<p>Juvenile nucleus pulposus (NP) cells of the intervertebral disc (IVD) are large, vacuolated cells that form cell clusters with numerous cell-cell interactions. With maturation and aging, NP cells lose their ability to form these cell clusters, with associated changes in NP cell phenotype, morphology and proteoglycan synthesis that may contribute to IVD degeneration. Studies demonstrate healthy, juvenile NP cells exhibit potential for preservation of multi-cell clusters and NP cell phenotype when cultured upon soft, laminin-containing substrates; however, the mechanisms that regulate metabolism and phenotype of these NP cells are not understood. N-cadherin is a cell adhesion molecule that is present in juvenile NP cells, but disappears with age. The goal of this dissertation was to reveal the role of N-cadherin for NP cells in multi-cell clusters that contribute to the maintenance of the juvenile NP cell morphology and phenotype in vitro, and to evaluate the potential for laminin- functionalized poly(ethylene glycol) (PEG-LM) hydrogels to promote human NP cells towards a juvenile NP cell phenotype. </p><p>In this dissertation, juvenile porcine IVD cells were promoted to form cell clusters in vitro, and analyzed for preservation of the juvenile NP phenotype on soft, laminin-rich hydrogels. In the first part of this dissertation, preservation of the porcine juvenile NP cell phenotype and presence of N-cadherin was analyzed by culturing porcine NP cells on soft, laminin-rich or PEG-LM hydrogels. Secondly, cadherin-blocking experiments were performed to prevent cluster formation in order to study the importance of cluster formation in NP cell signaling. Finally, human IVD cells were cultured on PEG-LM hydrogels to investigate the potential to revert degenerate, human NP cells toward a juvenile NP cell phenotype and morphology. </p><p>Findings reveal soft (<500 Pa), laminin-rich substrates promote NP cell clustering, a key feature of the juvenile NP cell that is associated with N-cadherin positive expression. Additionally, N-cadherin-mediated cell-clustering regulates NP cell matrix production and gene expression of NP-specific and NP-matrix related markers. Inhibition of N-cadherin-mediated contacts resulted in decreased expression of juvenile NP cell features. Finally, juvenile human NP cells are also able to form N-cadherin positive cell clusters on soft, PEG-LM hydrogels with higher expression of juvenile NP cell features compared to culturing on stiff PEG-LM hydrogels. Some degenerate, human NP cells are also able to form N-cadherin positive cell clusters with some features of the juvenile NP cell. </p><p>The studies presented in this dissertation support the proposed hypothesis and establish the importance of soft, laminin-rich substrates in promoting NP cell clustering behaviors with associated features of a juvenile cell phenotype and morphology. Additionally, these studies establish a regulatory role for N-cadherin in juvenile NP cells and suggest that preservation of N-cadherin-mediated cell-cell contacts is important for preserving the juvenile NP cell phenotype and morphology. Furthermore, findings from this dissertation reveal the ability to promote degenerate, mature human NP cells towards a juvenile NP cell phenotype, demonstrating the potential to use PEG-LM hydrogels as a means for autologous cell delivery for the restoration of healthy IVD.</p> / Dissertation

Neste trabalho foi investigado o efeito exercido por microambientes sobre a estabilidade química e fotoquímica de corantes. Em particular, estudaram-se os fatores responsáveis pelo aumento da estabilidade química e fotoquímica de índigo quando em interação com paligorsquita, que compõem o pigmento histórico Azul Maia, sobre o qual ainda havia controvérsias na literatura. Os corantes investigados foram índigo, dehidroíndigo, alizarina, purpurina, luteolina e &#946;-caroteno; os microambientes foram proporcionados pelas argilas paligorsquita, sepiolita, montmorilonita, laponita e HDL de Al3+ e Mg2+ (3:1). Paligorsquita e a sepiolita são as únicas argilas que apresentam microporos em sua estrutura. As técnicas de caracterização empregadas neste trabalho foram: espectroscopia vibracional (Raman e absorção no infravermelho), espectroscopia de absorção no UV-VIS, difratometria de raios X, análise térmica (TG e DSC), CG-MS, HLPC-MS, medidas de área superficial por isoterma de adsorção de N2 e SEM. Duas técnicas com resolução temporal em escala de sub-picosegundos (absorção de transiente e infravermelho resolvido no tempo) foram utilizadas. O sistema índigo+paligorsquita corresponde à mistura dos dois sólidos, seguida de aquecimento, sendo que a partir de 70 °C a coloração da mistura adquire tonalidade esverdeada e também apresenta maior estabilidade química e fotoquímica. Essa estabilidade e também a alteração na cor aumentam com a temperatura de aquecimento da mistura e o intervalo considerado engloba as temperaturas de perda de água zeolítica (70 - 150 °C) e coordenada (170 - 280 °C) da estrutura da argila. Os resultados de espectroscopia vibracional e eletrônica dos simulantes de Azul Maia indicam que o índigo interage através de ligações de hidrogênio com as moléculas de água coordenada. Essa interação, entretanto, só é possível com a remoção da água zeolítica, o que ocorre a partir de 70 °C. Com aquecimento em temperaturas acima de 170 °C o comportamento do espectro eletrônico e vibracional se altera, indicando a formação direta de complexos com os metais presentes nas bordas internas dos microporos. Os resultados de espectroscopia Raman indicam que com a interação por ligação de hidrogênio a simetria molecular do índigo diminua. Os estudos por espectroscopia com resolução temporal mostraram que o índigo apresenta transferência de próton no estado excitado (ESIPT) de um dos amino grupos para a carbonila adjacente; após esta transferência, há a formação da espécie mono-enol a qual relaxa ao estado fundamental após 120 ps, através de intersecção cônica, o que explica a alta fotoestabilidade do corante. No caso da mistura aquecida a 130 °C os resultados, obtidos pela primeira vez para uma molécula imobilizada em argila, confirmam que o índigo encontra-se em um ambiente hidrofílico, considerando o tempo de vida de decaimento do estado excitado (3,0 ps), comparável ao do índigo carmim em solução aquosa (2,7 ps). O tempo de vida também é muito curto, comparado ao em solução de DMSO (120 ps) o que pode explicar a alta estabilidade do corante quando dentro do microcanal da argila. Finalmente, constatou-se que o dehidroíndigo não é responsável pela coloração de simulantes de Azul Maia, a qual resulta de alterações no espectro de absorção no visível do corante que ocorrem com a interação com a argila / In this work, the role played by the microenvironment on the chemical and photochemical stability of dyes was investigated. The factors responsible for the enhanced stability of indigo when interacting with palygorskite were detailed studied; the indigo and palygorskite system constitutes a simulant of Maya Blue, a historical pigment with properties which are controversially described in the literature. The dyes here investigated were indigo, dehydroindigo, alizarin, purpurin, luteolin and &#946;-carotene; the microenvironment was provided by palygorskite, sepiolite, montmorillonite, laponite and a layered double hydroxide (Al3+ e Mg2+, 3:1). Palygorskite and sepiolite are the only clays with micropores in their structure. Several characterization techniques were employed, namely vibrational spectroscopy (Raman and infrared), UV-VIS electronic absorption spectroscopy, X-ray diffractometry, thermal analysis (TG and DSC), CG-MS, HPLC-MS, surface area and porosity determination (N2 isotherm adsorption) and scanning electron microscopy. Two sub-picosecond time resolved techniques (transient absorption and infrared absorption) were also used. The indigo+palygorskite system corresponds to the intimate mixture of both solids, followed by heating; from 70 °C the mixture attains a greenish hue and an enhanced chemical and photochemical stability. Both stability and color change increase with the heating temperature, which also leads to loss of zeolitic and coordinated water (70 to 150 °C and 170 to 280 °C, respectively). Vibrational and electronic spectroscopies indicate that, in the Maya Blue simulants, the dye interacts with the clay through hydrogen bonds with the coordinated water molecules. Such interaction, however, is only possible with the removal of the zeolitic water, which starts at 70 °C. At temperatures above 170 °C, both vibrational and electronic spectral profiles change, indicating that the interaction is now proceeding directly with the metals that are at the internal borders of the micropores. Results from Raman spectroscopy suggest that with the hydrogen bond and metal interaction a symmetry lowering occurs. Time resolved spectroscopy results show that indigo present an excited state intramolecular proton transfer from one of the NH to the adjacent carbonyl group, originating a mono-enol species, which decays to the ground state after 120 ps through a conical intersection. Such fast decay explains the high photochemical stability of indigo. In the case of the ind+paly mixture heated at 130 °C, the time resolved data obtained for the first time for a dye+clay system confirms that indigo is in a hydrophilic environment, taking into account the excited state lifetime (3.0 ps), comparable to indigo carmine in aqueous solution (2.7 ps). The excited state lifetime of indigo in the clay is also very short when compared to the experimental data for the dye in DMSO solution (120 ps), which is possibly an explanation for the dye high stability when inside the clay micropores. Finally, dehydroindigo was not found to be responsible for the color of Maya Blue simulants, which results from the spectral changes in the dye absorption spectrum originated by the interaction with the clay.

Projetos desenvolvidos para instalações de animais de laboratório adotam limites estreitos de ventilação e de climatização de ambientes a fim de assegurar um ambiente definido na sala de animais, denominada de macroambiente. Entretanto o tratamento do ar de uma sala não atinge, de forma eficiente, a gaiola em que o animal se encontra alojado (microambiente). O desenvolvimento de sistemas de ventilação microambiental (VMA), que possibilita um fluxo unidirecional de ar forçado para o interior das gaiolas dos animais; associado à modernas tecnologias de termoeletricidade, termosensores e termocontroladores permitem um acurado controle de variáveis climáticas em sistemas microambientais para roedores. No entanto, nota-se carência de dados no que se refere ao binômio temperatura e velocidade do ar para estes sistemas. O objetivo deste estudo foi o desenvolvimento de uma Câmera de Preferencia Climática (CPC) do tipo labiritno que permitisse analise da escolha de ambientes (binômio temperatura e velocidade do ar) para roedores de laboratório. O sistema de Ventilação Microambiental (VMA), possibilitou a climatização da CPC, que foi dividida em quatro sub-camaras, em temperaturas pré-determinadas de 22, 26, 30 e 34oC, todas à uma velocidade de ar de 0,3m/s. A CPC foi mantida numa sala climatizada a 18 &plusmn; 2 Co, por um sistema convencional de compressão do freon&reg; para conforto humano. A massa de ar destinada a cada uma das sub-camaras era aquecida via resistência elétrica, controlada eletronicamente, para cada uma das temperaturas pré-determinadas. O comportamento exploratório dos ratos na CPC foi registrado pelo sistema de analise comportamental Ethovision Color-Pro 2.3® e analisado pelos parâmetros de tempo de permanência (Tp) (min.), freqüência de entrada (N) dos animais em cada sub-câmara climatizada e distancia percorrida (cm) dentro da CPC, em um período de 48 horas. Os testes físicos realizados com o equipamento mostraram que este foi capaz de manter as temperaturas pré-determinadas na faixa de &pludmn; 0,1 oC e velocidade de ar constante. Os resultados parciais da analise estatística do comportamento exploratório de ratos demonstraram que, no grupo controle, ou seja, onde as quatro sub-camaras estavam climatizadas em 22 Co, o Tp dos animais em cada uma das sub-camaras foi igual, evidenciando que as condições climáticas eram idênticas e portanto não houve preferência. No grupo experimental, ou seja, com as quatro camaras climatizadas em 22, 26, 30 e 34oC, aleatoriamente, o ratos demonstraram o maior Tp, ou seja, sua preferencia pelas menores temperaturas, 22 e 26oC; à despeito da velocidade de ar de 0,3 m/s, no interior das camaras.A CPC mostrou-se eficiente para estabelecer simulações do microambiente, em termos de preferência climática de roedores mantidos em laboratório. A CPC se mostra como um sistema eficiente também para estudos sobre a termoregulação em ensaios biológicos com animais, como os estudos de neurotoxinas do veneno de aranha Phoneutria nigriventer / Projects concerned with laboratory animals facilities usually adopt narrow range of ventilation and climatization variables to assure the define ambient. Such projects are concerned to the animal room (macro-environmental ambient) and this technology have a limitated operation to reach the micro-environmental (animal cages) ambients. The development of the intracage ventilation systems (ICV), which operating by an air influx inside the cages, beside the new technology on thermoelectric modules, thermosensitive and thermocontrolers materials, allows an accurate control of the microclimatic variables for animal enclosure. For this purpose we developed an experimental prototype, a climatic preference chamber (CPC) that allows to laboratory animal (e.g. rats) choose their preferred ambient under a different temperature conditionated chambers by the ICV thecnology, simulating the animal cage enclosure condition. That technology is characterized by delivery of air insufflation of 0.3 m/s, and regulated with differents temperatures in each chamber (22, 26, 30 and 34oC). The exploratory behavior of the rat at multiplechoise preference test was registered by Ethovision Color-Pro 2.3® system. This system allowed to the analysis of the climatic preference defined by the parameter time spent in different microclimates conditions. The analyses of the exploratory behavior demonstrated that rats spent much time in smaller temperature microclimates, as a 22 and 26 oC, indicating a preference for cooler ambients, in spite of the ICV air speed of 0,3m/s. The aim of this work is simulate laboratory animal housing condition to study the microclimate preference and also in termoregulation studies with laboratory rodents assays, like neurotoxins of the the spider Phoneutria nigriventer venom, that is an important tool to biochemical studies

Neste trabalho foi investigado o efeito exercido por microambientes sobre a estabilidade química e fotoquímica de corantes. Em particular, estudaram-se os fatores responsáveis pelo aumento da estabilidade química e fotoquímica de índigo quando em interação com paligorsquita, que compõem o pigmento histórico Azul Maia, sobre o qual ainda havia controvérsias na literatura. Os corantes investigados foram índigo, dehidroíndigo, alizarina, purpurina, luteolina e &#946;-caroteno; os microambientes foram proporcionados pelas argilas paligorsquita, sepiolita, montmorilonita, laponita e HDL de Al3+ e Mg2+ (3:1). Paligorsquita e a sepiolita são as únicas argilas que apresentam microporos em sua estrutura. As técnicas de caracterização empregadas neste trabalho foram: espectroscopia vibracional (Raman e absorção no infravermelho), espectroscopia de absorção no UV-VIS, difratometria de raios X, análise térmica (TG e DSC), CG-MS, HLPC-MS, medidas de área superficial por isoterma de adsorção de N2 e SEM. Duas técnicas com resolução temporal em escala de sub-picosegundos (absorção de transiente e infravermelho resolvido no tempo) foram utilizadas. O sistema índigo+paligorsquita corresponde à mistura dos dois sólidos, seguida de aquecimento, sendo que a partir de 70 °C a coloração da mistura adquire tonalidade esverdeada e também apresenta maior estabilidade química e fotoquímica. Essa estabilidade e também a alteração na cor aumentam com a temperatura de aquecimento da mistura e o intervalo considerado engloba as temperaturas de perda de água zeolítica (70 - 150 °C) e coordenada (170 - 280 °C) da estrutura da argila. Os resultados de espectroscopia vibracional e eletrônica dos simulantes de Azul Maia indicam que o índigo interage através de ligações de hidrogênio com as moléculas de água coordenada. Essa interação, entretanto, só é possível com a remoção da água zeolítica, o que ocorre a partir de 70 °C. Com aquecimento em temperaturas acima de 170 °C o comportamento do espectro eletrônico e vibracional se altera, indicando a formação direta de complexos com os metais presentes nas bordas internas dos microporos. Os resultados de espectroscopia Raman indicam que com a interação por ligação de hidrogênio a simetria molecular do índigo diminua. Os estudos por espectroscopia com resolução temporal mostraram que o índigo apresenta transferência de próton no estado excitado (ESIPT) de um dos amino grupos para a carbonila adjacente; após esta transferência, há a formação da espécie mono-enol a qual relaxa ao estado fundamental após 120 ps, através de intersecção cônica, o que explica a alta fotoestabilidade do corante. No caso da mistura aquecida a 130 °C os resultados, obtidos pela primeira vez para uma molécula imobilizada em argila, confirmam que o índigo encontra-se em um ambiente hidrofílico, considerando o tempo de vida de decaimento do estado excitado (3,0 ps), comparável ao do índigo carmim em solução aquosa (2,7 ps). O tempo de vida também é muito curto, comparado ao em solução de DMSO (120 ps) o que pode explicar a alta estabilidade do corante quando dentro do microcanal da argila. Finalmente, constatou-se que o dehidroíndigo não é responsável pela coloração de simulantes de Azul Maia, a qual resulta de alterações no espectro de absorção no visível do corante que ocorrem com a interação com a argila / In this work, the role played by the microenvironment on the chemical and photochemical stability of dyes was investigated. The factors responsible for the enhanced stability of indigo when interacting with palygorskite were detailed studied; the indigo and palygorskite system constitutes a simulant of Maya Blue, a historical pigment with properties which are controversially described in the literature. The dyes here investigated were indigo, dehydroindigo, alizarin, purpurin, luteolin and &#946;-carotene; the microenvironment was provided by palygorskite, sepiolite, montmorillonite, laponite and a layered double hydroxide (Al3+ e Mg2+, 3:1). Palygorskite and sepiolite are the only clays with micropores in their structure. Several characterization techniques were employed, namely vibrational spectroscopy (Raman and infrared), UV-VIS electronic absorption spectroscopy, X-ray diffractometry, thermal analysis (TG and DSC), CG-MS, HPLC-MS, surface area and porosity determination (N2 isotherm adsorption) and scanning electron microscopy. Two sub-picosecond time resolved techniques (transient absorption and infrared absorption) were also used. The indigo+palygorskite system corresponds to the intimate mixture of both solids, followed by heating; from 70 °C the mixture attains a greenish hue and an enhanced chemical and photochemical stability. Both stability and color change increase with the heating temperature, which also leads to loss of zeolitic and coordinated water (70 to 150 °C and 170 to 280 °C, respectively). Vibrational and electronic spectroscopies indicate that, in the Maya Blue simulants, the dye interacts with the clay through hydrogen bonds with the coordinated water molecules. Such interaction, however, is only possible with the removal of the zeolitic water, which starts at 70 °C. At temperatures above 170 °C, both vibrational and electronic spectral profiles change, indicating that the interaction is now proceeding directly with the metals that are at the internal borders of the micropores. Results from Raman spectroscopy suggest that with the hydrogen bond and metal interaction a symmetry lowering occurs. Time resolved spectroscopy results show that indigo present an excited state intramolecular proton transfer from one of the NH to the adjacent carbonyl group, originating a mono-enol species, which decays to the ground state after 120 ps through a conical intersection. Such fast decay explains the high photochemical stability of indigo. In the case of the ind+paly mixture heated at 130 °C, the time resolved data obtained for the first time for a dye+clay system confirms that indigo is in a hydrophilic environment, taking into account the excited state lifetime (3.0 ps), comparable to indigo carmine in aqueous solution (2.7 ps). The excited state lifetime of indigo in the clay is also very short when compared to the experimental data for the dye in DMSO solution (120 ps), which is possibly an explanation for the dye high stability when inside the clay micropores. Finally, dehydroindigo was not found to be responsible for the color of Maya Blue simulants, which results from the spectral changes in the dye absorption spectrum originated by the interaction with the clay.

Projetos desenvolvidos para instalações de animais de laboratório adotam limites estreitos de ventilação e de climatização de ambientes a fim de assegurar um ambiente definido na sala de animais, denominada de macroambiente. Entretanto o tratamento do ar de uma sala não atinge, de forma eficiente, a gaiola em que o animal se encontra alojado (microambiente). O desenvolvimento de sistemas de ventilação microambiental (VMA), que possibilita um fluxo unidirecional de ar forçado para o interior das gaiolas dos animais; associado à modernas tecnologias de termoeletricidade, termosensores e termocontroladores permitem um acurado controle de variáveis climáticas em sistemas microambientais para roedores. No entanto, nota-se carência de dados no que se refere ao binômio temperatura e velocidade do ar para estes sistemas. O objetivo deste estudo foi o desenvolvimento de uma Câmera de Preferencia Climática (CPC) do tipo labiritno que permitisse analise da escolha de ambientes (binômio temperatura e velocidade do ar) para roedores de laboratório. O sistema de Ventilação Microambiental (VMA), possibilitou a climatização da CPC, que foi dividida em quatro sub-camaras, em temperaturas pré-determinadas de 22, 26, 30 e 34oC, todas à uma velocidade de ar de 0,3m/s. A CPC foi mantida numa sala climatizada a 18 &plusmn; 2 Co, por um sistema convencional de compressão do freon&reg; para conforto humano. A massa de ar destinada a cada uma das sub-camaras era aquecida via resistência elétrica, controlada eletronicamente, para cada uma das temperaturas pré-determinadas. O comportamento exploratório dos ratos na CPC foi registrado pelo sistema de analise comportamental Ethovision Color-Pro 2.3® e analisado pelos parâmetros de tempo de permanência (Tp) (min.), freqüência de entrada (N) dos animais em cada sub-câmara climatizada e distancia percorrida (cm) dentro da CPC, em um período de 48 horas. Os testes físicos realizados com o equipamento mostraram que este foi capaz de manter as temperaturas pré-determinadas na faixa de &pludmn; 0,1 oC e velocidade de ar constante. Os resultados parciais da analise estatística do comportamento exploratório de ratos demonstraram que, no grupo controle, ou seja, onde as quatro sub-camaras estavam climatizadas em 22 Co, o Tp dos animais em cada uma das sub-camaras foi igual, evidenciando que as condições climáticas eram idênticas e portanto não houve preferência. No grupo experimental, ou seja, com as quatro camaras climatizadas em 22, 26, 30 e 34oC, aleatoriamente, o ratos demonstraram o maior Tp, ou seja, sua preferencia pelas menores temperaturas, 22 e 26oC; à despeito da velocidade de ar de 0,3 m/s, no interior das camaras.A CPC mostrou-se eficiente para estabelecer simulações do microambiente, em termos de preferência climática de roedores mantidos em laboratório. A CPC se mostra como um sistema eficiente também para estudos sobre a termoregulação em ensaios biológicos com animais, como os estudos de neurotoxinas do veneno de aranha Phoneutria nigriventer / Projects concerned with laboratory animals facilities usually adopt narrow range of ventilation and climatization variables to assure the define ambient. Such projects are concerned to the animal room (macro-environmental ambient) and this technology have a limitated operation to reach the micro-environmental (animal cages) ambients. The development of the intracage ventilation systems (ICV), which operating by an air influx inside the cages, beside the new technology on thermoelectric modules, thermosensitive and thermocontrolers materials, allows an accurate control of the microclimatic variables for animal enclosure. For this purpose we developed an experimental prototype, a climatic preference chamber (CPC) that allows to laboratory animal (e.g. rats) choose their preferred ambient under a different temperature conditionated chambers by the ICV thecnology, simulating the animal cage enclosure condition. That technology is characterized by delivery of air insufflation of 0.3 m/s, and regulated with differents temperatures in each chamber (22, 26, 30 and 34oC). The exploratory behavior of the rat at multiplechoise preference test was registered by Ethovision Color-Pro 2.3® system. This system allowed to the analysis of the climatic preference defined by the parameter time spent in different microclimates conditions. The analyses of the exploratory behavior demonstrated that rats spent much time in smaller temperature microclimates, as a 22 and 26 oC, indicating a preference for cooler ambients, in spite of the ICV air speed of 0,3m/s. The aim of this work is simulate laboratory animal housing condition to study the microclimate preference and also in termoregulation studies with laboratory rodents assays, like neurotoxins of the the spider Phoneutria nigriventer venom, that is an important tool to biochemical studies

A study of the microclimate of erosion control treatments was carried out in two habitat types in a semiarid southeastern Utah, salt desert shrub area. The soils are highly eroded Mancos shale and support a low density of salt desert shrub species which offers little protection against high intensity summer convectional rainstorms. Gully plugs and contour furrows had been installed by the Bureaus of Reclamation and Land Management to prevent runoff of soil laden water which results from these summer rainfalls. Formerly this sediment was carried to the Colorado River and deposited in Lake Powell. The purpose to t he study was to measure some parameters that influenced establishment and survival of seedlings which would stabilize the structures and increase the productivity of the area. Mature, indigenous species close to the structures had greater vigor and provided a larger, more constant seed source. This seed source was important since original seedlings of introduced grasses have failed to establish and stabilize the structures . Following favorable late winter and early spring precipitation, high numbers of seedlings emerged, but few survived into mid-summer. Those that did survive were found only at or near the high water line of the gully plugs and in the bottom of contour furrows. To aid in the explanation of the differential seedling survival, soil surface moisture following rainstorms, physical and chemical soil characteristics, net radiation, soil surface temperature, and evaporation were investigated. Sampling of the 15 centimeter soil surface in the environs of the structures showed that two days after either heavy or light rainfalls, essentially no available water remained on the sloping throw positions of the structures. The bottom of the gully plugs remained flooded for several days. As a result, the seedlings were drowned. Only at the high water line of the gully plugs and in the bottom of furrows was there enough water to support seedling growth in summer. Runoff water from summer storms carried fine material into the structures, which covered and destroyed seedlings, plus lowered infiltration and permitted much of the water to be lost through evaporation. The runoff water also carried salts brought to the surface by the desert shrubs or by the upward movement of water during evaporation from the soil surface. Sampling of soil in the environs of the structures showed no substantial build up of salt, indicating that leaching had occurred, or still was occurring. This does not mean that a build up will not result in the future. Seasonal salt distribution showed that lowest salinity levels did not always coincide with springtime when seedlings generally emerged. Established seedlings were also exposed to a harsh environment of high soil surface temperatures (over 60 C) and high soil water evaporation rates that were detrimental to their survival. In summary, the study showed that the erosion control structures have created a microenvironment much different from the undisturbed soil, which only permits the establishment of species with much different tolerances from the indigenous species.

Complementary greenhouse and field studies investigated the effects of ambient environmental conditions on cattle dungpat moisture, temperature, nutrient concentration, and crust formation dynamics, which in turn influence seed germination and seedling establishment in dungpats. 'Hycrest' crested wheatgrass [Agropyron desertorum (Fisch. ex Link) X A. cristatum (L.) Gaert.] was used as a representative revegetation species. After collecting feces from Holstein steers that had been fed crested wheatgrass seeds, uniform dungpats were prepared and placed on two soil types (loam and coarse sand) in containers under three watering treatments (field capacity, 1/2 field capacity, and no water) in the greenhouse. Dungpat and underlying soil microenvironmental factors, and germination and seedling development, were monitored for 14 weeks. Moisture and temperature were favorable for germination during the first 4 weeks, but increasing crust thickness prevented most of the developing seedlings from emerging from dungpats. Seedling emergence, development, and survival were greatest at the peripheral region of dungpats on the loam soil at moisture contents of 1/2 field capacity or higher. Uniform dungpats containing passed seeds and unpassed seeds were placed on a silt loam soil in the field in the spring (late April 1993) under natural and above-normal precipitation regimes and in the fall (late October 1993) under natural precipitation. Unpassed seeds were also broadcast and drill-seeded into soil seedbeds at the same times. Microenvironmental factors and germination and plant establishment were monitored for 49 weeks in the spring experiment and 17 weeks in the fall experiment. For dungpat treatments, seedling emergence and survival were greater for unpassed than passed seeds in both precipitation regimes; however, sufficient numbers of seedlings established from passed seeds, and these plants had greater biomass and similar or greater seed production than plants from unpassed seeds. Slight changes in nutrients in dungpats and underlying soil indicated that partial decomposition and mineralization of dungpats had occurred by the end of the spring experiment. Both studies indicate that dungpat microenvironmental factors are greatly influenced by ambient moisture, temperature, and insolation, and by the nature of the underlying soil. These studies support the claim that plants established in dungpats could serve as nuclei of seed production for surrounding areas.

Le cancer ovarien est la 1ère cause de décès par cancer gynécologique. A ce jour, le traitement le plus efficace consiste en une exérèse la plus complète possible de la tumeur associée à une chimiothérapie à base de sel de platine et de taxanes. Cependant, la survie globale des patientes à 5 ans reste faible (environ 40%), due à un fort taux de récidive et au développement d'une résistance aux traitements. Les interactions entre les cellules tumorales ovariennes (CTO) et le microenvironnement semblent être impliquées dans la progression de la maladie et l'acquisition de cette chimiorésistance. Parmi les cellules du microenvironnement tumoral, nous avons pu isoler des cellules stromales mésenchymateuses (MSC) à partir de biopsies de carcinome ovarien. Ces MSC associées au cancer (CA-MSC) ont la capacité d'induire une résistance des CTO au traitement par carboplatine. Afin de mieux comprendre les mécanismes conduisant des MSC à sécréter des facteurs pro-tumoraux, dans un contexte de cancer ovarien, nous avons mis en place un modèle à partir de MSC issues de donneurs ne présentant pas de cancer. Nous avons observé que la présence d'un environnement tumoral ovarien modifie le phénotype de MSC de moelle osseuse (BM-MSC) physiologiques, en entrainant notamment la sécrétion par ces CA-MSC " induites " de facteurs chimioprotecteurs pour les CTO. Parmi les facteurs sécrétés, nous avons identifié des chimiokines telles que CXCL1, CXCL2 et IL-8 comme des cibles thérapeutiques intéressantes pour lutter contre la résistance à la chimiothérapie. En effet, les CA-MSC et les CA-MSC " induites " sécrètent plus de CXCL1, CXCL2 et d'IL-8 que les BM-MSC. En outre, l'inhibition des récepteurs de ces chimiokines (CXCR1 et CXCR2) sensibilise les CTO au carboplatine, même en présence des sécrétions des CA-MSC et des CA-MSC " induites ". Ces expériences in vitro ont été confirmées in vivo dans un modèle expérimental murin. En effet, la co-injection à des souris immunodéprimées de MSC avec des CTO humaines entraine une protection des CTO vis-à-vis du carboplatine comparée à l'injection de CTO seules. Le co-traitement avec un inhibiteur de CXCR1 et de CXCR2 a permis de sensibiliser les CTO au carboplatine et d'empêcher la chimiorésistance induite par les MSC. En effectuant une étude rétrospective évaluant la concentration de ces chimiokines au moment du diagnostic, nous avons confirmé que les patientes qui seront a posteriori " résistantes " au carboplatine ont une concentration sérique de ces chimiokines qui est plus élevées que les patientes du groupe " sensibles " au carboplatine. Outre leur rôle direct dans l'acquisition de la chimiorésistance, les chimiokines comme CXCL1, CXCL2 et IL-8 peuvent être impliquées dans la régulation du système immunitaire. Nous avons montré que les CA-MSC étaient capables de modifier le phénotype des macrophages vers un phénotype M2 décrit comme pro-tumoral. En effet, ces macrophages ainsi polarisés ont un pouvoir cytotoxique dirigé contre les CTO inférieur aux macrophages non stimulés. L'ajout d'un inhibiteur de CXCR1 et de CXCR2 restaure le pouvoir cytotoxique initial des macrophages, même en présence des sécrétions des CA-MSC. Ainsi nos travaux suggèrent que les CA-MSC pourraient provenir de MSC physiologiques qui, au contact d'un environnement tumoral ovarien, vont acquérir un phénotype capable d'induire la sécrétion de facteurs chimioprotecteurs pour les CTO et de polariser les macrophages vers un phénotype moins cytotoxique pour les CTO. Ces deux phénomènes pro-tumoraux peuvent être inhibés par l'utilisation d'un inhibiteur des récepteurs de CXCR1 et de CXCR2. Ainsi ces récepteurs des chimiokines, semblent être des cibles thérapeutiques intéressantes afin de sensibiliser les CTO au carboplatine et traiter plus efficacement la tumeur. Ceci pourrait permettre d'éviter les récidives des cancers ovariens qui sont, à l'heure actuelle, observées chez plus de 70% des patientes. / Ovarian cancer is the leading cause of gynecological cancer death. To date, the most effective treatment consists of the complete excision of the tumor associated with chemotherapy based on platinum salts and taxanes. However, the 5-year overall survival remains low (close to 40%) due to a high rate of recurrence and development of resistance to treatments. Disease progression and the acquisition of this chemoresistance seem to be due to interactions between ovarian tumor cells (OTC) and the microenvironment. Amidst the cells of the tumor microenvironment, we were able to isolate mesenchymal stromal cells (MSC) from tumor biopsies of patients with ovarian adenocarcinoma. These cancer-associated MSC (CA-MSC) have the ability to induce resistance to carboplatin in OTC. In order to understand the mechanisms leading to the secretion of pro-tumoral factors by the CA-MSC in the context of ovarian cancer, we have developed a model based on the in vitro MSC culture of from healthy donors in tumor conditioning media. We have observed that an ovarian tumor environment modifies the physiological phenotype of bone marrow MSC (BM-MSC), leading in particular to the secretion by these "induced" CA-MSC of chemoprotective factors for OTC. Among these secreted factors, we have identified chemokines such as CXCL1, CXCL2 and IL-8 as therapeutic targets in order to control drug resistance. In fact, CA-MSC and "induced" CA-MSC secrete more CXCL1, CXCL2 and IL-8 than BM-MSC and the use of an inhibitor of their receptors (CXCR1 and CXCR2) sensitized OTC to carboplatin even in the presence of CA-MSC and " induced " CA-MSC secretions. These in vitro experiments have been confirmed in an experimental mouse model in vivo. Indeed, the co-injection of MSC with OTC yielded a greater protection of OTC to carboplatin compared with the OTC injection alone. Co-treatment with a CXCR1 and CXCR2 inhibitor resulted in sensitization of OTC to carboplatin and prevention of MSC-induced chemoresistance. We conducted a retrospective study evaluating the concentration of these chemokines at the time of diagnosis. We thus showed that patients who are a posteriori "resistant" to carboplatin have a higher concentration of chemokines than patients belong to the "sensitive" group to carboplatin. In addition to their direct role concerning the acquisition of chemoresistance, chemokines such as CXCL1, CXCL2 and IL-8 may be involved in the immune system regulation. In this context, we showed that CA-MSC were able to modify the phenotype of macrophages into a M2 phenotype described in literature to have a pro-tumoral activity. Indeed, these polarized macrophages present a lower cytotoxic capacity against OTC than unstimulated macrophages. CXCR1 and CXCR2 inhibitor restores the initial cytotoxic activity of macrophages even in the presence of CA-MSC secretions. Thus, our work suggests that CA-MSC could originate from physiological MSC which, in contact with an ovarian tumor environment, acquire a phenotype capable of inducing the secretion of chemoprotective factors for CTO and of polarizing macrophages into a less cytotoxic phenotype for OTC. These two pro-tumoral mechanisms can be inhibited by the use of CXCR1 and CXCR2 receptor inhibitors emphasizing the role of these chemokines in the development of a chemoresistance and showing how important is to go further is this study. Finally, these chemokines receptors seem to be therapeutic targets in order to sensitize OTC to carboplatin and to potentialize actual treatments. This could prevent the recurrence of ovarian cancers that are presently observed in more than 70% of patients.

T-cells are present in the immune system to fight against invaders. Once their job is done, suppressing their activity is an important step in maintaining a proper immune response. Myeloid derived suppressor cells (MDSCs) are immune cells that suppress T-cell activity. Currently, MDSCs are defined as a heterogeneous population of immature cells that are derived in the bone marrow and travel to the site of inflammation or cancer. Two major subtypes of MDSCs have been identified in mice and humans, monocyte-like MDSCs (M-MDSC) and granulocyte MDSCs (G-MDSC). G-MDSCs typically make up the majority of the total population of MDSCs but are less T-cell suppressive than M-MDSCs. One of the major problems in the study of MDSCs is that the current marker system for subtypes does not differentiate between precursor MDSCs (lacking suppressive ability) and functional MDSCs (those with suppressive ability). Therefore, using cancer models in mice, we investigated the development and potential to classify precursor MDSCs from functional MDSCs. While MDSCs have been highlighted as a target cell to inhibit in cancer, in other conditions, such as pregnancy, MDSCs have been shown to be beneficial in maintaining a normal pregnancy. Therefore, targeting the increase of MDSCs in abnormal pregnancy conditions like pre-eclampsia may act as a prevention or therapeutic strategy. Finally, it is known that many dietary components can act as modulators of immune cells. Specifically, the polyphenol like phytochemical, curcumin has been shown to act as an anti-inflammatory agent with the potential to modulate multiple immune cells. Therefore, we propose two different studies to investigate the potential of curcumin as either an inhibitor and/or promotor of MDSCs in a disease-specific context. Together the role of phytochemicals as immunomodulators of MDSCs is still very young, in part due to the complexity of phytochemicals themselves, but the studies cited here provide evidence that the field is ripe for additional questions to be asked.

Tumor microenvironmental conditions play a vital role in promoting metastasis and tumor recurrence. Due to inefficient vasculature, cancer cells experience hypoxia, glucose deprivation and low pH even during the early stages of tumor growth. Tumor cells are proposed to adapt to these microenvironmental conditions by acquiring increased migratory and invasion potential and tumor initiating ability. Our research addresses the effect of these biochemical factors of the tumor microenvironment (TME) on motility, epithelial to mesenchymal transition (EMT) and stemness of non-small cell lung cancer (NSCLC). NCI-H292 and NCI-H1650 NSCLC cell lines were used to measure the effect of the above mentioned TME conditions. Apart from acidic pH, low glucose and hypoxia, the effect of high glucose conditions was also measured on H292 and H1650 cell lines. Acidic pH, high and low glucose conditions were observed to have no effect on the motility, EMT and stemness of H1650 cell line. Hence, use of this cell line was discontinued and no further treatment conditions were tested on this cell line. In H292 cell line, acidic pH, low glucose and tumor like conditions combined together (acidic pH + low glucose + hypoxia) [AP+LG+HYP] significantly decreased motility whereas hypoxia significantly increased the motility of H292 cells. High glucose did not affect the motility of H292 cells. Although N-cadherin, a mesenchymal marker, expression was significantly upregulated by acidic pH, high and low glucose conditions, no direct correlation was observed between N-cadherin expression and motility. E-cadherin expression was not affected by acidic pH, high and low glucose conditions. An increase in N-cadherin expression and no change in E-cadherin expression under these conditions might be an indication of partial EMT. Hypoxia and AP+LG+HYP did not alter the expression of E-cadherin and N-cadherin. Although expression of vimentin, another mesenchymal marker, and Sox2, a cancer stem cell marker (CSC), was observed at the mRNA level, no expression of vimentin and Sox2 proteins was observed in H292 cells under any of these treatment conditions. The expression of OCT4, another CSC marker, was also not observed at the protein level in H292 cells. HIF-1α expression was observed in H292 cells under normoxic conditions and was unaffected by hypoxia and AP+LG+HYP. Therefore our research indicates that the effect of these TME conditions might be different on different cancer cell lines or cancer types. Not all cancers may depend on EMT for metastasis. An increase in metastasis under hypoxia may be independent of HIF-1α.