Growth hormone (GH) is a vital pituitary hormone controlling somatic cell growth and development. GH has a multitude of effects on the body: deficiency can lead to dwarfism while excess can cause conditions such as gigantism. Human patients with mutations in the transcription factor Pit-1 show decreased GH and prolactin levels. However, Pit-1 is known to control multiple pituitary hormones, so what other factors lead to the specificity of transcriptional regulation of the GH gene via its promoter? In order to study this, our lab has been analyzing rat pituitary cell lines to understand the role of ZNF292 (formerly called Zn-16), a selective GH transcription regulator with 16 zinc fingers that bind to the GH promoter DNA. This work has used rat MtT/S cells that are unique in that they exclusively express GH. MtT/S cells were procured from Riken Cell Bank in Japan, cultured, and examined for GH hormone and RNA expression. Results confirmed that the MtT/S cells are terminally differentiated as somatotrophs. To understand the role of ZNF292 in transcription of the GH gene, recent rat genomic data was analyzed to determine the positions of 7 exons upstream from the large exon 8 that contains the important zinc finger-encoding portions of the protein. First, MtT/S RNA was reverse transcribed into DNA, then PCR amplification was performed using primer pairs encompassing various sections of the exon 1 – 7 region. Specific PCR products were found with distinct products ranging in size from 130 to 960, all of which agreed with the predicted sizes of these exons. It had previously been theorized that ZNF292 contained a single large exon; however, these results show that splicing of the primary transcript does occur in this upstream region. Characterizing this exonic region was performed because it has been shown that ZNF292 produces circular RNA (circRNA) of unknown function in human endothelial cells and in certain types of cancer. CircRNAs are thought to be created by the “back-splicing” of exons, so that rather than a linear transcript, the ends are circularized for a portion of the transcript. Having determined the sequence and organization of these upstream exons, we are now testing primer sets that will demonstrate productive amplification only from circRNAs. Further, we are removing linear RNAs using RNAse R treatment to selectively enhance circRNA presence in the reactions. Finally, data from RNA-Seq analysis of the MtT/S cells will be scrutinized to determine if additional exon/intron boundaries or alternative splice sites exist in this upstream 7 exon region. The study of circular RNAs could be very important in understanding its role in acting as a microRNA sponge or RNA-binding protein sponge during their regulation of downstream gene transcription. Also, analysis of this mechanism shows potential as a clinical tool in cancer treatment because ZNF292 functions as a tumor suppressor in colorectal and chronic lymphatic leukemia.

Additional Hydroxyl group on CT6 (3,4-dihydroxy-5,7-dimethoxyflavone), a flavone extracted from Chromolaena Tacotana potentially confers additional activity against pancreatic cancer as compared to CT7 (4-hydroxy-5,7-dimethoxyflavone) Parker Wade1, Miranda Green1, April Weaver1, Omri Coke1, Ruben D. Torrenegra2, and Victoria Palau1 1Department of Pharmaceutical Sciences, College of Pharmacy, East Tennessee State University, Johnson City, TN. 2Department of Chemistry, Universidad de Ciencias Aplicadas y Ambientales, Bogota, Colombia and Pancreatic cancer is one of the deadliest types of cancers, with a mortality rate of about 95%. This high mortality rate signifies there is a need for further research into finding treatment options for those affected by pancreatic cancer. Recent studies have found cytotoxic effects on cancerous cells elicited from compounds, such as flavones, in plants indigenous to Western South America, specifically Colombia. The flavones 3,4-dihydroxy-5,7-dimethoxyflavone (CT6) and 4-hydroxy-5,7-dimethoxyflavone (CT7) were isolated from Chromolaena Tacotana, member of the asteraceae family. The molecular structures of the flavones differ only by an additional hydroxyl group on CT6. Both of these compounds were tested on MIA PaCa2 and Panc28 pancreatic cancer cells at concentrations ranging from 5μM to 80μM. Cell viability after dosing of CT6 and CT7 was determined using MTT and spectrophotometry analysis. MIA PaCa2 is more poorly differentiated than Panc28. CT6 conferred greater activity on both cell lines compared to CT7. Percent cell viability of the Panc28 cell line reached a low of 35.55% (p=0.0001) with CT6, compared to 84.25% (p=0.0275) with CT7. Percent cell viability of the MIA PaCa2 cell line reached a low of 46.72% (p=0.000001)with CT6. However, CT7 showed no significant difference, with percent cell viability reaching 103.73% (p=0.5605) when compared to the control for this cell line. While CT6 exerted cytotoxic activity on both Panc28 and MIA PaCa2, CT6 had significantly more cytotoxic activity on Panc28, which could be related to the greater differentiation status of this cell line. More in depth studies will need to be conducted to determine the exact reasons for greater activity of CT6 on Panc28 cells. This could be due to the compound’s target, mitochondrial activity of the cell lines, and the minor structural differences between the two compounds.

A simple method is reported to synthesize an anticancer drug, NCX 4040, conveniently in mild conditions using silicon chemistry. A starting material, 4-hydroxybenzyl alcohol, was silylated selectively first to give t-butyldimethylsilyl 4-hydroxybenzyl ether, which was then converted to NCX 4040 by esterification, desilylation, hydrochlorination and nitration.

Chlamydial attachment and infectivity in vitro and ascending disease and sequelae in vivo have been reported to be enhanced/modulated by estrogen. Endometrial carcinoma cell lines Ishikawa and HEC-1B and the breast cancer lines MCF-7 and HCC-1806 were examined for Chlamydia trachomatis E infectivity. Estrogen receptor (ER) presence was confirmed by Western blot and qRT-PCR analyses. FACS analysis was used to determine the percent of plasma membrane-localized ERs (mERs), and their activity was tested by estrogen binding and competitive estrogen antagonists assays. Chlamydiae grew in all cell lines with HEC (90%) ≫ MCF-7 (57%) > Ishikawa (51%) ≫ HCC-1806 (20%). The cell line ER isoform composition was re-defined as: ERα + ERβ + for MCF-7, HCC-1806 and Ishikawa; and ERβ only for HEC-1B. HeLa cells were also tested and found to express ERβ, but not ERα. A small percentage of both ERs were surface-exposed and functionally active. The endometrium- predominant ERβ isoform was found in all cell lines, including those most representative of the common sites of C. trachomatis infection. Thus, the role of chlamydial attachment/infectivity will now be analyzed in ERβ + and - isogenic HEC-1B cells.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Studentka: Lucie Vašková Školitel: RNDr. Jana Maixnerová, Ph.D. Název diplomové práce: Study on cytotoxicity of compounds in vitro The subject of this diploma thesis was to assess the effect of newly synthesized antimycobacterial substances on the viability of human hepatocellular carcinoma (HepG2) cells. The tested substances were esters (HE-nMe, HE-4PHOPH, HE-KARVA, HE-2NAFT, HE-METRO, HE-CH2PY, HE-8CHIN) and thioesters (HES-4H, HES- nETH) of antituberculotic isoniazid. Experiments performed with these substances have shown, that like isoniazid, the substances inhibit InhA enzyme in mycobacteria and therefore interfere with cell wall biosynthesis. Isoniazid is a drug standardly used in the first line of TB treatment. Together with other first-line antituberculotics, some hepatotoxic potential has been reported during treatment. To assess the possible cytotoxic effect of the tested isoniazid derivatives, the standard human hepatocyte cell line HepG2 was chosen as the cell model. Cell viability was assessed by a colorimetric method that measures the metabolic activity of cells based on the reduction of the tetrazolium compound MTS. Obtained values were quantitatively compared using the toxicological...

Cardiovascular disease is the major cause of death in the United States, with 80 million people suffering from some form of heart disease each year. One major limitation is the inability of the heart to repair the damaged tissue. Stem cell therapy holds enormous promise to repair and regenerate the damaged myocardium, but there are many technical difficulties that must first be overcome. One such difficulty is the present lack of ability to track and assess transplanted stem cells over time in vivo. The central hypothesis of this thesis is that in vivo bioluminescence imaging is a safe and useful method for monitoring transplanted stem cells in mouse hearts. To evaluate this hypothesis, two aims were performed. In aim 1, stable recombinant mES cell lines expressing the firefly luciferase (fLUC) reporter gene under the control of constitutive and cardiac-specific promoters were generated and characterized in vitro. In aim 2, these fLUC-expressing recombinant cell lines were evaluated following transplantation into neonatal mouse hearts. The major findings are: (1) Novel stable recombinant mES reporter cell lines were developed for in vivo bioluminescence imaging; (2) One of these cell lines was created using the glyceraldehyde 3-phosphodehydrogenase (GAPDH) promoter fused to the fLUC reporter and it showed similar levels of fLUC expression in undifferentiated (pluripotent) compared to cardiac-differentiated mES cells; (3) Another cell line was produced using the cardiac-specific sodium-calcium exchanger 1 (NCX1) promoter fused to the fLUC reporter and this cell line showed markedly increased fLUC expression following induction of cardiac differentiation in culture when compared to the pluripotent cells. (4) Transplantation of the recombinant fLUC-expressing cells into neonatal mouse hearts produced bioluminescent signals that persisted for at least 24 days, the maximum timepoint analyzed in this study; (5) Transplantation of 100,000 or more mES cells to the heart consistently produced teratoma and tumor formations, regardless of which recombinant clone was used or whether the mES cells were injected as pluripotent or cardiac-differentiated cells, (6) Transplantation of between 10,000 and 50,000 cardiac-differentiated NCX1-fLUC mES cells(containing mixed population of other cells) per heart resulted in measurable bioluminescent image signals in vivo with low incidence of tumor formation, and (7) Some of the transplanted NCX1-fLUC mES cells were identified in ventricular muscle tissue in postmortem histological sections where it was found that they had developed cardiomyocyte characteristics. In summary, I developed stable recombinant mES cell lines suitable for non-invasive bioluminescence imaging to study the survival and proliferation of the cells in vivo. These results demonstrate that bioluminescence imaging in the neonatal mouse heart model is an effective strategy for non-invasive monitoring of transplanted stem cells over time in vivo, and minimizes animal usage through elimination of the need for animal sacrifice at multiple timepoints.

Laser-induced fluorescence (LIF) has been shown to be potentially useful for identifying microorganisms in real time. It is a selective and sensitive technique because the excitation is performed at one wavelength while the emission is monitored at longer wavelengths so that background from the excitation source can be eliminated. This specialized optical property of LIF can be applied to development of an optical sensor capable of quickly, non-invasively, and quantitatively probing complex biochemical transformations in microorganisms. Various bio-photonic optical fiber sensors based on laser-induced fluorescence (LIF) spectroscopy were developed as diagnostic tools for microorganisms. In the first phase, the enhancement of the sensitivity and selectivity of the optical sensor system focused on diagnosis of human breast cancer cell lines and Azotobacter vinelandii (an aerobic soil-dwelling organism). Autoluorescence spectra from human breast cancer cell lines and Azotobacter vinelandii corresponding to different growth environments were investigated. Then, the study has expanded to include the use of gold nanoparticles for specific DNA detection. The use of gold nanoparticales opens a door into construction of a compact, highly specific, inexpensive and userriendly optical fiber senor for specific DNA detection. An optical fiber laser-induced fluorescence (LIF) sensor based has been developed to detect single-strand (ss) DNA hybridization at the femtomolar level. Effects of various experimental parameters and configuration were investigated in order to optimize sensor performance and miniaturize sensor size.

No description available.

Metallic and metal oxide nanoparticles (Nps) have a wide range of applications in various settings including household, cosmetics and chemical industries, as well as for coatings. Nevertheless, an in-depth study of the potential toxic effects of these Nps is still needed, in order to fulfill the mandatory requirement of ensuring the safety of workers, patients and the general public. In this study, Quick Cell colorimetric assays were used to evaluate the in vitro toxicity of different metal oxide Nps [Fe(II,III)Ox, TiOx, ZnO and CeO2] in several cell lines. The ZnO Nps were found to be highly toxic, with a lethal dose ≥100 μg/ml for all the cell lines studied. Western blot was also used to test the ability of the different Nps to activate the complement pathway. However, no activation of this cascade was observed when the Nps were added. In addition, the aggregation state and charge of the Nps in culture media was studied by dynamic light scattering (DLS) and measurement of zeta potential. Transmission Electron Microscopy was used to analyze Np uptake and localization at the cellular level.

<p> It has been suggested that combination treatment of high dose rate (HDR) intraluminal brachytherapy and PDT (Photodynamic therapy) in non-small cell lung cancer (NSCLC) may improve the efficacy of treatment, reduce the toxicity and improve quality of life for patients. To provide a cellular basis for this approach we have examined the in vitro sensitivity of normal lung fibroblasts (MRC5) and four NSCLC cell lines (SKMES-1, A549, NCIH460 and NCIH23) following, UVC treatment, HDR radiation, HDR radiation with Photofrin alone, PDT and combined HDR radiation and PDT. Cell sensitivity was measured using clonogenic survival. HDR radiation was cobalt-60 gamma rays (1.5-1.9 Gy/min). For PDT treatment, cells were exposed to 2.5 J.lg/ml Photofrin for 18-24 h followed by light exposure (20mW/cm2). D37 values calculated from the survival curves indicated a 2-fold difference in sensitivity to UVC, 6-fold difference in HDR radiation sensitivity and an 8-fold difference in PDT sensitivity. All cell lines showed a similar Photofrin uptake per cell when measured by flow cytometry using 488nm excitation and 620-675 nm emission wavelengths. Photofrin alone at concentrations up to 10 J.lg/ml had no significant effect on the survival of the NSCLC cell lines, whereas 10 J.lg/ml ofPhotofrin alone reduced survival significantly in MRC5 cells. A radiosensitizing effect of Photofrin was detected in MRC5 and NCIH460 cells, but not in A549, SKMES-1 and NCI-H23 cells. For combined treatment cells were exposed to Photofrin and then either exposed to light and 15-30 minutes later exposed to HDR radiation or exposed to HDR radiation and 15-30 minutes later exposed to light. Results showed that although light followed by gamma rays resulted in a somewhat greater tumor cell kill compared to gamma rays followed by light this difference was not significant for any of the cell lines tested. However, this difference was significant when data for all NSCLC cell lines were pooled. The combined treatment with high dose rate HDR radiation and PDT was not significantly different from an additive effect of the individual treatment modalities for in vitro survival of 4 NSCLC cells. In contrast the combined treatment was less than additive for the MRCS cells suggesting that the combined treatment would have the potential advantage of doing less damage to the normal lung cells and suggests that equivalent tumour cell kill in vivo may be possible at reduced systemic effects to patients. In preliminary experiments we have started to examine the effects of Photofrin-mediated PDT on the extra cellular signal-activated protein kinase (ERK) signaling pathway in NSCLC cells. The use of multiple NSCLC cell lines allows for the possible identification of cell line specific changes involved in resistance to PDT and HDR radiation and this will be explored in future work. </p> / Thesis / Master of Science (MSc)