One of the major limitations on research into the control of eukaryotic DNA replication has been the lack of any cell-free system that initiates DNA replication in vitro. The first part of the disseration describes the establishment of a eukaryotic system, derived from the activated eggs of the South African clawed toad, Xenopus laevis, that efficiently initiates and completes DNA replication in vitro. Using a variety of biochemical techniques I show that DNA added to the extract in the form of sperm nuclei is efficiently replicated over a period of 4 - 6 hours. Replication of nuclear DNA represents a single round of semiconservative, semidiscon-tinuous replication. The extract will also replicate naked DNA incubated in it, regardless of sequence, though less efficiently than nuclear templates. This is probably related to the unusual ability of the egg extract to assemble apparently normal interphase nuclei from any DNA molecule incubated in it Evidence is presented that initiation, rather than chain elongation, is the rate-limiting step for replication in vitro. In this and in other ways the cell-free system behaves as though it were an early embryo blocked in a single cell cycle. The second part of the dissertation describes experiments that examine the control of DNA replication in the extract The first set of experiments suggest that on replication, DNA is marked in some way so that it can no longer act as a substrate for further initiation. This provides a mechanism by which the template DNA is replicated precisely once per incubation in vitro (or per cell cycle in vivo). The second set of experiments investigate the relationship between nuclear assembly and the initiation of DNA replication in vitro. A novel method for quantifying DNA replication in intact nuclei using the nucleotide analogue biotin-11-dUTP is described. This technique reveals that although they are in the common cytoplasm of the egg extract, different nuclei start to replicate at different times. Entry into S-phase is characterised by a burst of many synchronous or near-synchronous initiations within individual nuclei. This means that nuclei act as independent and integrated units of replication in the cell-free system, and suggests a fundamental role for nuclear assembly in controlling DNA replication in vitro.

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Dose limiting haematotoxicity has been associated with a range of therapeutic agents used for the treatment of a number of different conditions. Haematotoxicity is usually assessed as part of the preclinical safety studies in experimental animals, where changes in peripheral blood cell numbers and bone marrow cellularity are determined at the end of the study. Often no information on the mechanism of the haematotoxicity is revealed. This thesis demonstrates how in vitro bone marrow cultures can be utilized to assist in the assessment of haematotoxicity by two different approaches; firstly, in vitro bone marrow cultures can be used to assess the haematopoietic lineage specificity of vincristine sulphate, vinblastine sulphate, hydroxyurea and anagrelide hydrochloride using clonogenic cultures, enabling ranking of these compounds according to their haematotoxicity. Secondly, using in vitro assays only, elucidate the mechanism(s) of the megakaryocytic lineage specific inhibition of anagrelide hydrochloride. To this end both clonogenic cultures and LTBMC offer the ability to elucidate mechanisms of action on multipotent stem cells, lineage specific cells and effects on the bone marrow microenvironment following single and repeated administration. In addition, the combination of cell identification techniques flow cytometry and light microscopy was shown to provide a more detailed understanding of the different cell populations within the non-adherent cell layer. In vivo AN reduces platelet counts only, however, the mechanism of the megakaryocyte specific toxicity by AN is not understood. In these studies, the mechanism (s) of the megakaryocytic lineage haematotoxicity of AN was examined using the established human clonogenic and LTBMC. The action of AN was shown to be focused at a late stage in megakaryocyte (Mk) colony development. Ranking the potential mechanisms of action of AN by concentration at which they were noted, the inability to organize the microtubules appears to be secondary to 1) alteration in cell cycling, 2) surface receptor expression and 3) inhibition in achieving high (greater than 8N) ploidy number. However, identification of the primary mechanism based solely on concentration seems to be very crude and most probably reflects a limitation of in vitro systems. The inhibition of platelet production by AN is most likely a result from a combination of mechanisms; inhibition of cell cycling, disruption in the expression of cell surface receptors, inhibition of the ability of the cells to increase ploidy number and an associated inability to organize microtubules leading to a reduction in platelet release. This work also demonstrated the importance of the selection of the source of bone marrow used in the cultures. The concentration at which 50 percent of Mk colony growth was inhibited (IC50) by AN for murine cells was markedly (46 fold) different (88.6μM) compared to the IC50 with human cord blood (hCB) (1.92μM). This disparity is indicative of differences in species sensitivity possibly related to AN having a greater affinity towards the human c-mpl chrombopoietin (TPO) receptor than the equivalent murine receptor as suggested by McCarty et al (2006). This work highlights the utility of in vitro bone marrow cultures as a tool for investigating the lineage specific haematotoxicity by evaluating compounds used in the treatment of ET. In addition in vitro haematopoietic cultures can successfully be used as a tool to investigate potential mechanism(s) of haematotoxicity as demonstrated herein by providing an insight to mechanism of platelet count reduction by AN.

Memoria para optar al título Profesional de Médico Veterinario / En este trabajo se investigó mediante fecundación in vitro, la capacidad de espermatozoides caninos refrigerados a 4 °C y frescos (controles), de atravesar la ZP, y penetrar el citoplasma ovular, de ovocitos de perra en estado inmaduro y madurados in vitro, a través de diferentes tiempos de coincubación gamética. Se trabajó con eyaculados de 5 perros adultos, el semen se obtuvo por estimulación digital, utilizando la segunda fracción espermática. Luego de la evaluación del semen, se retiró el plasma seminal por centrifugación, los espermatozoides utilizados como controles (frescos) fueron resuspendidos en medio Fert TALP y los espermatozoides que se sometieron a refrigeración se resuspendieron en extensor o diluyente en base a TRIS-yema de huevo-ácido cítrico y fueron posteriormente refrigerados a 4 °C, por 24 horas. Posterior a ese tiempo, se retiró el diluyente por centrifugación y el pellet espermático fue resuspendido en medio Fert TALP. Ambos tipos de espermatozoides -frescos y refrigerados- fueron coincubados con ovocitos de perra tanto inmaduros como madurados in vitro, en forma separada. Los ovocitos utilizados provinieron de ovarios macerados, obtenidos previamente por ovariectomía. Los ovocitos fueron seleccionados bajo la lupa estereoscópica y lavados en PBS. Posteriormente, los utilizados en estado inmaduro se ambientaron en medio de coincubación Fert TALP y luego se coincubaron con espermatozoides frescos o refrigerados. Los ovocitos que se sometieron a maduración in vitro fueron incubados en medio TCM 199 suplementado con 10% de suero fetal bovino, 10 UI/mL de hCG, 2.5 μl/mL de solución piruvato y 5 μl/mL de solución antibiótica por 72 y 96 horas. Posteriormente fueron coincubados separadamente con ambos tipos de espermatozoides. La coincubación gamética se realizó por períodos de 1 a 10 horas en medio Fert TALP. Cada hora de incubación se retiraron ovocitos, se fijaron en una solución en base a ácido acético, metanol y cloroformo (3:6:2) por tres minutos, y posteriormente en ácido acético y metanol (1:3) por 72 horas a 4 °C y teñidas con 0,1% de Yoduro de Propidio para la 4 posterior evaluación de la penetración espermática y fecundación en microscopia de epifluorecencia. Un ovocito penetrado se determinó cuando se encontró espermatozoides en zona pelúcida (ZP), en espacio perivitelino (PV) o en el citoplasma ovular (C). Se utilizó análisis de varianza y de regresión para determinar diferencias en los diferentes parámetros estudiados. Se evaluaron un total de 3391 ovocitos inmaduros (n= 1765) y madurados in vitro (n= 1626). La penetración espermática fue directamente proporcional al tiempo de coincubación. El aumento del porcentaje de penetración total con espermatozoides frescos fue significativo a partir de las 3 horas en ovocitos inmaduros y 2 horas en ovocitos madurados in vitro (p< 0,05). Con espermatozoides refrigerados, los ovocitos inmaduros muestran diferencias en la penetración total a partir de las 5 horas de coincubación, en ovocitos madurados in vitro es posible observar diferencias a partir de las 2 horas. Los mayores porcentajes de penetración total se obtuvieron a partir de las 6 y 7 horas de coincubación y hacia finales del cultivo. Los espermatozoides refrigerados tuvieron un menor porcentaje de penetración que los frescos, durante todo el periodo de cultivo, excepto a la primera hora de coincubación con ovocitos madurados in vitro y espermatozoides refrigerados (p<0,05). Los ovocitos madurados in vitro fueron penetrados en mayor proporción que los inmaduros (p< 0,05), considerando todo el periodo de cultivo. Se concluye que al aumentar el tiempo de cultivo, un mayor número espermatozoides logró atravesar las cubiertas ovocitarias. Los ovocitos inmaduros y madurados in vitro son penetrados en mayor porcentaje por espermatozoides frescos respecto a aquellos refrigerados, sin embargo, los espermatozoides sometidos a refrigeración serían capaces de penetrar antes los ovocitos madurados in vitro. No obstante los ovocitos de perra inmaduros y madurados in vitro pueden ser penetrados por espermatozoides caninos frescos y refrigerados, la utilización de ovocitos madurados sería más apropiado para evaluar la capacidad fecundante de los espermatozoides frescos y criopreservados. / Proyecto FONDECYT 1030380

Class of 2017 Abstract / Objectives: Evaluate thymoquinone (TQ) and thymol (THY) inhibitory activities against α-glucosidase enzyme by using an in vitro assay and determine the IC50 (concentration of TQ/THY to inhibit 50% of maximum enzyme activity). Methods: Various concentrations of thymoquinone and thymol were incubated, separately, with one concentration of the substrate - p-nitrophenol-α-D-glucopyranoside (PNPG) (<<Km) in presence of α-glucosidase enzyme. A positive and a negative control consisting of acarbose, and buffer, respectively, were included in the incubation as well. The incubation time was set at 30 min in a 37 °C controlled water bath. The enzyme activity was determined by detecting and quantitating the levels of p-nitrophenol using a spectrophotometer set at 405 nm. The percent inhibition exhibited by any studied drug was calculated as shown in equation 1. % inhibition = Absorbance Substrate Alone – Absorbance of Substrate + Inhibitor Absorbance Substrate Alone Results: Results of the in vitro incubation of thymoquinone, thymol and acarbose revealed “statistically” significant inhibition of -glucosidase (p<0.05). At 400 g/ml, thymoquinone inhibited the enzyme activity by ~52 % whereas the enzyme inhibition by thymol and acarbose were calculated to be ~84% and 57%, respectively. IC50 were tentatively determined although the maxima inhibitions of the inhibitors were not reached fully. IC50s were calculated as 234 μg/ml, 304 μg/ml and 157 μg/ml for each of thymoquinone, thymol and acarbose, respectively. Conclusions: Thymoquinone and thymol do exhibit antagonistic pharmacological activity against α-glucosidase.

Nitrogen (N) belongs among necessary elements for plant growth and development. In the past attention was paid mostly to the inorganic forms - nitrate and ammonium. In soil N is also present in organic forms, including proteins, for which plants could compete with soil microorganisms. Recently two ways have been considered - the hydrolysis of proteins by secreted proteases and endocytosis of native proteins, possibly their confluence. Tobacco plants were grown in vitro under sterile conditions in modified Murashige-Skoog medium with casein as the only source of N (CAS), decreased concentration of inorganic forms of N (AD) or in complete Murashige-Skoog medium as control plants (MS). After the 12 weeks growth, the standard growth parameters were measured. The CAS plants were able to grow without inorganic N, and protein content in the leaves was higher than in other experimental plants. Proteomic analysis documented differences in protein expression in plant roots in the dependence on the form of N. In total 185 proteins were identified, 75% of proteins were less and 14% more abundant in the CAS plants. The uptake of casein conjugated with fluorescein was followed and the proteolytic activity was analyzed by confocal microscopy. Among proteins secreted from roots to the medium aspartic protease...

In vitro cultures of medicinal plants - XI. Elicitation is one of the few strategies that can be used in enhancement of secondary metabolites production from explant cultures. The effect of abiotic elicitor (silver nitrate) on flavonolignan and flavonoid taxifolin production in suspension culture of Silybum marianum L. (Gaertn.) and on isoflavones production in suspension culture of Genista tinctoria L. was tested. Silver nitrate in various concentrations (5.887.10-3 mol/l; 5.887.10-4 mol/l; 5.887.10-5 mol/l) was used as elicitor. Content of secondary metabolites in suspension cultures was determinated by high performance liquid chromatography (HPLC). The samples were taken after 6, 12, 24, 48, 72 and 168 hours after elicitor treatment. The highest content of taxifolin production (0.02 %) in suspension culture of Silybum marianum L. (Gaertn.) after silver nitrate (5.887.10-4 mol/l) treatment and 72 h sampling was detected. The highest content of genistin (0.05 %) in suspension culture of Genista tinctoria L. was found after silver nitrate (5.887.10-4 mol/l) treatment and 48 h sampling. The highest content of daidzein (0.09 %) was detected after elicitor (5.887.10-4 mol/l) treatment and 168 h sampling.

Martina Janoutová In vitro cultures of medicinal plants - XII. Abstract The effect of ultrasound (US) as abiotic elicitor on the flavonolignans production in Silybum marianum L suspension culture was investigated. The culture was cultivated in Murashige and Skoog nutritive medium with ( - NAO) (g/l) as growth regulator at 25o C and luminous period 16 h of light and 8 h of darkness. The elicitor - ultrasound by frequency 35kHz and intensity 0,1Wcm-3 for a period 1, 2, 3, 4 and 5 min has been used. The samples were taken in 0, 6, 12, 24, 48, 72 and 168 h after US exposition. The control samples were taken in 0 and 48 h. The quantity of flavonolignans was determined by High performance liquid chromatography (HPLC). The highest increase of taxifolin content was apparent after 5 min of US elicitation and 48 h sampling (0,04%) - 400%, other increase was apparent after 5 min of elicitation and 72 h sampling. The higher content of silychristin was found after 1 min of US elicitation and 72 h sampling, the same level was observed after 2 min of elicitation and 24 h sampling. The higher level of silydianin was detected after 2 min of US elicitation and 6 h sampling and the silybin B after 2 min of elicitation and 12 h sampling after exposition. Taxifolin and flavonolignans release to the nutrient medium was...

Jan Kubeš Genista tinctoria in vitro - abiotická elicitace Cultures of medicinal plants in vitro - XIII. The plants cultures in vitro contain lesser amounts of secondary metabolites in compare with intact plants. The elicitors can affect these metabolites production. The effect of electric current (50mA) of different voltage (5, 7, 8, 10, 15, 20 and 24 V) and different time exposition (10, 30, and 60 minutes) on content of isoflavones was studied on (Genista tinctoria) suspension culture. The highest concentration of genistin (0.17 %) was measured in suspension culture after 30 min of elicitation by 10 V after 6 hours of cultivation. The highest concentration of daidzein (0.35 %) was found in suspension culture after 60 min of elicitation by 5 V after 24 hours of cultivation. The highest concentration of genistin (1.6 mg/100ml) was measured in medium after 30 min of elicitation by 5 V after 24 hours of cultivation. The highest concentration of daidzein (1.77 mg/100ml) was found in medium after 10 min of elicitation by 24 V after 6 hours of cultivation.