Adenovirus-based exogenous gene expression in mammalian cells

Thesis (Ph.D.)--Brock University, 2010.Brock University. Dept. of Biological Sciences. ThesisAdenoviruses have been used as a model system for understanding gene expression, DNA replication, gene delivery and other molecular biological phenomenon. In this project, adenovirus was used as a model to study exogenous gene expression in mammalian cells. More specifically, several adenoviral components were identified to enhance gene expression together with components needed for viral DNA replication. The adenoviral elements that enhance gene expression were assembled in an expression vector (pEl). These include the viral inverted terminal repeats (ITRs), the El region, the major late promoter (MLP) and the tripartite leader sequence (TPL). The green florescence protein (GFP) was used as a reporter gene. Various aspects of gene expression were examined including DNA delivery and stability inside the cells as well as mRNA transcription and protein expression. First, the effect of DNA quality on its delivery, stability and expreSSIOn III mammalian cells was studied. Five different conditions of the major DNA contaminants were used in this investigation including ethidium bromide (EtBr) , cesium chloride (CsCl), EtBr/CsCl, endotoxins and ethanol. CsCl, EtBr/CsCl and endotoxins affected the delivery process while EtBr affected the expression process but not the delivery. The used EtOH had no significant effect on both. In addition, the effect of all the contaminants was reversible. Next, we looked at the factors that enhance mRNA transcription and translation levels. Three approaches were tested, the first was the co-transfection of pEl and a plasmid that contains adenoviral genes involved in replication (PE2: contains E2 and viral protease). The second was the establishment of a cell line expressing these adenoviral genes involved in replication and the third approach was the super-infection with the wild type adenovirus. The co-transfection did not show any significant increase in gene expression or vector stability. On the other hand, the construction of CHO-E2 cell lines yielded five cell lines but none of them showed expression of all the integrated adenoviral E2 genes or enhancement of stability. Adenoviral super-infection enhanced gene expression. CHO cells showed higher enhancement in intensity and time than human embryonic kidney (HEK) 293 cells. In addition, such enhancement was dependent on the multiplicity of infection (MOl). Finally, this study emphasizes the importance of DNA quality on gene expression. However, the use of adenoviral elements to enhance exogenous gene expression is successful only when the complementary viral proteins and sequences are present. Active expression of the adenoviral proteins does not depend on a few major elements, but depends on the combination of different elements that work in cis or trans to activate gene expression

RESPONSE OF JATROPHA CURCAS L. SHRUBS TO COMPLETE (NPK) FERTILIZER, BIO AND ORGANIC FERTILIZERS UNDER SANDY SOIL CONDITIONS

The present work was carried out during the two successive seasons of 2015 and 2016 at the Experimental Farm of El-Quassassin Horticultural Research Station, Ismailia Governorate, Egypt, to study the effect of complete NPK fertilization rates (control, 50, 75 and 100% from the recommended dose), bio and organic fertilizers (control, EM1 applied as the rate of 100 ml/shrub and active dry yeast with the concentration of 4 g/las a rate of 2 liters/shrub) and the interaction treatments between them on the growth, (fruits and seeds yields), fixed oil content and its components in seeds of Jatropha (Jatropha curcas) shrubs.The results showed that, the best vegetative growth parameters (increasing percentage of shrub height, number of branches and canopy width/shrub), number and weight of fruits (kg)/shrub, number and weight of seeds (kg)/shrub, the yield of fruits and seeds (ton)/fed, fixed oil percentage, weight of fixed oil (g)/shrub and yield of fixed oil (kg)/fed were recorded from the interaction treatment of 75% complete NPK fertilization + EM1. The analysis of fixed oil of Jatropha shrub showed that, the treatment of 50% NPK gave the best palmitic acid percentage and the treatment of 50% NPK + EM1 recorded maximum percentages of stearic and oleic acids, while the treatment of 100% NPK + EM1 gave the highest percentage of linoleic acid

Pre-harvest foliar application of mineral nutrients to retard chlorophyll degradation and preserve bio-active compounds in broccoli

Foliar application of micronutrients has become a common farm management practice to increase the overall yield of various crops. However, the effects of foliar fertilization on shelf life and postharvest quality of the crops are rather under-researched. The aim of this field experiment was to evaluate the effect of foliar application of individual mineral nutrients (calcium (Ca), zinc (Zn), manganese (Mn), and iron (Fe) on pre and postharvest quality of broccoli. The broccoli plants were subjected to single foliar sprays of either Ca, Fe, Zn, or Mn, which was repeated four times during plant growth at a 1 g/kg concentration. Once harvested, the broccoli heads were refrigerated at 4 °C for 28 days. Our results indicated that foliar application of Ca, Zn, Mn, and Fe did not have a significant effect on plant growth parameters, apart from enhancing Soil Plant Analysis Development (SPAD) chlorophyll meter values. However, during postharvest, foliar application treatment showed a positive response on weight loss during storage and reduction in yellowing of the broccoli heads. Foliar treatments increased the concentration of Nitrogen (N), Phosphorus (P), Ca, Zn, Mn and Fe significantly in the broccoli head tissue. Total chlorophyll content, total phenolic compound, ascorbic acid, peroxidase activity, glucoraphanin and glucobrassicin and flavonoids were significantly increased by all foliar treatments. Crude protein content and sulforaphane were enhanced by Ca and Mn treatments. Overall, foliar application of the investigated mineral nutrients may prove beneficial in improving the shelf-life and nutrient content of broccoli during postharvest handling and storage

RESPONSE OF CORIANDER VEGETATIVE GROWTH, YIELD COMPONENTS, PIERCING–SUCKING PESTS INFESTATION AND ASSOCIATED NATURAL ENEMIES TO NITROGEN FERTILIZER RATES AND FOLIAR SPRAY WITH L-TRYPTOPHAN, PHENYLALANINE ACIDS UNDER SANDY SOIL CONDITIONS

The present work was conducted at the Experimental Farm of EL-Quassasin Horticultural Research Station, Ismailia Governorate, Egypt, during the two winter consecutive seasons of 2016/2017 and 2017/2018. The aim was, to investigate the effect of different nitrogen fertilization rates (control, 50, 75 and 100% of recommended N rate), amino acids type (control, L-tryptophan, phenylalanine and L-tryptophan + phenylalanine) as a foliar spray at 150 mg/l for each one and their interactions on growth and production of coriander andpiercing–sucking pests infestation. The obtained results indicated that, nitrogen fertilization rate at 300 kg ammonium sulfate/feddan (recommended rate) significantly increased growth parameters (plant height, number of branches/plant and fresh and dry herb weights/plant), root system (fresh and dry weights of roots/plant and root length), yield components (number of umbels/plant and number of florets/umbel/plant, fruit yield/plant and /feddan), volatile oil productivity (volatile oil percentage, volatile oil yield/plant and /feddan) and chemical constituents (N, P, K and total carbohydrates percentages) compared to control. Moreover, the highest values of the above mentioned characters were registered by foliar spray of L-tryptophan + phenylalanine with significant differences between the other rates under study. Generally, the better growth characters and root system, higher yield components and volatile oil production, chemical constituents of fruits could be obtained by spraying L-tryptophan + phenylalanine at 150 mg/l on fertilized coriander plants with  nitrogen at the recommended rate, followed by the combined treatment of nitrogen fertilization as 75% plus L-tryptophan + phenylalanine. In the same trend, highly significant differences (≤ 0.05) between the mean numbers of the piercing–sucking pests; i.e., Hyadaphis coriandri (Das), Bemisia tabaci (Genn.), Thrips tabaci (Lind.), Tetranychus urticea Koch., and their associated natural enemies i.e., Coccinella spp, Chrisoprella carnea (Steph.), aphids parasitoids and predacious mite which were resulted as the variation in nitrogen fertilization rates, and foliar spray of amino acids types. Also, the simple correlation calculation revealed significant coefficients for the relation between each of the surveyed  pests and their associated natural enemies correlated with growth parameters (plant height and number of branches/plant) and seed yield/plant

Agronomical, physiological and molecular evaluation reveals superior salt-tolerance in bread wheat through salt-induced priming approach

Salt stress significantly limit wheat crop productivity worldwide. Exposure to non-lethal levels of salt stress, referred to as "salt-priming", allows plants to persist subsequent lethal conditions; the priming effect continues even after an extended salt stress-free period. This study attempted to evaluate the effectiveness of the salt-induced priming approach to cope with the toxic effects of long-term salinity stress in wheat. After 22 days of gradual salt acclamation to reach 250 mM NaCl, plants were recovered for eight days and finally shocked with 250 mM NaCl (priming+shock) for 7 days. After that, physiological parameters and gene expression of six salt-responsive genes were assessed. Additionally, 120 days after germination (at the end of the season), agronomic traits were recorded. Analysis of the agronomical traits revealed higher productivity in the salt-pretreated group (priming+shock) plants than the non-pretreated (shock only). Consistently, salt-pretreated plants maintained higher photosynthetic pigments level and decreased proline and MDA content than non-pretreated, suggesting enhanced salt tolerance. Moreover, salt-pretreated plants sustained high expressional levels of salt-responsive genes (TaNHX1, TaSOS1, TaSOS4, TaHKT1, TaHKT2, and TaAKT1) comparing with non-pretreated, indicating a vital role in ion homeostasis and conferring salt tolerance. Ultimately, this finding could facilitate novel smart approaches to improve wheat productivity under salt stress

Structural and Functional Abnormalities in the Caudate Nucleus of Schizophrenic Patients with and without Obsessive Symptoms

Background: Obsessive\u2013compulsive symptoms (OCS) have been frequently described in schizophrenic populations. There is a controversy on whether this co-occurrence is more than just comorbidity or represents a distinct subgroup in schizophrenia. Aim: The aim of this study was to investigate whether there are possible underlying structural and functional differences between schizophrenic patients with and those without OCS. Patients and methods: Participants in this study were divided into three groups: 20 patients having schizophrenia with OCS; 20 patients having schizophrenia without OCS (both were diagnosed according to DSM-IV-TR); and 20 matching controls. All participants underwent an MRI for volumetric measurement of the caudate nucleus and a magnetic resonance spectroscopy to measure choline (Cho), creatine, and N-acetyl aspartate (NAA) in the caudate nucleus. Results: Caudate volume was found to be reduced in schizophrenic patients with or without OCS (P 640.05). Magnetic resonance spectroscopy findings showed that schizophrenic patients without OCS had significant increase in Cho concentration (P 640.05), but average NAA concentration. In contrast, schizophrenic patients with OCS had significant reduction in NAA concentration (P 640.05) with average Cho concentration. Conclusion: Schizophrenic patients with OCS may have an atypical set of neuroradiological characteristics that would specifically categorize it within the schizophrenia spectrum

Role of Magnetic Resonance Spectroscopy in Follow up Brain Tumors after Treatment

Background: 1 H magnetic resonance spectroscopy (MRS) is an analytical method that enables the identification and quantification of metabolites in samples. It differs from conventional Magnetic Resonance Imaging (MRI) in that spectra provide physiological and chemical information instead of anatomy. MRS imaging allows a valuable insights into brain tumors characteristics, grads, and progression then follow up during treatments. Typically in MRS a single spectrum is acquired by averaging enough spectra over a long acquisition time. Averaging is necessary because of the complex spectral structures and relatively low concentrations of many brain metabolites, which result in a low signal-to-noise ratio (SNR) in MRS of a living brain. Objective: In this paper, acquiring and analyzing multivoxel MRS data are reviewed by calculating the areas under different peaks then compared with that obtained directly from 1H-MRS machine. 1H-MRS measurements of amounts of Choline (Cho), creatine (Cr) and Nacetylaspartate (NAA) relative to Cho, NAA and Cr in healthy brain tissue of a normal control brain tissue, and in the tissue of tumor of patient who had taken radiation therapy sessions. Results: The obtained results show a good agreement between the data obtained directly from MRS machine and that calculated from their spectra. This method is now used for to insure that these obtained spectra are calibrated with that obtained directly from MRS machine. So these may reflect the small changes in metabolites during treatment and follow up. Conclusion: The MRS data are seen to provide unique information that when combined with high-quality anatomical MR images has implications for defining tumor type and grade, directing biopsy or surgical resection, planning focal radiation or biological therapies, and understanding the mechanisms of success and failure of new treatments

Application of potassium humate and salicylic acid to mitigate salinity stress of common bean

In the current study, we investigated the effect of potassium humate (Kh) and salicylic acid (SA) in mitigating the salinity stress of common bean plants. Common bean seedlings were treated with 0.2 g/L SA as a foliar application and 0.3 g/L Kh as a soil application individually or in combination. After 7 days of germination, plants were treated with 50 mM NaCl and normal water as a control. Our results indicate that salt treatment reduced the plant growth (fresh and dry shoots and roots), leaf pigments (total chlorophyll and carotenoids), ascorbic acid (AA), glutathione (GSH), and potassium (K) contents. On the contrary, proline content; sodium (Na); hydrogen peroxide (H2O2); superoxide anion (O2•−); and antioxidant enzymes, including catalase (CAT), peroxidase (POX), and superoxide dismutase (SOD), were increased by saline stress. However, applying either individual Kh and SA or their combination stimulated seedling growth under salinity stress by increasing growth parameters, leaf pigment contents, AA, GSH, proline content, K content, and antioxidant enzymes compared with the control. Additionally, Na content, H2O2, and O2•− were reduced by all applications. The application of the Kh (0.3 g/L) + SA (0.2 g/L) combination was more effective than using the individual compounds. In conclusion, applications of Kh + SA can mitigate salt stress and improve the seedling growth of common bean