<p>Ovarian stimulation in assisted reproduction often leads to the production of a high number of oocytes. After fertilization of these oocytes, the resulting embryos can be cryopreserved for later use. Vitrification is a recently introduced method for cryostoring embryos, showing high survival rates for both cleavage stage embryos and blastocysts. Characteristic of vitrification are high concentrations of cryoprotectants and ultra fast freezing which makes the material glassily. A major concern with vitrification has been the direct contact of the cryo-solutions with liquid nitrogen. Therefore, sealed containers have been developed and one of these is the Rapid-i™ made by Vitrolife Sweden AB.</p><p>We evaluated this new device using embryos not suitable for embryo transfer or cryopreservation for clinical purposes. Embryos at cleavage stages were first vitrified and then warmed. Outcome parameters were cryosurvival and development to the blastocyst stage. Blastocysts were randomised between the established VitroLOOP™ and the Rapid-i™ as carriers. Outcome parameters were cryosurvival and further development. Our results show that Rapid-i™ gives good survival rates in vitrification for cleavage stage embryos and blastocysts.</p>

An infectious cDNA clone of Vero cell adapted Beaudette strain of IBV was constructed using in vitro assembly of cDNA fragments. The entire genome of IBV was RT-PCR amplified into seven fragments, with each piece overlapping about 10 nucleotides. The fragments were ligated and transcribed to synthesize RNA, which was transfected into BHK-21 cells. These cells were then overlaid onto IBV susceptible Vero cells. After five days transfection, the virus was successfully rescued from the transfected cells. The cDNA clone from our laboratory strain has a five nucleotide insertion not present in the originally sequenced virus, resulting in total genome size of 27,613 nucleotides. The infectious cDNA clone was further manipulated to demonstrate its potential as a gene transfer vector, by replacing the ORF5a open reading frame with enhanced green fluorescent protein. The recombinant infectious cDNA clone was also successfully rescued after three days transfection of BHK-21 cells followed by co-culturing with Vero cells. This study showed that the 5a protein, whose function is not known, is not necessary for in vitro IBV replication. This study also showed that the 5a ORF is a good candidate for an insertion site of recombinant genes for the development of IBV infectious cDNA clone as a gene transfer vector.

Proteases play a key role in the cascade of tumor-associated proteolysis leading to extracellular matrix degradation, stromal invasion and blood vessel recruitment and inroad. Protease systems are widely described as implicated in the formation of new blood vessels. Until now, only few datas are available concerning their role in lymphangiogenesis. We successfully transposed the aorta ring assay to a mouse lymphatic thoracic duct assay. By immunochemistry and transmission electron microscopy, we characterized the outgrowing cells as being lymphatic cells that organize into microvessels containing a lumen and that conserved lymphatic endothelial cell features. This quantifiable model responds to several well-known lymphangiogenic factors as the VEGF-C but not to specific angiogenic factors. This model is so suitable to screen growth factors and inhibitors as well as conditioned media. Plasminogen activator inhibitor-1 is a component of the plasminogen cascade and, though it was critical for angiogenesis, it comes out that it is dispensable for lymphatic outgrowth. In sharp contrast, synthetic and physiological inhibitors of matrix metalloproteases inhibit lymphangiogenesis, and thoracic duct rings derived from MMP-2- but not MMP-9-deficient mice showed an impaired lymphatic cell outgrowth. These data identify MMP2 as an important player in lymphangiogenesis and was confirmed by an in vivo experiments. Proteases are thus also implicated in lymphangiogenesis and the lymphatic ring assay seems to be helpful to discover novel genes and mechanisms that underly the lymphangiogenesis process, including by comparing with angiogenesis in a similar system.

Con el fin de mejorar la producción in vitro de embriones (PIVE) desde ovocitos de cabra perpúber, fueron diseñados tres estudios en esta investigación. El objetivo del primer estudio fue determinar en ovocitos seleccionados mediante el test azul de cresol brillante (BCB), el efecto de la adición de gutatión (GSH) solo o en combinación con glucosa al medio de cultivo in vitro (CIV), sobre el desarrollo embrionario de ovocitos de cabra perpúber. Los ovocitos fueron expuestos al test de BCB y fueron clasificados como: ovocitos con citoplasma azul (BCB+) y ovocitos sin el citoplasma azul (BCB-). Los ovocitos BCB+ mostraron mayor porcentaje de maduración nuclear que los ovocitos BCB- y grupo control (82.6%, 55.7% y 74.7% respectivamente). El porcentaje de ovocitos poliespérmicos fue mayor en ovocitos BCB- que en los BCB+. La suplementación del medio de cultivo (CIV) con 1 mM de GSH, no afectó el desarrollo embrionario, pero el porcentaje de embriones totales desarrollados después del cultivo fue mayor en ovocitos BCB+ que en los BCB-, independientemente de la suplementación con GSH. La adición de glucosa, sola o con GSH no afectó el desarrollo embrionario. La finalidad del segundo estudio era evaluar el efecto de agregar diferentes concentraciones de cisteamina (100&#956;M, 200&#956;M o 400&#956;M) al medio de MIV y al medio de CIV (50 &#956;M o 100 &#956;M) sobre el desarrollo embrionario de ovocitos de cabra perpúber seleccionados por el test BCB. La adición de 400 &#956;M de cisteamina al medio MIV mejoró la fecundación normal y desarrollo embrionario de ovocitos BCB- a los mismos niveles de los ovocitos BCB+. Las proporciones de mórulas mas blastocistos desarrollados no fueron afectados por los tratamientos. Finalmente, fue estudiado el efecto de la adición de cisteamina (400 &#956;M) para el medio de MIV, glutatión (1mM) al medio FIV e ionomicina al medio de capacitación espermática. Este tratamiento mejoró la fecundación normal, cigotos con pronúcleos masculinos y el desarrollo embrionario de ovocitos de cabra prepuber, sin embargo no mejoró el desarrollo de blastocistos. / With the aim of trying to improve in vitro embryo production (IVEP) from prepubertal goat oocytes, three studies were designed in this investigation. The objetive of first study was to assess, in oocytes selected by the brillant cresyl blue (BCB) test, the effect of the addition to in vitro culture (IVC) medium of either glutathione (GSH) alone or GSH in combination with glucose on the embryo development. Oocytes were exposed to BCB and were classified as: oocytes with a blue cytoplasm (BCB+) and oocytes without blue cytoplasm (BCB-). BCB+ oocytes showed higher percentage of nuclear maturation than the BCB- and control group (82.6%, 55.7% and 74.7%, respectively). The percentage of polyspermic oocytes was higher in BCB- than BCB+ oocytes. Supplementation of in vitro culture (IVC) medium with 1mM de GSH did not affect embryo development, but the porcentage of total embryos developed after culture was higher in BCB+ oocytes than in BCB- oocytes independently of the GSH supplementation. The addition of glucose, alone or with GSH, did not affect embryo development. The aim of the second study was to evaluate the effect of adding different concentrations (100&#956;M, 200&#956;M and 400 &#956;M) of cyteamine to the IVM medium and to the in vitro embryo culture (IVC) medium (50 &#956;M or 100 &#956;M) on the embryo development of prepubertal goat oocytes BCB-selected. The addition of 400 &#956;M cysteamine to the IVM improved normal fertilisation and embryo development of BCB- oocytes at the same rates as those obtained from BCB+ oocytes. The proportions of morulae plus blastocyst development were not affects by the treatments. Finally, was studied the effect of adding cysteamine (400 &#956;M) to IVM medium, glutathione (1mM) to IVF medium and ionomycin to the sperm capacitation medium. This treatment improved normal fertilisation, zygotes with male pronucleus and embryo development of prepubertal goat oocytes, however did not improve blastocyst development.

La importància de les anomalies cromosòmiques en els problemes d'infertilitat ha estat àmpliament confirmada per diversos autors. S'ha estimat que la freqüència d'aberracions cromosòmiques dels zigots humans pot anar des d'un 8-10% (Kajii i Mikamo, 1978) fins a un 50% (Boué i col., 1975) al moment de la concepció. Una estimació directa de la incidència primària d'aparició de les anomalies cromosòmiques després de la fecundació en humans és molt difícil d'obtenir pels problemes de tipus tècnic i ètic que comporta. De la mateixa manera és difícil de realitzar una anàlisi directa de les característiques cromosòmiques dels gametes, fonamentalment els femenins, a causa de la seva inaccessibilitat inherent. De tota manera s'ha suggerit (Bond i Chandley, 1983) que l'espècie humana presenta un major nombre d'anomalies cromosòmiques al moment de la fecundació que no pas altres espècies de mamífers. Aquest fet ha passat malgrat que les noves tècniques de fertilització in vitro (FIV), cada cop més exteses en el tractament de determinats tipus d'infertilitat, podrien carregar l'espècie humana amb una font extra d'aberracions cromosòmiques derivades dels processos de fecundació fora del tracte genital femení. L'estudi de la freqüència primària d'aparició d'anomalies cromosòmiques en una població de mamífers (els ratolins), mitjançant l'anàlisi de la primera divisió embrionària, ha permès abordar aquest problema a través d'un enfocament experimental lliure de les limitacions ètiques i tècniques a les que ja hem fet esment. Per aquesta raó s'ha desenvolupat un mètode (Fraser i Maudlin, 1979) consistent en el cultiu dels zigots de ratolí recent fecundats en un medi amb antimitòtic que impedeix la singàmia, de manera que els complements d'origen matern i patern romanen separats i fàcilment distingibles l'un de 1'altre. Mercès a aquest sistema es pot atribuir l'origen, masculí i femení, de cada anomalia cromosòmica observada alhora que s'obté la incidència d'aberracions cromosòmiques en el moment de la concepció. Mitjançant la comparació de les característiques cromosòmiques de la primera divisió zigòtica d'una població d'embrions de ratolí fecundats in vivo amb una de fecundats in vitro, s'ha pogut esbrinar quin és l'efecte de la tècnica de FIV sobre la dotació cromosòmica dels embrions que se'n deriven. D'altra banda la possibilitat de manipular els donants dels gametes amb substàncies de suposat efecte genotòxic ofereix l'oportunitat d'utilitzar aquests estudis com a test de mutagenicitat per a certs tipus de compostos amb unes característiques concretes. Seguint aquest disseny experimental s'han detectat un total d'anomalies cromosòmiques al moment de la concepció que representen un 20.74% dels zigots fecundats in vivo, dels quals un 8.41% són aneuploides, un 10.37% poliploides i un 1.76% presenten anomalies estructurals.Pel que fa a l'aportació dels gamètes, la incidència d'aneuploïdies (un 4%) i d'anomalies estructurals (al voltant del 0.7%) s'ha observat que és igual en ambdós sexes.D'altra banda l'anàlisi dels embrions fecundats in vitro mostra que un 21.39% presenten anomalies cromosòmiques, dels quals el 5.42% són aneuploïdies, el 14"23% són poliploïdies i el 1.74% tenen anomalies estructurals. Aquest resultats indiquen que, en general, les tècniques de FIV no augmenten el nombre d'anomalies detectades en la primera divisió, tret d'un increment significatiu del nombre de dispèrmies i del nombre d'espermato zoides diploides que intervenen en la fecundació. Aquesta darrera observació confirma l'existència d'una selecció pre-zigòtica exercida pel tracte genital femení en contra dels espermatozoides morfològicament anòmals. Pel que fa a la utilització de la primera divisió embrionària com a test de mutagenicitat es confirma llur interès, en detectar-se un tipus d'acció de la substància emprada (la I3PL) difícilment observable a través d'altres proves clàssiques de detecció del caràcter genotòxic d'un compost, com és la formació d'oòcits diploides a causa.de la no-extrusió del segon corpuscle polar. / The importance of chromosomal abnormalities in reproductive failures has been confirmed by several authors. It is estimated that the frequen cy of chromosomally abnormal human zygotes at the time of conception may range from 8-10% (Kajii and Mikamo, 1978) to 50% (Boue et al.,1975). A direct estimate of the number of chromosomally abnormal human conceptus is difficult to obtain because of ethical and technical difficulties. Likewise, a direct study of human gametes has been limited, mainly for human oocytes, because of the problems involved in their obtention. It has been suggested that more conceptions are lost through chromosome imbalance in man than in other mammalian species (Bond and Chandley, 1983). This has led to the concern that in vitro fertilization (IVF) procedures, which have become a common technique for some infertility problems, would load the human species with an extra source of chromosome abnormalities. Fraser & Maudlin (1979) developed a mouse experimental system, of first embryonic cleavage chromosome analysis which can be used as a model for man. It involves the culture of mouse embryos in a medium containing antimitotic to arrest the cleaving process before singamy is reached.Thus the male and female chromosome sets do not mingle, but remain in separate clusters, which permits to assess the parental origin and incidence of chromosomal abnormalities at conception. Comparing the characteristics of first cleavage in vivo and in vitro fertilized embryos, it is possible to evaluate whether IVF techniques enhance the proportion of chromosomally abnormal embryos.On the other hand, the possibility of injecting genotoxic compounds to gametic donors, permit the use of the first cleavage system as a mutagenic test for specific substances. After the analysis of 1022 in vivo fertilized embryos we have seen that, in the mouse, 20.74% are chromosomally abnormal, among them, 8 show aneuploidy, 10.37% polyploidy and 1.76% structural rearrangments. Comparing the origin of the abnormalities, the incidence of aneuplody (4%) and structural rearrangments (0.7%) is the same for both sexes. After the analysis of 1033 in vitro fertilized embryos, we have seen that 21.39% have a chromosome abnormality, among them 5.42% are aneuploid 14.23% are polyploid and 1.74% have structural rearrangments. These results indicate that, in mice, the exposure of the fertilizing gametes to an in vitro environment is not a source of extra chromosomal abnormalities, except for an increase in the level of polispermy and of diploi spermatozoa that fertilize in vitro. The later confirms the existence of a form of prezygotic selection exerted by the genital female tract against the morphologically abnormal spermatozoa. Upon the use of first cleavage analysis as a mutagenic test, our data stress its importance. We have detected a type of action that would be hard to assess through a classic mutagenic test: the production of diploid oocytes due to the non extrusion of second polar body.

Las fluoroquinolonas se han convertido en agentes importantes en quimioterapia, especialmente en pacientes con infecciones graves que requieran fármacos potentes administrados por vía oral. Sin embargo, la aparición de resistencias a las quinolonas ha conducido a la búsqueda de nuevas moléculas, principalmente incluyendo modificaciones en el núcleo base de la benzopiridona, lo que ha llevado al hallazgo y comercialización de nuevas quinolonas, más activas pero, en muchos casos, más tóxicas, hasta el punto de que algunas han dejado de utilizarse o han sido retiradas del mercado tras haberse demostrado su peligrosidad a medio o largo plazo.En este contexto, el ciprofloxacino es una de las quinolonas más clásicas y de uso más extendido por su amplio espectro de acción, su eficacia, su excelente tolerancia y por permitir su administración tanto por vía parenteral como por vía oral, si bien presenta el problema de poseer una biodisponibilidad oral relativamente baja e irregular. Por este motivo y tomando esta quinolona como referencia de actividad, se han sintetizado nuevos derivados homólogos pertenecientes a dos series: 4'N-alquilciprofloxacino y 3'metil,4'N-alquilciprofloxacino que podrían mejorar la biodisponibilidad y la actividad. En esta línea se incluye el Proyecto de Investigación SAF 96-1710 titulado "Predicción de la biodisponibilidad en los estudios de desarrollo de fármacos: nuevas quinolonas" que pretende comprobar si el aumento de biodisponibilidad esperado en los derivados afecta a la actividad antimicrobiana.En resumen, se ha estudiado y comparado la actividad de estos homólogos con la del ciprofloxacino. Para ello, se ha determinado la CMI de los derivados, sobre 160 cepas bacterianas de origen clínico, utilizando el método de diluciones dobles seriadas en MHA según las especificaciones del NCCLS.Se demuestra que la adición del sustituyente alquílico en 4'N no altera el espectro de acción del patrón utilizado. Sin embargo, los compuestos sintetizados presentan diferente actividad frente a los gérmenes ensayados. En1conjunto los más activos son 4'N-metilciprofloxacino, 4'N-etilciprofloxacino, 3'metilciprofloxacino(97-100) y 3'metil,4'N-metilciprofloxacino (97-101).De forma general, se puede afirmar que respecto a la serie 4'N-alquilciprofloxacino, los compuestos con un radical metilo o etilo son los más activos: 4'N-metilciprofloxacino es el más activo frente a los Gram negativos (a excepción de E. coli) y 4'N-etilado el más activo frente a los Gram positivos. No obstante, 4'N-propilciprofloxacino también presenta mejor actividad que el patrón en los microorganismos Gram positivos (a excepción de E. faecalis), y en A. calcoaceticus. 4'N-butilciprofloxacino no presenta mejor actividad que el compuesto de referencia en ningún caso, si bien en los microorganismos ya mencionados la actividad es la misma.Respecto a la serie 3'metil, 4'N-alquilciprofloxacino, se puede colegir que los compuestos 97-100 y 97-101 son los más activos. En resumen, el derivado 97-101 presenta la mayor actividad frente a A. calcoaceticus, M. catarrhalis, S. aureus, SCN y S. pneumoniae. Por otro lado, 97-100 es el más activo frente a Achromobacter spp. No obstante, 97-102 y 97-103 también presentan mejor actividad que el ciprofloxacino en los microorganismos A. calcoaceticus, S. aureus y S. pneumoniae. El derivado 97-104 no presenta mejor actividad que el patrón en ningún caso, si bien en los microorganismos ya mencionados la actividad es la misma.En lo que se refiere a los heterólogos estudiados, en general, el grepafloxacino es el compuesto más activo. El ofloxacino y el sarafloxacino presentan una actividad muy similar a la del ciprofloxacino, y el flumequino es el menos activo.También se ha estudiado la cinética de letalidad de algunos de estos compuestos en Staphylococcus aureus ATCC 25923. Este estudio proporciona una serie de parámetros de gran interés en lo que se refiere al mecanismo de acción. Los resultados obtenidos demuestran que la dinámica de crecimiento y letalidad que presentan los homólogos es la misma que su patrón, siguen la misma cinética bactericida y muestran un efecto paradógico similar. Si se2considera la concentración absoluta, el antibiótico con mayor velocidad de letalidad es el 97-101. Se planteó si el grupo metilo adicional que presenta este derivado en posición 3' con respecto a ciprofloxacino y los compuestos de la otra serie, podría ser el responsable de su mayor letalidad. Dado que la selección de la girasa como primera diana por parte de las quinolonas se asocia con un aumento en la velocidad de letalidad, se determinó la diana de primera elección para este derivado. Se comprobó que la mayor letalidad de este compuesto no se debe a un cambio en la selección de la diana intracelular; pues se demostró, mediante estudios de CMI sobre cepas mutantes, que la topoisomerasa IV es su primera diana en S. aureus, al igual que para el ciprofloxacino.Por otro lado, debido a que los antibióticos estudiados pertenecen a series homólogas, se ha estudiado la relación entre la lipofilia, como parámetro estructural, y la inversa de la media geométrica de la CMI, como índice de actividad. Esta correlación proporciona ajustados bilineales y permite identificar el coeficiente de reparto óptimo para una especie dada, que es en general más bajo para los microorganismos Gram negativos que para los Gram positivos.Además, se ha investigado acerca del mecanismo de acción de estas quinolonas a nivel molecular. En concreto, la investigación se ha centrado en Streptococcus pneumoniae, determinando los efectos de inhibición de las quinolonas ensayadas en la girasa y topoisomerasa IV bacteriana in vitro, con el propósito de conocer la diana específica de los derivados alquilados en comparación con el ciprofloxacino, proporcionar información acerca del modo de acción de estos compuestos y comprobar si ello permite explicar las diferencias en la actividad observadas sobre este microorganismo. Esta parte del trabajo se ha desarrollado en el St. George's Hospital Medical School, University of London, bajo la dirección del Dr LM Fisher.3Se demostró por estudios de CMI sobre cepas mutantes de S. pneumoniae que los homólogos mantienen la diana principal en Topoisomerasa IV, como el patrón. Por otra parte, la determinación de la CC25 mediante electroforesis en gel de agarosa demuestra que los compuestos estudiados no provocan mayor formación del complejo ternario ni con la topoisomerasa IV ni con la girasa. Sólo con el derivado 97-100 éste es igual que ciprofloxacino, lo que indica que la alquilación en 4'N del anillo piperacínico del ciprofloxacino no es favorable, si bien afecta a las interacciones entre fármaco y enzima. El efecto del sustituyente en C7 sobre la bomba de secreción PmrA no se conoce puesto que los resultados de este estudio no fueron concluyentes. No se puede descartar, pues, la penetración intracelular como posible responsable de la diferencia de actividad de los homólogos, debido a que para un microorganismo dado, este parámetro está bajo el control de las propiedades fisicoquímicas de la quinolona.Por último, al considerar el equilibrio actividad-biodisponibilidad oral comparada para la selección de los candidatos, existen tres compuestos que presentan las mejores propiedades en conjunto: 4'N-metilciprofloxacino, compuesto de elección en todos los microorganismos Gram negativos a excepción de E. coli. 4'N-etilciprofloxacino, muy eficaz frente a los microorganismos Gram positivos, entre los que es el derivado de elección en todos los casos, y además presenta buenas propiedades en los microorganismos Gram negativos. 3'-metilciprofloxacino (97-100) presenta una eficacia en general mejor a la del patrón en todas las especies estudiadas, sin embargo sólo resulta el candidato de primera elección en E. coli, en el que la mayoría de compuestos son menos activos que el ciprofloxacino.No obstante, aunque estos tres compuestos son los candidatos de elección, los demás derivados también podrían resultar de interés en algunos de los microorganismos estudiados como A. calcoaceticus, S. aureus y S. pneumoniae. / The thesis entitled "In vitro antimicrobial activity of new 6-fluoroquinolones for oral administration. Structure-activity relationship" is a part of the research Project SAF 96-1710 "Bioavailability prediction in drug development studies: new quinolones". In this study, we have synthesized new ciprofloxacin homolog derivatives belonging to two series: 4'N-alkylciprofloxacin and 3'methyl,4'N-alkylciprofloxacin, whose antimicrobial activity (minimum inhibitory concentration, MIC) we have studied and compared with that of ciprofloxacin against 160 bacterial strains from clinical sources. MICs were determined by the standard NCCLS, M7-A5 method. The results showed that 4'N alkyl substitution does not modify the reference drug antibacterial spectrum. Nevertheless, the synthesized compounds present different activity against the assayed microorganisms.Time-kill kinetics of some homolog compounds against Staphylococcus aureus ATCC-25923 were determined. These derivatives present the same lethality kinetics than ciprofloxacin. The 3'methyl,4'N-methylciprofloxacin derivative shows the quickest lethality, which is not due to a change in the primary target selection, as topoisomerase IV is its primary target for S. aureus, as it is for ciprofloxacin.Furthermore, we have studied the relationship between the lipophilicity, as a structural parameter, and the inverse of the geometric mean MIC, as an activity index. The activity-lipophilicity correlations can be fitted to a bilineal equation and allow the selection of the optimum partition coefficient for each species studied. This optimum tends to be lower for gramnegative microorganisms than for grampositive ones.We have also studied the mechanisms of action of these quinolones by determining the in vitro inhibition effects through Streptococcus pneumoniae gyrase and topoisomerase IV (carried out in St. George's Hospital Medical School, London). Topoisomerase IV is the primary target of ciprofloxacin homologs for S. pneumoniae. Ciprofloxacin C7 substitution affects drug-enzyme interaction. The cleavage through gyrase or topoisomerase IV is low, suggesting that 4'N ciprofloxacin alkylation is not favourable.Finally, the activity-bioavailability balance has been considered, in order to select the best drug candidates. Three compounds are pointed out: 4'N-methylciprofloxacin and 3'methylciprofloxacin against gramnegative microorganisms and 4'N-ethylciprofloxacin against grampositive ones. These finding are consistent with the established activity-lipophilicity correlations and the selected candidates against gramnegatives are more hydrophilic than those against grampositives.

Microsporidia are a group of obligate intracellular fungal parasites that infect a wide range of vertebrates and invertebrates, and are of economic and academic interest. Some areas of their economic impact are in aquaculture where they can infect salmon and other fish species. In agriculture they have been considered as control agents for insect pests, but more importantly as likely contributing to colony collapse disorder of bees. As an academic topic, microsporidia are fascinating because they are the smallest and simplest eukaryotic cells and require eukaryotic host cells in order to complete their life cycle. Therefore one research avenue that moves forward both economic and academic interests is to use cultures of animal cells to support the growth and development of the microsporidia life cycle, including the production of spores. Although the use of animal cell cultures for studying the microsporidia of insect and mammals has a fairly large literature, fish cell cultures have been employed less often but have had some successes as reviewed in this thesis. Very short-term primary cultures have been used to show how microsporidia spores can modulate the activities of phagocytes. The most successful microsporidia/fish cell culture system has been relatively long-term primary cultures of salmonid leukocytes for culturing Nucleospora salmonis. Surprisingly, this system can also support the development of Enterocytozoon bienusi, which is of mammalian origin. Some modest success has been achieved in growing Pseudoloma neurophilia on several different fish cell lines. The eel cell line, EP-1, appears to be the only published example of any fish cell line being permanently infected with microsporidia, in this case Heterosporis anguillarum. These cell culture approaches promise to be valuable for describing the growth and development of the microsporidia and for documenting the responses of fish cells to infection. In this thesis, cell lines from warm water fish, goldfish, fathead minnow and zebrafish, and a coldwater species, rainbow trout, were explored as potential cellular hosts of two microsporidia species that have never been grown or associated with fish before. One is Anncaliia algerae, which is an aquatic microsporidium that most commonly infects mosquitoes. This microsporidia is one of the easiest species to grow in mammalian cells, with the rabbit kidney cell line, RK 13, being the most documented culture system. The other is Nosema apis, which is a pathogen of bees and for which few cell culture systems exist. The ability of warm water fish cell lines to support the life cycle of A. algerae was investigated first. Spores were purified from RK-13 cultures and added to cell lines from three warm water species as well as to an insect cell line. The cell lines were GFSK-S1 and GFB3C- W1 from goldfish skin and brain respectively, ZEB2J from zebrafish embryos, FHMT-W1 from fathead minnow testis, and Sf9 from ovaries of a fall armyworm moth. All cultures were maintained at 27 °C. Infection was judged to have taken place by the appearance of sporonts and/or spores in cells and occurred in all cell lines. Spores were also isolated from ZEB2J cultures and used to successfully infect new cultures of ZEB2J, RK-13 and Sf9. These results suggest that cells of a wide range of vertebrates support A. algerae growth in vitro and fish cells can produce spores infectious to cells of mammals, fish and insects. As ZEB2J was the most characterized of the fish cell lines and supported good A. algerae growth, this cell line was used in further studies described below to compare the efficacy of antimicrosporidial drugs and to test whether fish cells could support N. apis growth, but first A. algerae growth at lower temperatures was explored with cell lines from a coldwater fish. Cultures of cell lines from rainbow trout gill, RTgill-W1, and brain, RTbrain-W1, at 9, 18 and 21°C were evaluated for their ability to support the development of A. algerae. For up to 8 days after the addition of spores, living and DAPI stained cultures were examined by phase-contrast microscopy, allowing the identification of the meront, sporont, and spore stages in cultures at 18 and 21 °C. Meronts and sporonts were both spindle-shaped, but relative to meronts, sporonts were darker under phase contrast and brighter after DAPI staining. Spores were egg-shaped, phase- bright and intensely DAPI stained. These stages could not be identified conclusively in cultures at 9 °C, but their appearance at 18 °C sets a new low temperature for the growth of this species. The growth of A. algerae at room temperature allowed living cultures to be observed conveniently and videoed with a proprietary instrument, the Riveal microscope (www.quorumtechnologies.com). With this microscope, the development of A. algerae life cycle stages at room temperature was confirmed plus for the first time meront division and intracellular germination were captured on video. Spore germination in the absence of host cells and in response to 3 percent hydrogen peroxide was also observed by Riveal microscopy and for first time an abnormal germination phenomenon was clearly documented: polar tubes were extruded but the spore bodies retained the nuclei. ZEB2J cultures that had been infected with Anncaliia algerae spores were used as an in vitro test system to evaluate the curative actions of albendazole, fumagillin, and three fluoroquinolones; ciprofloxacin, norfloxacin, and ofloxacin. For each drug at concentrations above 50 µg/ml, the viability of ZEB2J cell declined sharply so concentrations of 10 and 20 µg/ml were studied. At these concentrations the drugs had little effect on the morphology and germination A. algerae spores. Each of the fluoroquinolones failed to prevent A. algerae from infecting ZEB2J cells and from growing to the same extent as in untreated ZEB2J cultures. Adding albendazole or fumagillin to cultures did not prevent A. algerae from infecting ZEB2J cells but impeded the growth and accumulation of A. algerae life-cycle stages. However, albendazole treatments caused a significant fraction of the ZEB2J cells to have nuclear abnormalities. Fumagillin reduced the intensity of infections within a ZEB2J cell, although the number of infected cells in a culture was not reduced. Over 5 days of infection with A. algerae the accumulation of ZEB2J cells in cultures was reduced but fumagillin treatment restored the accumulation to control levels. These results suggest that fumagillin has some potential as a treatment for A. algerae infections. ZEB2J was exposed to Nosema apis spores from the western honey bee (Apis mellifera). Bees were collected from hives that had been naturally infected and confirmed polymerase chain reaction (PCR) to have N. apis. Frozen bees were crushed in water to yield a mixture of bee parts, pollen grains, yeast, and microsporidial spores. The mixture was filtered and then centrifuged through Percoll to produce a pellet of spores that was resuspended in L-15 with 10 percent fetal bovine serum (FBS). Aliquots of this were added to ZEB2J cultures. Cultures were observed periodically for up to 24 days with a combination of phase contrast microscopy and of fluorescence microscopy, usually after staining with 4’,6-diamidino-2-phenylindole (DAPI). Although earlier life cycle stages were not observed, structures that were concluded to be either sporonts, sporoblasts and/or spores were seen, but these were in less than 5 percent of the fish cells. These N. apis life cycle stages had grown in ZEB2J because some appeared to be inside the cells and often they were arranged around the nucleus of the host cell rather than being randomly distributed in cultures. Despite repeated rinsing over a three week period, all cultures were ultimately lost due to yeast from the original spore preparations over growing the fish cell cultures. The overarching observation of this thesis is that fish cells in culture have been shown for the first time to support the growth A. algerae, and possibly N. apis. This suggests that the cells of vertebrates might support the growth of a wide range of microsporidia species that normally are associated with insects. In turn this suggests restriction of a microsporidial species to a particular animal group is unlikely accomplished at the cellular level but through physiological systems expressed at the organismal level and disturbances in these systems might lead to infections in new groups of animal hosts. The overarching observation of this thesis has two general implications for future studies. Firstly, for studying the expression of antimicrosporidia mechanisms in fish cells, the ZEB2J/A. algerae co-culture system promises to be useful. Secondly, for microsporidia species that are difficult to grow in culture, cell lines from a wide range of vertebrate and invertebrate species should be explored and one possibility for N. apis is fish cells.

Ovarian stimulation in assisted reproduction often leads to the production of a high number of oocytes. After fertilization of these oocytes, the resulting embryos can be cryopreserved for later use. Vitrification is a recently introduced method for cryostoring embryos, showing high survival rates for both cleavage stage embryos and blastocysts. Characteristic of vitrification are high concentrations of cryoprotectants and ultra fast freezing which makes the material glassily. A major concern with vitrification has been the direct contact of the cryo-solutions with liquid nitrogen. Therefore, sealed containers have been developed and one of these is the Rapid-i™ made by Vitrolife Sweden AB. We evaluated this new device using embryos not suitable for embryo transfer or cryopreservation for clinical purposes. Embryos at cleavage stages were first vitrified and then warmed. Outcome parameters were cryosurvival and development to the blastocyst stage. Blastocysts were randomised between the established VitroLOOP™ and the Rapid-i™ as carriers. Outcome parameters were cryosurvival and further development. Our results show that Rapid-i™ gives good survival rates in vitrification for cleavage stage embryos and blastocysts.

To evaluate the anticancer potential of sorghum phenolic compounds, different experiments including in vitro and in vivo tests were performed. A set of 25 sorghum samples was evaluated for phenolic (total phenols, flavonoids, anthocyanins and tannins) content, hydrophilic and lipophilic antioxidant capacity using de Oxygen Radical Absorbance Capacity assay (ORAC), and screened for citotoxic properties in mammary, colon and hepatic mammalian cancer cell lines in vitro. Results indicated that there was a wide variability in the phytochemical profile among the different sorghums. Among the 25 samples, sumac sorghum bran had the highest amount of phenolic compounds, flavonoids, tannins and the highest ORAC values. It exerted the highest percent inhibition (near 100%) in mammary, colon and liver cancer cell lines. Sumac sorghum bran was selected for further investigation. Methanolic extracts from sumac whole grain, bran and tannin removed bran were tested in vitro at different concentrations in hormone dependent MCF-7 mammary cancer cells and non hormone dependent Caco2 and HepG2 colon and liver cancer cells. Results indicated that the methanolic extract from sumac bran inhibited 100% of MCF-7 cancer cells at a concentration of 0.5 mg/ml and that the citotoxic effect could be partially due to the tannin content of the extract. Concentrations of 0.5 and 1.5 mg/ml were selected for an in vivo preventive cancer study with 7,12-dymethylbenz(a)-anthracene (DMBA) induced female rats. Bran at low and high concentrations and the correspondent amount of methanol extracts were included in the diet. It was observed that sumac methanol extract at low concentration promoted tumor appearance and development, whereas sumac bran had a preventive effect, however, there were no significant differences in rats treated and un-treated with sumac. Differences between in vitro and in vivo results could be due to the degree of absorption of tannins during the in vivo experiment. To obtain additional data about the effect of sumac extracts on cancer development, a quinone reductase enzyme bioassay was performed. Methanol and hexane extracts from sumac bran induced phase II enzymes in vitro. Phytochemicals of sumac bran sorghum including phenolic compounds and lipid like compounds appeared to have potential for cancer prevention.

Listeria monocytogenes is a foodborne pathogenic bacterium responsible for ~500 deaths and a financial burden of ~$2.3 billion each year in the United States. Though a zero tolerance policy is enforced with regard to its detection in cooked ready-to-eat foods, additional preemptive control alternatives are required for certain products. Among these alternatives are strategies permitting the usage of food antimicrobial combinations to control the pathogen. Research on antimicrobial combinations can provide insight into more efficient control of the pathogen, but is currently lacking. The purpose of this study was to evaluate the in vitro inhibition of L. monocytogenes exposed to the antimicrobials e-Poly-L-Lysine (EPL), lauric arginate ester (LAE), and sodium lactate (SL) at pH 7.3, octanoic acid (OCT) at pH 5.0, and nisin (NIS) and acidic calcium sulfate (ACS) at both pH 5.0 and 7.3. A broth dilution assay was used to determine single antimicrobial minimum inhibitory and bactericidal concentrations for L. monocytogenes Scott A, 310, NADC 2783, and NADC 2045. Optical density differences (delta<0.05 at 630 nm) were used to denote inhibition. Concentrations producing population decreases of greater than or equal to 3.0 log10 CFU/ml after incubation were considered bactericidal. Inhibition resulting from combinations of antimicrobials (NIS+ACS, EPL+ACS, SL+ACS, NIS+LAE, OCT+ACS, and OCT+NIS) was assessed using a checkerboard assay, and fractional inhibitory concentrations (FIC) were determined. FIC values were plotted on isobolograms and were used to create FIC indices (FICI). Isobologram curvature was used to classify combinations as synergistic, additive, or antagonistic. From FIC indices, interactions were defined as antagonistic (FICI >1.0), additive (FICI =1.0), or synergistic (FICI &lt;1.0). Strain-dependent factors had a bearing on MIC and MBC values for NIS and EPL. At pH 7.3, NIS+ACS displayed synergistic inhibition, NIS+LAE and EPL+ACS demonstrated additive-type interactions, and the SL+ACS pairing was unable to be defined. At pH 5.0, interpretation of the OCT+NIS interaction also presented challenges, while the OCT+ACS combination resulted in synergistic behavior. Additional studies are needed to validate in vitro findings on surfaces of ready-to-eat meats. Future in vivo studies should investigate the ability of synergistic combinations (NIS+ACS and OCT+ACS) to control the pathogen. Better characterizations of inhibitory mechanisms should also be performed.