Dissertations / Theses on the topic 'RNA therapeutic'

Neurodegenerative diseases are linked to altered splicing mechanisms (Mills et al., 2012). Fronto temporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) is one such disease that stems from the differential splicing caused due to mutations in Microtubule associated protein tau (MAPT) gene (Esther et al., 2002). This PhD thesis focuses on developing RNA-based therapeutic approaches to address FTDP-17. CHAPTER 1 introduces a broad range of topics such as splicing mechanism, neurodegenerative diseases associated with splice defects, therapeutic tools to modulate such splice defects in the context of neurogenetic diseases and possible applications of available tools for FTDP-17. CHAPTER 2 explores an exon skipping strategy to modulate splice defects in the context of FTD-17 using small nuclear RNAs (snRNAs). CHAPTER 3 is based on a short interfering RNA (siRNA) approach to modulate post-transcriptional gene silencing of specific isoform associated to FTDP-17. CHAPTER 4 employs long non coding RNA (lncRNA) to mediate post transcriptional repression of tau protein associated to FTDP-17 and deciphers its auxiliary role in splicing of exon 10 CHAPTER 5 elaborates on the future perspectives of all the above mentioned approaches to find a cure for FTDP-17.

Neurodegenerative diseases are linked to altered splicing mechanisms (Mills et al., 2012). Fronto temporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) is one such disease that stems from the differential splicing caused due to mutations in Microtubule associated protein tau (MAPT) gene (Esther et al., 2002). This PhD thesis focuses on developing RNA-based therapeutic approaches to address FTDP-17. CHAPTER 1 introduces a broad range of topics such as splicing mechanism, neurodegenerative diseases associated with splice defects, therapeutic tools to modulate such splice defects in the context of neurogenetic diseases and possible applications of available tools for FTDP-17. CHAPTER 2 explores an exon skipping strategy to modulate splice defects in the context of FTD-17 using small nuclear RNAs (snRNAs). CHAPTER 3 is based on a short interfering RNA (siRNA) approach to modulate post-transcriptional gene silencing of specific isoform associated to FTDP-17. CHAPTER 4 employs long non coding RNA (lncRNA) to mediate post transcriptional repression of tau protein associated to FTDP-17 and deciphers its auxiliary role in splicing of exon 10 CHAPTER 5 elaborates on the future perspectives of all the above mentioned approaches to find a cure for FTDP-17.

Prion diseases are fatal, transmissible neurodegenerative disorders characterised by accumulation throughout the brain of PrPSc, an abnormally folded isoform of the normal cellular prion protein, PrP. PrPSc is associated with infectivity but is not directly neurotoxic and targeting it is of limited efficacy in prion therapeutics. However, PrP-null mice are resistant to prion infection and neurotoxicity. Transgene-c mediated depletion of neuronal PrP in mice with established prion infection reverses early spongiosis, neuronal loss and cognitive deficits, and prevents clinical disease progression. Thus, reducing PrPC expression in the brain through extrinsic means is likely to be an effective therapy for prion diseases. RNA interference can be exploited to mediate gene silencing and can be stably achieved in non-dividing cells such as neurons by incorporation of the small interfering RNAs into replication-deficient lentiviruses. The work described in this thesis strongly validates the use of lentiviral-mediated RNA interference as a therapeutic approach in prion disease. Reducing PrPc expression with siRNA duplexes enabled clearance of PrPSc and infectivity from prion-infected cells in vitro. Lentiviruses constructed to express the interfering sequences demonstrated effective reduction of PrPc expression in vitro. Stable expression of the interfering RNA molecules in vivo through lentiviral transduction of the hippocampus reduced local pathology and significantly prolonged survival in a mouse model of prion disease. This represents an important and novel advance in the treatment of established prion disease with relevance for all prion strains.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, 2019
Cataloged from PDF version of thesis.
Includes bibliographical references.
RNA interference (RNAi) presents a highly promising approach for cancer therapeutics via specific silencing of disease-implicated genes, but its clinical translation remains severely limited by barriers in delivering short interfering RNA (siRNA). Numerous delivery vehicles have been developed to protect siRNA from degradation, promote target cell uptake, and facilitate endosomal escape into the cytoplasm, where RNAi occurs. However, in vivo instability, low silencing efficiency, undesired toxicity, and immunogenicity remain challenges for current siRNA delivery systems, particularly as the low valency and high rigidity of siRNA make it difficult to condense into stable nanoparticles. Here we engineer the siRNA cargo to make it more amenable to stable encapsulation by using a polymeric form of siRNA, or periodic short hairpin RNA (p-shRNA), as well as design a biodegradable polycationic carrier for efficient in vivo delivery of p-shRNA.
Consisting of tens of linked siRNA repeats, p-shRNA is synthesized by the repeated action of T7 RNA polymerase around a circular DNA template. We first leverage molecular engineering design an open-ended p-shRNA structure that is efficiently processed inside cells into siRNAs, greatly enhancing its silencing potency. Furthermore, the much higher valency and flexibility of p-shRNA compared to siRNA enable more stable complexation with delivery materials. To exploit these advantages of p-shRNA, we optimize biodegradable polycations with hydrophobic regions that promote stable condensation and efficient intracellular release. Our approach unveils key design rules governing p-shRNA delivery, and we develop stabilized p-shRNA complexes that show in vivo therapeutic efficacy in a syngeneic melanoma mouse model. Finally, we extend our p-shRNA platform to act as a dual therapeutic agent, harnessing innate immune activation together with gene silencing.
By modulating the surface of the p-shRNA complexes with an anionic polypeptide, we dramatically enhance innate immune recognition of p-shRNA by pattern recognition receptors while maintaining high silencing efficiency. These dually acting complexes can target ovarian tumors in vivo and prolong survival in a syngeneic ovarian cancer mouse model. Our findings establish a potent, multifunctional RNAi platform that can potentially move RNAi therapeutics closer to clinical translation by addressing the delivery and in vivo efficacy challenges faced by current siRNA systems.
National Science Foundation Graduate Research Fellowshipgrant #1122374
Koch Institute Ludwig Center for Molecular Oncology Graduate Fellowship
Congressionally Directed Medical Research Program Ovarian Cancer Research Program Teal Innovator Award from the Department of Defense (13-1-0151)
by Connie Wu.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Chemical Engineering

The theoretical framework in which this study is framed concerns the internationalization of production dynamics of firms abroad. The international literature in this field argues that there is not a conventional definition nor a universally tested theory that can explain all forms of foreign-owned production (see, among others, Dunning, 1988b and 1993a, Ietto-Gillies, 2005 and 2007). In this complex context, a large amount of Italian literature has been published around the theoretical framework of internationalization of production of Italian companies abroad (Tattara, Corò and Volpe, 2006, Mariotti and Mutinelli, 2005, et.al.). Nevertheless, a limited number of quantitative studies are available about the topic of Italian firms “migration” in Romania - as a particular expression of productive internationalization of our companies in this Country (Majocchi, 2002, Unimpresa Romania, 2005 and Antenna Veneto Romania, 2005). Thus, in order to investigate it by an industrial policy perspective a multilevel modelling approach has been applied. In particular, a two level model has been used to determine the effects of Romanian regions (romanian judets) in which Italian manufacturing firms internationalized the production in 2009 on their industrial performance. Based on a business register-based survey a business register of the Italian business community in Romania has been created. The data on Italian manufacturing firms located in Romania in 2009 have been selected from this register. The dataset used for the analysis includes 796 cases of firms internationalized in Romania. Therefore, a two level model has been carried out in order to determine the effects of intra-industry characteristics of these firms (i.e. Firm size and Firm Industrial specialization) on their industrial performance in 2009 both at individual and area level. The model results have shown that: as the firms dimension increase they have to be extremely sensible in the choice of the romanian region in which they want to (de)localize the production. Furthermore, the choice of the romanian region of productive delocalization has to be chosen by firms according to their sectors of activity.

Chronic myeloid leukaemia (CML) was one of the first cancers to be linked to a chromosomal abnormality, the Philadelphia chromosome. This chromosome results in a translocation between chromosomes 9 and 22, where the ABL gene on chromosome 9, a tyrosine kinase, is translocated to the BCR gene region on chromosome 22 giving rise to an abnormal BCR/ABL fusion gene. The resultant fusion gene has an abnormally upregulated tyrosine kinase activity that results in an increase in the proliferation of immature white blood cells, thus leading to the development of CML. There are several breakpoints that can occur in the BCR gene two of which give rise to 95% of CML cases. These fusion points are called the β3α2 and β2α2 depending on where the chromosomal breakages occur in the BCR gene. The aim of the project was to establish a new method of treatment for CML through the use of RNAi to abolish the increase in tyrosine kinase activity of the abnormal fusion gene product. The K562 and KCL22 cell lines incorporating the β3α2 and β2α2 fusion points respectively were used in this project. The fusion points were cloned and sequenced. The human U6 and H1 promoters, were selected for the production of the antisense molecules and were obtained by PCR of K562 cDNA. Short hairpin RNA molecules were designed to the sequences of the β3α2 and the β2α2 fusion points. These designed shRNA molecules were synthesized as oligonucleotides and were incorporated into a reverse PCR primer. Cassettes containing shRNA molecules and a respective promoter were produced by means of PCR and the products cloned into pB12mcs-­‐eGFP vector, which was used as a GFP reporter system. Constructs were then transfected into the appropriate cell lines, and expression studies including qPCR and Western blot analysis were conducted, to examine the effects of the designed shRNA constructs to their target sites on both mRNA and protein levels. In addition, these experiments also indicate the efficiency of the construct and also their specificity to their targets. qPCR and Western blot analysis, show that both the shRNA molecules designed against the β3α2 and the β2α2 fusion points, efficiently induced RNAi based gene silencing to their target sites. In addition, the designed constructs show high specificity to only its target sites and not to other unrelated or related genes. These results point towards the use of molecular modulation of gene expression as a promising strategy for potential CML therapy.

The discovery of the bacterial Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated Cas9 protein, and its ability to edit genes in mammalian cells is poised to revolutionise our ability to treat genetic diseases, particularly in the cancer setting where the driver genes are known. Indeed, CRISPR/Cas9 was shown to effectively edit any gene of interest with high efficiency and at low cost. However, a major challenge to treating cancer is the heterogeneity of the genetic alterations and the ongoing accumulation of new mutations that enable cancers to survive and drug resistance to emerge. With the increasing knowledge of cancer biology, thanks to the advances in DNA sequencing technologies, the low cost, and the ability to edit genes with CRISPR/Cas9 with high efficiency, it is now possible to develop more targeted anticancer therapeutics with promising outcomes. Despite the exceptional CRISPR/Cas9 gene-editing features, the early studies found several limitations to its potential use as a human gene therapy, such as the targeting specificity of CRISPR/Cas9, the in vivo delivery of the treatment with minimal systemic toxicity, the in vivo efficacy under immunocompetent conditions, the immunogenicity of CRISPR/Cas9 and the delivery vehicle when delivered systemically, and whether the introduced edits would induce an immune response. Particularly for cancer treatment, challenges can be broadly categorized into: the in vivo efficacy of CRISPR/Cas9 therapeutics, the in vivo delivery of the treatment to target tissue with high transfection efficiency, and the heterogeneity of genetic mutations in cancer, and thus targeting a known gene may not be enough to cure cancer. To enable the utilisation of CRISPR/Cas9 as an anticancer therapy, the above-mentioned challenges need to be addressed. We utilised the well-established Human Papillomavirus (HPV)-driven cervical cancer models due to their addiction on the expression of HPV oncogenes, namely E6 and E7, for their survival and progression, and thus enabled us to assess the efficacy and the delivery of CRISPR/Cas9 therapies, independent of cancer heterogeneity. To improve the on-target specificity, we assessed the feasibility of using the highly specific variant of Cas9, the catalytically inactive Cas9 fused to the dimerization-dependent cutting domain, FokI, or FokI-dCas9, and compared its editing efficiency on target genes and the effect on downstream protein expression compared to the wild type (WT) Cas9. Our results proved that the FokI-dCas9 is ineffective as a cancer therapeutic, particularly when the target genes are short. We further explored the repair mechanism of the CRISPR/Cas9-mediated double-stranded breaks (DSB) and found that the high-fidelity homology-directed repair (HDR) was modest, accounting for ≈ 8%, compared to the random non-homology end joining (NHEJ) repair, which accounted for ≈ 80% of the edited cells, with a significant inhibition of cancer cell proliferation. We also showed that the cell death was apoptotic, mediated by the reactivation of the tumour suppressor p53 protein when E6 gene was targeted, or the restoration of retinoblastoma protein (Rb) when E7 gene was targeted. To test the feasibility of the intravenous delivery of CRISPR/Cas9, we optimised a protocol to package the treatment in PEGylated liposomes by using the Hydration-of-Freeze-Dried-Matrix (HFDM) method and showed that these liposomes effectively protected the payload against serum nucleases. Furthermore, the intravenously administered CRISPR/Cas9 against HPV 16E7 and HPV 18E7 oncogenes, coated in PEGylated liposomes, effectively cleared established cervical cancer xenografts in immunocompromised mouse models. Next, we aimed to explore if the in vivo efficacy would be affected by the immunogenicity of the treatment under immunocompetent conditions. We showed for the first time that CRISPR/Cas9 therapies eliminated HPV16E7-driven tumour xenografts entirely in syngeneic mice, with no significant inflammation or hepatic toxicity. In addition, an ideal therapeutic outcome would be the induction of an immunogenic cell death (ICD), such that recurrent tumours would be eliminated by the host immune system. Therefore, we explored the immunogenicity of cell death and showed for the first time that CRISPR/Cas9-mediated cell death was not immunogenic. Overall, this research demonstrates that CRISPR/Cas9 therapeutics are very effective for the treatment of oncogene-addicted cancers. We showed that the PEGylated liposomes can be an ideal delivery vehicle for CRISPR/Cas9 therapies despite the large payloads, with no significant immune response or toxicity, and provided new insights into harnessing CRISPR/Cas9 technology as an anticancer therapeutic.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
Full Text

The dysfunction of autophagy pathways has been linked to the development and progression of numerous human diseases, in particular neurological disorders and cancer. Investigating these pathological autophagy mechanisms is essential to gain insights into the underlying disease mechanisms, identify novel biomarkers, and develop targeted therapies. In this thesis, I present three manuscripts that investigate the regulatory mechanisms of autophagy machinery in human diseases. In the first manuscript (Chitiprolu et al., 2018), we investigated the mechanism of p62-mediated selective autophagic clearance of RNA stress granules implicated in Amyotrophic Lateral Sclerosis (ALS). Repeat expansions in C9ORF72, the major cause of ALS, reduce C9ORF72 levels but how this impacts stress granules is uncertain. By employing mass spectrometry, high resolution imaging and biochemical assays, we demonstrated that the autophagy receptor p62 associates with C9ORF72 to eliminate stress granules by autophagy. This requires p62 to associate with proteins that are symmetrically methylated on arginines. Patients with C9ORF72 repeat expansions accumulate symmetric arginine dimethylated proteins which co-localize with p62. This suggests that C9ORF72 initiates a cascade of ALS-linked proteins (C9ORF72, p62, SMN, FUS) to recognize stress granules for degradation by autophagy and hallmarks of a defect in this process are observable in ALS patients. The second manuscript (Guo, Chitiprolu et al., 2014) describes the mechanism by which autophagy degrades retrotransposon RNA from both long and short interspersed elements, thereby preventing new retrotransposon insertions into the genome. By employing quantitative imaging tools, we demonstrated that retrotransposon RNA localizes to RNA granules that are selectively degraded by the autophagy receptors NDP52 and p62. Mice lacking a copy of Atg6/Beclin1, a gene critical for autophagy, also accumulate both retrotransposon RNA and genomic insertions. This suggests a mechanism for the increased tumorigenesis upon autophagy inhibition and therefore a role for autophagy in tempering evolutionary change. Finally, the third manuscript (Guo, Chitiprolu et al., 2017) examines the intersection of autophagy machinery with exosome release and function in cancer metastasis. By employing dynamic light scattering, Nanosight particle tracking, electron microscopy, super-resolution imaging and Western blotting, we robustly quantified exosome identity and purity in multiple cell lines. We demonstrated that exosome production is strongly reduced in cells lacking Atg5 and Atg16L1, but this is independent of Atg7 and canonical autophagy. The effect of Atg5 on exosome production promotes the migration and in vivo metastasis of orthotopic breast cancer cells. These findings delineate autophagy-independent pathways by which autophagy-related genes can contribute to metastasis. Taken together, data presented in the three manuscripts highlight the molecular mechanisms of autophagy core machinery proteins and selective receptors such as Atg5, p62 and NDP52, in the pathogenesis of cancer and neurodegeneration. In these diseases characterized by mutations in autophagy pathways, the mechanisms we uncover provide insights into their causes and serve as potential therapeutic targets.

Spinal muscular atrophy is caused by mutation of the SMN1 gene resulting in the selective loss of spinal cord motor neurons. HuD has been shown to interact with SMN and to localize to RNA granules along axons. In conditions where SMN is decreased, like in SMA, HuD’s localization to RNA granules affected. Overexpression of HuD in an SMA cell culture model was shown to rescue SMA-like axonal defects. Here, existence of a signaling pathway downstream of PKC leading to the activation of HuD was investigated in MN-1 cells. Stimulation of this pathway using a pharmacological agonist of PKC increased HuD levels and enhanced its binding to GAP-43 and Tau mRNAs. An scAAV9 viral expression system to overexpress HuD in vivo was established, laying the foundation for the next phase of the study. Overall, modulating HuD expression and activity would be beneficial and could constitute an attractive therapeutic approach for SMA.

Synthetic antisense oligoribonucleotides can be used to modulate gene splicing by masking key motifs on the pre-mRNA required for spliceosome assembly. Yet, intracellular expression of oligoribonucleotides generates only a transitory effect whereas stable delivery of antisense sequences can be achieved by linking them to chimeric small RNAs delivered and expressed by viral vectors. In the murine model of Duchenne Muscular Dystrophy a chimeric U7 snRNA (U7Dtex23) induces skipping of the mutated exon 23 and restores the Dystrophin mRNA reading frame. The main limitation of this approach remains the large amount of snRNA vector needed to be produced and administered to patients. To optimize this system we used self-complementary AAV vectors (scAAV) to express the U7snRNA shuttles. ScAAV vectors were tested in mouse myoblast cultures and we observed an increase in U7Dtex23 expression and in dystrophin exon 23 skipping compared to single-stranded AAV, highlighting the potential for this strategy to reduce the vector dose. Alternatively, we have used a muscle and heart-specific enhancer (MHCK) to drive the expression of U7Dtex23 cassettes delivered with AAV vectors and our results showed that MHCK improves chimeric U7snRNA expression and increases dystrophin exon 23 skipping in vitro and in vivo. However, additional U7snRNA species were produced following gene transfer, pointing at a possible limitation of the cellular processing machinery capability with saturating levels of U7 shuttles. We have also explored the possibility of using small nucleolar (sno) RNAs as novel molecular platforms for antisense delivery. We replaced the original antisense of MBII-52 snoRNA with the Dtex23 sequence and observed low levels of exon 23 skipping in AAV-transduced myotubes. While our observation validates the approach, the efficiency of skipping is still considerably lower than with the U7snRNA cassette. As a last approach, we engineered the human C/D box U24snoRNA to specifically target the methylation of an adenosine branch point in a luciferase reporter pre-mRNA in order to induce the skipping of the downstream exon. We were not able to observe any modulation of splicing using this strategy.

Clustered regularly interspaced short palindromic repeats (CRISPR) are derived from a bacterial and archaeal adaptive immune system. The core enzymes of CRISPR are RNA-guided endonucleases that sequence-specifically cleave foreign double-stranded DNA. Improving and controling the properties of the CRISPR system is a crucial step in advancing the therapeutic potential of CRISPR technology. Several classes of these enzymes exist and are being adapted for biotechnology, such as genome engineering. Cas12a (Cpf1) is a Type V CRISPR-associated (Cas) enzyme that naturally uses only one guide RNA, in contrast to Type II CRISPR-Cas9 enzymes. Thus, Cpf1 may represent a simpler, more practical tool for applications such as gene editing and therapeutics. This dissertation comprises four related studies in this area. To better understand the functional requirements for Cpf1-crRNA interaction and develop modified crRNAs suitable for synthetic biology and therapeutic applications, the first study performed nucleotide substitutions in the crRNA. It focused on the protein-interaction motif of the crRNA by incorporating base changes at the 2ʹ position that alter hydrogen-bonding capacity, sugar pucker, and flexibility. DNA substitutions in RNA can probe the importance of A-form structure, 2ʹ-hydroxyl contacts, and conformational constraints within RNA-guided enzymes. In addition, Chemical modifications include 2'-deoxy, 2'-fluoro, 2'- deoxy-arabinonucleic acid, and oxepane. Our study discovered that 2'-fluoro maintains the A-form structure and is compatible with AsCpf1 activity. Biochemical endonuclease activity, gene editing efficiency, Cpf1 binding affinity, and ribonucleoprotein stability were used to assess the tolerance and effects of modification. Characterizing structure-function requirements for Cpf1-crRNA interaction will facilitate better design and tuning of Cpf1 enzymes. The second study established a FRET-based assay in collaboration with a computational collaborator to identify small molecule inhibitors predicted by virtual docking and simulations. This study aims to lay the foundation for efficient, safe implementation of CRISPR-Cpf1. The third study used chemically modified Cas9-guide RNAs to offset known weaknesses of CRISPRi. It takes advantage of the high binding affinity and nuclease resistance of modified guides to potentially reduce the required components for CRISPRi.

Thesis (Ph. D.)--University of Missouri-Columbia, 2009.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "May 2009" Includes bibliographical references.

Adenosine deaminase acting on transfer RNA (ADAT) is a human heterodimeric enzyme that catalyzes the deamination of adenosine (A) to inosine (I) at the first position of the anticodon of transfer RNAs (tRNAs) (position 34, or wobble position); one of the few essential post-transcriptional modifications on tRNAs (1-5). Inosine 34 allows the recognition of three different nucleotides: cytidine, uridine and adenosine, at the third position of the codon, thus increasing the decoding capacity of tRNAs to more than one messenger RNA (mRNA) codon (adenosine 34 can in principle only pair with codons with uridine at the third position) (6, 7). This alters the tRNA pool available for each codon and it has been proved to align the correlation between codon usage and tRNA gene copy number (8). It has also been suggested to improve fidelity and efficiency of translation (8, 9), especially for mRNAs enriched in codons translated by modified tRNAs (10, 11). Monitoring ADAT-mediated deamination is crucial for the characterization of the enzyme in terms of activity, substrates, regulation, as well as for drug discovery purposes. However, this analysis is often challenging, laborious and lacks quantitativeness. We developed an in vitro deamination assay based on restriction fragment length polymorphism (RFLP) analyses to monitor ADAT activity in an efficient, cost-effective, and semiquantitative manner (12). To overcome a limitation of the method being the need of reverse transcription and amplification of the tRNA, we designed a direct method to quantify I34 formation in vitro using the first fluorescent analogs of nucleic acids that have been reported to undergo enzymatic deaminations (13-15). ADAT has been conserved over the evolution with the acquisition of multi-substrate specificity. Whereas its bacterial homolog TadA deaminates exclusively tRNAArg (2), the human enzyme deaminates eight different tRNAs (3, 16). However, the mechanisms that drove this evolution remain unknown. While the substrate recognition in TadA has been well studied, in the eukaryotic ADAT is poorly understood. Through in vitro enzymatic activity assays with different variants of tRNAArg and tRNAAla, we elucidated the most important features for efficient A34-to-I34 conversion and characterized the substrate recognition of the human enzyme. We also proposed a new potential mechanism of control of ADAT deamination activity by human tRNA-derived fragments, which provides new insights into the regulation of ADAT function and may open a door for the development of new strategies to modulate ADAT activity. A missense mutation (V128M) in one of the two subunits of the human ADAT enzyme causes intellectual disability and strabismus, but the molecular bases of the pathology are unknown (17, 18). We characterized human ADAT in terms of kinetics and structure, and investigated the effect of the V128M mutation. We found that this substitution decreases ADAT deamination activity, and severely affects the stability of the quaternary structure of the enzyme. In this regard, we discovered small molecules with the ability to activate the enzyme, which could potentially recover the defective tRNA editing caused by the mutation. References 1. Gerber AP, Keller W. An adenosine deaminase that generates inosine at the wobble position of tRNAs. Science. 1999;286(5442):1146-9. Epub 1999/11/05. 2. Wolf J, Gerber AP, Keller W. tadA, an essential tRNA-specific adenosine deaminase from Escherichia coli. The EMBO journal. 2002;21(14):3841-51. Epub 2002/07/12. 3. Torres AG, Pineyro D, Rodriguez-Escriba M, Camacho N, Reina O, Saint-Leger A, et al. Inosine modifications in human tRNAs are incorporated at the precursor tRNA level. Nucleic acids research. 2015;43(10):5145-57. Epub 2015/04/29. 4. Zhou W, Karcher D, Bock R. Identification of enzymes for adenosine-to-inosine editing and discovery of cytidine-to-uridine editing in nucleus-encoded transfer RNAs of Arabidopsis. Plant physiology. 2014;166(4):1985-97. Epub 2014/10/16. 5. Tsutsumi S, Sugiura R, Ma Y, Tokuoka H, Ohta K, Ohte R, et al. Wobble inosine tRNA modification is essential to cell cycle progression in G(1)/S and G(2)/M transitions in fission yeast. J Biol Chem. 2007;282(46):33459-65. Epub 2007/09/19. 6. Crick FH. Codon--anticodon pairing: the wobble hypothesis. Journal of molecular biology. 1966;19(2):548-55. Epub 1966/08/01. 7. Torres AG, Pineyro D, Filonava L, Stracker TH, Batlle E, Ribas de Pouplana L. A-to-I editing on tRNAs: biochemical, biological and evolutionary implications. FEBS Lett. 2014;588(23):4279-86. Epub 2014/09/30. 8. Novoa EM, Pavon-Eternod M, Pan T, Ribas de Pouplana L. A role for tRNA modifications in genome structure and codon usage. Cell. 2012;149(1):202-13. Epub 2012/04/03. 9. Schaub M, Keller W. RNA editing by adenosine deaminases generates RNA and protein diversity. Biochimie. 2002;84(8):791-803. Epub 2002/11/30. 10. Rafels-Ybern A, Attolini CS, Ribas de Pouplana L. Distribution of ADAT-Dependent Codons in the Human Transcriptome. International journal of molecular sciences. 2015;16(8):17303- 14. Epub 2015/08/01. 11. Rafels-Ybern A, Torres AG, Grau-Bove X, Ruiz-Trillo I, de Pouplana LR. Codon adaptation to tRNAs with Inosine modification at position 34 is widespread among Eukaryotes and present in two Bacterial phyla. RNA biology. 2017:0. Epub 2017/09/08. 12. Wulff TF, Arguello RJ, Molina Jordan M, Roura Frigole H, Hauquier G, Filonava L, et al. Detection of a Subset of Posttranscriptional Transfer RNA Modifications in Vivo with a Restriction Fragment Length Polymorphism-Based Method. Biochemistry. 2017;56(31):4029-38. Epub 2017/07/14. 13. Sinkeldam RW, McCoy LS, Shin D, Tor Y. Enzymatic interconversion of isomorphic fluorescent nucleosides: adenosine deaminase transforms an adenosine analogue into an inosine analogue. Angew Chem Int Ed Engl. 2013;52(52):14026-30. Epub 2013/11/30. 14. McCoy LS, Shin D, Tor Y. Isomorphic emissive GTP surrogate facilitates initiation and elongation of in vitro transcription reactions. Journal of the American Chemical Society. 2014;136(43):15176-84. Epub 2014/09/26. 15. Rovira AR, Fin A, Tor Y. Chemical Mutagenesis of an Emissive RNA Alphabet. J Am Chem Soc. 2015;137(46):14602-5. Epub 2015/11/03. 16. Juhling F, Morl M, Hartmann RK, Sprinzl M, Stadler PF, Putz J. tRNAdb 2009: compilation of tRNA sequences and tRNA genes. Nucleic acids research. 2009;37(Database issue):D159-62. Epub 2008/10/30. 17. Alazami AM, Hijazi H, Al-Dosari MS, Shaheen R, Hashem A, Aldahmesh MA, et al. Mutation in ADAT3, encoding adenosine deaminase acting on transfer RNA, causes intellectual disability and strabismus. Journal of medical genetics. 2013;50(7):425-30. Epub 2013/04/27. 18. El-Hattab AW, Saleh MA, Hashem A, Al-Owain M, Asmari AA, Rabei H, et al. ADAT3- related intellectual disability: Further delineation of the phenotype. American journal of medical genetics Part A. 2016;170A(5):1142-7. Epub 2016/02/05.
L’adenosina deaminasa específica per RNA de transferència (ADAT) és un enzim humà heterodimèric que catalitza la reacció de deaminació de l’adenosina (A) a inosina (I) a la primera posició de l’anticodó de RNAs de transferència (tRNAs) (també anomenada posició 34 o posició de balanceig); una de les poques modificacions post-transcripcionals essencials en tRNAs (1-5). La inosina 34 permet el reconeixement de tres nucleòtids diferents: citidina, uridina i adenosina, a la tercera posició del codó, augmentant per tant la capacitat de descodificació dels tRNAs a més d’un codó en l’RNA missatger (mRNA) (l’adenosina 34 en principi únicament pot aparellar-se amb uridina en la tercera posició) (6, 7). Això altera els nivells de tRNAs disponibles per cada codó i s’ha demostrat que alinea la correlació entre l’ús de codons i número de còpies gèniques de cada tRNA (8). També s’ha suggerit que millora la fidelitat i eficiència de la traducció (8, 9), especialment per mRNAs enriquits en codons traduïts per tRNA modificats (10, 11). Monitoritzar la deaminació produïda per ADAT és clau per la caracterització de l’enzim en termes d’activitat, substrats, regulació, així com també pel disseny de fàrmacs. No obstant, aquest anàlisi és sovint complex, laboriós i poc quantitatiu. Per això, hem desenvolupat un assaig de deaminació in vitro basat en el polimorfisme de longitud dels fragments de restricció (RFLP) amb el propòsit de monitoritzar l’activitat d’ADAT de manera eficient, cost-efectiva i semiquantitativa (12). Per superar una limitació del mètode essent la necessitat de transcripció reversa i amplificació del tRNA, hem dissenyat un mètode directe per quantificar la formació d’I34 in vitro utilitzant els primers anàlegs fluorescents d’àcids nucleics que són substrats de deaminacions enzimàtiques descrits fins al moment (13-15). ADAT ha estat conservat en l’evolució amb l’adquisició de multi-especificitat de substrat. Mentre que el seu homòleg bacterià TadA deamina exclusivament tRNAArg (2), l’enzim humà deamina vuit tRNAs diferents (3, 16). Tot i així, els mecanismes que van conduir a aquesta evolució romanen desconeguts. Mentre que el reconeixement de substrat en TadA es coneix bé, en ADAT eucariòtic ha estat poc estudiat. A través d’assajos enzimàtics in vitro amb diferents variants de tRNAArg i tRNAAla, hem elucidat els trets més importants per a una conversió eficient d’A34 a I34 i hem caracteritzat el reconeixement de substrat per part de l’enzim humà. També proposem un nou potencial mecanisme de control de l’activitat d’ADAT per part de fragments derivats de tRNAs humans, el qual ofereix noves perspectives en la regulació de la funció d’ADAT i que pot obrir la porta al desenvolupament de noves estratègies per modular-ne l’activitat. Una mutació sense sentit (V128M) en una de les dues subunitats en l’enzim ADAT humà s’ha associat a retard mental i estrabisme, encara que les bases moleculars de la patologia es desconeixen (17, 18). Hem caracteritzat ADAT humà en termes de cinètica enzimàtica i estructura, i n’hem investigat l’efecte de la mutació V128M. Hem descobert que la substitució de valina 128 per metionina redueix l’activitat deaminatòria d’ADAT i que altera severament l’estabilitat de l’estructura quaternària de l’enzim. En aquest aspecte, hem descobert molècules amb l’habilitat d’activar l’enzim, la qual cosa podria revertir potencialment la reducció en l’activitat enzimàtica causada per la mutació. References 1. Gerber AP, Keller W. An adenosine deaminase that generates inosine at the wobble position of tRNAs. Science. 1999;286(5442):1146-9. Epub 1999/11/05. 2. Wolf J, Gerber AP, Keller W. tadA, an essential tRNA-specific adenosine deaminase from Escherichia coli. The EMBO journal. 2002;21(14):3841-51. Epub 2002/07/12. 3. Torres AG, Pineyro D, Rodriguez-Escriba M, Camacho N, Reina O, Saint-Leger A, et al. Inosine modifications in human tRNAs are incorporated at the precursor tRNA level. Nucleic acids research. 2015;43(10):5145-57. Epub 2015/04/29. 4. Zhou W, Karcher D, Bock R. Identification of enzymes for adenosine-to-inosine editing and discovery of cytidine-to-uridine editing in nucleus-encoded transfer RNAs of Arabidopsis. Plant physiology. 2014;166(4):1985-97. Epub 2014/10/16. 5. Tsutsumi S, Sugiura R, Ma Y, Tokuoka H, Ohta K, Ohte R, et al. Wobble inosine tRNA modification is essential to cell cycle progression in G(1)/S and G(2)/M transitions in fission yeast. J Biol Chem. 2007;282(46):33459-65. Epub 2007/09/19. 6. Crick FH. Codon--anticodon pairing: the wobble hypothesis. Journal of molecular biology. 1966;19(2):548-55. Epub 1966/08/01. 7. Torres AG, Pineyro D, Filonava L, Stracker TH, Batlle E, Ribas de Pouplana L. A-to-I editing on tRNAs: biochemical, biological and evolutionary implications. FEBS Lett. 2014;588(23):4279-86. Epub 2014/09/30. 8. Novoa EM, Pavon-Eternod M, Pan T, Ribas de Pouplana L. A role for tRNA modifications in genome structure and codon usage. Cell. 2012;149(1):202-13. Epub 2012/04/03. 9. Schaub M, Keller W. RNA editing by adenosine deaminases generates RNA and protein diversity. Biochimie. 2002;84(8):791-803. Epub 2002/11/30. 10. Rafels-Ybern A, Attolini CS, Ribas de Pouplana L. Distribution of ADAT-Dependent Codons in the Human Transcriptome. International journal of molecular sciences. 2015;16(8):17303- 14. Epub 2015/08/01. 11. Rafels-Ybern A, Torres AG, Grau-Bove X, Ruiz-Trillo I, de Pouplana LR. Codon adaptation to tRNAs with Inosine modification at position 34 is widespread among Eukaryotes and present in two Bacterial phyla. RNA biology. 2017:0. Epub 2017/09/08. 12. Wulff TF, Arguello RJ, Molina Jordan M, Roura Frigole H, Hauquier G, Filonava L, et al. Detection of a Subset of Posttranscriptional Transfer RNA Modifications in Vivo with a Restriction Fragment Length Polymorphism-Based Method. Biochemistry. 2017;56(31):4029-38. Epub 2017/07/14. 13. Sinkeldam RW, McCoy LS, Shin D, Tor Y. Enzymatic interconversion of isomorphic fluorescent nucleosides: adenosine deaminase transforms an adenosine analogue into an inosine analogue. Angew Chem Int Ed Engl. 2013;52(52):14026-30. Epub 2013/11/30. 14. McCoy LS, Shin D, Tor Y. Isomorphic emissive GTP surrogate facilitates initiation and elongation of in vitro transcription reactions. Journal of the American Chemical Society. 2014;136(43):15176-84. Epub 2014/09/26. 15. Rovira AR, Fin A, Tor Y. Chemical Mutagenesis of an Emissive RNA Alphabet. J Am Chem Soc. 2015;137(46):14602-5. Epub 2015/11/03. 16. Juhling F, Morl M, Hartmann RK, Sprinzl M, Stadler PF, Putz J. tRNAdb 2009: compilation of tRNA sequences and tRNA genes. Nucleic acids research. 2009;37(Database issue):D159-62. Epub 2008/10/30. 17. Alazami AM, Hijazi H, Al-Dosari MS, Shaheen R, Hashem A, Aldahmesh MA, et al. Mutation in ADAT3, encoding adenosine deaminase acting on transfer RNA, causes intellectual disability and strabismus. Journal of medical genetics. 2013;50(7):425-30. Epub 2013/04/27. 18. El-Hattab AW, Saleh MA, Hashem A, Al-Owain M, Asmari AA, Rabei H, et al. ADAT3- related intellectual disability: Further delineation of the phenotype. American journal of medical genetics Part A. 2016;170A(5):1142-7. Epub 2016/02/05.

L'ELA és una malaltia neurodegenerativa multifactorial. Atès que actualment és una malaltia incurable sense una causa coneguda, els esforços per descobrir noves dianes terapèutiques estan ben justificats. L'evidència disponible indica que la fisiopatologia de l'ELA pot estar composta parcialment d'interaccions entre alteracions de lípids, estrès oxidatiu, deslocalització de TDP-43 i metabolisme de l'ARN. Per explorar l'impacte de la deslocalització de TDP-43, quantifiquem per primera vegada l’splicing críptic derivat de la disfunció de TDP-43 en teixit humà, models cel·lulars i ratolins. Els exons críptics són candidats a biomarcadors en l'ELA, i els quantifiquem en altres patologies relacionades amb alteracions de TDP-43, com la demència tipus Alzheimer-LATE. Un dels ARNm controlats per TDP-43 codifica per ATG4B, una proteïna essencial en la formació de la maquinària de membrana de l'autofàgia. D'altra banda, les membranes de el sistema nerviós central estan enriquides en àcids grassos poliinsaturats (PUFA), molt sensibles a l'estrès oxidatiu. Les publicacions anteriors de el grup demostren una disminució en l'àcid docosahexaenoic (DHA) -un PUFA- a la medul·la espinal dels donants d'ELA. El DHA és un regulador crucial de la inflamació i s'ha relacionat amb malalties neurodegeneratives relacionades amb l'edat. Aquesta tesi mostra que la intervenció dietètica pot augmentar eficaçment el contingut de DHA de la medul·la espinal mitjançant l'ús d'una dieta enriquida amb DHA, que té un efecte beneficiós en la supervivència dels ratolins transgènics mascles. Aquesta millora es va associar amb una disminució dels marcadors inflamatoris i de dany de l'ADN. A causa de que s'observa un dany en l'ADN similar i un perfil d'alliberament de citocines en el perfil secretor associat a la senescència (SASP) en la neuroinflamació, també examinem el paper potencial de la senescència en l'ELA. Vam detectar nivells augmentats de marcadors SASP i signes de senescència cel·lular (expressió augmentada de p16-INK i p21) en etapes presimptomàtiques i simptomàtiques en la medul·la espinal dels ratolins hSOD1-G93A. No obstant això, el tractament senolític amb Navitoclax (un inhibidor de Bcl-2) no va oferir cap benefici en la supervivència dels ratolins ni una disminució dels nivells d'expressió de gens de senescència cel·lular i marcadors SASP. També vam demostrar que l'splicing críptic augmenta en ratolins hSOD1-G93A en l'etapa final i es correlaciona amb el marcador de senescència p16-INK. Finalment, per millorar el potencial de transferència, vam explorar les alteracions de l'splicing críptic i canvis metabolòmics com a potencials biomarcadors perifèrics. Desafortunadament, els exons críptics mesurats en teixit nerviós no s'expressen en cèl·lules mononuclears de sang perifèrica. No obstant això, les anàlisis del metaboloma plaquetari van revelar canvis significatius en els enfocaments de casos-controls i de pronòstic. En conclusió, aquesta tesi destaca les noves troballes relacionades amb possibles intervencions dietètiques i senolítics, i proposa nous biomarcadors metabolòmics i transcriptòmics.
La ELA es una enfermedad neurodegenerativa multifactorial. Dado que actualmente es una enfermedad incurable sin una causa conocida, los esfuerzos para descubrir nuevas dianas terapéuticas están bien justificados. La evidencia disponible indica que la fisiopatología de la ELA puede estar compuesta parcialmente de interacciones entre alteraciones de lípidos, estrés oxidativo, deslocalización de TDP-43 y metabolismo del ARN. Para explorar el impacto de la deslocalización de TDP-43, cuantificamos por primera vez el splicing críptico derivado de la disfunción de TDP-43 en tejido humano, modelos celulares y ratones. Los exones crípticos son candidatos a biomarcadores en la ELA, y los cuantificamos en otras patologías relacionadas con alteraciones de TDP-43, como la demencia tipo Alzheimer-LATE. Uno de los ARNm controlados por TDP-43 codifica para ATG4B, una proteína esencial en la formación de la maquinaria de membrana de la autofagia. Por otro lado, las membranas del sistema nervioso central están enriquecidas en ácidos grasos poliinsaturados (PUFA), muy sensibles al estrés oxidativo. Las publicaciones anteriores del grupo demuestran una disminución en el ácido docosahexaenoico (DHA) -un PUFA- en la médula espinal de los donantes de ELA. El DHA es un regulador crucial de la inflamación y se ha relacionado con enfermedades neurodegenerativas relacionadas con la edad. Esta tesis muestra que la intervención dietética puede aumentar eficazmente el contenido de DHA de la médula espinal mediante el uso de una dieta enriquecida con DHA, que tiene un efecto beneficioso en la supervivencia de los ratones transgénicos machos. Esta mejora se asoció con una disminución de los marcadores inflamatorios y de daño del ADN. Debido a que se observa un daño en el ADN similar y un perfil de liberación de citocinas en el perfil secretor asociado a la senescencia (SASP) en la neuroinflamación, también examinamos el papel potencial de la senescencia en la ELA. Detectamos niveles aumentados de marcadores SASP y signos de senescencia celular (expresión aumentada de p16-INK y p21) en etapas presintomáticas y sintomáticas en la médula espinal de los ratones hSOD1-G93A. Sin embargo, el tratamiento senolítico con Navitoclax (un inhibidor de Bcl-2) no ofreció ningún beneficio en la supervivencia de los ratones ni una disminución de los niveles de expresión de genes de senescencia celular y marcadores SASP. También demostramos que el splicing críptico aumenta en ratones hSOD1-G93A en la etapa final y se correlaciona con el marcador de senescencia p16-INK. Por último, para mejorar el potencial de transferencia, exploramos las alteraciones del splicing críptico y cambios metabolómicos como potenciales biomarcadores periféricos. Desafortunadamente, los exones crípticos medidos en tejido nervioso no se expresan en células mononucleares de sangre periférica. No obstante, los análisis del metaboloma plaquetario revelaron cambios significativos en los enfoques de casos-controles y de pronóstico. En conclusión, esta tesis destaca los hallazgos novedosos relacionados con posibles intervenciones dietéticas y senolíticos, y propone nuevos biomarcadores metabolómicos y transcriptómicos.
ALS is a multifactorial neurodegenerative disease. As it is currently an incurable disease without a known cause, the efforts in discovering novel targets are well justified. Available evidence indicates that ALS pathophysiology can be partially comprised of interactions between lipid alterations, oxidative stress, TDP-43 mislocalization, and RNA metabolism. In order to explore the impact of TDP-43 mislocalization, we quantified for the first time the cryptic exon splicing derived from TDP-43 dysfunction in human tissue, cell models, and mice. Cryptic exons are candidate biomarkers in ALS, and we quantified them in other pathologies related to TDP-43 disturbances, such as the Alzheimer-LATE type dementia. One of the TDP-43 controlled mRNAs is codifying for ATG4B, an essential protein at the build-up of autophagy's membranal machinery. On the other hand, the central nervous system membranes are enriched in polyunsaturated fatty acids (PUFAs), which are very sensitive to oxidative stress. The group's previous publications indicated a decrease in docosahexaenoic acid (DHA) -a PUFA- in ALS donors' spinal cord. DHA is a crucial regulator of inflammation and has been implicated in age-related neurodegenerative diseases. This thesis shows that dietary intervention can effectively increase DHA content of the spinal cord by using a DHA enriched diet, which has a beneficial effect on male survival time. This improvement was associated with decreased inflammatory and DNA damage markers. Because a similar DNA damage and cytokine release profile are seen in senescence-associated secretory profile (SASP) in neuroinflammation, we also examined the potential role of senescence in ALS. We detected increased levels of SASP markers and signs of cell senescence (increased expression of p16-INK and p21) in pre- and symptomatic stages in the hSOD1-G93A ALS spinal cord. However, a senolytic treatment with Navitoclax (a Bcl-2 inhibitor) did not offer any benefit in mice survival nor a decreased level of cell senescence and SASP markers. We also demonstrate that cryptic exon splicing is increased in the hSOD1-G93A ALS model at the end-stage, and it is correlated with senescence marker p16-INK mRNA. Finally, to enhance transference potential, we explored both metabolomic and cryptic exon splicing alterations in peripheral biomarkers. Unfortunately, cryptic exons measured in nervous tissue are not expressed in peripheral blood mononuclear cells. Notwithstanding, platelet metabolome analyses revealed significant changes in case-control and prognostic approaches. Globally, this thesis highlights novel findings related to potential dietary interventions, open the door for new therapeutic agents such as senolytic and propose new metabolomics and transcriptomic biomarkers.

Ovarian cancer is the most lethal gynaecological malignancy, responsible for over 4,000 deaths each year in the UK. There is growing evidence that mRNA-binding proteins (RBPs) can be post-transcriptional drivers of cancer progression. Here, I investigated the expression of the RBP LARP1 in ovarian malignancies and role of the protein in ovarian cancer cell biology. LARP1 is highly expressed at both an mRNA and protein level in ovarian cancers compared with benign tumours and normal ovarian tissue. I show that higher levels of LARP1 in tumour tissue are predictive of poor patient survival. Consistent with this clinical finding, in xenograft studies knockdown of LARP1 expression causes a dramatic reduction in tumour growth. In vitro, LARP1 knockdown is associated with increased apoptosis, and is sufficient to restore platinum sensitivity in chemotherapy-resistant cell lines. Furthermore, LARP1 is required to maintain cancer stem cell marker-positive populations, and knockdown decreases tumour-initiating potential, as demonstrated by in vivo limiting dilution assays. Transcriptome deep-sequencing following LARP1 knockdown revealed altered expression of multiple genes linked to survival and evasion of apoptosis, including BCL2 and BIK. Transcripts of both genes are in complex with LARP1 protein, and LARP1 maintains the stability of BCL2 mRNA, whilst actively destabilising BIK transcripts. This effect is mediated at the level of the 3' untranslated region. I therefore conclude that by differentially regulating mRNA stability, LARP1 is a key post-transcriptional driver of tumourigenicity and cell survival in ovarian cancer.

Influenza A is a single-stranded, multi-segmented, negative sense RNA virus of the family Orthomyxoviridae and is the causative agent of seasonal Influenza. Influenza viruses cause significant impacts on a global scale regarding public health and economics. Annual influenza virus infections in the United States account for over 200,000 hospitalizations, up to 49,000 deaths, and an $87.1 billion economic burden. Influenza A virus has caused several pandemics since the turn of the 20th century. The effects of Influenza on public health and economics, compounded with low efficacy of the annual vaccine and emerging antiviral resistance, brings to light the need for an effort to stem these impacts, prevent pandemics, and protect public health by developing novel treatments. This project proposes an alternative approach to combatting Influenza by targeting host factors hijacked during infection that, if inhibited, significantly impair viral RNA expression, but result in low host toxicity. The host factors we examined include RNA export factors (XpoT and Xpo5) and RNA helicases (UAP56 and URH49). We selected paralogs URH49 (DDX39A) and UAP56 (DDX39B) because previous studies suggest differing roles during infection, but we theorize that their high degree of sequence similarity, similar function, and association with many of the same cellular factors may allow them to substitute for one another if one is inhibited. CRISPR was considered as the primary method to evaluate the effect of knockout of these factors on viral RNA expression and host cell toxicity. CRISPR is an RNA-guided mechanism for gene editing and can be used to make null mutations in targeted host genes. However, CRISPR proved to be a significant challenge and, while we could not conclusively confirm whether the CRISPR plasmids were effective at targeting our genes of interest, our initial results were not promising and we did not pursue this approach further. As an alternative, host RNA export factors were evaluated using siRNA to knockdown the factor prior to influenza infection. RNA was analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The potential of inhibiting UAP56 or URH49 as a novel therapeutic target was determined using a visual assessment of cell death. We found that siRNA-mediated knockdown of XpoT and Xpo5 did not have any impact on viral RNA synthesis early during infection. siRNA against UAP56 and DDX39 (targets both UAP56 and URH49) resulted in significant impairment in viral RNA synthesis, confirming previously established work suggesting that UAP56 and URH49 have important roles during infection. Importantly, these helicases play an interferon (IFN) independent role to enhance viral replication, as indicated by analysis in IFN deficient VERO cells. A viability assay relying on trypan blue exclusion did not yield trustworthy results, so a visual assessment of cell death was done. The visual assessment confirms previously-established observations that Nxf1 siRNA treatments result in a high degree of cell death, indicating the toxic nature of Nxf1 inhibition. Cells treated with UAP56 or DDX39 siRNAs demonstrated little to no additional toxicity compared to the non-target control, suggesting they can be inhibited to serve as antiviral targets.

Huntington’s disease (HD) is an autosomal dominant, progressive neurodegenerative disorder. Invariably fatal, HD is caused by expansion of the CAG repeat region in exon 1 of the Huntingtin gene which creates a toxic protein with an extended polyglutamine tract 1. Silencing mutant Huntingtin messenger RNA (mRNA) is a promising therapeutic approach 2-6. The ideal silencing strategy would reduce mutant Huntingtin while leaving the wild-type mRNA intact. Unfortunately, targeting the disease causing CAG repeat expansion is difficult and risks targeting other CAG repeat containing genes. We examined an alternative strategy, targeting single nucleotide polymorphisms (SNPs) in the Huntingtin mRNA. The feasibility of this approach hinges on the presence of a few common highly heterozygous SNPs which are amenable to SNP-specific targeting. In a population of HD patients from Europe and the United states, forty-eight percent were heterozygous at a single SNP site; one isoform of this SNP is associated with HD. Seventy-five percent of patients are heterozygous at least one of three frequently heterozygous SNPs. Consequently, only five allele-specific siRNAs are required to treat three-quarters of the patients in the European and U.S. patient populations. We have designed and validated siRNAs targeting these SNPs. We also developed artificial microRNAs (miRNAs) targeting Huntingtin SNPs for delivery using recombinant adeno-associated viruses (rAAVs). Both U6 promoter driven and CMV promoter driven miRNAs can discriminate between matched and mismatched targets in cell culture but the U6 promoter driven miRNAs produce the mature miRNA at levels exceeding those of the vast majority of endogenous miRNAs. The U6 promoter driven miRNAs can produce a number of unwanted processing products, most likely due to a combination of overexpression and unintended export of the pri-miRNA from the nucleus. In contrast, CMV-promoter driven miRNAs produce predominantly a single species at levels comparable to endogenous miRNAs. Injection of recombinant self complementary AAV9 viruses carrying polymerase II driven Huntingtin SNP targeting miRNAs into the striatum results in expression of the mature miRNA sequence in the brain and has no significant effect on endogenous miRNAs. Matched, but not mismatched SNP-targeting miRNAs reduce inclusions in a knock-in mouse model of HD. These studies bring us closer to an allele-specific therapy for Huntington’s disease.

Les DIPG représentent les tumeurs cérébrales pédiatriques les plus sévères. Aucun progrès dans leur prise en charge n’a été accompli au cours des 50 dernières années et la radiothérapie ne demeure que transitoirement efficace. Récemment, une mutation somatique de l’histone H3 (K27M) spécifique des DIPG a été trouvée chez environ 95% des patients. Elle est aujourd’hui considérée comme l'événement oncogénique initiateur de ces tumeurs. Deux sous-groupes majeurs de patients présentant des programmes oncogéniques et une réponse à la radiothérapie distincts peuvent être définis en fonction du gène dans lequel l’altération survient, codant les variantes protéiques H3.1 ou H3.3. Nous avons réalisé deux cribles de létalité synthétique par ARN interférence ciblant le kinome humain afin d'identifier d’une part les gènes nécessaires à la survie des DIPG et d’autre part les gènes dont l’inhibition sensibilise ces tumeurs à la radiothérapie. Le double objectif de ce projet était de mieux comprendre la biologie sous-jacente à l’oncogenèse des DIPG et de découvrir de nouvelles cibles thérapeutiques.Nous avons mis en évidence 41 gènes requis pour la survie des DIPG sans effet délétère majeur sur des cellules contrôles normales. Parmi eux, nous avons identifié VRK3 codant une serine thréonine kinase dont les fonctions restent peu décrites à ce jour et qui n'avait jamais été associée préalablement à l'oncogenèse de DIPG. Nous avons pu confirmer par la suite que son inhibition conduit à un arrêt total de la prolifération des cellules de DIPG associé à d’importants changements morphologiques, plus particulièrement dans les tumeurs mutées pour H3.3-K27M. VRK3 constitue par conséquent une nouvelle cible thérapeutique prometteuse dans cette pathologie à l’issue fatale pour la totalité des patients.En parallèle, un crible de survie similaire a été réalisé en conjonction avec l’irradiation des cellules. Très peu d’ARN interférents ont permis de sensibiliser les cellules H3.3-K27M à la radiothérapie contrairement aux cellules H3.1-K27M. Ce travail nous a permis de mettre en évidence une différence significative de radiosensibilité des modèles vitro de DMG en fonction du sous-groupe de tumeurs considéré, H3.1- ou H3.3-K27M muté, conformément à la survie des patients observée suite à la radiothérapie. Ces résultats inédits laissent entrevoir des perspectives d’amélioration du traitement de référence des patients atteints de DIPG actuellement identique quelle que soit leur génotype
DIPG is one of the most severe paediatric brain tumours. No progress has been made in their management over the past 50 years and radiotherapy remains only transiently effective. Recently, a specific somatic mutation in the histone H3 (K27M) has been found in approximately 95% of DIPG patients and can be considered as the oncogenic driver of these tumours. Two major subgroup of patients with distinct oncogenic program and response to radiotherapy can be defined according to the gene in which the alteration occurs, encoding the H3.1 or H3.3 protein variants. We performed two synthetic lethality screens by RNA interference targeting the human kinome in order to identify the genes responsible for DIPG cell survival, as well as those sensitizing tumour cells to radiotherapy after inhibition. The dual purpose of this project was to better understand the biology underlying oncogenesis of DIPGs and to discover new therapeutic targets.We identified 41 genes required for DIPG cell survival with no major deleterious effect on normal control cells. Among them, we identified VRK3, a serine threonine kinase never involved in DIPG oncogenesis with functions remaining poorly described to date. We have shown that its inhibition leads to a complete arrest of DIPG cell proliferation and is additionally associated with important morphological changes, more particularly in H3.3-K27M mutated tumours. VRK3 is therefore a promising new therapeutic target for all patients in this fatal pathology.In parallel, a similar survival screen was performed in conjunction to cell radiation and very few interfering RNAs enhance H3.3-K27M cell radiosensitivity, in contrast to H3.1-K27M cells. These data highlighted a significant difference in radiosensitivity of the DMG in vitro models in H3.1- versus H3.3-K27M mutated tumours, in a concordant way with patient survival following radiotherapy. These unprecedented results suggest new opportunities for improving the current treatment of DIPG patients regardless of their genotype

Background: Due to its continuous exposure to food antigens and microbes, the gastrointestinal tract (GIT) is in a constant state of low level immune activation and contains an abundance of activated CCR5+CD4+ T lymphocytes, the primary target HIV-1. As a result, the GIT is a site of intense viral replication and severe CD4+ T cell depletion, a process that begins during primary HIV-1 infection and continues at a reduced rate during chronic infection in association with increased production of pro-inflammatory cytokines, a breakdown in the epithelial barrier, microbial translocation, systemic immune activation and the continued recruitment and infection of new target cells. AntiRetroviral Therapy (ART) is only partially effective in reversing these pathogenic changes. Despite the importance of the GIT in HIV-1 pathogenesis, and as a reservoir of persistent virus during ART, little is known about the diversity of HIV-1 in the GIT, or how different tissues in the GIT respond to ART. Objectives: Primary objectives of this thesis were to: 1) characterize the diversity of HIV-1 RNA variants in different parts of the GIT; 2) determine whether there is compartmentalized evolution of HIV-1 RNA variants in the GIT and whether these variants are likely to have different biological properties; 3) investigate the impact of ART on immune restoration in the GIT. Methods: A prospective study of the duodenum, jejunum, ileum and colon of African AIDS patients with chronic diarrhea and/or weight loss, sampled before and during 6 months of ART. RNA extracted from gut biopsies was reverse transcribed and PCR amplified. Env and gag PCR fragments were cloned, sequenced and subjected to extensive phylogenetic analysis; pol PCR fragments were analyzed for drug resistance. CD4+, CD8+ and CD38+CD8+ T cells levels in biopsies collected at baseline (duodenum, jejunum, ileum and colon) and after 3 (duodenum) and 6 (duodenum and colon) months of ART were quantified by flow cytometry and immunohistochemistry, plasma and tissue VL by the Nuclisens assay. Results: Viral diversity varied in different regions of the GIT with env HIV-1 RNA variants being significantly more diverse than gag variants. Gag HIV-1 RNA variants were widely dispersed among all tissue compartments. Some env variants formed tight monophyletic clusters of closely related viral quasispecies, especially in the colon, a finding that is suggestive of compartmentalized viral replication and adaptive evolution. CD4+ T cell and VL levels were significantly lower, while CD8+ including activated CD38+CD8+ T cell levels were higher in the duodenum and jejunum versus the colon. After 6 months of ART, a significant but incomplete recovery of CD4+ T cells was observed in the colon but not in the duodenum. Failed restoration of CD4+ T cells in the duodenum was associated with non-specific enteritis and CD8+ T cell activation. Conclusions: These results advance our understanding of the GIT as a host-pathogen interface by providing new insights into the diversity, evolution and dissemination of HIV-1 variants in the GIT. Strategies aimed at decreasing immune activation, especially in the small intestine, may be highly beneficial in enhancing the therapeutic efficacy of ART.
Thesis (PhD)--University of Pretoria, 2014.
lk2014
Immunology
PhD
Unrestricted

This project investigated the role of FGFR2 isoforms (FGFR2b/FGFR2c) and their cognate FGF ligands in endometrial cancer development, prognosis, and treatment response via designing and validating an innovative BaseScope RNA in-situ hybridization assay and generating patient tumour-derived organoids. FGFR2c and high FGF18 expression were significantly associated with aggressive tumour characteristics and poor survival outcome. It was also noted FGFR2c expression is associated with progestin treatment failure in atypical hyperplasia and well-differentiated endometrial cancers. Overall, FGFR2c and FGF18 are independent prognostic biomarkers that could improve our ability to predict patient prognosis and predict response to FGFR inhibitor treatment in endometrial cancer.

The emerging field of RNA nanotechnology necessitates creation of functional RNA nanoparticles, but has been limited by particle instability. Previously, it was found the three-way junction (3WJ) of the Phi29 DNA packaging motor pRNA was found to be ultra-stable and assemble in solution without the presence of metal ions. The three-way junction is composed of three short oligo RNA strands and proven to be thermodynamically stable. Here the assembly mechanism, thermodynamic and enzymatic stabilities, and kinetics are examined in order to understand the stability behind this unique motif. Thermodynamic and kinetics studies found that the pRNA 3WJ formed out of three components at a rapid rate creating a single-step three component collision with a lack of dimer intermediate formation while being governed by entropy, instead of the commonly seen enthalpy. Furthermore, the pRNA 3WJ proved to be stable at temperatures above 50 °C, concentrations below 100 pM, and produced a free energy of formation well below other studied RNA structures and motifs. With the high stability and folding efficiency of the pRNA 3WJ, it serves as an ideal platform for multi-branched RNA nanoparticles constructed through bottom-up techniques. RNA nanoparticles were constructed for the specific targeting of prostate cancer cells expressing Prostate Specific Membrane Antigen (PSMA) by receptor mediated endocytosis through the addition of an RNA aptamer; and the delivery of anti-miRNA sequences for gene regulation. The resulting nanoparticles remained stable while showing highly specific binding and entry in PSMA positive cells through cell surface receptor endocytosis. Furthermore, the entry of the nanoparticles allowed for the knockdown of against onco-miRNAs. Nanoparticles harboring antimiRNAs led to the upregulation of tumor suppressor genes, and signaling of apoptotic pathways. These findings display RNA nanotechnology can result in the production of stable nanoparticles and result in the specific treatment of cancers, specifically prostate cancer.

Our genome is constantly challenged by sources that cause DNA damage. To repair DNA damage and maintain genomic stability eukaryotes have evolved a complex network of pathways termed the DNA damage response (DDR). The DDR consists of signal transduction pathways that sense DNA damage and mediate tightly coordinated reactions to halt the cell cycle and repair DNA with a collection of different enzymes. In this manner, the DDR protects the genome by preventing the accumulation of mutations and DNA aberrations that promote cellular transformation and cancer development. Loss of function mutations in DDR genes and genomic instability occur frequently in many tumor types and underlie numerous cancer-prone hereditary syndromes such as Fanconi Anemia (FA). My thesis research applies candidate-based and unbiased experimental approaches to investigate the role of several tumor suppressor genes (TSGs) in the DDR. My dissertation will first describe a novel function for the breast and ovarian cancer tumor suppressor and FA-associated gene FANCJ in the DDR to ultraviolet (UV) irradiation. In response to UV irradiation FANCJ supports checkpoint induction, the arrest of DNA synthesis, and suppresses UV induced point mutations. Suggesting that FANCJ could suppress UV induced cancers, in sequenced melanomas from multiple databases I found somatic mutations in FANCJ previously associated with breast/ovarian cancer and FA syndrome. The second part of my dissertation will describe an RNA interference screen to identify genes modulating cellular sensitivity to the chemotherapeutic drug cisplatin. The hereditary breast/ovarian cancer tumor suppressor BRCA2 is essential for DNA repair, thus BRCA2 mutant ovarian cancer cells are initially sensitive to cisplatin chemotherapy that induces DNA damage. However, drug resistance develops and remains a major problem in the clinic. My screen identified the chromatin remodeling factor CHD4 as a potent modulator of cisplatin sensitivity and predictor of response to chemotherapy in BRCA2 mutant cancers. Taken together, my investigations highlight the important contribution of the DDR and the role they play in tumorigenesis and predicting therapeutic response.

Our genome is constantly challenged by sources that cause DNA damage. To repair DNA damage and maintain genomic stability eukaryotes have evolved a complex network of pathways termed the DNA damage response (DDR). The DDR consists of signal transduction pathways that sense DNA damage and mediate tightly coordinated reactions to halt the cell cycle and repair DNA with a collection of different enzymes. In this manner, the DDR protects the genome by preventing the accumulation of mutations and DNA aberrations that promote cellular transformation and cancer development. Loss of function mutations in DDR genes and genomic instability occur frequently in many tumor types and underlie numerous cancer-prone hereditary syndromes such as Fanconi Anemia (FA). My thesis research applies candidate-based and unbiased experimental approaches to investigate the role of several tumor suppressor genes (TSGs) in the DDR. My dissertation will first describe a novel function for the breast and ovarian cancer tumor suppressor and FA-associated gene FANCJ in the DDR to ultraviolet (UV) irradiation. In response to UV irradiation FANCJ supports checkpoint induction, the arrest of DNA synthesis, and suppresses UV induced point mutations. Suggesting that FANCJ could suppress UV induced cancers, in sequenced melanomas from multiple databases I found somatic mutations in FANCJ previously associated with breast/ovarian cancer and FA syndrome. The second part of my dissertation will describe an RNA interference screen to identify genes modulating cellular sensitivity to the chemotherapeutic drug cisplatin. The hereditary breast/ovarian cancer tumor suppressor BRCA2 is essential for DNA repair, thus BRCA2 mutant ovarian cancer cells are initially sensitive to cisplatin chemotherapy that induces DNA damage. However, drug resistance develops and remains a major problem in the clinic. My screen identified the chromatin remodeling factor CHD4 as a potent modulator of cisplatin sensitivity and predictor of response to chemotherapy in BRCA2 mutant cancers. Taken together, my investigations highlight the important contribution of the DDR and the role they play in tumorigenesis and predicting therapeutic response.

Les Dystrophies Myotoniques de type 1 (DM1) et 2 (DM2) sont des maladies neuromusculaires autosomiques dominantes causées par l’expansion anormale de séquences microsatellites C(C)TG situées respectivement dans la région 3’UTR du gène DMPK et le premier intron du gène ZNF9. Les ARNs mutés contenant les expansions de répétitions sont retenus dans le noyau des cellules sous formes d’agrégats riboprotéiques et séquestrent les protéines de liaison à l’ARN de la famille MBNL conduisant à des dérégulations de l’épissage alternatif associés aux symptômes cliniques. Bien que différentes approches thérapeutiques pour la DM soient en développement, il n’existe à ce jour aucune thérapie. Dans ce travail de thèse, nous avons développé une stratégie innovante de thérapie génique basée sur une protéine MBNL1 modifiée. Ce dérivé MBNL∆ agit comme un leurre pour libérer les facteurs MBNL endogènes anormalement séquestrés par les ARN mutés, et restaurer leurs fonctions. Ainsi l’expression de MBNL∆ dans des modèles DM1 permet la correction des anomalies moléculaires et phénotypiques. Nous avons également réalisé différentes optimisations de cet outil dans le but d’accroître ces capacités fonctionnelles. Enfin, nous avons développé un système d’autorégulation basé sur un « senseur d’épissage » dans le but de contrôler l’expression protéique d’un transgène. La preuve de concept de ce système a été faite à l’aide de MBNL∆ et validée dans des modèles DM1. En conclusion, mon travail de thèse a permis d’établir le potentiel d’une approche de thérapie génique pour la DM de type « leurre » basée sur l’ingénierie de protéine de liaison à l’ARN
Myotonic dystrophy (DM) is an autosomal neuromuscular disease encompassing two distinct forms, type 1 (DM1) and type 2 (DM2), which are caused by abnormal microsatellite expansions of C(C)TG repeats in the 3’UTR of the DMPK and first intron of ZNF9 genes, respectively. Mutant RNAs carrying expanded repeats are retained in the nucleus as riboprotein aggregates that abnormally sequester MBNL splicing factors leading to alternative splicing misregulations associated with clinical symptoms. Although various therapeutic approaches for DM are under development, there is no effective therapy available so far. In this study, we designed a novel gene therapy strategy with the use of an engineered MBNL RNA-binding protein derivative that acts as a CUGexp-decoy to release sequestered endogenous MBNL factors and restore their proper functions. Expression of the decoy results in the correction of DM1-associated features in both in vitro and in vivo models of the disease. Subsequent optimization processes were applied to the engineered decoy and the most potent derivate that increases its functional capacity was selected for further therapeutic application. Additionally, we developed an autoregulatory system based on a splice-sensor strategy to control transgene product expression and provided a proof-of-concept of its efficacy in both in vitro and in vivo systems. In conclusion, my work establishes the potency of gene therapy treatment for DM and support the use of the decoy-based approach as an alternate or complementary therapeutic intervention for DM

Les mutations dans le génome mitochondrial humain sont souvent associées à de graves maladies neuromusculaires. Mon projet de thèse a consisté tout d’abord au développement d’une stratégie thérapeutique basée sur l’import mitochondrial de molécules d’ARN. J’ai pu démontrer que l’expression stable de molécules d’ARN recombinantes dans les cellules humaines permet de diminuer le taux de mutations pathogéniques de l’ADN mitochondrial. Dans une seconde partie, j’ai élaboré une nouvelle méthode, CoLoC-seq, permettant l’identification à grande échelle des ARN localisés dans les mitochondries. En appliquant cette méthode sur des cellules humaines, j’ai pu confirmer l’adressage mitochondrial de certains ARN cytosoliques non-codant et identifier de nouveaux ARN potentiellement importés. Ces données permettront d’élargir les connaissances sur les voies d’adressage mitochondrial des ARN, leurs mécanismes et leur régulation, et d’optimiser les stratégies thérapeutiques basées sur l’import d’ARN
Mutations in the human mitochondrial genome are often associated with severe neuromuscular disorders. The first part of my thesis project consisted in the development of a therapeutic strategy based on the mitochondrial import of RNA molecules. I demonstrated that the stable expression of recombinant RNA molecules in human cells induced the decrease of the pathogenic mutation load in mitochondrial DNA. In the second part, I developed a nex method, CoLoC-seq, for the large-scale identification of RNA species localized in the mitochondria. By applying this method to human cells, I confirmed the mitochondrial targeting of some non-coding cytosolic RNAs and identified new potentially imported RNAs. These data will broaden the knowledge on the pathway of RNA targeting into the mitochondria, its mechanisms and regulation, and will allow optimization of the therapeutic strategies based on RNA import

Malignant Pleural Mesothelioma (MPM) is a very aggressive cancer poorly responsive to current therapies. The prognosis is very poor as the development of MPM leads to a median survival of less than one year from the time of diagnosis (Bertino et al. 2009). Moreover, the rapidly growing nature of this cancer, the non-specific onset of symptoms and the lack of an accurate biomarker do not lead to a sufficiently early diagnosis for a radical treatment of the disease. Thus, the identification of novel diagnostic and prognostic biomarkers and therapeutic targets is needed. The knowledge of the genes involved in the triggering/progression of MPM is still limited. In recent years a number of aberrantly expressed genes were suggested, but with a poor consistency among studies. To identify the relevant genes potentially involved in MPM, recently, our group carried out an extensive literature review focused on transcriptome studies whose results were intersected with those from a data mining approach (Melaiu et al. 2012). The results underwent to validation on MPM tissues and cell lines (Melaiu et al. 2015). The study led to the identification of a group of 21 deregulated genes in MPM. Among the 21 genes identified we focused our attention on those up-regulated in MPM tissues and at least in one MPM cell line (i.e. THBS2, CCNO, CFB, TIMP3, SULF1, PDGFRB, ASS1, SOD1, RAN, CDH11, EIF4G1). In addition, we made an extensive literature search to identify the most promising novel diagnostic and therapeutic target for MPM and we undertook the study of IMP3 (Hanley et al.2008; Ikeda et al. 2010; Ikeda et al. 2011; Shi et al.2011; Lee et al. 2013; Okazaki et al. 2013; Minato et a. 2014) , MCT4 (Mogi et al. 2013), MCT1 (Mogi et al. 2013), BSG (Pinheiro et al. 2012) and CAIX (Ramsey et al. 2012; Capkova et al. 2014). In order to investigate the role of these genes in the carcinogenesis of MPM we performed a phenotypic screening of five MPM cell lines (Mero14, Mero25, IstMes2, NCI-H28, REN) and one non-malignant mesothelial cell line (Met5A) following gene-silencing. We analysed the changes in the proliferation rate, in the caspase3-7 activity, in the migration ability, in the colony formation ability, in the level of senescence and in the cell cycle after transient siRNA transfections. The RNAi screening highlighted a role in MPM malignant phenotype for ASS1, CDH11, EIF4G1, IMP3, MCT4, PDGFRB, RAN, SOD1, SULF1, THBS2, TIMP3. Moreover, we evaluated the potential prognostic role of THBS2, CDH11, ITGA4, MCT4, MCT1, BSG and CAIX through a tissue microarray (TMA) immunohistochemistry on a series of 135 MPM samples. None of the target analysed showed a statistical significant association, after multiple correction, between protein expression and the overall survival. The weak staining of THBS2 and the negative staining of MCT4 on six normal pleura samples compared to MPM tissues prompted us to further investigate on protein expression levels: we increased the analysis of protein expression to 15 normal pleura samples. These results highlights the role of a group of genes, overexpressed in MPM, in the carcinogenetic process suggesting potential novel therapeutic approaches for the treatment of MPM.

Clinical laboratory test results are integral to patient management. Important aspects of laboratory tests’ contributions are the use of the test information and the role they have in facilitating efficient and effective use of healthcare resources. Methods of measuring those contributions were examined using quantitative HCV RNA test results (HCV VL) in therapeutic management decisions as a model. Test precision is important in those decisions; therefore, the clinical use was evaluated by studying the impact that knowledge of inherent assay imprecision had on clinicians’ decisions. A survey describing a simulated patient at a decision point for HCV triple-combination therapy management was sent to 1491 hepatology clinicians. Participants saw HCV RNA results at five different levels and were asked to choose to: continue therapy, discontinue therapy, or repeat the test. Test results were presented both with and without the 95% confidence intervals (CIs). Three of the VLs had CIs that overlapped the therapeutic decision level. Participants saw both sets of results in random order. Demographics and practice preferences were also surveyed. One-hundred-thirty-eight responses were received. Adherence to clinical guidelines was demonstrated in self-reported behaviors and in most decisions. However, participants chose to repeat the test up to 37% of the time. The impact of the knowledge of assay imprecision did not have a statistically significant effect on clinicians’ decisions. To determine economic value, an analytic decision-tree model was developed. Transition probabilities, costs, and Quality of Life values were derived from published literature. Survey respondents’ decisions were used as model inputs. Across all HCV VL levels, the calculated test value was approximately $2600, with up to $17,000 in treatment-related cost savings per patient at higher HCV VLs. The test value prevailed regardless of the presence or absence of CIs, and despite repeat testing. The calculated value in cost savings/patient was up to 100 times the investment for HCV VL testing. Laboratory tests are investments in efficient uses of healthcare resources. Proper interpretation and use of their information is integral to that value. This type of analysis can inform institutional decisions and higher level policy discussions.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterised pathologically by the accumulation of amyloid-β plaques and neurofibrillary tangles within the brain. Currently, there are no disease-modifying therapeutic interventions available. This thesis describes the development and delivery of novel RNA-based therapeutics for AD. Recently, significant advances have occurred in the field of RNA interference (RNAi)-based therapeutics, allowing targeted silencing of disease-relevant genes through the use of short interfering RNAs (siRNAs) or microRNAs (miRNAs). However, development has been impeded by poor penetrance of the blood-brain-barrier (BBB) in vivo. Exosomes, as secreted extracellular vesicles (EVs), may overcome this problem as they naturally deliver RNA between cells and cross the BBB. Furthermore, recent evidence suggests that they may be loaded with exogenous RNA and preferentially targeted to neuronal tissue. Thus, this study investigated EV-mediated delivery of RNAi-based therapeutics shown to regulate AD-relevant genes. A comparison of EV-loading methods revealed that electroporation was confounded by the formation of large siRNA aggregates and indicated that endogenous loading strategies may be preferable. Endogenous loading of RNAi molecules was shown to be enhanced by co-expression with RNA-binding proteins, particularly following inclusion of an acylation domain. These findings highlight the potential for EV-mediated delivery of RNAi-based therapeutics. A combinatorial therapeutic approach is increasingly recognised to hold the most promise for the future of AD. Thus, this study also focused upon the development of novel multi-gene targeting locked nucleic acid-modified antisense oligonucleotides (LNA-ASOs) as a combinatorial approach towards AD therapy. Two multi-gene LNA-ASOs each produced potent silencing of two separate target genes simultaneously, resulting in a significant reduction in amyloid-β production in vitro. A murine-specific multi-gene LNA-ASO was also identified, paving the way for future in vivo studies. These findings suggest that multi-gene targeting LNA-ASOs may represent a promising novel therapeutic strategy for AD. In summary, this thesis has investigated novel strategies for both RNA delivery and multi-gene targeting, demonstrating the potential of RNA-based therapeutics for the treatment of AD.

Prostate cancer is the most common noncutaneous cancer among men, yet current diagnostic methods are insufficient and more reliable diagnostic markers need to be developed. The answer that can bridge this gap and enable more efficient diagnoses may lie in microRNAs. These small, single stranded RNA molecules impact protein expression at the translational level and regulate important cellular pathways. Dysregulation of these small RNA molecules can have tumorigenic effects on cells and lead to many types of cancers. Currently the Prostate-Stimulating Antigen (PSA) is used as a diagnostic marker for prostate cancer. However, many factors can elevate PSA levels such as infections and certain medications, consequently leading to false positive diagnoses and unnecessary concern and over treatment with dire outcomes for the patient. Even worse, are the chances of false negative diagnoses, which result in prostate cancer not being diagnosed until its later stages. Therefore, although the use of the PSA level has had its uses in the clinic, it has failed to sufficiently bridge the gap or to distinguish indolent from aggressive disease. It has long been suggested in the literature that microRNAs are drastically altered throughout the course of cancer progression. Here, RNA sequencing was used to identify changes in miR expression profiles diagnostic for prostate cancer patients compared to non-patient controls. The RNA sequencing results were also used to identify normalization miRs to be used as endogenous controls. Confirmatory qRT-PCR was then used to corroborate these results for the top seven dysregulated miRs found from the RNA sequencing data. Data analysis of the Area Under the Curve (AUC) of the Receiver Operating Curves (ROC) of the selected miRs exhibited a better correlation with prostate cancer (AUC Range= 0.819- 0.950) than PSA (AUC of PSA=0.667). In summary, a panel of seven miRs are proposed, many of which have prostate specific targets, which would represent a significant improvement over current testing methods.

Les mitochondries sont impliquées dans de nombreuses voies métaboliques, et des mutations au sein de leur génome (ADNmt) conduisent à l’apparition de nombreuses pathologies. A l’heure actuelle, il n’existe aucun traitement contre ces affections mais différentes pistes thérapeutiques sont envisagées. L’objectif de ce travail a consisté en la mise au point d’une telle stratégie, dite anti-réplicative via l’exploitation de la voie d’import naturelle des ARN dans les mitochondries. De petits ARN artificiels importables dans les mitochondries humaines ont ainsi été utilisés comme vecteurs pour y importer une séquence capable de s’hybrider spécifiquement à l’ADNmt mutant et d’en stopper sa réplication. Les résultats obtenus ont permis de prouver la validité de cette stratégie vis-à-vis d’une large délétion et de mutations ponctuelles liées à divers cas pathologiques et de caractériser l’effet de modifications chimiques sur la stabilité, l’import et l’efficacité de ces ARN recombinants
Mitochondria are involved in many metabolic pathways, and mutations in their genome (mtDNA) can cause a wide range of human disorders. No efficient treatment against these pathologies is currently available. The objective of this work consisted in the development of a therapeutic approach, called anti-replicative, based on the use of the natural pathway of RNA import into mitochondria. Small artificial RNA molecules able to be imported into human mitochondria have been used as vectors to address oligoribonucleotides capable to hybridize specifically to mutant mtDNA and to stop its replication. The effect of various chemical modifications on the stability, import and efficiency of these recombinant RNA has been characterized. All the data obtained prove the validity of the anti-replicative strategy for mtDNA containing a large deletion or pathogenic point mutations and can be considered as an important step to further develop an efficient therapy of mitochondrial diseases

O carcinoma diferenciado de tireóide (CDT) abrange 95% de todas as doenças malignas da tireóide. Nos EUA, aumentou em 2,4 vezes nos últimos anos (1973-2002). O seu tratamento inclui tireoidectomia total, seguido por terapia com radioiodo e supressão do TSH com L-tiroxina. A doença pode recidivar em ~20% dos casos, sendo necessária avaliação periódica através de exames de imagens e dosagem de tireoglobulina sérica (TGs). Os Anticorpos (Acs) anti-TG podem ser detectados em 15 a 25% dos pacientes, comprometendo, parcialmente, o uso da TGs como marcador de recidiva do câncer. Um método alternativo proposto para monitorar os pacientes é a detecção de células tireoidianas em sangue periférico, através da mensuração do RNA mensageiro de TG (RNAm-TG) pela técnica de RTPCR em tempo real. Esta nova metodologia aumenta a sensibilidade da detecção desta molécula. O objetivo deste estudo é verificar a significância da quantificação do RNAm-TG, como método diagnóstico complementar no acompanhamento a longo prazo de pacientes com CDT. Amostras de sangue de 45 pacientes (25 sem metástase, 14 com metástase ganglionar e 6 com metástase à distância) foram coletadas nos tempos: antes e 24, 48, 72 horas, 7 dias, 1, 3, 6, 9 meses, 1, 2, 4, 5, 6 e 7 anos após a dose ablativa de radioiodo. Foi realizada extensiva padronização da técnica com a finalidade de excluir interferências metodológicas, empregando dois genes controles interno (GAPDH e HPRT1) para o cálculo da concentração do RNAm-TG. Concomitantemente foi realizada a mensuração de TGs, perfil hormonal e de anticorpos anti-TG. A pesquisa de corpo inteiro, realizada 7 dias após a dose terapêutica, estabeleceu o estadio clínico inicial dos pacientes. Não foi possível estabelecer um valor de corte para o RNAm-TG. O RNAm-TG não diferenciou os estadios clínicos da doença ao longo do tempo, independente do gene controle interno utilizado, e tampouco quando analisaram-se os dados na presença de Acs anti-TG e TSH30ng/mL. A TGs diferenciou os estadios clínicos ao longo do tempo. Concluiu-se que, o RNAm-TG não é um bom marcador de recidiva do CDT, mesmo quando considerou-se critérios de padronização da técnica, avaliação em longo prazo e presença de Acs anti-TG, sendo assim não poderia ser utilizado como método diagnóstico complementar no acompanhamento de pacientes com CDT. Este estudo demonstra que a técnica de RT-PCR em tempo real é muito sensível perdendo especificidade, inviabilizando sua utilização no acompanhamento dos pacientes com CDT
The differentiated thyroid carcinoma (DTC) encloses 95% of all thyroid malignant disease. In USA, it increased 2,4 times in recent years (1973- 2002). The treatment includes total thyroidectomy, ablation with radioiodine (RAI) followed by TSH suppression with L-Thyroxine. The cancer recurrence occurs in 20% of the cases. Periodic evaluation through imaging examinations and serum thyroglobulin (TG) measurements by imunoassays method is recommended for careful follow-up of these patients. The anti-TG antibodies prevalence is 15-25% and would impair, partially, the serum TG use as a tumor marker. An alternative method to identify the recurrence of the tumor is the thyroid cells detection in peripheral blood, through the TG messenger RNA quantification (mRNA-TG) by real time RT-PCR. This new methodology increases the sensitivity detection for this molecule. The objective of this study was to verify the mRNA-TG peripheral blood quantification significance, as a complementary diagnostic method in the long term follow up of patients with DTC. Fourty five blood samples from patients with DTC have been collected before and 24, 48, 72 hours, 7 days, 1, 3, 6, 9 months, 1, 2, 4, 5, 6 and 7 years after the ablation therapy. Extensive technique standardization for mRNA-TG measurements was carried out to exclude methodological interventions and two housekeeping genes (GAPDH and HPRT1) were used to calculate the mRNA-TG concentrations. Concomitantly, serum TG measurements, hormonal profile and antibodies anti-TG assays were performed. The whole body scan was performed 7 days after RAI ablation to determine the stage of the disease. It was not possible to establish a cut-point value for mRNA-TG. The mRNA-TG did not differentiated the clinical stage of the disease in the long term follow-up and neither in the presence of anti-TG antibodies and TSH30ng/mL. Serum TG was able to differentiate the clinical stage of the patients during the follow-up. In conclusion mRNA-TG is not a good marker for the DCT recurrence, even when technical standardization, long term evaluation and the presence of antibodies anti-TG were considered. Thus it could not be used as a complementary diagnostic method in the DTC patients follow-up. This study confirmed the high sensivity of the real time RT-PCR whereas with very low specificity, consequently is unviable to be used in the DTC patients follow-up

[Truncated abstract] Breast cancer is the cause of significant suffering and death in our community. It is now estimated that the risk of developing breast cancer for an Australian woman before the age of 85 is 1 in 8, with this risk rising for unknown reasons. While mortality rates from breast cancer are falling due to increased awareness and early detection, few new treatments have been developed from an advanced understanding of the molecular basis of the disease. From decades of scientific research it is clear that estrogen (E2) has a large role to play in breast cancer. However, the basic mechanism behind E2 action in breast cancer remains unclear. E2 plays a fundamental role in breast cancer cell proliferation and is highly expressed in breast cancers, thus, it is important to understand both E2 and its receptor, the estrogen receptor (ER). The ER is a member of the nuclear receptor (NR) superfamily. The NR superfamily consists of a large group of proteins which regulate a large number of homeostatic proteins together with regulator proteins termed coregulators and corepressors. SRA (steroid receptor RNA activator) is the only known RNA coactivator and augments transactivation by NRs. SRA has been demonstrated to play an important role in mediating E2 action (Lanz et al., 1999; Lanz et al., 2003) and its expression is aberrant in many human breast tumors, suggesting a potential role in breast tumorigenesis (Murphy et al., 2000). Despite evidence that an alternative splice variant of SRA exists as a protein (Chooniedass-Kothari et al., 2004), it has been conclusively shown that SRA can function as an RNA transcript to coactivate NR transcription (Lanz et al., 1999; Lanz et al., 2002; Lanz et al., 2003). The precise mechanism by which SRA augments ER activity remains unknown. However, it is currently hypothesized that SRA acts as an RNA scaffold for other coregulators at the transcription initiation site. Several SRA stem loops have been identified as important for SRA function, including structure (STR) 1, 5 and 7 (Lanz et al., 2002; Zhao et al., 2007). Previously, I sought to identify SRA-binding proteins using a specific stem-loop structure of SRA (STR7) that was identified as both important for its coactivator function (Lanz et al., 2002) and also as a target for proteins from breast cancer cell extracts (Hatchell, 2002). From a yeast E. Hatchell Abstract iii III hybrid screen using STR7 as bait, I identified a novel protein which was named SLIRP (Patent Number: WO/2007/009194): SRA stem-Loop Interacting RNA-binding Protein (Hatchell, 2002; Hatchell et al., 2006). '...' This thesis demonstrates that SLIRP modulates NR transactivation, provides mechanistic insight into interactions between SRA, SRC-1, HSP-60 and NCoR and suggests that SLIRP may regulate mitochondrial function. These studies contribute significantly to the growing field of NR biology, and contribute more specifically to the elucidation of estrogen action in breast cancer. Furthermore, it lays a strong and exciting foundation for further studies to evaluate SLIRP as a biomarker and potential therapeutic target in hormone dependent cancers.

Thesis (Ph. D. in Medical Engineering)--Harvard-MIT Program in Health Sciences and Technology, 2012.
Vita. Cataloged from PDF version of thesis.
Includes bibliographical references (p. 234-250).
Efforts to sequence cancer genomes have begun to uncover comprehensive lists of genes altered in cancer. Unfortunately, the number and complexity of identified alterations has made dissecting the underlying biology of cancer difficult, as many genes are not amenable to manipulation by small molecules or antibodies. RNA interference (RNAi) provides a direct way to assess and act on putative cancer targets. However, the translation of RNAi into the clinic has been thwarted by the "delivery" challenge, as small interfering RNA (siRNA) therapeutics must overcome clearance mechanisms and penetrate into tumor tissues to access cancer cells. This thesis sought to develop nanotechnology-based platforms to rapidly discover and validate cancer targets in vivo. First, we developed versatile surface chemistries for nanoparticle tumor targeting. Leveraging new discoveries in amplified transvascular transport, we designed a siRNA delivery system that integrates the tumor specificity and tissue-penetrating ability of tumor-penetrating peptides with membrane penetration properties of protein transduction domains to direct siRNA to tumors in vivo. Second, we utilized this delivery system to bridge the gap between cancer genomic discovery and in vivo target validation. Comprehensive analysis of ovarian cancer genomes identified candidate targets that are undruggable by traditional approaches. Tumor-penetrating delivery of siRNA against these genes potently impeded the growth of ovarian tumors in mice and improved survival, thereby credentialing their roles in tumor initiation and maintenance. Lastly, we described efforts extending this platform for clinical translation. Mechanistic studies identified functional properties that favored receptor-specific siRNA delivery. We also explored a strategy to improve the microdistribution of successively dosed siRNA therapeutics through modulating the tumor microenvironment. Finally, we investigated the utility of the system in primary human tumors derived from patients with ovarian cancer. Together, these findings illustrate that the combination of cancer genomics with the engineering of siRNA delivery nanomaterials establishes a platform for discovering genes amenable to RNAi therapies. As efforts in genome sequencing accelerate, this platform illustrates a path to clinical translation in humans.
by Yin Ren.
Ph.D.in Medical Engineering

Cis-diamminedichloroplatinum (II), or cisplatin, is a widely prescribed anticancer compound, currently one of only three platinum (II) complexes FDA approved for cancer treatment. Despite its widespread use, we lack a comprehensive picture of global drug targets, which would lend valuable insights into the molecular mechanisms of action and resistance in different tissues. Drug binding to genomic DNA is an accepted cause of downstream apoptotic signaling, but less than 10% of Pt (in the case of cisplatin) accumulates within genomic DNA. Non-genomic contributions to cisplatin's therapeutic action are also under active investigation. In particular, cisplatin treatment can disrupt RNA-based processes such as splicing and translation. Pt(II) targeting of non-DNA species such as RNA may contribute to or sensitize a cell to the downstream effects of this drug, including the induction of apoptosis. Chapter I summarizes the activity profile of Pt(II) therapeutics, describing cellular uptake, cellular localization, incidences of Pt(II) accumulation within RNA, and RNA processes affected following drug treatment. Chapter II reports our thorough investigation of the distribution of Pt species throughout messenger and ribosomal RNA, with the discovery that Saccharomyces cerevisiae ribosomes act as a de facto cellular Pt sponge. In Chapter III, we report the synthesis of an azide-functionalized platinum (II) species, picazoplatin, for post-treatment click labeling and isolation of drug targets in vivo. Picazoplatin was designed to circumvent mislocalization and misprocessing of Pt typically encountered when trying to track small molecules tethered to large, charged fluorophores. This chapter contains several proof-of-principle studies validating the use of this class of reagents for future purification and sequencing of Pt-bound nucleic acids. Chapter IV describes the first application of the click-capable Pt reagent technology: the demonstration of significant in-gel fluorescent detection of Pt-bound ribosomal RNA and transfer RNA extracted from picazoplatin-treated S. cerevisiae and the first evidence that cellular tRNA is a platinum substrate. Chapter V summarizes these data, which suggest a potential ribotoxic mechanism for cisplatin cytotoxicity and broadly describe a convenient click chemistry methodology that can be applied to identify other metal or covalent modification-based drug targets. This dissertation includes previously published and unpublished co-authored material.

Neuroblastoma (NB) is the most common solid tumour in childhood and accounts for 15% of childhood cancer deaths. It is known that high-risk NB is highly correlated with MYCN amplification. Overexpressed MYCN induces proliferation and cell growth and suppresses apoptosis and differentiation pathways in NB cells. Since RNA interference (RNAi) was first described, many research groups have investigated the application of RNAi with the use of short interfering RNA (siRNA). Our aim is to induce apoptosis and differentiation using RNAi as a novel therapeutic strategy for MYCN-amplified NB. Our hypothesis is that MYCN silencing by anti-MYCN siRNA induces apoptosis and differentiation at the mRNA and protein level. We are encapsulating siRNA with liposome and integrin-receptor targeting peptide to deliver MYCN siRNA into NB cells and optimising cationic and anionic polyethylene glycol (PEG)ylated receptor-targeting nanocomplexes (RTNs). In this project, we also aimed to optimise the methods to store RTNs for a long time in trehalose, which is known as a cryoprotectant. As a result, MYCN was silenced by the siRNA at both the mRNA and protein levels, and the siRNA-mediated MYCN reduction induced downstream effects, such as a neuronal differentiation marker TrkA upregulation and the morphological changes of the cells. The anti-MYCN siRNA delivered using RTNs successfully silenced MYCN mRNA in vivo as well. We used an NB cell line with non-functional p53 and resistance toward p53-pathway dependent anti-cancer drugs, probably induced by multiple sessions of chemotherapy and radiotherapy. Therefore, the results are promising for a novel therapy for relapse NB with MYCN amplification. In addition, we successfully demonstrated that trehalose maintains the biophysical properties and the function of RTNs, consisting of either DNA or siRNA at -80 °C. This allows us to make a large amount of RTN for many experiments, store it for the long term, and transport it to a place far from the laboratory.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder caused by absence of functional dystrophin protein. This thesis describes investigations into the role of small non-coding RNAs in both DMD pathology, and as potential therapeutic molecules. MicroRNAs (miRNAs) are a class of small RNAs that regulate gene expression and are implicated in wide-ranging cellular processes and pathological conditions. This study has compared differential miRNA expression in proximal and distal limb muscles, diaphragm, heart and serum in the mdx dystrophic mouse model relative to wild-type controls. Global transcriptome analysis revealed muscle-specific patterns of differential miRNA expression as well as commonalities between tissues, including previously identified dystromirs. miR-1, miR-133a and miR-206 were found to be highly abundant in mdx serum, suggesting that these miRNAs are promising disease biomarkers. Indeed, the relative serum levels of these miRNAs were normalised in response to peptide-PMO mediated dystrophin restoration therapy. This study has revealed further complexity in the miRNA transcriptome of the mdx mouse, an understanding of which will be valuable for the development of novel DMD therapeutics and for monitoring their efficacy. Myostatin is a secreted growth factor that negatively regulates muscle mass and is therefore a potential pharmacological target for the treatment of muscle wasting disorders such as DMD. This study describes a novel myostatin inhibition approach in which small interfering RNAs (siRNAs) complementary to a promoter-associated transcript induce transcriptional gene silencing (TGS) in cultured myotubes. Silencing was sensitive to treatment with the histone deacetylase inhibitor Trichostatin A, and the silent state chromatin mark H3K9me2 was enriched at the myostatin promoter following siRNA transfection, suggesting epigenetic remodelling underlies the silencing effect. These observations suggest that long-term epigenetic silencing may be feasible for myostatin and that TGS is a promising novel therapeutic strategy for the treatment of muscle wasting disorders. The work in this thesis therefore demonstrates the potential of small RNAs as therapeutic agents and as disease biomarkers in the context of DMD.

A Glutamina (Gln) melhora a regeneração hepática em ratos nutridos. Para avaliar o efeito molecular da Gln endovenosa no remanescente hepático, 52 ratos foram classificados em: 1. Nutridos com hepatectomia parcial (HP) e Gln (N-Gln=10) ou prolina (N-Pro=10); 2. Desnutridos com HP: (D-Gln=10) ou (D-Pro=10); 3. Nutridos e Desnutridos sem HP (N-Controle=6) e (D-Controle=6). Após 96 horas da HP os resultados foram: DNA = (D-Gln, D-Pro e D-Controle < N-Gln, N-Pro e N-controle), (N-Gln, N-Pro, D-Gln e D-Pro < N e D-Controle), (N-Gln > D-Gln); RNA= (N-Gln, N-Pro, D-Gln e D-Pro < N e D-Controle), (N-Gln > D-Gln e N-Pro). Não houve diferença nos genes transcritos (GT) para HGF, TGF-a e timidina kinase. Concluímos que: A desnutrição e HP reduzem DNA e RNA; A Gln não altera os GT estudados em ratos desnutridos 96 horas após HP
Glutamine dipeptide (Gln) improve hepatic regeneration of nourished rats. To evaluate molecular effect of Gln on liver remnant 52 rats was classified into: 1. Nourished with partial hepatectomy (PH) and Gln (N-Gln=10) or proline (N-Pro=10); 2. Malnourished (MN) with PH: (MN-Gln=10) or (MN-Pro=10); 3. Nourished and Malnourished rat without PH (N-Control=6) and (MN-Control=6). Results: DNA = (MN-Gln, MN-Pro and MN-Control < N-Gln, N-Pro and N-control), (N-Gln, N-Pro, MN-Gln and MN-Pro < N and MN-Control), (N-Gln > MN-Gln); RNA= (N-Gln, N-Pro, MN-Gln and MN-Pro < N and MN-Control), (N-Gln > MN-Gln and N-Pro). There was no difference on transcript genes (TG) for HGF, TGF-a and thymidine kinase. Conclusions: Malnutrition and PH decrease hepatic DNA and RNA; Gln does not modify TG studied 96 hours after PH in MN rats

Prostate cancer represents a serious health concern as the most diagnosed form of cancer in men and the second leading cause of cancer death in the Western world. Current treatments for prostate cancer are non-targeted and result in a number of undesirable, non-specific effects, highlighting the need for novel, targeted therapeutics in the treatment of prostate cancer. Prostate Specific Membrane Antigen (PSMA) offers great promise in the targeting of prostate cancer for imaging and therapy. PSMA is a transmembrane carboxypeptidase with cell surface expression several orders of magnitude higher in cancerous prostatic epithelia than found in other tissue and PSMA is constitutively internalized into cells. The unique expression profile of PSMA and its constitutive internalization offer great value in the targeted delivery of therapeutics to prostate cancer cell. In 2002, two synthetic RNA ligands, aptamers, were selected for their ability to inhibit the enzymatic activity of PSMA. In 2006, the utility of these aptamers in the delivery of cytotoxic siRNA across the cell membrane was demonstrated in vivo using aptamer-siRNA chimeras. However, those experiments were performed by intratumoral injection, and systemic administration will be necessary for use in the clinic. In this thesis, we improve PSMA targeted chimeras to serve as more powerful therapeutics in the treatment of prostate cancer. We optimize existing aptamer-siRNA chimeras for increased potency and stability and improved pharmacokinetics to enable systemic administration. We truncate the PSMA binding aptamers for amenability to large-scale chemical synthesis. With emerging roles for PSMA enzymatic activity in the prostate cancer disease we identify aptamers that are suitable for chemical synthesis and retain inhibitory properties against PSMA. Finally, we assess the use of aptamers as synthetic ligands in the functional inhibition of PSMA mediated motility in prostate cancer. Our results demonstrate the ability of aptamer-siRNA chimeras to specifically kill PSMA-expressing cells with cytotoxic siRNA upon systemic injection. We confirm a newly reported role for PSMA in the promotion of cell motility and demonstrate the ability of aptamers to effectively neutralize PSMA-mediated motility. The results presented within argue strongly for the functional utility of aptamers in the treatment of prostate cancer.

Pulmonary arterial hypertension (PAH) is a progressive disease of the small pulmonary arteries, characterised by pulmonary vascular remodelling due to excessive proliferation and resistance to apoptosis of pulmonary artery endothelial cells (PAECs) and pulmonary artery smooth muscle cells (PASMCs). The increased pulmonary vascular resistance and elevated pulmonary artery pressures result in right heart failure and premature death. Germline mutations of the bone morphogenetic protein receptor-2 (bmpr2) gene, a receptor of the transforming growth factor beta (TGF-β) superfamily, account for approximately 75%-80% of the cases of heritable form of PAH (HPAH) and 20% of sporadic cases or idiopathic PAH (IPAH). IPAH patients without known bmpr2 mutations show reduced expression of BMPR2. However only ~ 20% of bmpr2-mutation carriers will develop the disease, due to an incomplete penetrance, thus the need for a ‘second hit’ including other genetic and/or environmental factors is accepted. Diagnosis of PAH occurs most frequently when patients have reached an advanced stage of disease. Although modern PAH therapies can markedly improve a patient’s symptoms and slow the rate of clinical deterioration, the mortality rate from PAH remains unacceptably high. Therefore, the development of novel therapeutic approaches is required for the treatment of this multifaceted disease. Noncoding RNAs (ncRNAs) include microRNAs (miRNAs) and long noncoding RNAs (lncRNAs). MiRNAs are ~ 22 nucleotide long and act as negative regulators of gene ex-pression via degradation or translational inhibition of their target mRNAs. Previous studies showed extensive evidence for the role of miRNAs in the development of PAH. LncRNAs are transcribed RNA molecules greater than 200 nucleotides in length. Similar to classical mRNA, lncRNAs are translated by RNA polymerase II and are generally alternatively spliced and polyadenylated. LncRNAs are highly versatile and function to regulate gene expression by diverse mechanisms. Unlike miRNAs, which exhibit well-defined actions in negatively regulating gene expression via the 3’-UTR of mRNAs, lncRNAs play more diverse and unpredictable regulatory roles. Although a number of lncRNAs have been intensively investigated in the cancer field, studies of the role of lncRNAs in vascular diseases such as PAH are still at a very early stage. The aim of this study was to investigate the involvement of specific ncRNAs in the development of PAH using experimental animal models and cell culture. The first ncRNA we focused on was miR-143, which is up-regulated in the lung and right ventricle tissues of various animal models of PH, as well as in the lungs and PASMCs of PAH patients. We show that genetic ablation of miR-143 is protective against the development of chronic hypoxia induced PH in mice, assessed via measurement of right ventricular systolic pressure (RVSP), right ventricular hypertrophy (RVH) and pulmonary vascular remodelling. We further report that knockdown of miR-143-3p in WT mice via anti-miR-143-3p administration prior to exposure of mice to chronic hypoxia significantly decreases certain indices of PH (RVSP) although no significant changes in RVH and pulmo-nary vascular remodelling were observed. However, a reversal study using antimiR-143-3p treatment to modulate miR-143-3p demonstrated a protective effect on RVSP, RVH, and muscularisation of pulmonary arteries in the mouse chronic hypoxia induced PH model. In vitro experiments showed that miR-143-3p overexpression promotes PASMC migration and inhibits PASMC apoptosis, while knockdown miR-143-3p elicits the opposite effect, with no effects observed on cellular proliferation. Interestingly, miR-143-3p-enriched exosomes derived from PASMCs mediated cell-to-cell communication between PASMCs and PAECs, contributing to the pro-migratory and pro-angiogenic phenotype of PAECs that underlies the pathogenesis of PAH. Previous work has shown that miR-145-5p expression is upregulated in the chronic hypoxia induced mouse model of PH, as well as in PAH patients. Genetic ablation and pharmacological inhibition (subcutaneous injection) of miR-145-5p exert a protective against the de-velopment of PAH. In order to explore the potential for alternative, more lung targeted delivery strategies, miR-145-5p expression was inhibited in WT mice using intranasal-delivered antimiR-145-5p both prior to and post exposure to chronic hypoxia. The decreased expression of miR-145-5p in lung showed no beneficial effect on the development of PH compared with control antimiRNA treated mice exposed to chronic hypoxia. Thus, miR-143-3p modulated both cellular and exosome-mediated responses in pulmonary vascular cells, while the inhibition of miR-143-3p prevented the development of experimental pulmonary hypertension. We focused on two lncRNAs in this project: Myocardin-induced Smooth Muscle Long noncoding RNA, Inducer of Differentiation (MYOSLID) and non-annotated Myolnc16, which were identified from RNA sequencing studies in human coronary artery smooth muscle cells (HCASMCs) that overexpress myocardin. MYOSLID was significantly in-creased in PASMCs from patients with IPAH compared to healthy controls and increased in circulating endothelial progenitor cells (EPCs) from bmpr2 mutant PAH patients. Exposure of PASMCs to hypoxia in vitro led to a significant upregulation in MYOSLID expres-sion. MYOSLID expression was also induced by treatment of PASMC with BMP4, TGF-β and PDGF, which are known to be triggers of PAH in vitro. Small interfering RNA (siR-NA)-mediated knockdown MYOSLID inhibited migration and induced cell apoptosis without affecting cell proliferation and upregulated several genes in the BMP pathway in-cluding bmpr1α, bmpr2, id1, and id3. Modulation of MYOSLID also affected expression of BMPR2 at the protein level. In addition, MYOSLID knockdown affected the BMP-Smad and BMP-non-Smad signalling pathways in PASMCs assessed by phosphorylation of Smad1/5/9 and ERK1/2, respectively. In PAECs, MYOSLID expression was also induced by hypoxia exposure, VEGF and FGF2 treatment. In addition, MYOSLID knockdown sig-nificantly decreased the proliferation of PAECs. Thus, MYOSLID may be a novel modulator in pulmonary vascular cell functions, likely through the BMP-Smad and –non-Smad pathways. Treatment of PASMCs with inflammatory cytokines (IL-1 and TNF-α) significantly in-duced the expression of Myolnc16 at a very early time point. Knockdown of Myolnc16 in vitro decreased the expression of il-6, and upregulated the expression of il-1 and il-8 in PASMCs. Moreover, the expression levels of chemokines (cxcl1, cxcl6 and cxcl8) were sig-nificantly decreased with Myolnc16 knockdown. In addition, Myolnc16 knockdown decreased the MAP kinase signalling pathway assessed by phosphorylation of ERK1/2 and p38 MAPK and inhibited cell migration and proliferation in PASMCs. Thus, Myolnc16 may a novel modulator of PASMCs functions through anti-inflammatory signalling pathways. In summary, in this thesis we have demonstrated how miR-143-3p plays a protective role in the development of PH both in vivo animal models and patients, as well as in vitro cell cul-ture. Moreover, we have showed the role of two novel lncRNAs in pulmonary vascular cells. These ncRNAs represent potential novel therapeutic targets for the treatment of PAH with further work addressing to investigate the target genes, and the pathways modulated by these ncRNAs during the development of PAH.