Ames dwarf (df/df) mice are deficient in anterior pituitary hormones: growth hormone (GH), thyroid stimulating hormone (TSH), and prolactin (PRL) due to a spontaneous, homozygous mutation of prop1[superscript df] gene. These dwarf mice exhibit characteristics such as delayed growth and development coupled with delayed aging, increased lifespan, overall increased insulin sensitivity, as well as resistance to certain diseases and cancers. The mutant mice possess low blood glucose, low serum insulin, and lower body temperature. Their enhanced longevity (about 40-60% longer lifespan than normal mice) is associated with their GH deficiency and disruption in the somatotropic axis (GH/IGF-1 hormonal pathway) as well as increased insulin sensitivity, which is supported by other mutant mouse models for longevity like Snell dwarfs and growth hormone receptor knock-out (GHRKO) mice. When young male Ames dwarf mice were treated with GH replacement therapy, they showed increased body growth to nearly match the normal mouse phenotype. In conjunction to an increase in physical growth, however, GH treatment also decreases the longevity and insulin sensitivity that are characteristic of these mice to levels seen in normal mice. Because of the lack of TSH, they also have undetectable levels of Thyroxine (T4). While T4 treatment didn't increase bodyweight of dwarfs to the same extent as GH treatment, the T4 treated mice retained their enhanced lifespan. Although df/df mice have enhanced whole-body insulin sensitivity, the male skeletal muscle was previously shown to be less responsive to insulin than their liver. In our study we analyzed the insulin signaling pathway in skeletal muscle from female mice after treatment with GH or GH combined with T4. Gene expression and protein expression were investigated in the skeletal muscle of female Ames dwarf mice that were treated with GH or GH and T4 therapy. Real Time Polymerase Chain Reaction (RT-PCR) was used to analyze the expression of mRNA involved with insulin and GH signaling, while western blots were used to analyze protein expression. This project found that female Ames skeletal muscle didn't respond to GH treatment to the same extent as males, and that GH and T4 treatment tends to neutralize the effects seen in GH-only treatment.

Mammals are born on a precocial–altricial continuum. Altricial species produce helpless neonates with closed distant organs incapable of locomotion, whereas precocial species give birth to well-developed young that possess sophisticated sensory and locomotor capabilities. Previous studies suggest that distinct patterns of cortex development differ between precocial and altricial species. This study compares patterns of neocortex neurogenesis and maturation in the precocial guinea pig and altricial dwarf rabbit, both belonging to the taxon of Glires. We show that the principal order of neurodevelopmental events is preserved in the neocortex of both species. Moreover, we show that neurogenesis starts at a later postconceptional day and takes longer in absolute gestational days in the precocial than the altricial neocortex. Intriguingly, our data indicate that the dwarf rabbit neocortex contains a higher abundance of highly proliferative basal progenitors than the guinea pig, which might underlie its higher encephalization quotient, demonstrating that the amount of neuron production is determined by complex regulation of multiple factors. Furthermore, we show that the guinea pig neocortex exhibits a higher maturation status at birth, thus providing evidence for the notions that precocial species might have acquired the morphological machinery required to attain their high functional state at birth and that brain expansion in the precocial newborn is mainly due to prenatally initiating processes of gliogenesis and neuron differentiation instead of increased neurogenesis. Together, this study reveals important insights into the timing and cellular differences that regulate mammalian brain growth and maturation and provides a better understanding of the evolution of mammalian altriciality and presocialit

Mammals are born on a precocial–altricial continuum. Altricial species produce helpless neonates with closed distant organs incapable of locomotion, whereas precocial species give birth to well-developed young that possess sophisticated sensory and locomotor capabilities. Previous studies suggest that distinct patterns of cortex development differ between precocial and altricial species. This study compares patterns of neocortex neurogenesis and maturation in the precocial guinea pig and altricial dwarf rabbit, both belonging to the taxon of Glires. We show that the principal order of neurodevelopmental events is preserved in the neocortex of both species. Moreover, we show that neurogenesis starts at a later postconceptional day and takes longer in absolute gestational days in the precocial than the altricial neocortex. Intriguingly, our data indicate that the dwarf rabbit neocortex contains a higher abundance of highly proliferative basal progenitors than the guinea pig, which might underlie its higher encephalization quotient, demonstrating that the amount of neuron production is determined by complex regulation of multiple factors. Furthermore, we show that the guinea pig neocortex exhibits a higher maturation status at birth, thus providing evidence for the notions that precocial species might have acquired the morphological machinery required to attain their high functional state at birth and that brain expansion in the precocial newborn is mainly due to prenatally initiating processes of gliogenesis and neuron differentiation instead of increased neurogenesis. Together, this study reveals important insights into the timing and cellular differences that regulate mammalian brain growth and maturation and provides a better understanding of the evolution of mammalian altriciality and presociality.

Mergers are known to be essential in the formation of large scale structures and to have a significant role in the history of galaxy formation and evolution. Besides a morphological transformation, mergers induce important bursts of star formation. These starburst are characterised by high Star Formation Efficiencies (SFEs) and Specific Star Formation Rates, i.e., high Star Formation Rates (SFR) per unit of gas mass and high SFR per unit of stellar mass, respectively, compared to spiral galaxies. At all redshifts, starburst galaxies are outliers of the sequence of star-forming galaxies defined by spiral galaxies. We have investigated the origin of the starburst-mode of star formation, in three local interacting systems: Arp 245, Arp 105 and NGC7252. We combined high-resolution JVLA observations of the 21-cm line, tracing the Hi diffuse gas, with UV GALEX observations, tracing the young star-forming regions. We probe the local physical conditions of the Inter- Stellar Medium (ISM) for independent star-forming regions and explore the atomic-to-dense gas transformation in different environments. The SFR/H i ratio is found to be much higher in central regions, compared to outer regions, showing a higher dense gas fraction (or lower Hi gas fraction) in these regions. In the outer regions of the systems, i.e., the tidal tails, where the gas phase is mostly atomic, we find SFR/H i ratios higher than in standard Hi-dominated environments, i.e., outer discs of spiral galaxies and dwarf galaxies. Thus, our analysis reveals that the outer regions of mergers are characterised by high SFEs, compared to the standard mode of star formation. The observation of high dense gas fractions in interacting systems is consistent with the predictions of numerical simulations; it results from the increase of the gas turbulence during a merger. The merger is likely to affect the star-forming properties of the system at all spatial scales, from large scales, with a globally enhanced turbulence, to small scales, with possible modifications of the initial mass function. From a high-resolution numerical simulation of the major merger of two spiral galaxies, we analyse the effects of the galaxy interaction on the star forming properties of the ISM at the scale of star clusters. The increase of the gas turbulence is likely able to explain the formation of Super Star Clusters in the system. Our investigation of the SFR–H i relation in galaxy mergers will be complemented by highresolution Hi data for additional systems, and pushed to yet smaller spatial scales.

Nous présentons la découverte de quatorze nouveaux systèmes binaires ayant une séparation supérieure à 250 UA et dont au moins l'une des composantes est une naine M ou une naine L. Ces systèmes ont d'abord été identifiés en cherchant des objets ayant un mouvement propre commun autour d'étoiles connues possédant un mouvement propre élevé, grâce à une corrélation croisée de grands relevés du ciel dans l'infrarouge proche (2MASS, SDSS et SIMP). Un suivi astrométrique, afin de confirmer le mouvement propre commun, a été réalisé sur toutes les cibles avec la caméra SIMON et/ou la caméra CPAPIR à l'Observatoire du Mont-Mégatic (OMM) ou à l'Observatoire interaméricain du Cerro Tololo (CTIO). Un suivi spectroscopique a aussi été effectué sur la plupart des compagnons avec GMOS ou GNIRS à Gemini afin de déterminer leurs types spectraux. La probabilité que deux objets forment un système binaire par hasard a été évaluée afin de s'assurer que les couples candidats que nous présentons soient réellement liés.Un de nos nouveaux systèmes a un compagnon de masse sous-stellaire : 2M1259+1001 (L4.5). L'étude des systèmes que nous avons découverts pourra, entre autre, nous aider à mieux comprendre les mécanismes de formation des étoiles de très faible masse et des naines brunes. / We report the discovery of 14 new low-mass binary systems containing mid-M to mid-L dwarf companions with a separation larger than 250 AU. These systems were first identified by searching for common proper motion sources in the vicinity of known high proper motion stars, based on a cross-correlation of wide area near-infrared surveys (2MASS, SDSS, and SIMP). An astrometric follow-up, for common proper motion confirmation, was made with SIMON and/or CPAPIR at the Observatoire du Mont-Mégantic (OMM) and Cerro Tololo Inter-American Observatory (CTIO) telescopes for most of the candidates identified. A spectroscopic follow-up was also made with GMOS or GNIRS at Gemini to determine their spectral types. Statistical arguments are provided to show that all of the systems we report here are very likely to be truly bound. One of the new systems we discovered has a brown dwarf companion: 2M1259+1001 (L4.5). The study of the new systems we have discovered will be useful to help us better understand how very low mass stars and brown dwarfs form.

Master of Science / Department of Plant Pathology / William Bockus / Barley yellow dwarf (BYD) is one of the most important wheat diseases in the state of Kansas. Despite the development of cultivars with improved levels of resistance to BYD, little is known about the impact that this resistance has on yield loss from the disease. The intent of this research was to estimate yield loss in winter wheat cultivars in Kansas due to BYD and quantify the reduction in losses associated with resistant cultivars. During seven years, BYD disease incidence was visually assessed on numerous winter wheat cultivars in replicated field nurseries. Cultivars were planted about three weeks early to promote disease. When grain yields were regressed against BYD incidence scores, negative linear relationships significantly fit the data for each year and for the combined dataset covering all seven years. The models showed that, depending upon the year, 19-48% (average 33%) of the yields was explained by BYD incidence. For the combined dataset, 29% of the relative yield was explained by BYD incidence. The models predicted that cultivars showing high disease incidence had 25-86% (average 49%) less yield than a hypothetical cultivar that showed zero incidence. Using the models, the moderate level of resistance in the cultivar Everest was calculated to reduce yield loss from BYD by about 73%. Therefore, utilizing visual BYD symptom evaluations in Kansas, coupled with grain yields, is useful to estimate yield loss from the disease. Furthermore, linear models that incorporate those parameters can be used to calculate the impact of improving cultivar resistance to BYD on yield losses.

A deficiência de hormônio de crescimento (DGH) é tratada convencionalmente com repetidas injeções do hormônio recombinante. Este trabalho teve como objetivo estabelecer uma alternativa de tratamento baseada na transferência dos genes do hormônio de crescimento humano (hGH) ou de camundongo (mGH), em camundongos anões lit/lit ou lit/scid, mediante administração de DNA plasmidial associada à eletrotransferência, com a finalidade de atingir a máxima recuperação de crescimento em comparação ao camundongo normal (catch-up growth). Inicialmente foi realizada a administração do plasmídeo contendo o gene do mGH no músculo quadríceps exposto ou tibial anterior (TA) não exposto. Utilizando diferentes condições de eletrotransferência, baseadas em pulsos alternados de baixa (100 V/cm) e alta (1000 V/cm) voltagem (HV/LV, HV/8LV) ou em pulsos seguidos de baixa voltagem (8 pulsos de 150 V/cm), o músculo TA na condição HV/LV apresentou os maiores níveis de expressão de mGH: 6,7 ± 2,5 ng/mL. O tempo de exposição e a quantidade da enzima hialuronidase (HI) necessária para a eletrotransferência foram também analisados. O tempo de 30 minutos e a dose de 20 U de HI proporcionaram os melhores resultados de expressão. Diferentes quantidades de DNA foram também testadas, mas a administração de 50 &mu;g DNA/animal foi confirmada como a melhor. Na padronização do volume de solução do plasmídeo administrado no TA, foi observado que a injeção de 20 &mu;L de DNA apresentou expressão significativamente maior da proteína em comparação a de 10 &mu;L. Buscando uma maior expressão de GH, foi realizado experimento adicionando poli-L-glutamato ao diluente do DNA, comparando também diferentes condições de eletrotransferência (HV/LV e 375 V/cm). A condição de 375 V/cm, sem a adição do polímero, proporcionou as maiores concentrações, tanto de hGH como de mGH, no soro de camundongos lit/scid e lit/lit, respectivamente. Quando utilizados 3 pulsos de 375 V/cm e a administração do plasmídeo com o gene do mGH em dois locais de cada músculo TA, foram obtidos os mais altos níveis de expressão atingindo 14,7 ± 3,7 ng mGH/mL. Estes foram os parâmetros utilizados em um bioensaio, no qual foi também determinada a medida do comprimento inicial e final do fêmur por radiografia. Neste bioensaio de 36 dias, a curva de crescimento dos camundongos lit/lit tratados foi similar a de camundongos heterozigotos não tratados e os níveis de mGH do grupo DNA foram significativamente maiores (P<0,0002) em relação ao grupo controle. Os camundongos tratados também apresentarem concentração de mIGF-I no soro superior a do grupo controle. Considerando os parâmetros de crescimento avaliados, o grupo tratado com DNA apresentou percentuais de incremento altamente significativos em relação ao grupo controle, com P<0,001 para o peso corpóreo e P<0,002 para o comprimento do corpo, da cauda e para ambos os fêmures, com valores de catch-up da ordem de 79% para o comprimento dos fêmures. Podemos concluir que foi estabelecida uma metodologia eficiente de transferência gênica não viral, que poderá levar a uma completa normalização de crescimento de camundongos anões mediante utilização de animais mais jovens, como mencionado na literatura e em trabalho recente do nosso grupo. / Growth hormone deficiency (GHD) is conventionally treated with repeated injections of the recombinant hormone. This work aimed at establishing an alternative treatment based on the transfer of the human (hGH) or mouse growth hormone (mGH) genes into lit/lit or lit/scid dwarf mice, using plasmid DNA administration associated with electrotransfer, in order to achieve the maximum growth recovery compared to normal mice (catch-up growth). Administration of the plasmid containing the mGH gene was first carried out in the exposed quadriceps or non-exposed anterior tibialis (TA) muscle. Using different electrotransfer conditions, based on alternate pulses of high (1000 V/cm) and low (100 V/cm) voltage (HV/LV, HV/8LV) or consecutive pulses of low voltage (8 pulses of 150 V/cm), the TA muscle in the HV/LV condition showed the highest levels of mGH expression: 6.7 ± 2.5 ng/mL. Exposure time and amount of the enzyme hyaluronidase (HI) required for electrotransfer were also analyzed. The time of 30 minutes and the dose of 20 U HI provided the best results of expression. Different amounts of DNA were also tested, but the administration of 50 &mu;g DNA/animal was confirmed as the best. In the optimization of the volume of plasmid solution administered to TA, it was observed that injection of 20 &mu;L of DNA showed a significantly higher expression of the protein compared with 10 &mu;L. Aiming at a higher GH expression, an experiment was carried out by adding poly-L-glutamate to the DNA diluent, comparing also different electrotransfer conditions (HV/LV and 375 V/cm). The condition of 375 V/cm, without the polymer addition, provided the higher concentrations of both hGH and mGH in the serum of lit/scid or lit/lit mice, respectively. Using 3 pulses of 375 V/cm and administration of mGH-DNA in two locations on each TA muscle, the highest expression levels of up to 14.7 ± 3.7 ng mGH/mL were obtained. These were the parameters utilized in a bioassay, which was also carried out by measurement of the initial and final femur length by radiography. In this 36-day bioassay, the growth curve of treated lit/lit mice was similar to that of heterozygous untreated mice and the mGH levels of DNA group were significantly higher (P<0.0002) than the control group. Treated mice also showed a higher mIGF-I concentration in the serum compared to the control group. Concerning growth parameters, DNA-treated group showed percentages of increase highly significant compared to the control group, with P<0.001 for body weight and P<0.002 for body, tail and both femurs lengths, with catch-up values of the order of 79% for femur lengths. We can conclude that an efficient non-viral gene transfer methodology has been established, which lead to a complete growth normalization of the dwarf mice through the use of younger animals, as reported in the literature and in a recent paper of our group.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Empresa Brasileira de Pesquisa AgropecuÃria / Os efeitos dos regimes irrigado e de sequeiro foram avaliados em folhas sombreadas e a pleno sol nos clones de cajueiro anÃo precoce (Anacardium occidentale L) CCP 76 e BRS 189. Os experimentos foram conduzidos na EstaÃÃo Experimental da Embrapa AgroindÃstria Tropical, em Paraipaba, CearÃ, entre setembro de 2006 e agosto de 2007. Mensalmente, foram investigados a condutÃncia estomÃtica, a taxa transpiratÃria, a taxa fotossintÃtica, a relaÃÃo entre a concentraÃÃo interna e externa de CO2, o potencial osmÃtico. O teor de Ãgua, de proteÃnas solÃveis, de prolina, de N-aminossolÃveis, de carboidratos (aÃÃcares totais, redutores e nÃo redutores e de amido), a atividade das enzimas do metabolismo do carbono, sintase da sacarose fostato (SPS), sintase da sacarose (SuSy), pirofosforilase do ADP glicose (AGPase) e das amilases (alfa, beta e total) foram determinados nas folhas das plantas bi-mensalmente.AlÃm disso, foram analisadas a qualidade pÃs-colheita dos pedÃnculos dos clones em ambos os tratamentos. Em geral, nÃo foram encontradas diferenÃas significativas entre os dois tipos de cultivos para os dois clones e tipos de folha em relaÃÃo aos parÃmetros fisiolÃgicos, tais como, fotossÃntese, transpiraÃÃo e condutÃncia estomÃtica. Essas variÃveis foram afetadas apenas em funÃÃo da Ãpoca do ano. Carboidratos, tais como amido e aÃÃcares redutores, tiveram uma tendÃncia de acÃmulo nas folhas de sol e de sombra no mÃs de maio, exibindo teores menores no mÃs de setembro, Ãpoca em que os teores de aÃÃcares nÃo-redutores (sacarose) aumentaram para suprir a formaÃÃo de frutos. Sob o regime nÃo irrigado, as enzimas SPS, SuSy e AGPase tiveram suas atividades justificadas parcialmente quando relacionadas com as variaÃÃes nos teores de aÃÃcares totais e amido. A atividade amilÃsica total variou para os regimes de irrigaÃÃo, clone e folhas em relaÃÃo ao perÃodo do ano. A supressÃo da irrigaÃÃo para os clones CCP 76 e BRS 189 durante o experimento nÃo foi capaz de modificar a qualidade pÃs-colheita dos pedÃnculos de caju. As variaÃÃes ao longo do experimento comprovam o efeito da forÃa dos drenos nas diversas fases fenolÃgicas e para os ÃrgÃos reprodutivos das plantas de cajueiro. As plantas de cajueiro anÃo precoce possuem, portanto, uma plasticidade, quando cultivadas em condiÃÃes de sequeiro, conseguindo manter suas atividades metabÃlica e fisiolÃgica vitais sem maiores danos à planta como um todo. / Effects of irrigation and drought regimen were evaluated on shaded and sunny leaves in clones of early dwarf cashew trees (Anacardium occidentale L) CCP 76 and BRS 189. The experiments were conducted at the Embrapa Agro indÃstria Tropical experimental station in Paraipaba, CearÃ, between Setember of 2006 and August of 2007. The means for the obtained data were statistically analysed at a 5% level of significance. Stomatal conductance(gs), respiratory rate(E), photosynthetic rate (A), relation between inner and outer concentration of CO2 (Ci/Car), osmotic potential (&#936;s) and water usage efficiency were accessed monthly. The relative water cont ent, of soluble proteins, prolin, N-amino soluble, of sugars (soluble, reductants, non- reductants, starch) and activity of carbon metabolism enzymes, sucrose-phosphate synt hase (SPS), sucrose synthase (SuSy), ADP phosphorylase, glucose and amylases (alfa, beta and total) were determined on leaves each two months. Beyond that, the postharvest qual ity of pseudofruits of the clones on both treatments was accessed. In general, no significant differences were found among cultivations for both clones in relation to physiological parameters, like photosynthesis, transpiration and stomatal conductance. These variants were affected only in function of the time of year. Independently of irrigation regimen, wate r usage efficiency was lower during the drought (from June to December) in relation to the months with higher pluviometric precipitation (from January to May). Carbohydr ates such as starch and reductant sugars tend to accumulate on both sunny and shaded leaves on May, while their contents are smaller on September, a time when non-reductant sugars (sucrose) increase in order to supply fruit formation. Under a drought regimen, enzymes SPS, SuSy and ADP correlate partially with the variations of contents of soluble sugars and starch. Total amylasic activity varied for irrigation regimens, clones and kind of leaves in relation to the period of the year. The supression of the hydric regimen for the clones CCP 76 and BRS 189 during the experiment wasn't able to chan ge the postharvest quality of the cashew pseudofruits. The variations shown during the experiment confirm the effect of drainage strength on several phenological phases and for the reprodutive or gans of the cashew trees. The trees of early dwarf cashew have, therefore, some plasticity when exposed to drought conditions, being able to maintain metaboli cal and physiological activity without major damage to the individual as a whole.

A deficiência de hormônio de crescimento (DGH) é tratada convencionalmente com repetidas injeções do hormônio recombinante. Este trabalho teve como objetivo estabelecer uma alternativa de tratamento baseada na transferência dos genes do hormônio de crescimento humano (hGH) ou de camundongo (mGH), em camundongos anões lit/lit ou lit/scid, mediante administração de DNA plasmidial associada à eletrotransferência, com a finalidade de atingir a máxima recuperação de crescimento em comparação ao camundongo normal (catch-up growth). Inicialmente foi realizada a administração do plasmídeo contendo o gene do mGH no músculo quadríceps exposto ou tibial anterior (TA) não exposto. Utilizando diferentes condições de eletrotransferência, baseadas em pulsos alternados de baixa (100 V/cm) e alta (1000 V/cm) voltagem (HV/LV, HV/8LV) ou em pulsos seguidos de baixa voltagem (8 pulsos de 150 V/cm), o músculo TA na condição HV/LV apresentou os maiores níveis de expressão de mGH: 6,7 ± 2,5 ng/mL. O tempo de exposição e a quantidade da enzima hialuronidase (HI) necessária para a eletrotransferência foram também analisados. O tempo de 30 minutos e a dose de 20 U de HI proporcionaram os melhores resultados de expressão. Diferentes quantidades de DNA foram também testadas, mas a administração de 50 &mu;g DNA/animal foi confirmada como a melhor. Na padronização do volume de solução do plasmídeo administrado no TA, foi observado que a injeção de 20 &mu;L de DNA apresentou expressão significativamente maior da proteína em comparação a de 10 &mu;L. Buscando uma maior expressão de GH, foi realizado experimento adicionando poli-L-glutamato ao diluente do DNA, comparando também diferentes condições de eletrotransferência (HV/LV e 375 V/cm). A condição de 375 V/cm, sem a adição do polímero, proporcionou as maiores concentrações, tanto de hGH como de mGH, no soro de camundongos lit/scid e lit/lit, respectivamente. Quando utilizados 3 pulsos de 375 V/cm e a administração do plasmídeo com o gene do mGH em dois locais de cada músculo TA, foram obtidos os mais altos níveis de expressão atingindo 14,7 ± 3,7 ng mGH/mL. Estes foram os parâmetros utilizados em um bioensaio, no qual foi também determinada a medida do comprimento inicial e final do fêmur por radiografia. Neste bioensaio de 36 dias, a curva de crescimento dos camundongos lit/lit tratados foi similar a de camundongos heterozigotos não tratados e os níveis de mGH do grupo DNA foram significativamente maiores (P<0,0002) em relação ao grupo controle. Os camundongos tratados também apresentarem concentração de mIGF-I no soro superior a do grupo controle. Considerando os parâmetros de crescimento avaliados, o grupo tratado com DNA apresentou percentuais de incremento altamente significativos em relação ao grupo controle, com P<0,001 para o peso corpóreo e P<0,002 para o comprimento do corpo, da cauda e para ambos os fêmures, com valores de catch-up da ordem de 79% para o comprimento dos fêmures. Podemos concluir que foi estabelecida uma metodologia eficiente de transferência gênica não viral, que poderá levar a uma completa normalização de crescimento de camundongos anões mediante utilização de animais mais jovens, como mencionado na literatura e em trabalho recente do nosso grupo. / Growth hormone deficiency (GHD) is conventionally treated with repeated injections of the recombinant hormone. This work aimed at establishing an alternative treatment based on the transfer of the human (hGH) or mouse growth hormone (mGH) genes into lit/lit or lit/scid dwarf mice, using plasmid DNA administration associated with electrotransfer, in order to achieve the maximum growth recovery compared to normal mice (catch-up growth). Administration of the plasmid containing the mGH gene was first carried out in the exposed quadriceps or non-exposed anterior tibialis (TA) muscle. Using different electrotransfer conditions, based on alternate pulses of high (1000 V/cm) and low (100 V/cm) voltage (HV/LV, HV/8LV) or consecutive pulses of low voltage (8 pulses of 150 V/cm), the TA muscle in the HV/LV condition showed the highest levels of mGH expression: 6.7 ± 2.5 ng/mL. Exposure time and amount of the enzyme hyaluronidase (HI) required for electrotransfer were also analyzed. The time of 30 minutes and the dose of 20 U HI provided the best results of expression. Different amounts of DNA were also tested, but the administration of 50 &mu;g DNA/animal was confirmed as the best. In the optimization of the volume of plasmid solution administered to TA, it was observed that injection of 20 &mu;L of DNA showed a significantly higher expression of the protein compared with 10 &mu;L. Aiming at a higher GH expression, an experiment was carried out by adding poly-L-glutamate to the DNA diluent, comparing also different electrotransfer conditions (HV/LV and 375 V/cm). The condition of 375 V/cm, without the polymer addition, provided the higher concentrations of both hGH and mGH in the serum of lit/scid or lit/lit mice, respectively. Using 3 pulses of 375 V/cm and administration of mGH-DNA in two locations on each TA muscle, the highest expression levels of up to 14.7 ± 3.7 ng mGH/mL were obtained. These were the parameters utilized in a bioassay, which was also carried out by measurement of the initial and final femur length by radiography. In this 36-day bioassay, the growth curve of treated lit/lit mice was similar to that of heterozygous untreated mice and the mGH levels of DNA group were significantly higher (P<0.0002) than the control group. Treated mice also showed a higher mIGF-I concentration in the serum compared to the control group. Concerning growth parameters, DNA-treated group showed percentages of increase highly significant compared to the control group, with P<0.001 for body weight and P<0.002 for body, tail and both femurs lengths, with catch-up values of the order of 79% for femur lengths. We can conclude that an efficient non-viral gene transfer methodology has been established, which lead to a complete growth normalization of the dwarf mice through the use of younger animals, as reported in the literature and in a recent paper of our group.

Tong, Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 66-78). / Abstracts in English and Chinese; includes Chinese. / Acknowledgements --- p.i / Conferences & Academic Awards --- p.ii / Table of contents --- p.iii / List of figures --- p.vii / List of tables --- p.viii / List of abbreviations --- p.ix / Abstract --- p.x / Abstract (Chinese) / Chapter Chapter I --- Background of diabetes mellitus and DM complicating pregnancy …… --- p.1 / Chapter 1.1 --- Definitions and clinical manifestations of Diabetes Mellitus --- p.1 / Chapter 1.2 --- Diagnostic criteria of DM --- p.1 / Chapter 1.3 --- Classification of DM --- p.1 / Chapter 1.4 --- Prevalence of DM --- p.2 / Chapter 1.5 --- Aetiology and Pathogenesis of DM --- p.3 / Chapter 1.6 --- Treatment of DM --- p.3 / Chapter 1.7 --- Complications of DM --- p.4 / Chapter 1.8 --- DM complicating pregnancy --- p.4 / Chapter 1.8.1 --- Implications of DM complicating pregnancy --- p.4 / Chapter 1.8.2 --- Diabetic Embryopathy --- p.5 / Chapter 1.8.3 --- Incidences of the major congenital anomalies --- p.5 / Chapter 1.8.4 --- Possible pathogenesis of congenital anomalies in DM complicating pregnancy --- p.6 / Chapter 1.8.4.1 --- Apoptosis --- p.6 / Chapter 1.8.4.2 --- Oxidative stress --- p.7 / Chapter 1.8.4.3 --- Arachidonic acid and PGE2 --- p.7 / Chapter 1.8.5 --- Clinical management of DM complicating pregnancy --- p.8 / Chapter 1.8.5.1 --- Pre-pregnancy care --- p.8 / Chapter 1.8.5.2 --- Antenatal management of DM complicating pregnancy --- p.9 / Chapter Chapter II --- Background of Traditional Chinese Medicine in treatment of DM --- p.10 / Chapter 2.1 --- Definition and manifestations of DM in TCM theory --- p.10 / Chapter 2.2 --- Historical context of DM in TCM --- p.10 / Chapter 2.2.1 --- "Spring and Autumn Period and Warring States Period (770 B.C.一8 A.D.): The first nomenclature of ""Wasting Thirst""" --- p.10 / Chapter 2.2.2 --- "Han Dynasty (9 A.D.-280 A.D.)： monograph on ""Wasting Thirst""" --- p.11 / Chapter 2.2.3 --- "Sui and Tang Dynasty (581 A.D.-960 A.D.)： the diagnosing marker of ""Wasting Thirst""" --- p.11 / Chapter 2.2.4 --- Song Dynasty (960 A.D.-1270 A.D.): the Golden Time of developing the treatment on DM --- p.12 / Chapter 2.2.5 --- Ming and Qing Dynasty (1270 A.D. - 1911 A.D.): the integration period of TCM theory on DM --- p.15 / Chapter 2.3 --- Aetiology of DM in TCM theory --- p.15 / Chapter 2.3.1 --- Congenital weakness --- p.16 / Chapter 2.3.2 --- Improper diet --- p.16 / Chapter 2.3.3 --- Emotional disorders and overstrain --- p.17 / Chapter 2.3.4 --- Excessive sexual activities --- p.17 / Chapter 2.4 --- Pathogenesis of DM in TCM theory --- p.17 / Chapter 2.5 --- Prognosis of DM in TCM theory --- p.19 / Chapter 2.5.1 --- """Dual Qi-Yin Deficiency"" and ""Dual Yin-Yang Deficiency""" --- p.19 / Chapter 2.5.2 --- "Multi-systemic malfunction of ""Zang Fu""" --- p.19 / Chapter 2.6 --- Principle of treatment --- p.20 / Chapter 2.7 --- Commonly used herbal remedies and recent experimental studies --- p.20 / Chapter 2.8 --- TCM on relieving DM complications --- p.21 / Chapter 2.9 --- "Dwarf Lilyturf Tuber (Ophiopogonis Radix, Mai Dong,麥冬）" --- p.21 / Chapter 2.10 --- Objectives and hypothesis --- p.22 / Chapter 2.10.1 --- Objectives --- p.22 / Chapter 2.10.2 --- Hypotheses --- p.23 / Chapter Chapter III --- Methodology and Results --- p.24 / Chapter 3.1 --- Set up of mouse embryos --- p.24 / Chapter 3.1.1 --- Mouse strain --- p.24 / Chapter 3.1.2 --- Research animal ethnics and care guidelines --- p.24 / Chapter 3.1.3 --- Mouse sacrifice and embryo dissection --- p.24 / Chapter 3.1.4 --- Grouping of embryos --- p.25 / Chapter 3.2 --- Preparations of D-glucose --- p.25 / Chapter 3.3 --- Chinese medicine quality controls and preparations --- p.25 / Chapter 3.4 --- Whole mouse embryo culture --- p.26 / Chapter 3.5 --- Morphological scoring on mouse embryos and statistical analysis --- p.27 / Chapter 3.6 --- Establishment of cranial NTD by D-glucose --- p.28 / Chapter 3.6.1 --- Dosage of D-glucose to induce cranial NTD --- p.29 / Chapter 3.6.2 --- Result --- p.30 / Chapter 3.7 --- Experimental designs --- p.31 / Chapter 3.8 --- Part I: Efficacy and dose-response effects of Maidong extract --- p.32 / Chapter 3.8.1 --- Safety dose of Maidong extract on non-diabetic mouse embryos --- p.32 / Chapter 3.8.1.1 --- Dosage --- p.32 / Chapter 3.8.1.2 --- Result --- p.35 / Chapter 3.8.2 --- Efficacy and dose-effect response of Maidong extract on non-diabetic mouse embryos --- p.36 / Chapter 3.8.2.1 --- Dosage and grouping --- p.37 / Chapter 3.8.2.2 --- Result --- p.38 / Chapter 3.9 --- Part II: Efficacy and dose-response effects of serum from Maidong extract-treated rat serum --- p.40 / Chapter 3.9.1 --- Preparation of Maidong treated non-diabetic full rat serum --- p.41 / Chapter 3.9.1.1 --- Rats --- p.41 / Chapter 3.9.1.2 --- Dosage for feeding --- p.41 / Chapter 3.9.1.3 --- Administration --- p.42 / Chapter 3.9.1.4 --- Termination of rats and preparation of rat serum --- p.42 / Chapter 3.9.2 --- Safety dose of Maidong treated non-diabetic full rat serum non-diabetic mouse embryos --- p.43 / Chapter 3.9.2.1 --- Dosage --- p.43 / Chapter 3.9.2.2 --- Result --- p.44 / Chapter 3.9.3 --- Protective Effect of Maidong extract-treated full rat serum --- p.46 / Chapter 3.9.3.1 --- Dosage and grouping --- p.46 / Chapter 3.9.3.2 --- Result --- p.47 / Chapter 3.10 --- "Part III: Efficacy and dose-response effects of Ophiopogonin D, a major chemical component of Maidong in preventing hyperglycemia-induced cranial neural tube defect" --- p.49 / Chapter 3.10.1 --- Safety dose of Ophiopogonin D --- p.50 / Chapter 3.10.1.1 --- Preparation of Ophiopogonin D --- p.50 / Chapter 3.10.1.2 --- Dosage --- p.50 / Chapter 3.10.1.3 --- Results --- p.52 / Chapter 3.10.2 --- Efficacy and dose-response effects of Ophiopogonin D --- p.53 / Chapter 3.10.2.1 --- Dosage and grouping --- p.53 / Chapter 3.10.2.2 --- Results --- p.55 / Chapter Chapter IV --- Discussion --- p.58 / Chapter 4.1 --- Whole embryo culture system --- p.58 / Chapter 4.2 --- Quality control of Maidong extract --- p.58 / Chapter 4.3 --- "Therapeutic effect of single herb, formula and chemical components" --- p.59 / Chapter 4.4 --- Dosage of D-glucose to induce cranial NTD --- p.60 / Chapter 4.5 --- Dosage and efficacy of Maidong extract and Ophiopogonin D --- p.60 / Chapter 4.6 --- Administration of Maidong extract to non-diabetic female rats --- p.61 / Chapter Chapter V --- Conclusions --- p.63 / Chapter Chapter VI --- Future Study --- p.64 / References --- p.66