Thesis advisor: Vassilios Bezzerides / The aim of this study was to utilize a massively parallel reporter assay (MPRA) to identify organ-specific microRNA (miRNA) target sequences to refine the timing and expression of transgene expression for gene therapy. We previously had developed a cardiac gene therapy for Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) using a systemically delivered adeno-associated virus (AAV9) vector. We hypothesized that incorporation of organ specific miRNA target sites into our vector construct could improve our therapy’s tissue specificity due to the ability of miRNAs to silence transgene expression. Initially, we attempted to incorporate mir-124 target sequences into our vector to detarget the brain. Although these initial attempts were unsuccessful, the study allowed us to develop a protocol to test the effectiveness of miRNA target sequences. Thereafter, we developed a method to screen thousands of putative miRNA target sequences simultaneously. In this study, target sequences of miRNAs specific to the heart, brain and liver were incorporated into a plasmid library. This plasmid library was subsequently made into AAV and injected into mice from a CPVT transgenic line. Total DNA and RNA was later extracted from the target organs, converted into genomic DNA (gDNA) and complementary DNA (cDNA) libraries respectively, and sent for amplicon sequencing. We analyzed the results using Comparative Microbiome Analysis 2.0 software (CoMA) and a custom python script to count the occurrence of each specified barcode per sample. In doing so, we showed that the miRNA suppression mechanism is not only effective but also organ specific. Furthermore, we developed a second script to create a combinatorial library from a set list of miRNA target sequences enabling us to efficiently test thousands of target sequence combinations at once. In doing so, we will be able to identify effective miRNA target sequence combinations to further improve gene therapy specificity. / Thesis (BS) — Boston College, 2021. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Departmental Honors. / Discipline: Biology.

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Mucosa associated lymphoid tissue (MALT) lymphoma is characterized by t(11;18)(q21;q21)/API2-MALT1, t(1;14)(p22;q32)/BCL10-IGH andt(14;18)(q32;q21)/IGH-MALT1, which commonly activate the NF-κB pathway. Gastric MALT lymphomas harbouring such translocation do not respond to Helicobacter pylori eradication, while those without translocation can be cured by antibiotics. To understand the molecular mechanism of MALT lymphoma with and without chromosome translocation, 24 cases (15 translocation-positive and 9 translocation-negative) of MALT lymphomas together with 7 follicular lymphomas and 7 mantle cell lymphomas were analysed by Affymetrix gene expression microarray platform. Unsupervised clustering showed that cases of MALT lymphoma were clustered as a single branch. However, within the MALT lymphoma group, translocation-positive cases were intermingled with translocation-negative cases. Gene set enrichment analysis (GSEA) of the NF-κB target genes and 4394 additional gene sets covering various cellular pathways, biological processes and molecular functions showed that translocation-positive MALT lymphomas were characterized by an enhanced expression of NF-κB target genes, particularly TLR6, CCR2, CD69 and BCL2, while translocation-negative cases were featured by active inflammatory and immune responses, such as IL8, CD86, CD28 and ICOS. Separate analyses of the genes differentially expressed between translocation-positive and negative cases and measurement of gene ontology term in these differentially expressed genes by hypergeometric test reinforced the above findings by GSEA. The differential expression of these NF-κB target genes between MALT lymphoma with and without translocation was confirmed by quantitative RT-PCR and immunohistochemistry or Western blot. Expression of TLR6, in the presence of TLR2, enhanced both API2-MALT1 and BCL10 mediated NF-κB activation in vitro. In addition, there was cooperation between expression of BCL10, MALT1 or API2-MALT1, and stimulation of the antigen receptor or CD40 or TLR in NF-κB activation as shown by both reporter assay and IκBα degradation. Interestingly, expression of BCL10 but not API2-MALT1 and MALT1, in the presence of LPS stimulation, also triggered IκBβ degradation, suggesting activation of different NF-κB dimers between these oncogenic products. Study by co-immunoprecipitation showed that BCL10 directly interacts with MALT1. Sub-cellular localisation experiments in BJAB B-cells, showed that BCL10 localisation was affected by MALT1. When BCL10 was over-expressed, the protein was predominantly expressed in the nuclei, but when MALT1 was over-expressed, BCL10 was mainly localised in the cytoplasm. When both BCL10 and MALT1 were over-expressed, BCL10 was expressed in the cytoplasm in the early hours when the protein level was low, but in both the cytoplasm and nuclei after 9 hours when the protein level was high. Over-expression of API2-MALT1 did not shown any apparent effect on BCL10 sub-cellular localisation in vitro. Finally, comparison of MALT lymphoma expression microarray with other lymphomas showed lactoferrin to be highly expressed in MALT lymphoma. This was confirmed by qRT-PCR, showing lactoferrin to be significantly over-expressed in MALT lymphoma compared to FL and MCL. Thus lactoferrin may be a potential marker for MALT lymphoma.

No description available.

Targeting oncogene promoters: a novel heterocyclic cations as G-quadruplex stabilizing ligands Lenka Pohlová Abstract: The diploma thesis studies an effect of newly synthesized group of compounds - helquats - on the expression of c-myc as a major player in malignant transformation and tumorigenesis via the stabilization of G-quadruplex in c-myc promotor. The G-quadruplex c-myc stabilization ability was tested for 101 helquats using dual luciferase reporter assay. The G-quadruplex c-myc stabilization ability was found for 13 helquats by this method. 8 successful helquats was selected by a comparison of the results from dual luciferase reporter assay and FRET melting assay. Effect on cell viability of tumor (HeLa S3) and non-tumor (HUVEC) cell lines was evaluated for these 8 helquats. Three of them exhibited cytotoxic effect on tumor cells but no effect was observed on viability of non-tumor cells. Moreover, an effect of these 3 helquats on c-myc expression on both mRNA and protein level, where significant effect on c-myc mRNA expression was not found for most of incubation periods. The 30% decrease in mRNA level was observed only for 24 hours incubation period for two helquats (LS702 and MJ656). The decrease in the expression on protein level was observed for all tested helquats, and helquat LS702 had the...

La régulation des gènes est à l’origine de la diversité cellulaire en permettant aux cellules de se différencier et de se spécialiser. La régulation génique repose largement sur l’existence de séquences d’ADN non codantes dans le génome, appelées "éléments cis-régulateurs", qui vont permettre de recruter de nombreux facteurs de transcription afin de former d’importants complexes (nucléo)protéiques qui vont agir sur le niveau de transcription des gènes. Ce recrutement est notamment contrôlé par des modifications épigénétiques. Le développement des techniques de séquençage et des méthodes d’analyse bioinformatiques permettent d’intégrer de grandes quantités de données pour étudier le fonctionnement des éléments régulateurs. Dans un premier temps, l’intégration de l’ensemble des données ChIP-seq disponibles dans les bases de données nous a permis de créer un catalogue d’éléments régulateurs putatifs chez l’Homme. L’analyse de ce catalogue nous a alors mené à caractériser ces éléments et à mettre en évidence la complexité combinatoire des facteurs de transcription. Dans un deuxième temps, nous avons réalisé une étude basée sur l’analyse des éléments régulateurs impliqués dans la différenciation précoce des lymphocytes T chez la souris. Cette étude a permis de mettre en évidence deux niveaux de complexité impliqués dans la régulation des gènes : le premier est basé sur la combinatoire des facteurs de transcription au sein des éléments régulateurs et le second repose sur la combinatoire des éléments eux-mêmes. Finalement, nous avons développé une nouvelle technique d’analyse quantitative et à haut débit de l’activité régulatrice de régions génomiques chez les mammifères. / Gene regulation is responsible for cell diversity by allowing cell differentiation and specialisation. Gene expression regulation relies mainly on the existence of non-coding DNA sequences in the genome, called "cis-regulatory elements", which recruit numerous transcription factors to form (nucleo)protein complexes which act on the gene transcription level. This recruitment is controlled in particular by epigenetic modifications. The rapid development of sequencing technologies and bioinformatics methods makes possible the integration of large amounts of data to study regulatory elements. First, the integration of ChIP-seq data for all transcription factors available in public databases has allowed us to create an extensive catalogue of putative regulatory elements in the human genome. The overall analysis of this catalogue led us to further characterize these elements and to highlight the high level of combinatorial complexity of transcription factors in the genome. Secondly, we conducted a more specific study based on the analysis of the regulatory elements involved in the early differentiation of T-cells in mice. This study provided an opportunity to highlight two levels of complexity based on regulatory elements and involved in gene regulation: the first rests on the transcription factor combinatorial in regulatory elements and the second is based on the combinatorial of elements themselves within loci. Finally, to validate experimentally the regulatory elements, we have developed a new quantitative and high-throughput technique to assess the regulatory activity of genomic regions in mammals.

Hepatitis C virus (HCV) is a worldwide spread pathogen infecting up to 3 % of the human population. Nowadays, research of new drugs against this virus is focused on the individual steps in its life cycle, including the translation initiation. In the case of HCV translation initiation is dependent on the internal ribosome entry site (IRES). Besides of components of the translational machinery also other components of the cell, so called IRES trans-acting factors (ITAF), contribute to its proper progress. This work continues in previous research of our laboratory focused on searching for new ITAF. In order to search for potential ITAF increasing HCV IRES activity new recombinant plasmid vectors and reference strains were prepared and selection conditions of the selection system were optimized. The differences in the growth characteristics of the reference strains were analyzed and quantified under selective and non-selective conditions. A set of pilot high efficiency transformations of the yeast strain pJ69-4A carrying bicistronic construct with HCV IRES were conducted using human expression cDNA library in order to optimize the efficiency of transformation and selection conditions and to attempt to identify new ITAF. Several dozens of randomly selected clones from these transformations obtained under...

This PhD aimed to elucidate the mechanisms by which polymorphisms may alter androgen-induced transactivation of androgen receptor (AR) target genes which may be important in prostate cancer aetiology. The second aspect of this PhD focused on identifying and characterising functional polymorphisms that may have utility as predictive risk indicators for prostate cancer and which may aid in earlier therapeutic intervention and better disease management. Analyses were carried out on the kallikrein-related peptidase 3 (KLK3), also known as the prostate specific antigen (PSA), gene and the kallikrein-related peptidase 4 (KLK4) gene. The PSA and KLK4 genes are part of the serine protease family that have trypsin or chymotrypsin like activity and are thought to play a role in the development of hormone-dependent cancers in tissues such as those in the prostate, breast, endometrium and ovaries. In the prostate, PSA is regulated by androgens and three androgen response elements (AREs) have been described in the promoter and upstream enhancer region. The PSA ARE I harbours a polymorphism at -158 bp from the transcription initiation site (TIS) that results in a G to A transition (G-158A). This PhD investigated the functional significance of the PSA G-158A polymorphism which has been reported to be associated with prostate cancer risk. Electromobility shift assays (EMSAs) investigating the interaction of ARE I variants with the AR DNA binding domain (AR-DBD) demonstrated that the A allele had a two-fold increased binding affinity for the AR-DBD when compared with the G allele. This was confirmed with endogenous AR in limited proteolysis-EMSA experiments. The limited proteolysis-EMSA experiments also demonstrated differential sensitivities of PSA ARE I alleles to trypsin digestion, which suggests that the G-158A polymorphism has an allosteric effect on the AR that alters AR/ARE I complex stability. Furthermore, Chromatin Immunoprecipitation (ChIP) assays suggest that the A allele more readily recruited the AR in vivo when compared with the G allele and is consistent with the in vitro binding data. Luciferase reporter assays carried out in both LNCaP and 22Rv1 prostate cancer cells, and using the natural (dihydrotestosterone; DHT) ligand demonstrated that the A allele was more responsive to androgens in LNCaP cells. Hence, this study has elucidated the potential mechanisms by which the G-158A polymorphism may differentially regulate PSA expression (of which up-regulation of PSA is thought to be important in prostate cancer development and progression). KLK4 has similar tissue-restricted expression as PSA and is up-regulated by steroid hormones in many endocrine cells including those in the prostate. A putative ARE (KLK4-pARE) located at -1,005 to -1019 relative to the more predominantly used transcription initiation site, TIS3, was initially found in supershift assays using AR antibodies to interact with endogenous AR. However, subsequent EMSA analysis using purified AR-DBD suggest that KLK4-pARE may be interacting with the AR indirectly. To investigate this hypothesis, a tandem construct of KLK4-pARE was cloned into the pGL3-Promoter vector for hormone-induced reporter assays. However, reporter assays did not demonstrate any responsiveness of KLK4-pARE to androgens, estradiol or progestins. Consequently, Real-Time PCR was carried out to reassess the hormonal regulation of KLK4 at the mRNA level. Consistent with the literature, data from this study suggests that KLK4 may be up-regulated by androgens, progestins and estradiol in a cyclical manner. Hormone-induced luciferase reporter assays were then carried out on seven promoter constructs that span 2.8 kb of the KLK4 promoter from TIS3. However, none of the seven promoter constructs demonstrated any significant responsiveness to androgens, estradiol or progestins. This study suggests that hormone response elements (HREs) that may drive the hormonal regulation of KLK4 in prostate cancer may be located further upstream from the promoter region investigated in this PhD, or alternatively, may lie 3' of TIS3. The characterisation of KLK4 promoter polymorphisms and their flanking sequences were also carried out in parallel to the functional work with the intent to assess the functional significance of any polymorphisms that may be located within HREs. In total 19 polymorphisms were identified from the public databases and from direct sequencing within 2.8 kb of the KLK4 promoter from TIS3. However, the functional and clinical significance of these 19 polymorphisms were not further pursued given the negative findings from the functional work. The PSA AR enhancer region was also assessed for potential polymorphisms that may be associated with prostate cancer risk. A total of 12 polymorphisms were identified in the PSA enhancer of which two (A-4643G and T-5412C) have been reported to alter functionality of the enhancer region and thus, prioritised for further analysis. Association analysis for prostate cancer risk was then carried out on these PSA enhancer polymorphisms as none of the KLK4 promoter polymorphisms were found in functional HREs. No significant association for either the A-4643G or T-5412C polymorphism with prostate cancer risk was found at the P = 0.05 level. However, under an age-adjusted dominant model a 1.22- (95% CI = 1.16-1.26) and 1.23-fold (95% CI = 1.17-1.29) increased risk for prostate cancer was found for the A-4643G or T-5412C polymorphisms, respectively. Both polymorphisms were also assessed for association with tumour grade and stage and PSA levels. Genotypes were significantly different for the A-4643G and T-5412C polymorphisms with tumour stage and PSA levels, respectively. However, these results are likely to be biased by the case population which consist primarily of men who presented with incidental (pT1) and organ-confined (pT2) tumours. To summarise, the A-4643G and T-5412C polymorphisms are unlikely to be associated with prostate cancer risk, PSA levels or stage/grade of disease. However, further analyses in a larger cohort is warranted given that these polymorphisms alter androgen responsiveness of the PSA enhancer and that elevated PSA levels are indicative of men with prostate cancer. To summarise, this PhD has elucidated the functional significance of the PSA G-158A polymorphism in prostate cancer and which may be important in prostate cancer patho-physiology. This PhD has also furthered the understanding of the hormonal regulation of KLK4 in prostate cancer cells. Finally, this PhD has carried out a pilot study on two functional PSA enhancer polymorphisms (A-4643G and T-5412C) with prostate cancer risk.

Tick-borne encephalitis is a serious disease of the central nervous system. It is caused by tick-borne encephalitis virus, which is transmitted by ticks. The Czech Republic is one of the countries with the highest prevalence of this disease. Tick-borne encephalitis virus is able to replicate in several cell types. In this work we focused on macrophage line IC-21 and dendritic cells, because these cells are the first, which encounter the virus and support its spreading in the host at early stage of infection. So far there is not known any specific receptor for virus entry into cells or which signaling pathways activates. Therefore, we decided to investigate the activation of selected signaling pathways after infection with tick-borne encephalitis virus and influence of tick saliva on this activation. We employed methods of dual luciferase reporter assay, immunosandwich assay and western blot. The obtained results showed that in virus infected IC-21 cells are activated phosphatidyl-inositol pathway, NF-κB pathway, signaling molecule Erk1/2 and others. Testing of tick saliva effect revealed significantly decreased activity of NF-κB, AP-1 and CREB.

All viruses encode for at least one viral interferon (IFN) antagonist, which is used to subvert the cellular IFN response, a powerful antiviral innate immune response. Numerous in vitro and in vivo studies have demonstrated that IFN antagonism is crucial for virus survival, suggesting that viral IFN antagonists could represent promising therapeutic targets. This study focuses on Respiratory Syncytial Virus (RSV), an important human pathogen for which there is no vaccine or virus-specific antiviral drug. RSV encodes two IFN antagonists NS1 and NS2, which play a critical role in RSV replication and pathogenicity. We developed a high-throughput screening (HTS) assay to target NS2 via our A549.pr(ISRE)GFP-RSV/NS2 cell-line, which contains a GFP gene under the control of an IFN-stimulated response element (ISRE) to monitor IFN- signalling pathway. NS2 inhibits the IFN-signalling pathway and hence GFP expression in the A549.pr(ISRE)GFP-RSV/NS2 cell-line by mediating STAT2 degradation. Using a HTS approach, we screened 16,000 compounds to identify small molecules that inhibit NS2 function and therefore relinquish the NS2 imposed block to IFN-signalling, leading to restoration of GFP expression. A total of twenty-eight hits were identified; elimination of false positives left eight hits, four of which (AV-14, -16, -18, -19) are the most promising. These four hit compounds have EC₅₀ values in the single μM range and three of them (AV-14, -16, -18) represent a chemically related series with an indole structure. We demonstrated that the hit compounds specifically inhibit the STAT2 degradation function of NS2, not the function of NS1 or unrelated viral IFN antagonists. At the current time, compounds do not restrict RSV replication in vitro, hence hit optimization is required to improve their potency. Nonetheless, these compounds could be used as chemical tools to determine the unknown mechanism by which NS2 mediates STAT2 degradation and tackle fundamental questions about RSV biology.