Marine sponges are economically and environmentally valuable, but restoration, commercial, and biomedical demands exceed what wild populations and aquaculture can provide. In vitro culture of sponge cells is a promising alternative, but has remained elusive until recent breakthroughs involving improved nutrient medium M1 in two-dimensional culture. The advantages of three-dimensional over two-dimensional cell culture have been increasingly recognized. Here we report the successful 21-day culture of cells from the marine sponge Geodia neptuni using multiple three-dimensional cell culture methods: FibraCel© disks, thin hydrogel layers, gel micro droplets, and spheroid cell culture. These methods performed comparably to two-dimensional control cultures, and each method offers advantages for restoration or in vitro applications using sponge cells. Further optimization of these methods may lead to the ability to culture fully functioning sponges from dissociated, cryopreserved cells, which will reduce the need for wild harvest of sponge tissues for commercial, restoration, and biomedical purposes. / Includes bibliography. / Thesis (MS)--Florida Atlantic University, 2021. / FAU Electronic Theses and Dissertations Collection

Anaerobic digestion process which has long been known to successfully reduce the organic content of sludge is one of the most common alternatives to meet pathogen reduction requirements for particular classes of biosolids. However, it has recently been reported that, significantly higher densities of indicator bacteria have been measured in dewatered cake samples compared to samples collected right after anaerobic digestion. In addition, this increase in bacterial population has been commonly observed after centrifugation but not after belt filter dewatering. Even though several theories have emerged to explain this occurrence, with the use of molecular tools such as Quantitative Polymerase Chain Reaction (Q

The objectives of this study were (1) to determine the optimum in vitro culture conditions for in vitro fertilized bovine embryos among culture methods and (2) to investigate whether bovine fertilization and embryo development would occur in simulated microgravity conditions in vitro. In the first part of this study, the result showed that the microdrop method was the optimum culture method among groups; however, FBS supplementation did not significantly affect the bovine preimplantation embryo development in vitro. In the second part of this study, the result showed that bovine in vitro fertilization did not occur in simulated microgravity conditions. Moreover, none of the presumptive zygotes and 2-8 cell stage embryos were able to develop to further stages, while embryos cultured in standard microdrop method culture conditions developed normally.

The master’s thesis deals with organizational culture and its influence on the motivation of employees in the company Foreigners.cz, s.r.o. The aim of this thesis is to identify the organizational culture of the chosen company and its influence on employee motivation. On the basis of the data obtained, solutions are proposed to enhance the employee motivation.

Le corps humain est un ensemble de microflores où cohabitent bactéries, archées, virus et eucaryotes. Ces écosystèmes complexes sont appelés microbiotes. Parmi ceux-ci figure le microbiote intestinal qui compte 1011 à 1014 bactéries/g de selle. Les modifications de la flore intestinale peuvent être à l'origine de pathologies comme les diarrhées infectieuses. Il s'agit d'un véritable problème de santé publique puisqu'environ 2.16 millions de décès sont liés à cette pathologie chaque année. Les virus intestinaux jouent un rôle prépondérant mais les infections bactériennes restent également importantes. Le diagnostic de ces infections bactériennes reste compliqué puisque le microbiote intestinal comporte 75% d'espèces non cultivables. De plus, on ne dispose pas réellement d'une liste exhaustive des bactéries pouvant être responsables de diarrhées infectieuses. Nous avons donc choisi d'étudier le microbiote intestinal dans des selles normales et pathologiques, par une approche polyphasique alliant une étape préliminaire de concentration des selles diarrhéiques par la lyophilisation, à des techniques de culture et des méthodes de biologie moléculaire. Pour cela nous avons mis au point une nouvelle technologie pour la détection des entéropathogènes par hybridation sur puce à ADN permettant la détection des bactéries et des virus ADN entéropathogènes, en présence d'un témoin archae. Notre outil permet le diagnostic multiplexe des diarrhées infectieuses puisque nous avons correctement identifié un adénovirus et la bactérie Campylobacter jejuni présents dans une même selle. / The human body is a collection of microflora where cohabit bacteria, archaea, viruses and eukaryotes. These complex ecosystems are called microbiota. Among these is the intestinal microbiota that counts 1011 to 1014 bacteria/g of stool. Changes in the intestinal flora can cause of pathologies such as infectious diarrhea. This is a real public health problem since about 2.16 million deaths are related to this disease each year. Enteric viruses play a preponderant role but bacterial infections are also important. The diagnosis of bacterial infections is complicated because the intestinal microbiota includes 75% non-cultivable species. In addition, there is not really a list of bacteria could be responsible for infectious diarrhea. We therefore decided to study the intestinal microbiota in normal stool and also pathological stools by a polyphasic approach combining a preliminary step of diarrheal stools concentration by lyophilization, with cultivation techniques and molecular biology methods. We developed a new technology for the detection of enteropathogens by hybridization on DNA microarray for the detection of bacteria and enteric viruses (DNA) in the presence of a control archaea. Our tool allows multiplexed diagnostic of infectious diarrhea since we correctly identified an adenovirus and Campylobacter jejuni present in a same sample. This is the first DNA microarray for multiplex detection of bacteria and viruses (DNA) enteropathogens. An improvement of our protocol for nucleic acid extraction is proposed to allow the detection of RNA viruses such as rotavirus and calicivirus which are currently dominant.

Micropropagation of Aloe polyphylla, an endangered species with a high ornamental and medicinal value, is an important part of its conservation. However, the in vitro culture was hindered by the phenomenon of hyperhydricity. The research reported in this thesis was undertaken for two reasons. Firstly, to understand the role of various culture factors involved in the process of hyperhydricity in A. polyphylla and to identify the in vitro conditions, under which this disorder can be prevented. Secondly, we conducted an investigation into the underlying mechanisms of this phenomenon by probing if it was mediated through internal cytokinins. Ammonium (NH4 +) ions, applied cytokinins (CKs) and CK concentrations were tested in multifactorial combinations and significantly influenced the regeneration rate and occurrence of hyperhydricity. Shoots were grown on media with different NH4 + concentrations (10.3, 20.6 and 61.8 mM) and supplemented with BA, zeatin or TDZ at 0, 5 or 15 ìM. Elevating the levels of NH4 +, in the absence of CKs, could not induce hyperhydricity. Similarly, very low hyperhydricity was observed when CKs were added to media containing low NH4 + (10.3 mM). However, in the presence of higher NH4 + concentrations, CKs increased hyperhydricity in a concentrationdependant manner, suggesting that they were capable of inducing this syndrome only when other factors in the culture system were not optimised. High numbers of healthy looking shoots were produced on media with low NH4 + and low BA or zeatin (5 ìM). The use of TDZ resulted in the formation of buds, which did not develop into shoots. In view of the fact that NH4 + was supplied in the form of NH4NO3, it was difficult to determine if NH4 + or nitrate (NO3 -) ions were associated with the increase in hyperhydricity. We further examined the role of nitrogen (N) supplied as inorganic NH4 + or NO3 -, or organic glutamine. The omission of total N from the culture medium resulted in low multiplication and hindered shoot growth. Ammonium as the sole source of N depressed shoot regeneration and growth and escalated the frequency of hyperhydricity to ca. 50%. When NO3 - was used as the sole N source, shoots of fine quality were produced and hyperhydricity was completely eliminated. Overall, the MS N mix was superior to any single N source for multiplication and growth of shoots, suggesting a synergistic effect between NH4 + and NO3 - on shoot regeneration. Furthermore, not only the absolute amount of N, but also the relative amounts of NH4 + and NO3 - influenced the multiplication rate, frequency of hyperhydricity and shoot quality. The highest regeneration was obtained with NH4 + : NO3 - ratios (mM) of 20 : 40, 30 : 30 and 40 : 20. Decreasing the ratio of NH4 + : NO3 - lowered the occurrence of hyperhydricity. The potential of glutamine as the sole source of N was also demonstrated, since its application resulted in the production of good quality shoots and almost no hyperhydricity. Shoot explants grown in static liquid media became hyperhydric and lost their ability to regenerate. The type of gelling agent used to solidify the medium affected greatly hyperhydricity and shoot multiplication. Gelrite resulted in a significantly lower multiplication rate and four times higher hyperhydricity (64.7%) compared to when agar was used. Gelrite was further selected to test the hypothesis if hyperhydricity can be overcome by decreasing the relative matric potential of the media, and respectively the availability of water, as represented by increasing gelrite concentrations. Satisfactory reduction in hyperhydricity was achieved only at 16 g l-1 gelrite, however the regeneration also decreased. The nature of the gelling agent is therefore essential for the successful control of this phenomenon. It appears that a crucial prerequisite for the reduction of hyperhydricity in tissue cultures of A. polyphylla is the gaseous exchange between the in vitro atmosphere and the outside environment. In ventilated cultures, achieved by using a modified lid with a hole (d = 7 mm) covered with polyester or cotton mesh, hyperhydricity was completely eliminated, irrespectively of the type of gelling agent. Ventilation was further advantageous for the in vitro regenerants by increasing their leaf chlorophyll content as well as epicuticular wax deposition, the last one being indicative of the development of the water loss regulation mechanisms of explants. The increased culture ventilation, however, was negatively correlated with the regeneration rate and shoot growth. Endogenous CKs were measured in in vitro regenerants after an eight-week cycle to examine whether the hyperhydricity-inducing effect of exogenous CKs and gelling agents is associated with changes in the endogenous CK content. The content of endogenous CKs, determined by HPLC-mass spectrometry, in the shoots grown on CK-free media comprised isopentenyladenine-, trans-zeatin- and cis-zeatin-type CKs. The application of exogenous CKs resulted in an increase in the CK content of the shoots. Following application of zeatin, dihydrozeatin-type CKs were also detected in the newly-formed shoots. Application of BA to the media led to a transition from isoprenoid CKs to aromatic CKs in the shoots. Shoots grown on gelrite media contained higher levels of endogenous CKs compared to those on agar media. Total CK content of hyperhydric shoots was higher than that of normal shoots grown on the same medium. We suggest that the ability of exogenous CKs and gelrite to induce hyperhydricity in shoots of Aloe polyphylla is at least partially due to up-regulation of endogenous CK levels. However, hyperhydricity is a multifactor process in which different factors intervene. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

Células mononucleares de sangue de cordão umbilical (SCU) e sangue periférico mobilizado (SPM) com G-CSF, foram cultivadas in vitro com citocinas, na presença ou não de estroma de medula óssea. Os objetivos foram avaliar a capacidade proliferativa de células progenitoras, a ocorrência de apoptose e expressão de integrina. Nas culturas sem estroma, a celularidade aumentou 5 vezes (SCU) e não se alterou nas de SPM. O total de células CD34+ caiu em ambas culturas. Com estroma, o total de células nucleadas aumentou 7 vezes (SCU) e 2,3 vezes (SPM). O total de células CD34+ permaneceu o mesmo. A apoptose foi menor nas culturas de SCU. A expressão de integrina caiu, na população de células CD34+ e de CD45+ / Mononuclear cells from umbilical cord blood (UCB) and G-CSF mobilized peripheral blood (MPB), were cultured in vitro, in the presence of cytokines, with or without bone marrow stroma. The aims were to evaluate the proliferative response of progenitor cells, occurrence of apoptosis and expression of adhesion molecule. In cultures without stroma, cellularity increased 5-fold for UCB, but has not changed for MPB. The number of CD34+ cells has dropped in both culture. With stroma, total nucleated cells had a 7-fold increse (UCB) and a 2,3-fold (MBP), however, CD34+ cells number has not changed. Apoptosis was lower in UCB culture. The expression of integrin decreased, in the CD34+ and CD45+ population

Células mononucleares de sangue de cordão umbilical (SCU) e sangue periférico mobilizado (SPM) com G-CSF, foram cultivadas in vitro com citocinas, na presença ou não de estroma de medula óssea. Os objetivos foram avaliar a capacidade proliferativa de células progenitoras, a ocorrência de apoptose e expressão de integrina. Nas culturas sem estroma, a celularidade aumentou 5 vezes (SCU) e não se alterou nas de SPM. O total de células CD34+ caiu em ambas culturas. Com estroma, o total de células nucleadas aumentou 7 vezes (SCU) e 2,3 vezes (SPM). O total de células CD34+ permaneceu o mesmo. A apoptose foi menor nas culturas de SCU. A expressão de integrina caiu, na população de células CD34+ e de CD45+ / Mononuclear cells from umbilical cord blood (UCB) and G-CSF mobilized peripheral blood (MPB), were cultured in vitro, in the presence of cytokines, with or without bone marrow stroma. The aims were to evaluate the proliferative response of progenitor cells, occurrence of apoptosis and expression of adhesion molecule. In cultures without stroma, cellularity increased 5-fold for UCB, but has not changed for MPB. The number of CD34+ cells has dropped in both culture. With stroma, total nucleated cells had a 7-fold increse (UCB) and a 2,3-fold (MBP), however, CD34+ cells number has not changed. Apoptosis was lower in UCB culture. The expression of integrin decreased, in the CD34+ and CD45+ population

O aumento da prevalência de doenças alérgicas como a asma, tem sido atribuído à falta de estímulos infecciosos. A atopia, que embasa as manifestações alérgicas, caracteriza-se por uma disfunção imune com predomínio da resposta do tipo Th2. Experimentos em modelos animais com micobactérias e seus produtos têm demonstrado resultados promissores na proteção e reversão de resposta imune do tipo Th2. A imunoterapia para alérgenos inalatórios tem mostrado resultados positivos e o uso do BCG como adjuvante poderia trazer benefícios adicionais. Em estudo randomizado duplo cego foram avaliados 21 indivíduos de ambos os sexos, com idades de 8 a 17 anos sensibilizados ao Dermatophagoides pteronissynus (Dpt) e portadores de asma leve ou moderada persistentes. Os pacientes foram divididos em dois grupos e tratados com imunoterapia específica (ITE) para Dpt. O Grupo A recebeu como adjuvante, no início da ITE, uma aplicação do diluente do BCG e o Grupo B uma dose da vacina BCG. Na avaliação realizada após o período de indução da ITE constatou-se nos dois grupos diminuição significativa dos sintomas, da necessidade de medicação para asma, da hiperreatividade brônquica inespecífica, da reatividade cutânea ao Dpt juntamente com a melhora da função pulmonar. Houve uma redução no índice de estimulação da cultura de células mononucleares do sangue periférico estimuladas com Dpt, acompanhada de uma elevação da IL-10 no sobrenadante e dos níveis da IgG específica para Dpt e da IgE total. Os eosinófilos, a IgE específica para Dpt do sangue e o óxido nítrico no ar exalado não se alteraram. Não houve diferença na comparação dentre os grupos, exceto na proliferação das células mononucleares do sangue periférico estimuladas com PPD, que foi maior no Grupo B. Conclusão: a ITE levou a resultados satisfatórios com melhora clínica e alterações imunológicas já ao final do período de indução. A avaliação do uso do BCG como adjuvante, não mostrou benefícios adicionais. / The increase of allergic diseases, such as asthma, has often been explained by a decline in infectious stimulation. Atopy that is characterized as an immune dysfunction promotes a strong type Th2 immune response and underlines allergic diseases. Experimental animal models researches with mycobacteria and its products had led to promising results in prevention or reversion of type Th2 response. Positive results have been obtained to allergens specific immunotherapy (SIT) and its association with BCG vaccine, as an adjuvant, could promote additional benefits. In a randomized double blind study 21 patients sensitized to mite Dermatophagoides pteronyssinus (Dpt) with mild or moderate persistent asthma, aged 8 to 17 years of both sex were evaluated. Patients were divided in two groups. Group A received at the beginning of SIT one dose of BCG diluent and Group B one dose of BCG vaccine. After the SIT induction period we observed in both groups a significant decrease in asthma symptoms, bronchial hyperreactivity, immediate cutaneous reactivity to Dpt and the necessity of drugs intake. Specific lymphoproliferation against Dpt significantly decreased while IL-10 levels, specific IgG and total IgE increased. Blood eosinophils, specific IgE, others immunoglobulins levels and nitric oxide in exhaled air didn\'t change. There were no differences in all parameters evaluated between the two groups except on specific lymphoproliferation against PPD that was higher in Group B. Conclusions: improvement in symptoms and immunologicals changes where observed at the end of SIT induction period. BCG as adjuvant didn\'t add additional benefits.

O aumento da prevalência de doenças alérgicas como a asma, tem sido atribuído à falta de estímulos infecciosos. A atopia, que embasa as manifestações alérgicas, caracteriza-se por uma disfunção imune com predomínio da resposta do tipo Th2. Experimentos em modelos animais com micobactérias e seus produtos têm demonstrado resultados promissores na proteção e reversão de resposta imune do tipo Th2. A imunoterapia para alérgenos inalatórios tem mostrado resultados positivos e o uso do BCG como adjuvante poderia trazer benefícios adicionais. Em estudo randomizado duplo cego foram avaliados 21 indivíduos de ambos os sexos, com idades de 8 a 17 anos sensibilizados ao Dermatophagoides pteronissynus (Dpt) e portadores de asma leve ou moderada persistentes. Os pacientes foram divididos em dois grupos e tratados com imunoterapia específica (ITE) para Dpt. O Grupo A recebeu como adjuvante, no início da ITE, uma aplicação do diluente do BCG e o Grupo B uma dose da vacina BCG. Na avaliação realizada após o período de indução da ITE constatou-se nos dois grupos diminuição significativa dos sintomas, da necessidade de medicação para asma, da hiperreatividade brônquica inespecífica, da reatividade cutânea ao Dpt juntamente com a melhora da função pulmonar. Houve uma redução no índice de estimulação da cultura de células mononucleares do sangue periférico estimuladas com Dpt, acompanhada de uma elevação da IL-10 no sobrenadante e dos níveis da IgG específica para Dpt e da IgE total. Os eosinófilos, a IgE específica para Dpt do sangue e o óxido nítrico no ar exalado não se alteraram. Não houve diferença na comparação dentre os grupos, exceto na proliferação das células mononucleares do sangue periférico estimuladas com PPD, que foi maior no Grupo B. Conclusão: a ITE levou a resultados satisfatórios com melhora clínica e alterações imunológicas já ao final do período de indução. A avaliação do uso do BCG como adjuvante, não mostrou benefícios adicionais. / The increase of allergic diseases, such as asthma, has often been explained by a decline in infectious stimulation. Atopy that is characterized as an immune dysfunction promotes a strong type Th2 immune response and underlines allergic diseases. Experimental animal models researches with mycobacteria and its products had led to promising results in prevention or reversion of type Th2 response. Positive results have been obtained to allergens specific immunotherapy (SIT) and its association with BCG vaccine, as an adjuvant, could promote additional benefits. In a randomized double blind study 21 patients sensitized to mite Dermatophagoides pteronyssinus (Dpt) with mild or moderate persistent asthma, aged 8 to 17 years of both sex were evaluated. Patients were divided in two groups. Group A received at the beginning of SIT one dose of BCG diluent and Group B one dose of BCG vaccine. After the SIT induction period we observed in both groups a significant decrease in asthma symptoms, bronchial hyperreactivity, immediate cutaneous reactivity to Dpt and the necessity of drugs intake. Specific lymphoproliferation against Dpt significantly decreased while IL-10 levels, specific IgG and total IgE increased. Blood eosinophils, specific IgE, others immunoglobulins levels and nitric oxide in exhaled air didn\'t change. There were no differences in all parameters evaluated between the two groups except on specific lymphoproliferation against PPD that was higher in Group B. Conclusions: improvement in symptoms and immunologicals changes where observed at the end of SIT induction period. BCG as adjuvant didn\'t add additional benefits.