Investigation of the role of long non-coding RNAs in oncogene induced cellular senescence

Cellular senescence is a stable arrest of cell proliferation induced by several factors such as activated oncogenes, oxidative stress and shortening of telomeres. Senescence acts as a tumour suppression mechanism to halt the progression of cancer. However, senescence may also impact negatively upon tissue regeneration, thus contributing to the effects of ageing. The eukaryotic genome is controlled by various modes of transcriptional and translational regulation. Focus has therefore centred on the role of long non- coding RNAs (lncRNAs) in regulating the genome. Accordingly, understanding how lncRNAs function to regulate the senescent genome is integral to improving our knowledge and understanding of tumour suppression and ageing. Within this study, I set out to investigate the expression of lncRNAs’ expression within models of senescence. Through a custom expression array, I have shown that expression of multiple different lncRNAs is up-regulated and down regulated in IMR90 replicative senescent fibroblasts and oncogene-induced senescent melanocytes. LncRNA expression was determined to be specific to stable senescence-associated cell arrest and predominantly within the nucleus of senescent cells. In order to examine the function of lncRNA expression in senescence, I selected lncRNA transcript ENST0000430998 (lncRNA_98) to focus my investigations upon. LncRNA_98 was robustly upregulated within multiple models of senescence and efficiently depleted using anti-sense oligonucleotide technology. Characterisation and unbiased RNA-sequencing of lncRNA_98 deficient senescent cells highlighted a list of genes that are regulated by lncRNA_98 expression in senescent cells and may regulate aspects of the senescence program. Specifically, the formation of SAHF was impeded upon depletion of lncRNA_98 expression and levels of total pRB protein expression severely decreased. Validation and recapitulation of consequences of pRB depletion was confirmed through lncRNA_98 knock-out cells generated using CRISPR technology. Surprisingly, inhibition of ATM kinase functions permitted the restoration of pRB protein levels within lncRNA_98 deficient cells. I propose that lncRNA_98 antagonizes the ability of ATM kinase to downregulate pRB expression at a post-transcriptional level, thereby potentiating senescence. Furthermore, lncRNA expression was detected within fibroblasts of old individuals and visualised within senescent melanocytes in human benign nevi, a barrier to melanoma progression. Conversely, mining of 337 TCGA primary melanoma data sets highlighted that the lncRNA_98 gene and its expression was lost from a significant proportion of melanoma samples, consistent with lncRNA_98 having a tumour suppressor functions. The data presented in this study illustrates that lncRNA_98 expression has a regulatory role over pRB expression in senescence and may regulate aspects of tumourigenesis and ageing.

RoMEO Studies 8: self-archiving: the logic behind the colour-coding used in the Copyright Knowledge Bank

Purpose – The purpose of this research is to show how the self-archiving of journal papers is a major step towards providing open access to research. However, copyright transfer agreements (CTAs) that are signed by an author prior to publication often indicate whether, and in what form, self-archiving is allowed. The SHERPA/RoMEO database enables easy access to publishers' policies in this area and uses a colour-coding scheme to classify publishers according to their self-archiving status. The database is currently being redeveloped and renamed the Copyright Knowledge Bank. However, it will still assign a colour to individual publishers indicating whether pre-prints can be self-archived (yellow), post-prints can be self-archived (blue), both pre-print and post-print can be archived (green) or neither (white). The nature of CTAs means that these decisions are rarely as straightforward as they may seem, and this paper describes the thinking and considerations that were used in assigning these colours in the light of the underlying principles and definitions of open access. Approach – Detailed analysis of a large number of CTAs led to the development of controlled vocabulary of terms which was carefully analysed to determine how these terms equate to the definition and “spirit” of open access. Findings – The paper reports on how conditions outlined by publishers in their CTAs, such as how or where a paper can be self-archived, affect the assignment of a self-archiving colour to the publisher. Value – The colour assignment is widely used by authors and repository administrators in determining whether academic papers can be self-archived. This paper provides a starting-point for further discussion and development of publisher classification in the open access environment.

Modulação da capacidade antioxidante e de detoxificação no peixe Cyprinus carpio (Cyprinidae) após tratamento com nanocápsulas de ácido lipóico

O metabolismo aeróbico é muito eficiente no processo de geração de energia, no entanto, é uma fonte de produção de espécies reativas de oxigênio (ERO). Para a prevenção dos efeitos potencialmente danosos dessas ERO, os organismos desenvolveram um sistema de defesa antioxidante (SDA), que inclui compostos enzimáticos e não enzimáticos. O ácido lipóico (AL) é uma molécula lipo e hidro solúvel, com capacidade de atravessar membranas celulares. Ele possui propriedades antioxidantes, auxiliando na eliminação de ERO, induzindo a expressão de genes importantes nas defesas antioxidantes, quelando metais e interagindo com outros antioxidantes. Trabalhos prévios demonstraram que nanocápsulas poliméricas de ácido lipóico favoreceram a proteção deste antioxidante, aumentando sua estabilidade físico- química em comparação com formulações contendo ácido lipóico livre. O objetivo deste estudo foi avaliar e comparar o efeito do AL livre e do AL em nanocápsulas sobre a atividade de enzimas antioxidantes (glutamato-cisteína ligase, GCL e glutationa-S- transferase, GST), a concentração de glutationa reduzida (GSH) e sub-produtos da peroxidação lipídica (malondealdeído, método TBARS) e da expressão de genes que codificam para as diferentes formas da enzima GST (alfa e pi). Para isso o peixe Cyprinus carpio (Cyprinidae) foi exposto a uma dose de 40 mg/kg a diferentes formas de AL (livre e em nanocápsulas) por injeção intraperitoneal (duas injeções, sendo a primeira no tempo 0 e a segunda após 24 h), sendo logo sacrificados a diferentes tempos da primeira injeção (48 h, 96 h e uma semana), sendo dissecados o cérebro, fígado e músculo dos peixes de cada tratamento. Os resultados obtidos indicam que os órgãos respondem de forma diferente. A curto prazo, o fígado foi o principal órgão a apresentar respostas antioxidantes após tratamento com AL, enquanto que a longo prazo o cérebro e o músculo se mostraram mais responsivos em termos antioxidantes quando 6 comparado ao fígado. Foi também importante a forma em que o AL é administrado, livre ou em nanocápsulas, sendo observado que um mesmo órgão em um mesmo tempo de exposição pode responder de forma diferente de acordo com o tipo de AL que está sendo utilizado. Além disso, o efeito antioxidante do AL nanoencapsulado parece ser mais efetivo quando utilizado a longo prazo, sugerindo que a forma nanoencapsulada libera o antioxidante em forma mais lenta. Os resultados também indicam que a composição da nanocápsulas deve ser levada em consideração, uma vez que foi observado um efeito antioxidante significativo nos tratamentos que continham apenas a nanocápsulas, sem o AL. Sugere-se que este efeito ocorra devido à produção endógena do próprio antioxidante em questão, favorecida pela composição da própria nanocápsula, que possui ácido octanóico, substrato para a síntese de AL. Também se observou um efeito pró-oxidante em alguns tratamentos onde foi utilizada esta formulação, sugerindo que alguns componentes da nanocápsula, como por exemplo, o surfactante que é utilizado para estabilizar a suspensão, possam aumentar a suscetibilidade dos órgãos ao estresse oxidativo.

The timing and interval of mate encounter affects investment during mating

The benefits obtained from mating are usually condition-dependent, favouring the evolution of flexible investment during copulation, for instance, in terms of invested time, energy, or sperm. Flexible investment strategies are predicted to depend on the likelihood of acquiring alternative mates and therefore they should depend on the timing of mate encounter. However, scarce experimental evidence for this hypothesis exists. Here we manipulated the time delay until first mating and the interval between first and second mating in the polygynandrous common lizard, Zootoca vivipara. We determined treatment effects on fertilisation success and copulation duration, the latter being a proxy for investment in mating and for quantity of transferred sperm. The duration of the second copulation decreased with increasing inter-mating interval and depended on the fertilisation success of first mates. The former provides evidence for time-dependent investment strategies, most likely resulting from the progression of the female's reproductive cycle. Fertilisation success of first mates increased with increasing inter-mating interval and was higher when females were closer to ovulation, showing that flexible investment strategies significantly affected male reproductive success. This points to fertilisation assurance, which may mitigate negative effects of low population density on reproductive success, e.g. Allee effects.

Time perception in mild cognitive impairment: Interval length and subjective passage of time

Objectives: Patients with mild cognitive impairment (MCI) may have difficulties in time perception, which in turn might contribute to some of their symptoms, especially memory deficits. The aim of this study was to evaluate perception of interval length and subjective passage of time in MCI patients as compared to healthy controls. Methods: Fifty-five MCI patients and 57 healthy controls underwent an experimental protocol for time perception on interval length, a questionnaire for the subjective passage of time and a neuropsychological evaluation. Results: MCI patients presented no changes in the perception of interval length. However, for MCI patients, time seemed to pass more slowly than it did for controls. This experience was significantly correlated with memory deficits but not with performance in executive tests, nor with complaints of depression or anxiety. Conclusions: Memory deficits do not affect the perception of interval length, but are associated with alterations in the subjective passage of time.

Influências genéticas nas características da carcaça e carne em bovinos

Tese de Doutoramento em Ciências Veterinárias - Especialidade de Produção Animal

Child injury in Brisbane South 1985-91: Implications for future injury surveillance

Specific childhood injury types are ranked by occurrence rate for mortality, hospital admission and emergency department attendance. Cases are drawn from the resident population of Brisbane South, 0-13 years of age, for the period 1 July 1985 to 30 June 1991. A total of 47,244 injuries, 7056 admissions and 99 deaths were analysed. The overall mortality rate was 12.6/100,000 per year (95% confidence interval (CI), 10.2-15.3), the overall admission rate was 911/100,000 per year (95% CI, 890-932) and the overall hospital attendance rate was 6013/100,000 per year (95% CI, 5958-6067). A fall was the most frequent injury mechanism for admissions and 65% of attendances involved injury in the child's own home. The surveillance data establish regional variation for childhood injury risk within Australia and identify an unexplained downward trend in head injury that requires further investigation. The future development of injury surveillance in Australia requires simplified coding which can be integrated into new computerized patient management information systems. Article in Journal of Paediatrics and Child Health 30(2):114-22 · May 1994

Contrast and lustre:a model that accounts for eleven different forms of contrast discrimination in binocular vision

Our goal here is a more complete understanding of how information about luminance contrast is encoded and used by the binocular visual system. In two-interval forced-choice experiments we assessed observers' ability to discriminate changes in contrast that could be an increase or decrease of contrast in one or both eyes, or an increase in one eye coupled with a decrease in the other (termed IncDec). The base or pedestal contrasts were either in-phase or out-of-phase in the two eyes. The opposed changes in the IncDec condition did not cancel each other out, implying that along with binocular summation, information is also available from mechanisms that do not sum the two eyes' inputs. These might be monocular mechanisms. With a binocular pedestal, monocular increments of contrast were much easier to see than monocular decrements. These findings suggest that there are separate binocular (B) and monocular (L,R) channels, but only the largest of the three responses, max(L,B,R), is available to perception and decision. Results from contrast discrimination and contrast matching tasks were described very accurately by this model. Stimuli, data, and model responses can all be visualized in a common binocular contrast space, allowing a more direct comparison between models and data. Some results with out-of-phase pedestals were not accounted for by the max model of contrast coding, but were well explained by an extended model in which gratings of opposite polarity create the sensation of lustre. Observers can discriminate changes in lustre alongside changes in contrast.

Coding Manuals

The VRAG-R is designed to assess the likelihood of violent or sexual reoffending among male offenders. The data set comprises demographic, criminal history, psychological assessment, and psychiatric information about the offenders gathered from institutional files together with post-release recidivism information. The VRAG-R is a twelve-item actuarial instrument and the scores on these items form part of the data set. Because one of the goals of the VRAG-R development project was to compare the VRAG-R to the VRAG, subjects' VRAG scores are included in this data set. Access to the VRAG-R dataset is restricted. Contact Data Services, Queen's University Library (academic.services@queensu.ca) for access.

General ledger account coding

The Supplier-Relationship Management system is used by SC Department of Motor Vehicles personnel to requisition most purchases of services and materials for DMV purposes. General ledger account codes are first assigned by the shopping cart preparer. Administrative departments such as procurement, payables and budget regularly correct general ledger codes during the purchasing cycle to ensure proper reporting. DMV goals are consistent with accurate reporting of expenditures by general ledger account code. Incorrect reporting would be contrary to DMV' s vision of promoting effective and efficient business processes. Journal entries increased from 34 to 66 in FY2014 and FY20152 ; with a notable amount correcting the general ledger code. This project examines the assignment or correction of general ledger account codes for DMV's planned purchases for the purpose of process improvement.

The role of non-coding RNA in the development of pulmonary arterial hypertension

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.

Transition matrices characterizing a certain totally discontinuous map of the interval

We study a totally discontinuous interval map defined in [0,1] which is associated to a deformation of the shift map on two symbols 0−1. We define a sequence of transition matrices which characterizes the effect of the interval map on a family of partitions of the interval [0,1]. Recursive algorithms that build the sequence of matrices and their left and right eigenvectors are deduced. Moreover, we compute the Artin zeta function for the interval map.

Role of non-coding RNA alterations in melanoma

Cutaneous melanoma (CM) is a potentially lethal form of skin cancer and its most important histopathologic factor for staging is Breslow thickness (BT). Its correct determination is fundamental for pathologists. A deeper understanding of the molecular processes guiding CM pathogenesis could improve diagnosis, treatment and prognosis. MicroRNAs (miRNAs) play a key role in CM biology. The firs aim was to investigate miRNA expression in reference to BT assessment. We found that the combined miRNA expression of miR-21-5p and miR-146a-5p above or below 1.5 was significantly associated with overall survival and successfully identified all superficially spreading melanoma (SSM) patients with relapsing suggesting that the combined assessment of these miRNAs expression could aid in SSM staging. Secondly, we focus on multiple primary melanoma (MPM) patients, which develop multiple primary melanomas in their lifetime, and represent a model of high-risk CM occurrence. We explored the miRNome of single CM and MPM: CM and MPM present several dysregulated miRNAs, including key miRNAs involved in epithelial-mesenchymal transition. A different miRNA profile was observed between 1st and 2nd melanoma from the same patient. MiRNA target analysis revealed a more differentiated and less invasive status of MPMs compared to CMs. This characterization of the miRNA regulatory network of MPMs highlights molecular features differentiating this subtype from CM. Recently, NGS experiments revealed the existence of miRNA variants (isomiRs) with different length and sequence. We identified a shorter 3’isoform as tenfold over-represented compared to the canonical form of miR-125a-5p. Target analysis revealed that miRNA shortening could change the pattern of target gene regulation. Finally, we study miRNA and isomiR dysregulation in benign nevi (BN) and CM and in CM and melanoma metastasis. The reported non-random dysregulation of specific isomiRs contributes to the understanding of the complex melanoma pathogenesis and serves as the basis for further functional studies.

Characterisation of RNase Y in H. pylori: a non-essential enzyme involved in the processing of cag-PAI non coding RNA 1 (CncR1) sRNA

The importance of Helicobacter pylori as a human pathogen is underlined by the plethora of diseases it is responsible for. The capacity of H. pylori to adapt to the restricted host-associated environment andto evade the host immune response largely depends on a streamlined signalling network. The peculiar H. pylori small genome size combined with its paucity of transcriptional regulators highlights the relevance of post-transcriptional regulatory mechanisms as small non-coding RNAs (sRNAs). However, among the 8 RNases represented in H. pylori genome, a regulator guiding sRNAs metabolism is still not well studied. We investigated for the first time the physiological role in H. pylori G27 strain of the RNase Y enzyme. In the first line of research we provide a comprehensive characterization of the RNase Y activity by analysing its genomic organization and the factors that orchestrate its expression. Then, based on bioinformatic prediction models, we depict the most relevant determinants of RNase Y function, demonstrating a correlation of both structure and domain organization with orthologues represented in Gram-positive bacteria. To unveil the post-transcriptional regulatory effect exerted by the RNase Y, we compared the transcriptome of an RNase Y knock-out mutant to the parental wild type strain by RNA-seq approach. In the second line of research we characterized the activity of this single strand specific endoribonuclease on cag-PAI non coding RNA 1 (CncR1) sRNA. We found that deletion or inactivation of RNase Y led to the accumulation of a 3’-extended CncR1 (CncR1-L) transcript over time. Moreover, beneath its increased half-life, CncR1-L resembled a CncR1 inactive phenotype. Finally, we focused on the characterization of the in vivo interactome of CncR1. We set up a preliminary MS2-affinity purification coupled with RNA-sequencing (MAPS) approach and we evaluated the enrichment of specific targets, demonstrating the suitability of the technique in the H. pylori G27 strain.