groups received either 0ng/ml or 50 ng/ml of melatonin, CG and MG respectively. On Day 7 out of 4 replicates all expanded blastocysts were dyed with Propidium Iodide and Hoechst. Remaining expanded blastocysts ass well as all the expanded blastocysts on Day 8 were vitrified. A medium sample was withdrawn for thiobarbituric acid assay (TBARS). Latter embryos were thawed and cultured aiming to evaluate melatonin effect over cryotolerance and post thaw culture system. Statistical analysis was performed using SAS system for Windows 9.2TM parametric data was submitted to student T-test and non parametric to Wilcoxon. Differences were considered meaningful when p<0,05. Expanded blastocysts from MG presented more cells and less cells with ruptured membrane than the ones from CG. But melatonin treatment neither influenced cleavage nor Blastocyst rates on Day 7, nor the rate of blastocysts that can be vitrified. Neither over embryos cryotolerance nor over post thaw culture system. Unexpectedly melatonin increased TBARS levels on medium. Melatonin improves bovine embryo quality however its antioxidant properties were not demonstrated.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-01-13T18:22:43Z No. of bitstreams: 1 nataliavelosomalta.pdf: 1376730 bytes, checksum: cdeb2bd74c09e4792776e296bd5f7f42 (MD5) / Approved for entry into archive by Diamantino Mayra (mayra.diamantino@ufjf.edu.br) on 2017-01-31T10:32:11Z (GMT) No. of bitstreams: 1 nataliavelosomalta.pdf: 1376730 bytes, checksum: cdeb2bd74c09e4792776e296bd5f7f42 (MD5) / Made available in DSpace on 2017-01-31T10:32:11Z (GMT). No. of bitstreams: 1 nataliavelosomalta.pdf: 1376730 bytes, checksum: cdeb2bd74c09e4792776e296bd5f7f42 (MD5) Previous issue date: 2016-06-09 / O objetivo desse estudo foi avaliar, a influência de duas diferentes técnicas de aplicação de glaze (pincél e spray) sobre a morfologia de superfície de uma cerâmica Y-TZP (IPS e.maxR®ZirCAD (IvoclarVivadent) antes e após a aplicação do ácido fluorídrico. Foram confeccionados 20 corpos de prova a partir de 4 blocos cerâmicos de zircônia parcialmente estabilizada por ítria pré-sinterizados, que foram divididos em 5 grupos (n=4) de acordo com o tratamento de superfície recebido: C= controle (nenhum tratamento de superfície); S: glaze spray VITA AKZENT( Vita Zanhfabrik )P/L: glaze pó/líquido VITA AKZENT (Vita Zanhfabrik,) S+HF: S + condicionamento com ácido fluorídrico 10% (HF)(FGM) (1min); P/L + HF: P/L + condicionamento com HF 10% (1min). As amostras forma então submetidas ao seguintes testes: 1) goniometria, para ánalise do ângulo de contato e da energia de superfície; 2) análise dos dados de rugosidade (Ra) por meio de um perfilômetro óptico digital; 3)identificação das fases cristalinas do material pela Difratometria de Raios X; 4) análise dos elementos químicos presentes no material cerâmico por Microscopia e espectrometria por energia dispersiva (EDS); 5) análise da superfície por meio de microscopia eletrônica de varredura (MEV). Os resultados demonstraram que: 1) a superfície das amostras tratadas com o glaze pó/líquido foram as que apresentaram uma camada mais espessa de glaze, com superfícies menos rugosas; 2)o grupo que recebeu o tratamento com o glaze pó/líquido foi o que apresentou a maior energia de superfície (69,83mN/m), e os demais grupos apresentaram elevados valores de ângulos de contato; 3) o tratamento térmico de vitrificação não causou alterações de fases na estrutura da zircônia; 4)As imagens de perfilometria e MEV revelaram um padrão irregular de disposição do glaze após o condicionamento com HF 10%, e um alto conteúdo de sílica foi observado somente nos grupos P/L e P/L + HF. O estudo concluiu que a utilização do glaze pó/líquido pela técnica do pincel promoveu uma maior padronização da camada de glaze e foi método de tratamento de superfície que apresentou uma maior energia de superfície e um maior conteúdo vítreo. Pelas imagens de microscopia, pode-se observar que a aplicação do ácido fluorídrico a 10% criou microretenções sobre a camada de glaze pó-liquido que podem favorecer a adesão a esta superfície. / The present study evaluated qualitatively the influence of two different types of glaze (brush and spray) on the surface of Y-TZP ceramics (IPS e.maxR® ZirCAD (IvoclarVivadent,) before and after use of hydrofluoric acid. Twenty specimens were made from 4 zirconia ceramic blocks partially stabilized by pre-sintered yttria, which were divided into 5 groups (n=5) according to the surface treatment received: C = Zirconia (control) (no surface treatment); S: glaze spray VITA AKZENT; P/L: glaze powder/liquid VITA AKZENT, S+HF: S + etching with hidrofluoridric acid 10% (HF)(FGM) (1min); P/L + HF: P/L + etching with HF 10% (1min).Samples were then subjected to the following tests: 1) Goniometry for analysis of the contact angle and the surface energy ; 2) Analysis of roughness data (Ra) by means of a digital optical profilometer; 3) Identification of the crystalline phases of the material by X-ray diffraction;4) Analysis of the chemical elements present in the ceramic material by microscopy and energy dispersive spectrometry (EDS); 5) analysis of the surface after each surface treatment by means of scanning electron microscopy (SEM).Partial results showed that: 1) The surface of the samples treated with glaze powder/liquid showed a thicker layer of glaze with less rough surfaces; 2) the group receiving treatment with glaze powder/liquid presented higher surface energy (69,83mN/m), while the other groups showed high values of contact angles; 3) thermal treatment of vitrification did not change the phases of zirconia surface; 4) The SEM and profilometry images revealed a glaze irregular pattern layout after etching with 10% HF, and a high silica content was observed only in GP / L and GP / L + HF groups. The study concluded that use of the glaze powder / liquid by brush technique promoted a greater standardization of the glaze layer and this surface treatment method was the one that showed a higher surface energy and a higher glassy content. For microscopy images, it can be seen that the application of hydrofluoric acid 10% created micro retentions on the glaze powder-liquid layer that can promote adhesion to this surface.

Au cours de cette thèse nous avons utilisé la théorie de la fonctionnelle de la densité et les calculs Monte Carlo quantiques pour analyser l'impact des effets de van der Waals sur la structure, l'énergie, et les propriétés des polymorphes de SiO2 et B2O3. Nous avons mis en évidence un phénomène de compensation d'erreur, lié à l'utilisation de fonctionnelle d'échange et corrélation incluant les effets de van der Waals, dans les polymorphes basse densité de SiO2 entre une sur-évaluation des longueurs Si-O et une sous-estimation des angles Si-O-Si. Nous avons effectué des calculs Monte-Carlo quantiques afin de prédire la structure et l'énergie d'un nouveau polymorphe de B2O3 avec une grande précision, ce qui nous a permis d'évaluer les performances de différentes fonctionnelles d'échange et corrélation sur B2O3. Nous avons ensuite utilisé la théorie de la fonctionnelle de la densité pour prédire la structure et l'énergie de 25 nouveaux polymorphes de B2O3 , ainsi que leurs propriétés mécaniques et électroniques. Cette étude permet de proposer une explication à l'anomalie de cristallisation de B2O3, et réconcilie le comportement de B2O3 avec celui des autres oxydes formateurs de réseaux. Elle souligne la possibilité de créer des borates cristallins aux propriétés mécaniques remarquables, et confirme qu'il existe lien entre polymorphisme de basse énergie et facilité de vitrification. / During this PhD I use density functional theory and quantum Monte Carlo to evaluate the importance of van der Waals effects on the structures, the energies, and the properties of SiO2 and B2O3 polymorphs. I show that exchange-correlation functionals including dispersion effects lead to an error cancellation between an overestimation of the Si-O distances and an underestimation of the Si-O-Si angles in low densities SiO2 polymorphs. By using quantum Monte Carlo calculations, I have predicted with high accuracy the relative energy of a new B2O3 polymorph, which allowed me to evaluate the performances of different exchange-correlation functionals on this material. I then use the best functional possible to compute the mechanical and electronic properties of 25 predicted B2O3 polymorphs. Some of the predicted polymorphs exhibit intriguing mechanical properties, such as negative linear compressibility, auxeticity and anisotropy. These calculations allow me to make a hypothesis explaining the crystallization anomaly in B2O3. They underline a seemingly universal link between low energy polymorphism and ease of vitrification.

The general aim of this thesis was to optimise the current methodologies of oocyte cryopreservation in order to obtain live offspring from cryopreserved rabbit oocytes. In chapter 1, meiotic spindle configuration, cortical granules (CGs) distribution and oocyte developmental competence were evaluated after cryopreservation with the current slow-freezing and vitrification procedures. The meiotic spindle organisation was dramatically impaired regardless of the method used. Nevertheless, altered CG distribution is more evident in vitrified oocytes than in slow-frozen ones and the developmental rate to blastocyst stage after parthenogenetic activation was only obtained using slow-freezing method. From this chapter it may be concluded that both methodologies equally affect oocyte structure. However, slow-freezing method seems to be the recommended option for this species as a consequence of the sensitivity to high levels of cryoprotectants in this species. The aim of the following two chapters was the optimisation of cryopreservation procedures using different strategies to modify the oocytes in order to make them more cryoresistant. In chapter 2, Taxol and Cytochalasin B were employed to stabilise the cytoskeleton system during vitrification. The effect of these two molecules on the meiotic spindle and chromosome configuration and development to blastocyst stage after parthenogenesis activation were also evaluated. There were no significant differences in the structural configuration between vitrified groups. Regarding cleavage and blastocyst developmental rate, no statistical differences were found between vitrified-non-treated and Taxol-treated oocytes, but no oocytes treated with Cytochalasin B reached this stage. Therefore, structural configuration and blastocyst development were not improved by this pre-treatment. Moreover, Cytochalasin B pre-treatment seems to cause a deleterious effect on developmental ability to blastocyst stage of these oocytes. In chapter 3, oocytes were incubated with cholesterol-loaded methyl-ß- cyclodextrin (CLC) to increase the membrane fluidity and stability and improve their developmental ability after parthenogenetic activation or intracytoplasmic sperm injection (ICSI). Cholesterol incorporation and its presence after cryopreservation were evaluated using confocal microscopy. Results showed that cholesterol was incorporated into the oocyte and remained, albeit in a lesser amount after cryopreservation procedures. However, no improvements on developmental competence were obtained after parthenogenetic activation or intracytoplasmic sperm injection. In the last three chapters of this thesis, the main objective was to develop a reliable technique which would allow us to obtain live offspring from cryopreserved oocytes. For that purpose, in vivo fertilisation using intraoviductal oocyte transfer assisted by laparoscopy was considered a good alternative to bypass the inadequacy of conventional in vitro fertilisation in rabbit. In chapter 4, two recipient models (ovariectomised or oviduct ligated immediately after transfer) were used to compare the ability of fresh oocytes to fertilise in vivo. This first work showed that embryo recovery rates in all transferred groups decreased significantly, but ligated oviduct recipients provided significantly higher results compared to ovariectomised ones. For that reason, in the second experiment the ligated oviduct recipient model was used to generate live births. Results obtained in this chapter suggested that it was possible to obtain offspring from cryopreserved oocytes using this technique, but this kind of animal models compromised the use of the reproductive tract in a high percentage of females. For that reason, chapter 5 was focused on the development of another type of animal model as an alternative. First, the ability of cyanoacrylate tissue adhesive to block the oviducts before the ovulation would take place was evaluated. Then, in vivo fertilisation ability of fresh transferred oocytes after blocking the oviduct with the adhesive was also assessed. Finally, slow frozen oocytes were transferred to generate live birth. Results showed that cyanoacrylate tissue adhesive was effective in blocking the oviduct, as no embryos were recovered in the blocked oviduct six days after artificial insemination (AI). Moreover, this method could fertilise fresh and also slowfrozen oocytes with a higher live birth rate than the previous recipient models. This study showed that successful production of live offspring using slow-frozen oocytes in combination with in vivo fertilisation was possible, which suggested that in vivo environment could help improve the results of oocyte cryopreservation. Thus, this method was employed in the last chapter of this thesis to generate live offspring from vitrified rabbit oocytes for the first time. Results obtained revealed that there were no differences in the rate of birth between vitrified and slow-frozen transferred oocytes. Nevertheless, based on the results with fresh oocytes, further experiments are still needed if the efficiency of cryopreservation procedures are to be improved. / Jiménez Trigos, ME. (2014). Generation of offspring from cryopreserved rabbit (Oryctolagus cuniculus) oocytes [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/37977 / TESIS

No description available.

Orientador: Marcelo Fábio Gouveia Nogueira / Resumo: Embora estágios iniciais de embriões de mamíferos tenham aparentemente uma grande plasticidade, eles, assim como o oócito em maturação, são bastante sensíveis a estresses exógenos. Responder a esses estresses é uma parte vital da fisiologia celular. Cada vez mais é aparente que um dos principais mecanismos, para iniciar a resposta celular a uma variedade de estressores exógenos, reside no retículo endoplasmático (RE). O RE é uma importante organela responsável pela síntese, processamento e transporte de proteínas e lipídeos. No entanto e in vitro, a perturbação do microambiente do RE pode causar alterações na estrutura das proteínas levando ao acúmulo de proteínas com o incorreto dobramento, uma condição denominada estresse do RE. Dependendo da intensidade do estresse, o RE pode desencadear a apoptose pela Unfolded Protein Response. Em várias espécies, estudos observaram uma melhoria da competência do oócito e no desenvolvimento do embrião quando foram adicionados inibidores do estresse do RE no meio de maturação e/ou meio de cultivo in vitro (CIV). Dentre os inibidores do estresse do RE, o ácido tauroursodesoxicólico (TUDCA) se mostrou benéfico na produção in vitro de embriões. Entretanto, pouco é conhecido sobre o seu efeito na maturação oocitária e no desenvolvimento do embrião bovino in vitro. Portanto, os objetivos gerais desta Tese foram: Capítulo 1 - investigar o efeito da adição de TUDCA durante a maturação in vitro (MIV) sobre a competência do oócito e do embrião [ma... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Although early stages of mammalian embryos appear to retain great plasticity, they and maturing oocytes are quite sensitive to exogenous stresses. Responding to those stresses is a vital part of cellular physiology, and it is increasingly apparent that one of the main mechanisms for initiating that response is based on the endoplasmic reticulum (ER). ER is an important organelle responsible for the synthesis, processing, folding and transport of proteins and lipids. However, in vitro, the disturbance of the ER microenvironment can cause changes in the structure and folding of proteins leading to the accumulation of misfolding proteins, a condition called ER stress. Depending on the intensity of the stress, the ER can trigger apoptosis by Unfolded Protein Response. In several species, studies have reported an improvement in oocyte and embryo competence when ER stress inhibitors have been added to the in vitro maturation (IVM) medium and/or in vitro culture (IVC) medium. Among the ER stress inhibitors, tauroursodeoxycholic acid (TUDCA) has been shown to be beneficial in in vitro embryo production. However, little is known about TUDCA’s effect on oocyte maturation and the development of bovine embryo in vitro. Therefore, the main objectives of this Thesis were: Chapter 1 - to investigate the effect of adding different TUDCA concentrations during IVM on the oocyte and embryo competence [nuclear maturation, mitochondrial activity, reactive oxygen species (ROS) production, pronucle... (Complete abstract click electronic access below) / Doutor

Introduction: Cryopreservation of human sperm is considered a routine practice in assisted reproduction laboratories. Semen samples are mainly cryopreserved as a back-up for procedures, donor sperm, and validation of samples from human immunodeficiency virus-positive patients. Human immunodeficiency virus semen samples generally result in a low yield of purified spermatozoa after decontamination. These samples need to be cryopreserved for later use. Unlike conventional cryopreservation, vitrification does not use harmful cryoprotectants, thereby potentially reducing sperm damage. Vitrification is not yet common practice for sperm cryopreservation in assisted reproduction. The aim of this study was to establish the feasibility of utilising vitrification as an alternative to current conventional cryopreservation of spermatozoa. Methods: Semen samples were collected from human immunodeficiency virus-negative patients seeking diagnostic assistance from the unit. All samples were processed according to the unit’s standard protocol. For Study 1A (n=10) washed samples were divided and cryopreserved using three different cryopreservation media, and two different freezing protocols. In Study 1B (n=15), washed samples were divided and preserved using cryoprotectant-free vitrification in 100 μl, 300 μl and 500 μl volumes. For Study 2 (n=35) washed samples were split and cryopreserved using cryoprotectant-free vitrification (utilizing the volume that resulted in the highest quality spermatozoa in Study 1B) and conventional slow freezing (using the medium and protocol that resulted in superior quality spermatozoa in Study 1A). Post thawing, motility and kinetic parameters (Studies 1 and 2), viability (Study 1), mitochondrial membrane potential (Study 2), and DNA fragmentation (Study 2) of the two groups were compared. vi Results: Study 1A indicated that cryopreserving spermatozoa using Freezing Medium resulted in the highest quality spermatozoa with regards to motility and viability (p<0.05). Comparing the two preservation protocols, no conclusion could be reached on which protocol yielded superior results (p>0.05). The RBL freezing method is shorter, simpler and requires less equipment, and was therefore deemed the preferred method. Study 1B showed that the larger vitrification volumes (300 μl and 500 μl) yielded better spermatozoa in terms of motility and viability (p<0.05). No significant difference was observed with respect to the 300 μl and 500 μl vitrification volume groups. For practical reasons, 300 μl volumes will provide sufficient sperm for any procedure and, the intermediate volume ensures that more than one straw can be preserved. Study 2 found that cryoprotectant-free vitrification resulted in spermatozoa with significantly higher mitochondrial membrane potential and significantly lower apoptosis post thawing (p<0.05). Discussion: Conventional cryopreservation methods may compromise various sperm parameters and final yield. In this study, cryopreservation and cryoprotectant-free vitrification had equivalent outcomes with respect to sperm motility. However, the latter method yielded superior results in terms of ΔΨ and DNA sperm fragmentation. In conclusion, vitrification is an easy, rapid and more affordable technique that requires no special equipment. Using vitrification for purified sperm samples of patients could potentially result in a better post thaw quality for ART procedures. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Obstetrics and Gynaecology / unrestricted

This thesis investigates enhancing the thermal stabilization of a human type 5 adenoviral vector (AdHu5) through spray drying. The spray drying process was used to dry and effectively immobilize the AdHu5 within a mixture of carbohydrate or amino acid excipients into a powder form, resulting in significantly increased thermal stabilization of the viral vector. Spray dried powders were characterized by scanning electron microscopy, differential scanning calorimetry, Karl Fischer titrations, X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS) to identify the effects of temperature and atmospheric moisture on the immobilizing matrix. The best performing spray dried powder in terms of thermal stability consisted of an excipient blend of mannitol and dextran. Response surface methodology was employed to optimize production of these mannitol/dextran powders; measured responses were those relevant to industrial processing of a therapeutic material, namely powder yield for maximizing quantity, particle size for maximizing production of inhalation-deliverable powders, and adenoviral vector response for minimizing the loss of therapeutic activity. The spray drying process parameters of inlet temperature, spray gas flow rate, liquid feed rate and solute concentration in the feed were optimized resulting in a powder yield of 90%, percentage of ideally-sized particles of 50% and a near-zero viral vector titre loss of 0.25 log loss median tissue culture infectious dose (TCID50). The spray dried mannitol/dextran powders proved to have exceptional thermal stability during long term storage as minimal viral vector activity loss was observed when stored at 20°C for 90 days at low relative humidity (0.7 ± 0.3 log TCID50) in comparison to the liquid control which exhibited complete activity loss under the same storage conditions. Furthermore, viral activity of mannitol/dextran powders was retained over short term exposure (72 hours) to temperatures as high as 55°C whereas the liquid control expectedly lost all AdHu5 activity after 30 minutes. Overall, this work provides a guideline for the production of thermally stable powders and active biopharmaceuticals, such as AdHu5 vectors for vaccine applications, using the spray drying process. / Thesis / Master of Applied Science (MASc) / Many vaccines and their base components inherently deteriorate in function at moderate temperatures. Storage by refrigeration at temperatures ranging between 4°C and -80°C is the norm. Such refrigeration is costly for long term storage and significantly limits where vaccines can be sent. This reduces the availability of vaccines in locations around the world where these storage conditions are infeasible but vaccines are needed most. Spray drying, a process which forms dry powders from solution, was used; the solution contained sugars or amino acids to surround and protect the sensitive vaccine component. The produced powders from this work exhibited enhanced thermal stability compared to the control, reducing the need for refrigeration during storage and transport. The spray drying process was further optimized for industrial use by maximizing the amount of powder recovered and ensuring the particle size was appropriate for inhalable use, but most importantly, minimizing losses in therapeutic effectiveness during processing. This production of a thermally stable vaccine is advantageous because is allows for better world-wide accessibility and reduces overall production and delivery costs.

Incineration of municipal solid waste (MSW) is common for energy recovery, and management of municipal solid waste incineration (MSWI) ashes has received a growing attention around the world. In the U.S., generation of MSW has increased up to 65% since 1980, to the current level of 251 million tons per year with 53.8% landfilled, 34.5% recycled and composted, and 11.7% incinerated with energy recovery. In the process of incineration, MSWI ash is being produced as byproducts; about 80 to 90% of the MSWI ash is bottom ash (BA) and 10 to 20% is fly ash (FA) by weight. The current practice of the U.S. is to combine both BA and FA to meet the criteria to qualify as non-hazardous, and all combined ashes are disposed in landfills. European countries have utilized MSWI BA as beneficial construction materials by separating it from FA. The FA is mostly limited to landfill disposal as hazardous material due to its high content of toxic elements and salts. BA has been actively recycled in the areas of roadbed, asphalt paving, and concrete products in many of European and Asian countries. In those countries, recycling programs (including required physical properties and environmental criteria) of ash residue management have been developed so as to encourage and enforce the reuse of MSWI ashes instead of landfill disposal. Moreover, many studies have demonstrated the beneficial use of MSWI ashes as engineering materials with minimum environmental impacts. On the other hand, the U.S. has shown a lack of consistent and effective management plans, as well as environmental regulations for the use of MSWI ashes., Due to persistent uncertainty of engineering properties and inconsistency in the Federal and State regulations in the U.S., however, the recycling of the MSWI ashes has been hindered and they are mostly disposed in landfills. In this research work, current management practice, existing regulations, and environmental consequences of MSWI ashes utilization are comprehensively reviewed worldwide and nationwide with an emphasis of the potential area of its utilization in asphalt paving and concrete product. This research also entails a detailed chemical and microstructural characterization of MSWI BA and FA produced from a Refuse Derived Fuel (RDF) facility in Florida so that the MSWI ash is well characterized for its beneficial uses as construction materials. The material characterization includes Scanning Electron Microscopy (SEM), Energy Dispersive X-ray Spectroscopy (EDS), and X-ray Diffraction (XRD) techniques. In addition, leaching experiments have been conducted to investigate the environmental properties (e.g. leachate concentration) of BA and ash-mixed hot mix asphalt (HMA) and Portland cement concrete (PCC). Leaching results reveals the reduced leaching potential of toxic material from MSWI ashes while incorporated in HMA and PCC. Lastly, a preliminary experimental approach has been devised for the vitrification of FA which is a promising thermal process of transferring material into glassy state with higher physical and chemical integrity to reduce toxicity so that utilization of FA can be possible.