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Fluorescent molecules as probes for characterization of amyloid β fibrilsMarginean, Denisse, Hellstrand, Ebba January 2021 (has links)
Alzheimer’s Disease (AD) is the leading cause of dementia in the world and the World Health Organization has recognized AD as a global public health priority. One of the pathological hallmarks of AD is amyloid plaques formed from amyloid β (Aβ) fibrils. Aβ is formed when amyloid precursor protein is cleaved by secretase enzymes. Cleavage by different secretases causes Aβ to occur in different forms, mainly as 40 and 42 residue long proteins, called Aβ1-40 and Aβ1-42, where Aβ1-42 is more likely to form amyloid fibrils and is therefore considered more harmful. Fluorescent probes are currently used to stain Aβ fibrils for their detection and characterization. We performed a literature study analysing which fluorescent probes are used for imaging of amyloid fibrils and present both the most commonly used probes but also newer probes that have been recently synthesized. Fluorescence spectra of a selection of probes were analysed in order to suggest some new combinations of probes for double-staining with the aim to be able to distinguish between Aβ1-40 and Aβ1-42. Microscopy images of the probe combinations were obtained in order to analyse the double staining results and the fluorescence intensities of the probes were plotted in different ways. All selected combinations were able to distinguish between Aβ1-40 and Aβ1-42, because of differently stained fibrils, and also displayed differences in fluorescence intensity at peak emission wavelength. The obtained results show that double-staining of amyloid fibrils with fluorescent probes can give additional information compared to staining fibrils with only one probe.
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Molekuly v buněčných membránách / Molecules in Cell MembranesTimr, Štěpán January 2017 (has links)
Biological membranes are actively involved in a multitude of processes in living cells; therefore, a detailed characterization of their structure, dynamics, and function is essential for an understanding of living organisms at the molecular level. In this work, we made use of the high spatial and temporal resolution offered by computer simulations to investigate the behavior of several molecular species which associate with cellular membranes. Using a combination of classical molecular dynamics simulations and ab initio electronic structure calculations, we were able to characterize nonlinear optical properties of membrane- embedded fluorescent probes and thus contribute to establishing two-photon polarization microscopy as a tool of structural biology. Moreover, our molecular dynamics simulations provided an atomistic picture of the reversible membrane binding of recoverin, a neuronal calcium-sensing protein involved in vision adaptation, and they also yielded an important insight into the mechanism of its calcium-induced myristoyl switch. In addition, we examined the biological role of cholesterol oxidation and compared two methods of representing transmembrane voltage in molecular dynamics simulations.
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Fluorescent Indicators for Disease BiomarkersLim, Soojin 01 January 2012 (has links)
Xanthene dyes are common fluorophores which have been widely used as molecular probes. The xanthene fluorophores can be used as highly selective optical sensors to detect disease biomarkers. A new fluorogenic dye containing an alpha, beta-unsaturated aldehyde moiety exhibits selective fluorescent signal enhancement in the presence of cysteine or peptides containing N-terminal cysteine residues. The mechanism is based on synergistic covalent and supramolecular interactions. A unique rhodamine boronic acid indicator is used in an optimized data collection protocol for wavelength- and time-dependent selectivity of various saccharides and nucleosides. One indicator is thereby capable of selectively distinguishing structurally related analytes in mixtures. Moreover, the rhodamine-based boronic acid responds linearly to increasing riboside concentrations in urine samples, potentially enabling the screening for inborn purine metabolism disorders.
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Towards the development of fluorescent probes targeting aldehyde dehydrogenase (ALDH) in cancer. Expression and epigenetic modulation of ALDH1A1, ALDH2 and ALDH3A1 in selected in vitro models.Cosentino, Laura January 2012 (has links)
The cancer stem cell (CSC) concept is still very controversial; therefore identification and isolation of this specific population remain challenging. A variety of putative markers have been described and measurement of high aldehyde dehydrogenase (ALDH) activity has been defined as a characteristic of stem cells (SCs). In this study, a library of novel small molecules (1,4-di-substituted acetalanthraquinones, AAQs), containing an acetal group as protected aldehyde functionality, was designed with the aim of probing affinity for ALDH metabolism and demonstrating their potential as molecular fluorescent probes to identify CSCs. The AAQs were shown to be subjective to acidic hydrolysis using 2M HCl at 37ºC; however compounds containing secondary or tertiary amine functionalities in their sidechain were only partly hydrolysed at 70 ºC. Metabolism studies were conducted using cytosolic fractions from rat liver enriched in ALDHs, yeast ALDH and human recombinant ALDH1A1. Some evidence was demonstrated which linked ALDH metabolism with aldehyde functionalities of hydrolysed AAQs (HAAQs). The AAQs were shown to emit far-red fluorescence (600-750 nm). A close relationship between structure modifications and alteration of cellular localisation, with gained specificity for selected sub-cellular compartments were achieved when assessed in A549 and U-2 OS cell lines. Thermal DNA denaturation and chemosensitivity assays were used to obtain information about DNA binding properties and cytotoxicity of AAQs and HAAQ congeners. All compounds were shown to be weak*to*moderately binding to DNA, and symmetrical 1,4-di-substituted compounds were shown to be non*toxic (IC50 = 100 :/! with non-symmetrical analogues generating IC50 values in the 1-100 :/ range. No fundamental variation in the biological activity was observed when comparing AAQs with HAAQs in the A549 (+ALDH) and MCF7 (-ALDH) cell lines. A pilot investigation revealed that aberrant gene methylation was cell-type dependent for three ALDH isoforms (1A1, 2, 3A1). Decitabine treatment led to enhanced protein expression for ALDH1A1 (A549), ALDH2 (MCF7) and ALDH3A1 (A549). In contrast, the protein level was reduced for ALDH1A1 in HT29 cells after decitabine treatment. ALDH1A1, ALDH2 and ALDH3A1 were highly expressed in prostate cell lines, with expression linked to promoter methylation. In contrast, low levels of DNA methylation were found in primary prostate cancer cells and benign prostatic hyperplasia. Interestingly, ALDH1A1, considered a SC marker, was found to be expressed at low levels in CD133+/ α2β1hi stem cell fraction and upregulated in CD133-= α2β1lo differentiated prostate cancer cells. In summary, the results in this thesis demonstrate the complexity and tumour type specificity of ALDH expression. This creates challenges for the development of selective probes for CSC isolation, such as the AAQs discussed in this thesis. Although inconclusive results were obtained in regard to AAQs and their potential in targeting ALDHs, selected AAQs were shown to reveal interesting biological features highlighting them as potential non-invasive cytometric probes for tracking molecular interactions in live cells. / EPSRC, Biostatus / The full text was made available at the end of the re-embargo period, 1st September 2017.
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Cyclotrivératrylènes fluorescents pour la détection d’ammoniums d’intérêt biologique / Fluorescent cyclotriveratrylenes for the detection of biological ammoniumsPeyrard, Lisa 17 December 2012 (has links)
Le développement de sondes moléculaires fluorescentes pour le suivi ex vivo de phénomènes biologiques (communication nerveuse, par exemple) est en plein essor. Dans cette optique, des dérivés fluorescents de cyclotrivératrylènes (CTV), cavitands propices à la complexation de petites molécules organiques, ont été synthétisés. La stratégie utilisée consiste à introduire des groupements électro-donneurs et électro-attracteurs conjugués sur chaque unité aromatique du CTV afin d’avoir des systèmes de type « push-pull ». L’extension de la conjugaison entre les groupements électro-attracteurs et électro-donneurs a été envisagée afin d’améliorer les propriétés spectroscopiques des sondes. Des réactions de couplage organométallique de Sonogashira, ainsi que des cycloadditions alcynes-azotures catalysées par le cuivre (CuAAc), ont été conduites sur le squelette CTV dans ce but. La caractérisation spectroscopique des différentes sondes fluorescentes développées a été réalisée en milieu organique et en conditions physiologiques, pour celles dont la solubilité le permettait. L’étude des propriétés de reconnaissance de ces dernières, pour des ammoniums d’intérêt biologique (tels que les neurotransmetteurs, acétylcholine, dopamine et dérivés) en conditions physiologiques, a également été menée par spectrofluorimétrie, ainsi que par d’autres techniques (RMN, calorimétrie). La formation d’assemblages moléculaires en milieu aqueux a également été mise en évidence (expériences de diffusion dynamique de la lumière, microscopie électronique en transmission), pour certaines sondes lors de ce travail. / The development of fluorescent probes for the ex vivo detection of biological phenomena (neuronal communication, for example) presents a growing interest. In this context, fluorescent cyclotriveratrylene (CTV) derivatives have been developed, as these cavitands are known to complex small organic molecules. The strategy used to get interesting spectroscopic properties, was to introduce conjugated electron-donating and electron-withdrawing groups on each aromatic unit of the CTV (leading to “push-pull” systems). To improve the spectroscopic properties of the probes, the conjugation between the electron-donating and the electron-withdrawing groups was extended. Hence, the Sonogashira organometalic coupling reaction and the copper catalyzed cycloaddition directly on CTV skeleton were used. The spectroscopic characterization of the new fluorescent probes synthesized was done, in organic solvent but also in physiological conditions when the solubility permits it. The recognition studies for biological ammoniums, such as neurotransmitters (acetylcholine, dopamine, and derivatives) were performed in physiological conditions by spectrofluorimetry but also other methods (like MNR or micro-calorimetry). The formation of molecular assemblies was also observed (by dynamic light scattering and transmission electron microscopy) during this work with some of the probes.
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Hemodinâmica uterina avaliada por ultrassom Doppler colorido e taxa de fertilidade de vacas submetidas à IATF com sêmen analisado por sondas fluorescentes / Uterine hemodynamics assessed by color Doppler ultrasound and fertility rate of cows submitted to FTAI with semen analyzed by fluorescent probesOliveira, Bruna Marcele Martins de 31 January 2012 (has links)
Após a inseminação artificial, as fêmeas apresentam reação inflamatória transitória e fisiológica, que pode resultar em alterações na vascularização uterina. A ultrassonografia Doppler colorido é uma técnica não invasiva, que vem sendo utilizada para estudar a hemodinâmica do trato reprodutivo de bovinos. Os objetivos deste trabalho foram verificar a resposta inflamatória uterina por ultrassonografia Doppler colorido e a taxa de fertilidade em bovinos após inseminação artificial (IA) com sêmen avaliado por sondas fluorescentes. Para isso foram delineados dois experimentos. O experimento 1 foi realizado para estudar a hemodinâmica uterina por ultrassonografia Doppler colorido em bovinos após a IA. Foram utilizadas 18 vacas da raça Nelore, submetidas a um protocolo para a inseminação artificial em tempo fixo (IATF) e divididas em dois grupos: Grupo não inseminado (controle, GC, n=9) e grupo submetido à inseminação artificial (GIA, n=9). Avaliações por ultrassonografia foram realizadas em cinco momentos em modo espectral (30 horas antes da IA, 4, 24, 48 e 168 horas após a IA) para avaliar o índice de resistência (RI) da artéria uterina e em quatro momentos em modo color Doppler (4, 24, 48 e 168 horas após a IA) para avaliar a vascularização dos cornos uterinos. O diagnóstico de gestação foi feito após 30 dias da IA. No experimento 2 objetivou-se verificar se o percentual de espermatozóides com integridade das membranas plasmática e acrossomal e função mitocondrial (PIAIC) avaliadas por sondas fluorescentes influencia a fertilidade e estudar a hemodinâmica uterina após a deposição do sêmen com diferentes percentuais de espermatozóides PIAIC. Foram utilizadas 182 vacas paridas da raça Nelore, submetidas a um protocolo para IATF e divididas em três grupos de acordo com a qualidade do sêmen: Bom (B) 44,5%, Médio (M) 23% e Regular (R) 8,5% de espermatozóides PIAIC/palheta. Uma amostra de 30% das fêmeas (54, sendo 18 de cada grupo) foram submetidas a avaliações com ultrassonografia Doppler colorido 30 horas antes da IA, 4 e 24 horas após a IA. Foram considerados os RI das artérias uterinas direita (AD) e esquerda (AE) e vascularização subjetiva dos cornos uterinos direito (CD) e esquerdo (CE). Posteriormente, foram separados levando em consideração o lado do ovário contendo o folículo dominante para comparar RI das artérias ipsi (AI) e contralateral (AC) e vascularização subjetiva dos cornos uterinos ipsi (CI) e contralateral (CC). O diagnóstico de gestação foi realizado 30 dias após a IA. Para a análise estatística foi utilizado o programa SAS versão 9.2 (SAS 2010). As variáveis foram analisadas pelo PROC MIXED, utilizando modelo linear misto para medidas repetidas no tempo. Foram considerados os efeitos fixos de grupo, tempo e interação grupo x tempo. Os dados obtidos foram submetidos à análise de variância e ao teste de Duncan, pelo PROC GLM. A análise de Qui-Quadrado foi utilizada para determinar a taxa de prenhez, de acordo com a qualidade do sêmen pelo PROC FREQ. O nível de significância considerado foi de 5%. No experimento 1 não foram observadas diferenças dos parâmetros avaliados por ultrassom Doppler entre GIA e GC, mas foram notados efeitos do tempo para RI, sendo mais baixos 4 horas após a IA. Quando os dados foram avaliados de acordo com o lado do folículo dominante foi observado que 4 e 24 horas após a IA, RI da AC do GIA foi mais baixo do que o GC. No experimento 2 não houve diferença entre os animais inseminados com sêmen B, M ou R quanto a hemodinâmica uterina, mas houve efeito do tempo, sendo que 4 horas após a IA verificou-se menor valor de RI e maior fluxo sanguíneo. A taxa de prenhez dos animais inseminados com o sêmen B (64,7%) foi maior quando comparada ao sêmen R (36,2%), sendo que ambos não diferiram do sêmen M (50,0%). Pode-se concluir que não é possível detectar por ultrassonografia Doppler alterações na hemodinâmica uterina devido à IA em fêmeas bovinas. A deposição de sêmen com diferentes qualidades não altera a hemodinâmica uterina de forma detectável por ultrassonografia Doppler em bovinos. Por outro lado, é possível detectar por ultrassonografia Doppler mudanças na perfusão sanguínea do útero em relação ao tempo da IA, o que pode ser devido a alterações hormonais que ocorrem durante o ciclo estral. Por fim, a IA com sêmen contendo menor percentual de espermatozoides PIAIC causa redução na taxa de fertilidade em bovinos. / After insemination, females have physiological and transient inflammatory reaction, which can result in changes in the uterine vasculature. The color Doppler ultrasound is a noninvasive technique that has been used to study the hemodynamics of cattle reproductive tract.The purposes of this work were to verify uterine inflammatory response by color Doppler ultrasound and fertility rate in cattle after artificial insemination (IA) with semen evaluated by fluorescent probes. For this, two experiments were delineated. The first experiment was conducted to study the uterine hemodynamic by color Doppler ultrasound in cattle after AI. Were used 18 Nellore cows that were subjected to a protocol for fixed timed artificial insemination (IATF). The animals were divided in two groups: Not inseminated group (control, GC, n=9) and inseminated group (GIA, n=9). Ultrasonografic evaluation were done in five moments on spectral mode (30 hours before AI, 4, 24, 48 e 168 hours after IA) to evaluate the resistence index (RI) of uterine artery and in four moments in color Doppler mode to evaluate the uterine horns vascularization. The pregnancy diagnostic was done 30 days after IA. In the second experiment the objective was to evaluate whether the percentage of sperm with plasma membrane integrity and acrossomal and mitochondrial function (PIAIC) assessed by fluorescent probes influences fertility and study the uterine hemodynamic after the deposition of semen with different percentages of PIAIC sperm. Were used 182 caved Nellore cows submitted to a protocol for IATF and divided into three groups according to semen quality: Good (B) 44.5%, Medium (M) 23% and Regular (R) 8,5% of PIAIC sperm/blade. A sample of 30% of female (54, 18 in each group) were submitted to evaluation by color Doppler ultrasound 30 hours before IA, 4 and 24 hours after IA. Were considered RI of right (AD) and left (AE) uterine arteries and subjective vascularization of right (CD) and left (CE) uterine horns. Later, they were separated taking into account the side of the ovary containing the dominant follicle to compare RI of ipsi (AI) and contralateral (AC) arteries and subjective vascularization of ipsi (CI) and contralateral (CC) uterine horns. The pregnancy diagnostic was done 30 days after IA. Statistical analysis was performed using the program SAS version 9.2 (SAS 2010). The variables were analyzed by PROC MIXED, using mixed linear model for repeated measures in time. We considered effects of group, time and group x time interaction. The data obtained were submitted to ANOVA and Duncan test, using PROC GLM. The chi-square analysis was used to determine pregnancy rate, according to the quality of semen by PROC FREQ. the significance level was 5%. In experiment 1 there were no differences of the parameters evaluated by Doppler ultrasound between GIA and GC, but time effects were noted for RI, it was lower four hours after IA. When the data were evaluated according to the side of the dominant follicle was observed that 4 and 24 hours after IA, RI of AC of GIA was lower when it was compared with GC. In experiment 2 there was no difference between animals inseminated with semen B, M or R about uterine hemodynamics, but there were effects of time, so that 4 hours after IA showed lower value of RI and increased blood flow. The pregnancy rate of animals inseminated with semen B (64.7%) was higher when compared with semen R (36.2%), both of them did not differ of semen M (50.0%). It can be concluded that is not possible to detect uterine hemodynamic changes by Doppler ultrasound due to IA in cattle. The deposition of semen with different qualities does not change the uterine hemodynamics enough to be detectable by Doppler ultrasonography in cattle. On the other hand, is possible to detect changes in blood perfusion of the uterus in relation to the time of IA by Doppler ultrasound, which may be due to hormonal changes that occur during the estrous cycle. Finally, IA with semen containing a lower percentage of PIAIC sperm causes a reduction in fertility rate in cattle.
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Hemodinâmica uterina avaliada por ultrassom Doppler colorido e taxa de fertilidade de vacas submetidas à IATF com sêmen analisado por sondas fluorescentes / Uterine hemodynamics assessed by color Doppler ultrasound and fertility rate of cows submitted to FTAI with semen analyzed by fluorescent probesBruna Marcele Martins de Oliveira 31 January 2012 (has links)
Após a inseminação artificial, as fêmeas apresentam reação inflamatória transitória e fisiológica, que pode resultar em alterações na vascularização uterina. A ultrassonografia Doppler colorido é uma técnica não invasiva, que vem sendo utilizada para estudar a hemodinâmica do trato reprodutivo de bovinos. Os objetivos deste trabalho foram verificar a resposta inflamatória uterina por ultrassonografia Doppler colorido e a taxa de fertilidade em bovinos após inseminação artificial (IA) com sêmen avaliado por sondas fluorescentes. Para isso foram delineados dois experimentos. O experimento 1 foi realizado para estudar a hemodinâmica uterina por ultrassonografia Doppler colorido em bovinos após a IA. Foram utilizadas 18 vacas da raça Nelore, submetidas a um protocolo para a inseminação artificial em tempo fixo (IATF) e divididas em dois grupos: Grupo não inseminado (controle, GC, n=9) e grupo submetido à inseminação artificial (GIA, n=9). Avaliações por ultrassonografia foram realizadas em cinco momentos em modo espectral (30 horas antes da IA, 4, 24, 48 e 168 horas após a IA) para avaliar o índice de resistência (RI) da artéria uterina e em quatro momentos em modo color Doppler (4, 24, 48 e 168 horas após a IA) para avaliar a vascularização dos cornos uterinos. O diagnóstico de gestação foi feito após 30 dias da IA. No experimento 2 objetivou-se verificar se o percentual de espermatozóides com integridade das membranas plasmática e acrossomal e função mitocondrial (PIAIC) avaliadas por sondas fluorescentes influencia a fertilidade e estudar a hemodinâmica uterina após a deposição do sêmen com diferentes percentuais de espermatozóides PIAIC. Foram utilizadas 182 vacas paridas da raça Nelore, submetidas a um protocolo para IATF e divididas em três grupos de acordo com a qualidade do sêmen: Bom (B) 44,5%, Médio (M) 23% e Regular (R) 8,5% de espermatozóides PIAIC/palheta. Uma amostra de 30% das fêmeas (54, sendo 18 de cada grupo) foram submetidas a avaliações com ultrassonografia Doppler colorido 30 horas antes da IA, 4 e 24 horas após a IA. Foram considerados os RI das artérias uterinas direita (AD) e esquerda (AE) e vascularização subjetiva dos cornos uterinos direito (CD) e esquerdo (CE). Posteriormente, foram separados levando em consideração o lado do ovário contendo o folículo dominante para comparar RI das artérias ipsi (AI) e contralateral (AC) e vascularização subjetiva dos cornos uterinos ipsi (CI) e contralateral (CC). O diagnóstico de gestação foi realizado 30 dias após a IA. Para a análise estatística foi utilizado o programa SAS versão 9.2 (SAS 2010). As variáveis foram analisadas pelo PROC MIXED, utilizando modelo linear misto para medidas repetidas no tempo. Foram considerados os efeitos fixos de grupo, tempo e interação grupo x tempo. Os dados obtidos foram submetidos à análise de variância e ao teste de Duncan, pelo PROC GLM. A análise de Qui-Quadrado foi utilizada para determinar a taxa de prenhez, de acordo com a qualidade do sêmen pelo PROC FREQ. O nível de significância considerado foi de 5%. No experimento 1 não foram observadas diferenças dos parâmetros avaliados por ultrassom Doppler entre GIA e GC, mas foram notados efeitos do tempo para RI, sendo mais baixos 4 horas após a IA. Quando os dados foram avaliados de acordo com o lado do folículo dominante foi observado que 4 e 24 horas após a IA, RI da AC do GIA foi mais baixo do que o GC. No experimento 2 não houve diferença entre os animais inseminados com sêmen B, M ou R quanto a hemodinâmica uterina, mas houve efeito do tempo, sendo que 4 horas após a IA verificou-se menor valor de RI e maior fluxo sanguíneo. A taxa de prenhez dos animais inseminados com o sêmen B (64,7%) foi maior quando comparada ao sêmen R (36,2%), sendo que ambos não diferiram do sêmen M (50,0%). Pode-se concluir que não é possível detectar por ultrassonografia Doppler alterações na hemodinâmica uterina devido à IA em fêmeas bovinas. A deposição de sêmen com diferentes qualidades não altera a hemodinâmica uterina de forma detectável por ultrassonografia Doppler em bovinos. Por outro lado, é possível detectar por ultrassonografia Doppler mudanças na perfusão sanguínea do útero em relação ao tempo da IA, o que pode ser devido a alterações hormonais que ocorrem durante o ciclo estral. Por fim, a IA com sêmen contendo menor percentual de espermatozoides PIAIC causa redução na taxa de fertilidade em bovinos. / After insemination, females have physiological and transient inflammatory reaction, which can result in changes in the uterine vasculature. The color Doppler ultrasound is a noninvasive technique that has been used to study the hemodynamics of cattle reproductive tract.The purposes of this work were to verify uterine inflammatory response by color Doppler ultrasound and fertility rate in cattle after artificial insemination (IA) with semen evaluated by fluorescent probes. For this, two experiments were delineated. The first experiment was conducted to study the uterine hemodynamic by color Doppler ultrasound in cattle after AI. Were used 18 Nellore cows that were subjected to a protocol for fixed timed artificial insemination (IATF). The animals were divided in two groups: Not inseminated group (control, GC, n=9) and inseminated group (GIA, n=9). Ultrasonografic evaluation were done in five moments on spectral mode (30 hours before AI, 4, 24, 48 e 168 hours after IA) to evaluate the resistence index (RI) of uterine artery and in four moments in color Doppler mode to evaluate the uterine horns vascularization. The pregnancy diagnostic was done 30 days after IA. In the second experiment the objective was to evaluate whether the percentage of sperm with plasma membrane integrity and acrossomal and mitochondrial function (PIAIC) assessed by fluorescent probes influences fertility and study the uterine hemodynamic after the deposition of semen with different percentages of PIAIC sperm. Were used 182 caved Nellore cows submitted to a protocol for IATF and divided into three groups according to semen quality: Good (B) 44.5%, Medium (M) 23% and Regular (R) 8,5% of PIAIC sperm/blade. A sample of 30% of female (54, 18 in each group) were submitted to evaluation by color Doppler ultrasound 30 hours before IA, 4 and 24 hours after IA. Were considered RI of right (AD) and left (AE) uterine arteries and subjective vascularization of right (CD) and left (CE) uterine horns. Later, they were separated taking into account the side of the ovary containing the dominant follicle to compare RI of ipsi (AI) and contralateral (AC) arteries and subjective vascularization of ipsi (CI) and contralateral (CC) uterine horns. The pregnancy diagnostic was done 30 days after IA. Statistical analysis was performed using the program SAS version 9.2 (SAS 2010). The variables were analyzed by PROC MIXED, using mixed linear model for repeated measures in time. We considered effects of group, time and group x time interaction. The data obtained were submitted to ANOVA and Duncan test, using PROC GLM. The chi-square analysis was used to determine pregnancy rate, according to the quality of semen by PROC FREQ. the significance level was 5%. In experiment 1 there were no differences of the parameters evaluated by Doppler ultrasound between GIA and GC, but time effects were noted for RI, it was lower four hours after IA. When the data were evaluated according to the side of the dominant follicle was observed that 4 and 24 hours after IA, RI of AC of GIA was lower when it was compared with GC. In experiment 2 there was no difference between animals inseminated with semen B, M or R about uterine hemodynamics, but there were effects of time, so that 4 hours after IA showed lower value of RI and increased blood flow. The pregnancy rate of animals inseminated with semen B (64.7%) was higher when compared with semen R (36.2%), both of them did not differ of semen M (50.0%). It can be concluded that is not possible to detect uterine hemodynamic changes by Doppler ultrasound due to IA in cattle. The deposition of semen with different qualities does not change the uterine hemodynamics enough to be detectable by Doppler ultrasonography in cattle. On the other hand, is possible to detect changes in blood perfusion of the uterus in relation to the time of IA by Doppler ultrasound, which may be due to hormonal changes that occur during the estrous cycle. Finally, IA with semen containing a lower percentage of PIAIC sperm causes a reduction in fertility rate in cattle.
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Síntese de novos heterocíclos benzazólicos fluorescentes e sua incorporação em matrizes de celuloseKuplich, Marcelo Domingues January 2007 (has links)
Neste trabalho é apresentada a síntese e a caracterização de compostos do tipo 2-(2’- hidroxifenil)benzazóis derivados da 1,3,5-triazina, fluorescentes devido a um mecanismo de transferência protônica intramolecular no estado excitado (TPIEE). Foram preparados quatro novos corantes diclorotriazinil-(hidroxifenil)benzazólicos, que apresentam emissão de fluorescência na região azul-verde do espectro visível e deslocamentos de Stokes compatíveis com substâncias que sofrem o fenômeno da TPIEE. Estas substâncias foram caracterizadas por diversas técnicas espectroscópicas (IR, RMN e HR-MS), enquanto que as análises fotofísicas (absorção no ultravioleta-visível e emissão de fluorescência) foram realizadas em solução e no estado sólido. Os ensaios preliminares realizados indicam a possibilidade de emprego desses corantes como sondas fluorescentes de material celulósico. / The synthesis and characterization of new 2-(2’-hydroxyphenyl)benzazoles derivatives from 1,3,5-triazine, fluorescent due to an excited state intramolecular proton transfer (ESIPT) mechanism, were presented in this work. The four new dichlorotriazinylbenzazole derivatives synthesized present fluorescence emission in the blue-green region of the spectra and Stokes shift compatible with ESIPT compounds. The characterization of the new fluorescent reactive dyes by means of spectroscopic (IR, RMN, UV-Vis and fluorescence) and spectrometric (HRMS) techniques, and the study of the possibilities to dye cellulose fibers were carried out. Fluorescent cellulose fibers were prepared using the dyes and a methodology used in the industry and show no color change when observed in the visible light (white cellulose) and a blue-green fluorescence when irradiated with UV light.
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Acides aminés phosphole ou silole : vers de nouvelles sondes fluorescentes pour un marquage de peptide innovant / Phosphole or silole amino acids : toward new fluorescent probes for innovative peptide labellingArribat, Mathieu 26 October 2018 (has links)
La première partie de ces travaux de thèse concerne la synthèse d’acides aminés phosphole par formation d’une liaison P-C. Les propriétés de fluorescence (absorption, émission et rendement quantique) sont modulées à la fois par les différents substituants présents sur le phosphore (BH3, O, S…) ainsi que par le squelette aromatique du phosphole. Des couplages peptidiques modèles réalisés en solution et sur support solide démontrent la possibilité d’intégrer ces acides aminés dans des peptides d’intérêts. La deuxième partie concerne la synthèse de nouveaux phospholes fonctionnalisés ainsi que d’une nouvelle méthode d’accrochage pour les introduire sur différents groupes pendants (SH, NH2, OH) d’acide aminés et peptides via la formation de liaisons P-S, P-N ou P-O. La troisième partie de ce travail a consisté en la synthèse d’une nouvelle classe d’acides aminés tétraphénylsilole fluorescents qui présentent des propriétés d’AIE (aggregation-induced emission) et pourront être utilisés pour le marquage de peptides d’intérêts. / The first part of this work is focused on phospholyl amino acids synthesis by formation of a P-C bond. The fluorescent properties (absorption, emission and quantum yield) are modulated either by the substituent on the phosphorus atom (BH3, O, S, …) or by the aromatic skeleton of the phosphole. Peptide coupling in solution or on solid support were performed and showed the possibility to introduce such amino acids into peptide of interest. The second part of this work is dedicated to the synthesis of new functionalized phospholes for a chemoselective grafting on amino acid and peptides pendant groups (SH, NH2, OH) via PS, P-N or P-O bonds. The third part consists into the synthesis of a new class of tetraphenylsilole amino acids which exhibit AIE (aggregation-induced emission) fluorescent properties. Those compounds were successfully incorporated into di- an tri- peptides in solution and on solid support.
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Molecular tools for elucidating copper biochemistry: Water-soluble fluorescent probes and robust affinity standardsMorgan, M. Thomas 09 April 2013 (has links)
Copper is an essential trace element for living organisms and has both known and additional suspected roles in human health and disease. The current understanding of copper metabolism is substantial but incomplete, particularly in regard to storage and exchange at the subcellular level, although available evidence indicates exchangeable intracellular copper is in the monovalent oxidation state. Selective fluorescent probes with sufficient sensitivity to detect Cu(I) availability at physiologically relevant levels and at subcellular resolution would be valuable tools for studying copper metabolism. As a contribution toward this goal, this work describes the development of Cu(I)-selective fluorescent probes with greatly improved aqueous solubility, contrast ratio, and fluorescence quantum yield. This work also describes the development of water-soluble, 1:1-binding chelators that form colorless, air-stable copper(I)-complexes. By acting as copper(I) buffering agents and affinity standards, these compounds can serve a complementary role to fluorescent probes in the study of copper biochemistry.
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