Este trabalho teve como objetivo a reprodução, avaliação e caracterização de manchamentos provocados por desodorante antitranspirante, suor, produtos do processo de lavagem como detergente em pó, amaciante, temperatura de passadoria em camisetas de malhas de algodão. Buscando uma melhor caracterização destes manchamentos, foram realizados três grupos de estudo com indivíduos selecionados, propensos à obtenção de manchas na região das axilas. Os indivíduos fizeram uso de desodorante com e sem perfume e utilizaram camisetas por um período de onze dias, sendo que um dos indivíduos fez uso apenas da camiseta sem desodorante e, uma das camisetas foi submetida ao desodorante sem uso por indivíduos. Ao final de cada dia, as camisetas foram lavadas com detergente em pó, amaciante, secas em secadora e passadas, simulando uma condição de uso doméstico. As camisetas manchadas foram submetidas às avaliações instrumentais de espectrofotometria (colorímetro), Microscopia Eletrônica de Varredura (MEV), análise por Dispersão de Energia (EDS) e medição da efusividade e condutividade térmica e Espectrometria de Emissão Atômica por Plasma Acoplado Indutivamente (ICP-OES). Essas camisetas também foram submetidas a dois processos de remoção: por lavagem a seco (LS) e por agentes químicos (RAQ). Após o processo de remoção, as camisetas foram submetidas à avaliação colorimétrica instrumental, condutividade e efusividade térmica, MEV, resistência à ruptura. As análises colorimétricas para todas as camisetas utilizadas pelos participantes dos grupos de estudo mostraram valores de E* de (0,87 a 32,43), indicando uma alteração de cor perceptível visualmente na região das axilas. As microscopias das amostras também mostram um acúmulo de alguns materiais nessa região que pode ter influenciado a troca de calor do material com o ambiente, como mostraram os resultados de efusividade e condutividade. Este material acumulado, analisado pela técnica de ICP-OES, mostrou que a camiseta manchada apresentou 99,7% de alumínio a mais do que na camiseta original na região da axila, o que pode ser oriundo do contato com o desodorante que também apresenta elevados teores de alumínio em sua formulação. Os métodos utilizados para remoção apresentaram resultados satisfatórios, promovendo a remoção dos manchamentos nas camisetas, observado através da diminuição dos valores de E* e nos índices da Escala Cinza de Alteração da Cor, na avaliação colorimétrica instrumental. Entretanto, o processo de remoção por agentes químicos apresentou melhores resultados, tendo cerca de 70% de eficácia quando comparados ao método de lavagem a seco, podendo ser um método mais efetivo na remoção deste tipo de manchamento. / This work was carried out to reproduction, evaluation and characterization of staining caused by antiperspirant deodorant, sweat, products of the washing process and detergent powder, fabric softener, temperature ironing shirts in cotton knits. Searching for a better characterization of these staining, three groups of studies selected, prone to getting stains on the underarm area individuals, were performed. Individuals made use deodorant and fragrance with shirts and used for a period of eleven days, and one of the subjects only used the shirt without deodorant and one of the shirts was submitted to without deodorant use by individuals. At the end of each day the shirts were washed with detergent powder, fabric softener, dryer dried and pressed, simulating a condition of household. The stained shirts were subjected to instrumental evaluations spectrophotometry (colorimeter), Scanning Electron Microscopy (SEM), Energy Dispersive analysis (EDS) and measuring the effusivity and thermal conductivity and Atomic Emission Spectrometry by Inductively Coupled Plasma (ICP-OES). These shirts were also subjected to two processes: removal by cleaning (LS) and chemicals (RAQ). After the removal process tees instrumental colorimetric evaluation, conductivity and thermal effusivity, SEM, tensile strength were submitted. colorimetric analyzes for all t used by the participants of the study groups showed values of E * (0.87 to 32.43), indicating a change of color visually noticeable in the underarm area. The microscopy of the samples also showed an accumulation of some materials in this region that may have influenced the heat exchange material with the environment as showed the results of effusivity and conductivity. This accumulated material, analyzed by ICP-OES technique, showed that the stained shirt showed 99.7% more aluminum than the original shirt, which can be derived from contact with the deodorant that also has a high concentration of aluminum in its formulation. The methods used for removal showed satisfactory results, promoting the removal of staining tees, observed by decreasing the values of E * and indexes of Gray Scale Change Colors, instrumental colorimetric evaluation. However, the process of removal by chemical agents showed better results, about 70% efficiency when compared to the method of cleaning, may be a more effective method in removing this type of stain.

A presente dissertação foi elaborada em dois experimentos. No primeiro experimento objetivou-se determinar o dia do parto através de alterações físicas e circulatórias locais, e avaliar o pH da secreção da glândula mamária comparando dois métodos em éguas no pré-parto. Foram utilizadas 30 éguas gestantes sem raça definida (SRD), durante duas estações de monta (2015 e 2016), com idade de 10±4 anos, e peso corporal de 560±57 kg. Foram observadas possíveis alterações na área pélvica, abdominal, edema vulvar e a coloração da mucosa vaginal até o dia do parto através de fotográfias sequênciais, e as fotos foram analisadas individualmente e classificadas usando um sistema de escore. Para mensuração do pH, foi utilizado o peagâmetro portátil (Quimis®) e o teste de pH em fita (Fusion®). As colheitas iniciaram a partir de 310 dias de gestação. Para às mensurações do pH, foi utilizado um modelo misto sendo a égua o efeito aleatório e o tempo como efeito fixo. As associações entre as diferentes mensurações de pH foram avaliadas por meio de correlação de Momento-produto de Pearson. Para avaliar o escores físicos e circulatórios locais os dados foram analisados pelo teste de razão de Probabilidade Qui-Quadrado e por regressão, para observar a frequência dos escores 1, 2 ou 3 até o dia do parto. O nível de significância utilizado foi de 5%. O pH da secreção mamária pode ser utilizada para determinar o dia do parto, independente do método utilizado. A coloração de mucosa vaginal e edema vulvar podem auxiliar na predição do dia do parto, porém não devem ser utilizados como único método. No segundo experimento, o objetivo foi estudar o relacionamento entre a égua e e sua cria no primeiro dia pós-parto, e relacionar o fator progênitor sobre o comportamento do potro. Foram utilizadas 22 éguas multíparas e seus respectivos potros em duas temporadas de parto (2015 e 2016). Os progenitores eram formados por jumento e equinos. O etograma foi delineado considerando-se os comportamentos característicos apresentados por éguas e potros, e divididos em atividades de interação entre égua e produto, atividades do produto, postura e outros comportamentos. As observações foram realizadas no primeiro dia pós-parto durante 10 horas a cada 2 minutos, através de observação direta com rota de amostragem focal da égua e sua cria. Foi realizada a análise de frequência dos comportamentos ao longo do tempo, considerando cada par (égua/potro) como uma unidade experimental. As frequências dos comportamentos de interação, postura e outros comportamentos das éguas foram analisadas pelo teste de Qui-Quadrado para proporções iguais. Para dependência do comportamento do potro sobre o tipo de progênitor foi realizado o teste de Qui-Quadrado de Pearson. O nível de significância utilizado foi 5%. Concluí-se que no primeiro dia pós-parto as éguas buscam auxiliar a mamada, enquanto os potros mamam e descansam. Potros e muares apresentam diferenças na postura. / The present dissertation was elaborated in two experiments. The first experiment aimed to determine the day of birth through local physical and circulatory changes, and to evaluate the pH of the mammary gland secretion comparing two methods of mares in preparturient. Thirty non-defined pregnant mares (SRD) were used during two mating seasons (2015 and 2016), aged 10 ± 4 years, and body weight of 560 ± 57 kg. Possible alterations in pelvic, abdominal, vulvar edema and vaginal mucosa staining until the day of delivery were observed through sequential photographies, and the photos were analyzed individually and classified using a scoring system. For pH measurement, the portable pHmeter (Quimis®) and the pH strip test (Fusion®) were used. Crops started at 310 days of gestation. For the pH measurements, a mixed model was used, the mare being the random effect and the time as a fixed effect. The associations between the different pH measurements were evaluated through Pearson\'s Moment-product correlation. In order to evaluate the local physical and circulatory scores, the data were analyzed by the chi-square probability and regression test to observe the frequency of scores 1, 2 or 3 until the day of delivery. The level of significance was 5%. The pH of the mammary secretion can be used to determine the day of delivery, regardless of the method used. The coloration of vaginal mucosa and vulvar edema may aid in the prediction of the day of delivery, but should not be used as the sole method. In the second experiment, the objective was to study the relationship between the mare and her offspring on the first postpartum day, and to relate the progenitor factor to the foal behavior. Twenty - two multiparous mares and their respective foals were used in two calving seasons (2015 and 2016). The parents were formed by donkeys and horses. The etogram was delineated considering the characteristic behaviors presented by mares and foals, and divided into activities of interaction between mare and product, product activities, posture and other behaviors. The observations were performed on the first day postpartum for 10 hours every 2 minutes, through direct observation with the focal sampling route of the mare and her offspring. The behavioral frequency analysis was performed over time, considering each pair (mare / foal) as an experimental unit. The frequencies of the interaction behaviors, posture and other behaviors of the mares were analyzed by the Chi-square test for equal proportions. For the dependence of the behavior of the foal on the type of progenitor was carried out the test of Chi-Square of Pearson. The level of significance was 5%. It was concluded that on the first day postpartum the mares seek to assist the feeding, while the foals suck and rest. Foals and mules show differences in posture.

INTRODUCTION: Les gouttières thermoplastiques orthodontiques doivent souvent être retirées par les patients avant de consommer des aliments ou des liquides autres que l’eau. Cette précaution évite notamment des changements de couleur au niveau du matériau les constituant. Des études antérieures ont concentré leurs recherches sur des changements de coloration chez des marques limitées d’aligneurs thermoplastiques en excluant certaines marques américaines populaires. OBJECTIFS: L’objectif principal de cette étude vise à évaluer la résistance au changement de teinte du polymère formant trois différentes marques d’aligneurs américaines. Un but secondaire est de comparer le pouvoir de retrait de taches de deux produits nettoyants conçus pour ces appareils amovibles. Un but tertiaire est d’évaluer le type de polymère qui constitue les surfaces interne et externe de chaque marque étudiée de gouttières. MATÉRIELS ET MÉTHODES : Les gouttières ont été exposées à l’un des divers liquides colorants de l’alimentation courante (thé, café, vin rouge et cola) ou à une solution de contrôle formée d’un gel de remplacement salivaire Biotène® Oral Balance. Les trois marques évaluées étaient Invisalign®, ClearCorrect® et Minor Tooth Movement®. Après leur immersion de douze heures ou de sept jours, les mêmes gouttières étaient nettoyées pendant quinze minutes avec soit les cristaux Invisalign®, soit le bain Cordless Sonic Cleaner combiné avec un comprimé Retainer Brite®. Les données de colorimétrie étaient obtenues à l’aide d’un numériseur calibré et du logiciel Adobe Photoshop® en utilisant l’espace chromatique CIELAB pour comparer les changements de coloration (E) entre les divers moments à l’étude. Les paramètres L*, a*, b* étaient mesurés au temps initial (T0), après douze heures d’immersion (T1), après sept heures d’exposition (T2) et après nettoyage (T3). Les deltas E étaient ensuite convertis en unités du National Bureau of Standards (NBS) pour faciliter l’interprétation clinique des données. Selon la section, les analyses statistiques employées (test de Levene, ANOVA, méthode de Brunner-Langer, test de Tukey ou test-t) permettaient d’évaluer les interactions entre les trois marques entre elles ou entre les marques et les nettoyeurs. Une valeur de p < 0,05 était considérée statistiquement significative. RÉSULTATS ET DISCUSSION: Les résultats suggèrent qu’une exposition de douze heures au café pour les aligneurs Invisalign® engendre un changement de couleur statistiquement significatif par rapport aux deux autres marques. Il en est de même pour une durée identique d’immersion dans le vin rouge ; les coquilles Invisalign® présentent un changement de teinte statistiquement significatif par rapport à ses compétiteurs. Après sept jours d’exposition au café ou au vin rouge, les coquilles de la marque Invisalign® ont aussi des changements de coloration statistiquement significatifs par rapport aux deux autres marques, mais plus prononcés qu’après douze heures. Le thé noir engendre des changements de couleur importants au niveau clinique chez les trois marques après sept jours d’exposition sous forme de taches extrinsèques. Les deux méthodes de nettoyage étudiées présentent un potentiel clinique de retrait de taches accru pour celles extrinsèques du thé noir par rapport aux taches intrinsèques des autres solutions étudiées. Les résultats par spectrophotométrie à lumière infrarouge démontrent des spectres similaires pour les surfaces interne et externe de chaque marque. Or, ce n’est pas le même polymère qui est employé par chaque compagnie. Des spectres ATR-FTIR d’un matériau à base de polyuréthane sont observés pour les marques Invisalign® et ClearCorrect®. Des spectres ATR-FTIR de matériau à base de polyéthylène téréphtalate glycolisé sont plutôt représentatifs de la marque Minor Tooth Movement®. CONCLUSION: Les aligneurs de la compagnie Invisalign® sont plus susceptibles de souffrir de pigmentation par le café et le vin rouge que les deux autres marques qui ont présenté une stabilité accrue de leur coloration au contact de ces solutions. Le thé a engendré des changements de couleur extrinsèques importants chez les trois marques étudiées. / INTRODUCTION: Manufacturers of orthodontic aligners suggest that users take off removable appliances every time they consume solid foods or any drink (except water). This is to avoid a color change within the clear thermoplastic material of which they are made. Prior studies have focused on a limited set of aligners, excluding some popular American brands on the market. OBJECTIVES: The main objective of this study is to evaluate the stain resistance of the polymer forming three different American aligner brands. A secondary aim is to evaluate the stain-removal potential of two cleaning techniques for these devices. A tertiary goal is to determine the type of polymer forming the internal and external surfaces of the studied brands of aligners. MATERIALS AND METHODS: The removable appliances were exposed to different staining agents common in a regular diet (coffee, red wine, black tea and cola) or to a control solution made of a Biotene® Oral Balance saliva replacement gel. The three brands evaluated were Invisalign®, ClearCorrect® and Minor Tooth Movement®. Following exposure, the same aligners were cleaned during fifteen minutes with either the Invisalign® cleaning crystals or the Cordless Sonic Cleaner combined with a Retainer Brite® tablet. Colorimetry data was acquired via a calibrated scanner and the Adobe Photoshop® software, using a CIELAB color space approach to compare color changes (E) in aligners exposed to various media. The CIE color parameters (L*, a*, b*) of each specimen were measured before immersion (T0), after a twelve-hour exposure (T1), after a seven-day exposure (T2) and after cleaning (T3). Those color differences were then converted into National Bureau of Standards (NBS) units to facilitate the clinical interpretation. Statistics methods (Levene’s test, ANOVA, Brunner-Langer model, Tukey’s range test and t-test) were used to identify interactions between the brands themselves or between the brands and the cleaning methods. A p value < 0.05 was considered statistically significant. RESULTS AND DISCUSSION: This study suggests that an exposure of twelve hours to instant coffee affects significantly the color of the Invisalign® aligners compared to the two other brands. A twelve-hour immersion into red wine also causes a statistically significant color change for the Invisalign® devices compared to their competitors. After seven days of exposure, a more conspicuous intrinsic color change is observed in the Invisalign® aligners exposed to instant coffee and red wine. In both media, the results are statistically significant for the Invisalign® appliances compared to the ClearCorrect® and the Minor Tooth Movement® aligners. The black tea creates an important extrinsic color change in the three brands after seven days. While normal exposure conditions would not typically consist of 12 h to 7 days of continuous exposure, these data indicate that repeated, cumulative exposure may be problematic. The two cleaning methods tend to show a better efficacy in removing extrinsic stains from black tea compared to intrinsic stains of other substrates. The infrared spectrophotometry confirms that the internal and external surfaces of each studied brand of aligners were made of the same polymer. ATR FTIR spectra of polyurethane-based material are observed for the Invisalign® and the ClearCorrect® devices. However, for the Minor Tooth Movement® aligners, the ATR FTIR analysis shows that the appliances are a polyethylene glycol terephthalate polyester. CONCLUSION: The Invisalign® aligners were more prone to pigmentation from coffee and red wine than the ClearCorrect® or the Minor Tooth Movement® devices. Black tea caused important extrinsic stains on the surface of the three tested brands.

La détection et l’identification des protéines sur gel d’électrophorèse dépend aussi bien du marqueur des protéines utilisés (généralement un marqueur fluorescent) que de ses propriétés physicochimiques propres (rendement quantique, Stokes’ shift et recouvrement des bandes d’absorption et d’émission, photoblanchiment), de sa stabilité (chimique et photochimique), et de son accessibilité. Créer une gamme de nouveaux fluorophores dont les propriétés physicochimiques peuvent être modulées en partant d’une structure bien connue et très avantageux dans l’optique d’anticiper les caractéristiques supposées des composées envisagés. Cependant, même si de nombreux fluorophores sont disponibles commercialement, ils souffrent généralement de différentes contraintes telles que leurs prix prohibitifs, leur manque de sensibilité, leur stockes’ shift trop faible ou bien encore leur stabilité. L’accès à une famille de fluorophores résolvant l’ensemble de ces contraintes pour la détection de protéines sur gel d’électrophorèse représenterait donc un réel défi. L’épicocconone, isolée du champignon Epicoccocum Nigrum, se lie de façon covalente aux amines (et donc également aux protéines) en conduisant à un adduit énaminique qui est très fluorescent. Cet adduit émet dans le rouge (610 nm) lorsqu’irradié dans l’UV (395 nm) ou le visible (520 nm). Ces propriétés ont donc conduit à l’utilisation de ce produit naturel comme marqueur fluorescent de protéines sur gel d’électrophorèse menant à une sensibilité de détection rarement atteinte et à une gamme de linéarité très importante (104). La première synthèse de l’épicocconone a alors été engagée au sein de notre laboratoire. Elle a finalement conduit à une chimiothèque d’analogues encore plus efficace ayant permis d’établir une véritable relation structure-fluorescence. / Detection and identification of proteins either in 2D electrophoresis gels or in biological media depends largely on the quality of protein sensitive dye used (generally a fluorescent dye) as well on the efficiency of their fluorescent properties (quantum yield, Stokes’ shift and overlap of excitation frequency with common laser sources), their stability (chemical and photophysical) and their accessibility. To be able to create a range of new fluorescent dyes whose profiles can be modulated starting from a proven scaffold is advantageous in terms of predicting the properties of the final products. Even if many fluorescent dyes are available on the market, they suffer from a prohibitive price, a lack of sensitivity or a restricted wavelength range (absorption/emission) or limited stability. Access to a family of fluorophores displaying similar physicochemical properties, but shifted excitation and emission wavelengths constitutes an invaluable tool Epicocconone, isolated from the fungus Epicoccum nigrum, covalently binds to primary amines (e.g. lysine of proteins), leading to a protein linked conjugate which is strongly fluorescent, emitting light in the red (610 nm) when submitted to UV-visible light (395 or 520 nm). This is a novel pro-fluorophore that allows protein quantification with unprecedented sensitivity and with an excellent linearity on a broad range of concentrations. A program of synthesis of epicocconone and analogues in order to provide a library of complementary fluorophores has been undertaken. The results concerning either the better efficiency of the analogs and the structure-fluorescence relation are presented in this dissertation.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18346号 / 農博第2071号 / 新制||農||1024(附属図書館) / 学位論文||H26||N4853(農学部図書室) / 31204 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 栗原 達夫, 教授 植田 充美, 教授 小川 順 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Irrigation of the municipal cemetery in Brigham City, Utah resulted in stained headstones in 2001 and 2002. The water used in the irrigation came from Mantua reservoir, a medium sized impoundment situated near the mouth of Box Elder Canyon. In order for Brigham City to establish a city wide secondary pressurized irrigation system using water from Mantua reservoir, the cause and the source of staining problem must be determined. Previous research (Wallace 2006) determined that the source of the staining was the reduction of iron found in Mantua Reservoir sediments that occurred when seasonal variations in the reservoir caused anaerobic conditions. The reduced iron then dissolved in the water and was used in the irrigation system, causing re-oxidation of the iron. The oxidized iron then precipitated out on the headstones causing the staining. The purpose of this investigation is to determine the iron oxidation kinetics after the re-aeration of the water which will help determine appropriate mitigation methods. A secondary purpose is to confirm the Mantua reservoir's capacity to become anaerobic, resulting in the conditions which cause staining. Using laboratory investigations and computer modeling, I determined that on re-aeration, fifty percent of the dissolved iron in the water precipitates in five hours. Using first-order kinetics to model this process, I found the rate constant of the kinetic reaction to be 0.0029 min-1. Fitting a geochemical computer model of the iron oxidation kinetics in Mantua reservoir, which uses a higher-order kinetics model to better model this process, to experimental kinetic data yielded a rate constant of 4x1013 /atm x min. I also recreated the staining process in the laboratory using concrete. This was successful and provided visual evidence that the iron precipitates out of the water and stained the concrete within a couple of hours of application. Field data collected from Mantua reservoir showed that the dissolved oxygen concentration in the reservoir drops regularly below levels consistent with equilibrium to the atmosphere. While my field measurements did not record anaerobic conditions, based on the patterns shown, this study shows that it would be possible for anaerobic conditions to occur during warmer weather.

Adoptive immunotherapy using chimeric antigen receptor (CAR)-T cells has achieved successful remissions in refractory B-cell leukemia and B-cell lymphomas. In order to estimate both success and severe side effects of CAR-T cell therapies, longitudinal monitoring of the patient’s immune system including CAR-T cells is desirable to accompany clinical staging. To conduct research on the fate and immunological impact of infused CAR-T cells, we established standardized 13-colour/15-parameter flow cytometry assays that are suitable to characterize immune cell subpopulations in the peripheral blood during CAR-T cell treatment. The respective staining technology is based on pre-formulated dry antibody panels in a uniform format. Additionally, further antibodies of choice can be added to address specific clinical or research questions. We designed panels for the anti-CD19 CAR-T therapy and, as a proof of concept, we assessed a healthy individual and three B-cell lymphoma patients treated with anti-CD19 CAR-T cells. We analyzed the presence of anti-CD19 CAR-T cells as well as residual CD19+ B cells, the activation status of the T-cell compartment, the expression of co-stimulatory signaling molecules and cytotoxic agents such as perforin and granzyme B. In summary, this work introduces standardized and modular flow cytometry assays for CAR-T cell clinical research, which could also be adapted in the future as quality controls during the CART cell manufacturing process.

In patients with Parkinson's disease (PD), the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS), the dorsal motor nucleus of the vagus (DMV), the intermediolateral nucleus of the spinal cord and the substantia nigra, providing the basis for the neuropathological staging of the disease. Here we report that intragastrically administered rotenone, a commonly used pesticide that inhibits Complex I of the mitochondrial respiratory chain, is able to reproduce PD pathological staging as found in patients. Our results show that low doses of chronically and intragastrically administered rotenone induce alpha-synuclein accumulation in all the above-mentioned nervous system structures of wild-type mice. Moreover, we also observed inflammation and alpha-synuclein phosphorylation in the ENS and DMV. HPLC analysis showed no rotenone levels in the systemic blood or the central nervous system (detection limit [rotenone]<20 nM) and mitochondrial Complex I measurements showed no systemic Complex I inhibition after 1.5 months of treatment. These alterations are sequential, appearing only in synaptically connected nervous structures, treatment time-dependent and accompanied by inflammatory signs and motor dysfunctions. These results strongly suggest that the local effect of pesticides on the ENS might be sufficient to induce PD-like progression and to reproduce the neuroanatomical and neurochemical features of PD staging. It provides new insight into how environmental factors could trigger PD and suggests a transsynaptic mechanism by which PD might spread throughout the central nervous system.

Background The inducible Cre-lox system is a valuable tool to study gene function in a spatial and time restricted fashion in mouse models. This strategy relies on the limited background activity of the modified Cre recombinase (CreER) in the absence of its inducer, the competitive estrogen receptor ligand, tamoxifen. The RIP-CreER mouse (Tg (Ins2-cre/Esr1) 1Dam) is among the few available β-cell specific CreER mouse lines and thus it has been often used to manipulate gene expression in the insulin-producing cells of the endocrine pancreas. Principal Findings Here, we report the detection of tamoxifen-independent Cre activity as early as 2 months of age in RIP-CreER mice crossed with three distinct reporter strains. Significance Evidence of Cre-mediated recombination of floxed alleles even in the absence of tamoxifen administration should warrant cautious use of this mouse for the study of pancreatic β-cells.

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2010. / Bibliography / ENGLISH ABSTRACT: Sperm morphology remains an important parameter in the prediction of fertility, both in vivo and in vitro. However, there remains a considerable level of concern surrounding the true potential of this parameter due to the lack of standardization of differential staining techniques used for the evaluation of sperm morphology. This study aimed at investigating two commonly used staining techniques, Rapidiff® (RD) and Papanicolaou (PAP), along with a new commercially available stain, SpermBlue® (SB), in the evaluation of sperm morphometry and morphology. Results indicated that significant differences in sperm morphometry exist due to the use of the staining techniques. Findings further indicated that RD causes sperm head swelling while PAP causes sperm head shrinkage. Results obtained using the SB staining technique have indicated measurements closest to that which would be obtained through the evaluation of fresh, unstained sperm. The lack of standardization and the different effects various stains have on sperm structure and overall sperm morphology evaluation should raise a level of concern, particularly when evaluating patients with borderline morphology. Based on this, the use of the SB staining technique is recommended over RD and PAP for effective and accurate morphology evaluation. In further support of this technique, SB was shown to be quick and simple in method, and allowed for the easy detection of sperm by computer aided sperm analysis (CASA) systems such as the Sperm Class Analyzer (SCA®). The second aim of this study was to examine the concentration, morphology and motility of the resultant sperm populations following semen preparation using the PureSperm® density gradient and swim-up techniques. Semen preparation is an essential step in any fertility treatment protocol, and it is important that the sperm obtained following semen preparation has sperm morphology and motility characteristics capable of improving assisted fertility success rates. Currently, the PureSperm® density gradient and sperm swim-up are the most widely employed techniques in fertility clinics. Although there is sufficient evidence to suggest they are each effective at extracting sperm with improved quality from neat semen, there remains insufficient evidence to suggest which of these two techniques is superior. The present investigation revealed that both sperm preparation methods were effective at improving sperm morphology and motility, however to varying degrees. The swimup method yielded a population of sperm with superior motility and morphology when assessed according to World Health Organisation (WHO) criteria, while the PureSperm® density gradient technique isolated a higher percentage of normal sperm, according to both WHO and Tygerberg strict criteria, with motility better than that of neat semen. Although results obtained via the swim-up method suggest it would be best for use in in vitro fertilization (IVF), the very low concentration of sperm isolated via this method remains a significant draw-back. The PureSperm® density gradient separation technique on the other hand is capable of isolating larger quantities of sperm, which is likely to be of more benefit with fertility treatments requiring larger quantities of sperm. Based on these findings, the use of PureSperm® density gradient technique is recommended, due to its ability to isolate large quantities of good quality sperm. However, a swim-up may still be of use when performing fertility treatment using a sperm sample which possesses a high concentration and motility. / AFRIKAANSE OPSOMMING: Sperm morfologie bly ‘n belangrike parameter in die voorspelling van vrugbaarheid, beide in vivo en in vitro. Tog is daar nogsteeds ‘n aansienklike vlak van kommer rondom die ware potensiaal van hierdie parameter weens die gebrek aan standardisering van verskillende kleuringstegnieke wat gebruik word vir die evaluering van spermmorfologie. Hierdie studie is daarop gemik om ondersoek in te stel na twee algemeen gebruikte kleurings tegnieke naamlik, Rapidiff® (RD) en Papanicolaou (PAP), asook ‘n nuwe kommersiëel beskikbare kleurstof, SpermBlue® (SB), vir die evaluering van spermmorfometrie en morfologie. Resultate dui aan dat beduidende verskille in sperm morfometriese afmetings ontstaan as gevolg van die gebruik van die verskillende kleurstowwe. Bevindinge dui verder daarop dat RD swelling van die sperm se kop versoorsaak, terwyl PAP die spermkop laat krimp. Resultate wat verkry is met behulp van die SB kleuringstegniek dui daarop dat hierdie kleurstof aanleiding gegee het tot afmetings naaste aan die verkry tydens die beoordeling van vars, ongekleurde sperme. Die gebrek aan standardisasie en die uiteenlopende effekte wat verskillende kleurstowwe het op die spermstruktuur en die evaluering van sperm morfologie ingeheel is kommerwekkend, veral tydens die evaluering van pasiënte met grensgeval morfologie. Op grond van hierdie resultate, word die gebruik van die SB kleuringstegniek, bo die gebruik van RD en PAP, vir effektiewe en akkurate morfologie evaluering aanbeveel. Verdere ondersteuning vir die gebruik van die SB kleuringstegniek is die feit dat daar bevind is dat SB ‘n vinnige en eenvoudige metode is, wat toelaat vir maklike visualisering van sperme deur rekenaargesteunde sperm analise sisteme soos die Sperm Class Analyzer (SCA®). Die tweede doel van hierdie studie was om die konsentrasie, morfologie en die motiliteit van spermpopulasies te ondersoek, soos verkry tydens die voorbereiding van semen deur gebruik te maak van die PureSperm® digtheidsgradiënt en op-swem tegnieke. Die voorbereiding van semen is ‘n noodsaaklike stap in enige vrugbaarheidsbehandeling protokol, aangesien dit belangrik is dat die sperme wat deur hierdie prosesse verkry word oor die nodige morfologiese en motiliteit eienskappe beskik wat in staat is om die sukses van vrugbaarheidsbehandelings te verbeter. Huidiglik is die PureSperm® digtheidsgradiënt en op-swem tegnieke die mees algemeen gebruikte tegnieke in vrugbaarheidsklinieke. Alhoewel daar voldoende bewyse is wat voorstel dat elke tegniek effektief is vir die ekstraksie van sperme met beter kwaliteit vanuit semen, bly daar steeds onvoldoende bewyse wat daarop dui dat een van hierdie twee tegnieke beter is as die ander een. Huidige navorsing het getoon dat beide sperm voorbereidings metodes daarin geslaag het om sperme met normale morfologie en beter motiliteit te selekteer. Die opswem metode het ‘n spermpopulasie met beter motiliteit en verbeterde morfologie gelewer, soos getoets volgens die WGO kriteria, terwyl die PureSperm digtheidsgradiënt tegniek sperme met verbeterde morfologie, volgens beide die WGO en Tygerberg Streng Kriteria, en ‘n redelike verbetering in sommige motiliteits parameters geselekteer het. Hoewel die resultate wat verkry word via die op-swem metode voorstel dat dit die beste metode vir die gebruik tydens in vitro bevrugting sou wees, bly die baie lae konsentrasie van sperme wat met hierdie metode verkry word ‘n belangrike nadeel. Die PureSperm® skeidingstegniek laat egter toe vir die isolering van groter hoeveelhede sperme, wat waarskynlik meer voordelig sal wees vir bevrugtingsbehandelings wat meer sperme benodig. Gebaseer op hierdie bevindinge, word die gebruik van die PureSperm® digtheidsgradiënt tegniek aanbeveel, as gevolg van hierdie tegniek se vermoë om groot hoeveelhede goeie gehalte sperm te isoleer. Daar kan egter nogsteeds van op-swem metodes gebruik gemaak word tydens vrugbaarheidsbehandeling indien die semenmonster beskik oor ‘n hoë konsentrasie sperme met goeie beweeglikheid.