Browsing by Subject "Microarray"

Now showing 1 - 9 of 9

Open Access
Cancer testis gene expression as a biomarker of one-carbon metabolic activity, drug sensitivity and phenotypic heterogeneity in non-small cell lung cancer and malignant melanoma
(2016-09) Şenses, Kerem Mert
Expression of cancer-testis (CT) genes on X chromosome (CT-X) is restricted to tumors, with very low or no expression in normal adult tissues. CT-X gene expression is one of the factors contributing to tumor heterogeneity and is variably observed in various types of cancers including non-small cell lung cancer (NSCLC) and malignant melanoma. Regulation of CT-X gene expression has been strongly linked to DNA methylation of promoter regions, however, mechanisms leading to re-expression of these genes in tumors, driven by promoter hypomethylation, is remained unsolved. Although tumors expressing CT-X genes are shown to be associated with higher tumor stage, larger tumors and aggressiveness, differential drug sensitivity of CT-X positive and negative tumors have not been investigated. In this thesis, we aimed to find the association between one-carbon pathway polymorphisms and CT-X gene expression. Moreover, we tested various tools and methods to find effective drugs for tumors in different phenotypic subgroups determined by CT-X gene expression or by other factors.
Open Access
Co-expression pairs and modules (CoEX-PM): a shiny application and an example case study on chromogranins
(2018-09) Kaya, Tuğberk
Gene expression signatures have been proved to be effective biomarkers of tumorigenesis and metastasis especially when alternative methods are inconvenient or ineffective. Nevertheless, handling very large datasets obtained via high-throughput protocols to extract gene expression signatures may prove challenging. A great number of software packages that facilitate such analyses have been written in R programming language are publicly available and free. However, the relatively steep learning curve that is required to use R proficiently prevents the utilization of these packages. I have developed the Shiny application Co-expression Modules and Pairs (CoEX-PM) using R programming language and the R package shiny. The CoEX-PM application handles human Affymetrix microarray data and enables users to generate pairwise correlation plots, conduct meta-correlation analysis with user-selected GEO datasets along with co-expression module generation by WGCNA program for genes of interest. The CoEX-PM application provides the user with a GUI, therefore, does not require any coding knowledge to perform the analyses. Pheochromocytoma (PCC) and neuroblastoma (NB) are neural-crest derived tumors, common in adults and children, respectively and are both associated with high-rate of morbidity and mortality. In addition, both tumor types display neuroendocrine tumor (NET) characteristics. Chromogranin A (CgA) has been linked with NETs as a moderately sensitive and non-specific tumor marker. The chromogranin family consists of up to seven members, three of which are chromogranin (CgA), chromogranin B (CgB) and secretogranin II (SgII) or occasionally named as chromogranin C (CgC). However, it is not known whether chromogranin/secretogranin family members are differentially co-expressed in PCC and NB. Here, I investigate the degree of co-expression in gene networks by analyzing gene expression signatures of the chromogranin/secretogranin paralogous gene family using CoEX-PM application on neuroendocrine tumor datasets. The findings indicate presence of concise and highly co-expressed functional components in PCC and NB driven by chromogranin expression signatures.
Open Access
Effects of aging and short-term dietary restriction on neurogenesis and cellular senescence in the zebrafish (Danio Reio) brain
(2016-09) Erbaba, Begün
Currently we know from rodent and fish studies that adult neuron generation is reduced but still continues in old animals with a dynamic change throughout aging. This process occurs mainly in hippocampal region, which is thought to be analogous to a region in telencephalon of the zebrafish brain. Changes in this neuron turnover are thought to be one contributing factor to cognitive change occuring with advanced age. Since we know that external factors can affect the process of neurogenesis, and as previous studies showed, dietary restriction (DR) extends life span; here, we hypothesized that DR should also alleviate several age associated alterations. In order to test this, we applied a 10-week feeding regimen to young (8-9 months) and old (26-32.5 months) male and female fish. We had two dietary regimen groups, one fed Ad libitum and one fed with a DR that was a pattern of every-other-day feeding, which is a widely accepted method of DR. A total of 124 animals were used in this study. As a result, a significant loss of body weight in both young and old DR groups was observed without an effect on body lengths. To be able to label actively dividing cells we used Bromodeoxyuridine (BrdU), which is a thymidine analog. It is injected into the fish intraperitoneally prior to euthanasia. Four hours later the brains were dissected and fixed for sectioning. We obtained cross-sectional slices of 50 m thickness with a vibratome, performed immunostaining with antibodies against BrdU, NeuN (neuronal marker), HuC (neuronal marker); and visualized the brain sections with confocal microscopy forming 3D reconstructed pictures. We counted the BrdU positive cells in all brain slices, forming a regional map of the telencephalic region of zebrafish brain, in which we documented the specific regions where the adult neurogenesis dominates the most and least. Our results confirmed that there are more BrdU positive cells in young animals than olds, and that age is correlated with an increased senescence associated fi-galactosidase (SA-fi-gal) activity, along with shortened telomere lengths. The 10-week diet was not found to be creating a significant change in cell proliferation rates, cellular senescence, or the differentiation pattern of glial cells. However, it was demonstrated to have a shortening effect on telomere lengths. Our data suggest that the potential effects of DR could be related to telomere regulation. Therefore, in order to detect differentially expressed genes that could be related to this mechanism between the groups, we performed microarray analysis with differing DR regimens. Initial data indicated no significant effects of a 4-week diet on gene expression differences among aged fish. Further analysis of the different periods of DR will be performed. Taken together, the effects of age are more robust than a short-term DR.
Open Access
Elucidating immunomodulatory effects of telomeric repeat mimicking synthetic A151 oligodeoxynucleotide on immune cell transcriptome
(2019-09) Yazar, Volkan
Recent evidence revealed that DNA is beyond just the blueprint of life; it is also involved in immunomodulation. Unmethylated Cytosine-phosphate-Guanine (CpG) motifs of prokaryotic DNA stimulate immune response by interacting with Toll-like receptor 9 (TLR9). This interaction is mimicked using synthetic oligodeoxynucleotides (ODN) bearing similar DNA motifs to boost vaccinedriven immune response in human. Conversely, mammalian telomeric ends expressing TTAGGG repeats suppress immune response and contribute to fine-tuning of delicate immune balance. In this respect, suppressive ODN A151 with such G-rich telomeric repeats has proven useful in downregulating immune response; an overly active immune response is just as harmful to the host, as in the case of autoimmune disorders. Both CpG ODN and A151 are currently under preclinical/clinical trials with the aim of averting or medically treating a wide range of conditions from cancer to infectious disease or from autoimmune to autoinflammatory conditions. Contrary to CpG ODN, A151 literature is very limited and its modus operandi at gene level remains more of a mystery. Additionally, the degree, duration and breath of A151-induced alterations in immune transcriptome appear partially understood. Given the medical potential A151 holds for immunosuppressive therapy in human as a “self-molecule”, understanding the underlying molecular mechanisms via which A151 operates is invaluable. Toward this end, we attempted to uncover the unidentified features lying behind A151 ODNs immunosuppressive effects on immune cell transcriptome using a combined analysis approach of microarray data in this thesis. We demonstrated for the first time that A151 ODN deprives the cells energy by ceasing cellular uptake of fundamental molecules into the immune cells after derailing the entire intracellular trafficking. Putting it another way, A151 does not directly act on immune system cells but actually suffocates the cells by messing with intracellular trafficking, thereby blocking cellular uptake of fundamental molecules like glucose and glutamine. As such, immune suppression is just an indirect consequence of this larger cellular chaos. Our results indicated that this phenomenon occurs independent of CpG ODN stimulation of the cells and in a timely manner. Most, if not all, regulators of intracellular trafficking, vesicle signaling, and membrane protein transportation were found downregulated after incubation of cells with A151 at a physiologically relevant concentration, as well, implying full-blown entry to this intracellular turmoil at cellular level. The A151 effect on immune transcriptome was not just restricted to setting off a chaos for intracellular dynamics; novel long non-coding RNAs (lncRNAs) with immunometabolic activities were identified within the scope of this study among elements potentially regulated by A151, such as Lncpint, Malat1 and H2-T10 just to name a few. The involvement of lncRNAs in immune regulation is a well-documented phenomenon. Finally, our data showed that as an epiphenomenon of the intracellular turmoil mentioned above A151 has a deep impact in immune cells on mTOR network, the cardinal network of cellular energetics, growth, proliferation, and survival. A major shift in expression profile of relevant genes, i.e. downregulation of many activators of mTOR signaling along with core mTOR components, was validated on the benchtop after different layers of experimental validation using a wide range of marker genes and functional assays, reflecting A151’s ability to vastly shape dynamics of metabolism in favor of a metabolically inert state in macrophages and in B-cells. This knowledge will expand the breadth of A151 therapy in the clinics.
Open Access
Functionally conserved effects of rapamycin exposure on zebrafish
(Spandidos Publications, 2016-03) Sucularli, C.; Shehwana, H.; Kuscu, C.; Dungul, D. C.; Ozdag, H.; Konu, O.
Mechanistic target of rapamycin (mTOR) is a conserved serine/threonine kinase important in cell proliferation, growth and protein translation. Rapamycin, a well-known anti-cancer agent and immunosuppressant drug, inhibits mTOR activity in different taxa including zebrafish. In the present study, the effect of rapamycin exposure on the transcriptome of a zebrafish fibroblast cell line, ZF4, was investigated. Microarray analysis demonstrated that rapamycin treatment modulated a large set of genes with varying functions including protein synthesis, assembly of mitochondrial and proteasomal machinery, cell cycle, metabolism and oxidative phosphorylation in ZF4 cells. A mild however, coordinated reduction in the expression of proteasomal and mitochondrial ribosomal subunits was detected, while the expression of numerous ribosomal subunits increased. Meta-analysis of heterogeneous mouse rapamycin microarray datasets enabled the comparison of zebrafish and mouse pathways modulated by rapamycin, using Kyoto Encyclopedia of Genes and Genomes and Gene Ontology pathway analysis. The analyses demonstrated a high degree of functional conservation between zebrafish and mice in response to rapamycin. In addition, rapamycin treatment resulted in a marked dose-dependent reduction in body size and pigmentation in zebrafish embryos. The present study is the first, to the best of our knowledge, to evaluate the conservation of rapamycin-modulated functional pathways between zebrafish and mice, in addition to the dose-dependent growth curves of zebrafish embryos upon rapamycin exposure.
Open Access
Gene expression changes in aging zebrafish (Danio rerio) brains are sexually dimorphic
(BioMed Central, 2014) Arslan-Ergül, Ayça; Adams, Michelle M.
Background: Brain aging is a multi-factorial process due to both genetic and environmental factors. The zebrafish has recently become a popular model organism for examining aging and age-related diseases because as in humans they age gradually and exhibit cognitive decline. Few studies have examined the biological changes in the aging brain that may contribute to these declines and none have examined them within individuals with respect to gender. Our aim was to identify the main genetic pathways associated with zebrafish brain aging across gender. We chose males and females from specific age groups (young, 7.5-8.5 months and old, 31-36 months) based on the progression of cognitive decline in zebrafish. RNA was isolated from individual brains and subjected to microarray and qPCR analysis. Statistical analyses were performed using a two-way ANOVA and the relevant post-hoc tests. Results: Our results demonstrated that in the brains of young and old male and female zebrafish there were over 500 differentially expressed genes associated with multiple pathways but most notably were those related to neurogenesis and cell differentiation, as well as brain and nervous system development. Conclusions: The gene expression of multiple pathways is altered with age and differentially expressed in males and females. Future studies will be aimed at determining the causal relationships of age-related changes in gene expression in individual male and female brains, as well as possible interventions that counteract these alterations.
Open Access
Simultaneous miRNA and mRNA transcriptome profiling of glioblastoma samples reveals a novel set of OncomiR candidates and their target genes
(Elsevier, 2018) Güllüoğlu, Ş.; Tüysüz, E. C.; Şahin, M.; Kuşkucu, A.; Yaltırık, C. K.; Türe, U.; Küçükkaraduman, Barış; Akbar, Muhammad Waqas; Güre, Ali Osmay; Bayrak, Ö. F.; Dalan, A. B.
Although glioblastomas are common, there remains a need to elucidate the underlying mechanisms behind their initiation and progression and identify molecular pathways for improving treatment. In this study, sixteen fresh-frozen glioblastoma samples and seven samples of healthy brain tissues were analyzed with miRNA and whole transcriptome microarray chips. Candidate miRNAs and mRNAs were selected to validate expression in fifty patient samples in total with the criteria of abundance, relevance and prediction scores. miRNA and target mRNA relationships were assessed by inhibiting selected miRNAs in glioblastoma cells. Functional tests have been conducted in order to see the effects of miRNAs on invasion, migration and apoptosis of GBM cells. Analyses were carried out to determine correlations between selected molecules and clinicopathological features. 1332 genes and 319 miRNAs were found to be dysregulated by the microarrays. The results were combined and analyzed with Transcriptome Analysis Console 3 software and the DAVID online database. Primary differential pathways included Ras, HIF-1, MAPK signaling and cell adhesion. OncomiR candidates 21-5p, 92b-3p, 182-5p and 339-5p for glioblastoma negatively correlated with notable mRNA targets both in tissues and in in vitro experiments. miR-21-5p and miR-339-5p significantly affected migration, invasion and apoptosis of GBM cells in vitro. Significant correlations with overall survival, tumor volume, recurrence and age at diagnosis were discovered. In this article we present valuable integrated microarray analysis of glioblastoma samples regarding miRNA and gene-expression levels. Notable biomarkers and miRNA-mRNA interactions have been identified, some of which correlated with clinicopathological features in our cohort.
Open Access
Strain-and region-specific gene expression profiles in mouse brain in response to chronic nicotine treatment
(Wiley-Blackwell Publishing, 2008) Wang, J.; Gutala, R.; Hwang, Y. Y.; Kim J. -M.; Konu, O.; Ma, J. Z.; Li, M. D.
A pathway-focused complementary DNA microarray and gene ontology analysis were used to investigate gene expression profiles in the amygdala, hippocampus, nucleus accumbens, prefrontal cortex (PFC) and ventral tegmental area of C3H/HeJ and C57BL/6J mice receiving nicotine in drinking water (100 μg/ml in 2% saccharin for 2 weeks). A balanced experimental design and rigorous statistical analysis have led to the identification of 3.5-22.1% and 4.1-14.3% of the 638 sequence-verified genes as significantly modulated in the aforementioned brain regions of the C3H/HeJ and C57BL/6J strains, respectively. Comparisons of differential expression among brain tissues showed that only a small number of genes were altered in multiple brain regions, suggesting presence of a brain region-specific transcriptional response to nicotine. Subsequent principal component analysis and Expression Analysis Systematic Explorer analysis showed significant enrichment of biological processes both in C3H/HeJ and C57BL/6J mice, i.e. cell cycle/proliferation, organogenesis and transmission of nerve impulse. Finally, we verified the observed changes in expression using real-time reverse transcriptase polymerase chain reaction for six representative genes in the PFC region, providing an independent replication of our microarray results. Together, this report represents the first comprehensive gene expression profiling investigation of the changes caused by nicotine in brain tissues of the two mouse strains known to exhibit differential behavioral and physiological responses to nicotine.
Open Access
A suppressive oligodeoxynucleotide expressing TTAGGG motifs modulates cellular energetics through the mTOR signaling pathway
(Oxford University Press, 2020) Yazar, Volkan; Kılıç, Gizem; Bulut, Özlem; Canavar-Yıldırım, Tuğçe; Yağcı, Fuat C.; Gamze, Aykut; Klinman, D. M.; Gürsel, M.; Gürsel, İhsan
Immune-mediated inflammation must be down-regulated to facilitate tissue remodeling during homeostatic restoration of an inflammatory response. Uncontrolled or over-exuberant immune activation can cause autoimmune diseases, as well as tissue destruction. A151, the archetypal example of a chemically synthesized suppressive oligodeoxynucleotide (ODN) based on repetitive telomere-derived TTAGGG sequences, was shown to successfully down-regulate a variety of immune responses. However, the degree, duration and breadth of A151-induced transcriptome alterations remain elusive. Here, we performed a comprehensive microarray analysis in combination with Ingenuity Pathway Analysis (IPA) using murine splenocytes to investigate the underlying mechanism of A151-dependent immune suppression. Our results revealed that A151 significantly down-regulates critical mammalian target of rapamycin (mTOR) activators (Pi3kcd, Pdpk1 and Rheb), elements downstream of mTOR signaling (Rps6ka1, Myc, Stat3 and Slc2a1), an important component of the mTORC2 protein complex (Rictor) and Mtor itself. The effects of A151 on mTOR signaling were doseand time-dependent. Moreover, flow cytometry and immunoblotting analyses demonstrated that A151 is able to reverse mTOR phosphorylation comparably to the well-known mTOR inhibitor rapamycin. Furthermore, Seahorse metabolic assays showed an A151 ODN-induced decrease in both oxygen consumption and glycolysis implying that a metabolically inert state in macrophages could be triggered by A151 treatment. Overall, our findings suggested novel insights into the mechanism by which the immune system is metabolically modulated by A151 ODN.