1,720,990 research outputs found
The Double Face of Nucleic Acids
Full text linkNucleic acids have been the object of intense and thorough observations for more than a century, leading to the understanding of their structure and function. Their role as genetic information carriers is well established but there are evidences that they are also involved in a series of other less known processes. RNA for example is a single strand biopolymer that, dependently from the primary sequence, can assume complex three-dimensional foldings that allow binding to diverse target molecules or the involvement in catalysis. Being closely related to several cellular events, they are also useful therapeutic targets.
This work is essentially divided in two parts, aimed at studying two different and complementary aspects of nucleic acids. While the first project goes into the understanding of the “RNA world” and of the applicability of extraordinary RNA conformational variability in a diagnostic and therapeutic system, the second topic is addressed to study the ability of two compounds to interfere with DNA integration, a genetic process that allows the viral DNA to be inserted into the host genome. Therefore nucleic acids demonstrate their “double face” meaning their ability to be leading actors in diagnosis and therapy but also indispensable supporting actors, being the main targets for several drugs.
The first part of the PhD program was focused on the use of SELEX to discover a new diagnostic or therapeutic tool. A protocol for the selection of RNA aptamers binding folic acid was developed. In particular, after 12 cycles of SELEX using affinity chromatography, it was possible to select an enriched pool of single stranded RNA oligonucleotides with affinity for the target. Subcloning and sequencing of the above-mentioned pool allowed the subsequent analysis of the oligonucleotide three-dimensional structure and the determination of the minimum binding sequence to folic acid. Computer tools and RNase protection assay were associated in the experimental protocol to obtain the most probable outcome. A clear region of ligand interaction on RNA was identified; this minimum sequence was chemically synthesized and Isothermal Titration Calorimetry (ITC) used for the determination of the binding constant of folic acid, that resulted in the nanomolar range. The approval from FDA and EMEA of Macugen®, a pegylated aptamer selective for the binding to VEGF and used in the treatment of age-related macular degeneration, is a proof that SELEX can be used to develop good drugs and diagnostics. RNA aptamers against folic acid could now find their way for diagnostic or therapeutic application. Therefore further studies must be addressed to allow their applicability to the desired purpose.
The second part of the PhD program was carried out in the Laboratory of Molecular Pharmacology of the National Cancer Institute (National Institute of Health - Bethesda -Maryland - USA) and it takes into consideration DNA integration as target of anti-HIV drugs. HIV-1 integrase is one of the viral enzymes encoded from the POL gene, that catalyses the insertion of the viral DNA into host chromosomes. Using synthetic oligonucleotides mimicking the terminal portion of the U5 viral LTR, it was analyzed the effect of two important drugs (raltegravir, the first integrase inhibitor approved by FDA last year, and elvitegravir, in advanced stage of human clinical trials) on the recombinant wild type integrase and on a series of resistant mutants. The study was addressed to compare raltegravir and elvitegravir on the same in vitro system, with the aim to understand how different aminoacidic point mutations of the protein sequence are responsible in the onset of drug resistance.Dal giorno della loro scoperta più di un secolo fa, gli acidi nucleici sono stati analizzati e studiati approfonditamente in tutti gli aspetti, cercando di svelare gli ancora numerosi segreti nascosti nella loro struttura per comprenderne appieno le funzioni. Il ruolo più assodato ed essi associato è sicuramente quello di trasportatori dell’informazione genetica ma numerose evidenze sperimentali confermano il coinvolgimento in una serie di altri processi meno noti. Basta pensare a come l’RNA, grazie alle complesse conformazioni tridimensionali che può assumere dipendentemente dalla sua sequenza primaria, risulti coinvolto nel legame a svariate molecole e risulti ancor più straordinariamente responsabile di processi catalitici. Essendo quindi correlati a numerosi eventi cellulari, gli acidi nucleici sono utili bersagli terapeutici.
Il presente lavoro è suddiviso essenzialmente in due parti ed è finalizzato alla comprensione di due aspetti differenti ma complementari legati agli acidi nucleici. Il primo progetto infatti si pone l’obiettivo di conoscere il vasto “mondo dell’RNA” e di applicare la straordinaria variabilità conformazionale di questo biopolimero ad un sistema terapeutico o diagnostico. La seconda parte della ricerca è invece indirizzata all’analisi dell’effetto di composti sul processo di integrazione del DNA, un meccanismo genetico che permette al DNA virale di essere correttamente inserito nel genoma dell’ospite. Gli acidi nucleici dimostrano quindi la loro “doppia faccia” intesa come la loro capacità di essere i protagonisti in sistemi di diagnosi o di terapia ma anche indispensabili attori co-protagonisti, essendo essi stessi i bersagli principali di numerosi farmaci.
La prima metà del ciclo di dottorato di ricerca si è concentrata sull’uso della tecnologia SELEX. Inizialmente si è sviluppato un protocollo per la selezione di aptameri contro l’acido folico. Un totale di 12 cicli di SELEX mediante cromatografia d’affinità, hanno condotto alla selezione di un pool di molecole di RNA a singolo filamento, aventi un’elevata predisposizione a legare il target d’interesse. Il subclonaggio e il sequenziamento del suddetto insieme di molecole ha poi permesso di approfondire l’analisi delle caratteristiche tridimensionali degli oligonucleotidi e della minima sequenza responsabile del legame all’acido folico. A questo scopo, è stata condotta un’analisi informatizzata elementare ed è stato sviluppato un protocollo per l’RNase protection assay. L’associazione dei risultati ottenuti con le due diverse metodiche ha permesso di definire una chiara regione di interazione tra RNA e ligando. Questa minima sequenza è stata quindi sintetizzata chimicamente e sottoposta, mediante Isothermal Titration Calorimetry (ITC), alla determinazione della costante di legame, che è risultata essere nell’intervallo nanomolare. L’approvazione da parte di EMEA e FDA di Macugen®, un aptamero peghilato selettivo per il legame a VEGF e usato nel trattamento della degenerazione maculare senile, è una prova di come la SELEX possa essere un utile strumento per lo sviluppo di farmaci e diagnostici. Gli aptameri ad RNA contro l’acido folico selezionati nel presente lavoro possono ora seguire la via dello sviluppo per l’applicazione desiderata e ulteriori studi saranno condotti a questo scopo.
La seconda metà del ciclo di dottorato di ricerca è stata invece svolta nel Laboratorio di Farmacologia Molecolare del National Cancer Institute (National Institute of Health - Bethesda - Maryland - USA) e ha preso in considerazione il processo di integrazione del DNA come bersaglio di farmaci anti-HIV. L’integrasi dell’HIV-1 è un enzima virale codificato dal gene POL e catalizza l’inserzione del DNA virale nei cromosomi della cellula ospite. Oligonucleotidi sintetici, con sequenza corrispondente alla porzione U5 terminale della LTR virale, sono stati utilizzati allo scopo di analizzare l’effetto di due importanti farmaci (raltegravir, il primo farmaco inibitore dell’integrasi approvato dall’FDA lo scorso anno, e elvitegravir, un composto in stadio avanzato di ricerca clinica) sull’enzima nativo ricombinante e su una serie di mutanti resistenti ai farmaci in questione. Lo studio è stato essenzialmente indirizzato a confrontare raltegravir e elvitegravir sullo stesso sistema in vitro, cercando di capire come differenti mutazioni aminoacidiche della proteina siano responsabili dell’insorgenza di resistenza al trattamento
Immunofluorescence microscopy of G-quadruplexes and R-loops
No full textA large variety of non-B secondary structures can be formed between DNA and RNA. In this chapter, we focus on G-quadruplexes (G4) and R-loops, which can have a close structural interplay. In recent years, increasing evidence pointed to the fact that they can strongly influence each other in vivo, both having physiological and pathological roles in normal and cancer cells. Here, we detail specific and accurate methods for purification of BG4 and S9.6 antibodies, and their subsequent use in immunofluorescence microscopy, enabling single-cell analysis of extent and localization of G4s and R-loops
Stimulation of cGAS-STING pathway as a challenge in the treatment of small cell lung cancer: a feasible strategy?
Full text linkLung cancer has a significant incidence among the population and, unfortunately, has an unfavourable prognosis in most cases. The World Health Organization (WHO) classifies lung tumours into two subtypes based on their phenotype: the Non-Small Cell Lung Cancer (NSCLC) and the Small Cell Lung Cancer (SCLC). SCLC treatment, despite advances in chemotherapy and radiotherapy, is often unsuccessful for cancer recurrence highlighting the need to develop novel therapeutic strategies. In this review, we describe the genetic landscape and tumour microenvironment that characterize the pathological processes of SCLC and how they are responsible for tumour immune evasion. The immunosuppressive mechanisms engaged in SCLC are critical factors to understand the failure of immunotherapy in SCLC and, conversely, suggest that new signalling pathways, such as cGAS/STING, should be investigated as possible targets to stimulate an innate immune response in this subtype of lung cancer. The full comprehension of the innate immunity of cancer cells is thus crucial to open new challenges for successful immunotherapy in treating SCLC and improving patient outcomes
Anthracyclines as Topoisomerase II Poisons: From Early Studies to New Perspectives
Full text linkMammalian DNA topoisomerases II are targets of anticancer anthracyclines that act by stabilizing enzyme-DNA complexes wherein DNA strands are cut and covalently linked to the protein. This molecular mechanism is the molecular basis of anthracycline anticancer activity as well as the toxic effects such as cardiomyopathy and induction of secondary cancers. Even though anthracyclines have been used in the clinic for more than 50 years for solid and blood cancers, the search of breakthrough analogs has substantially failed. The recent developments of personalized medicine, availability of individual genomic information, and immune therapy are expected to change significantly human cancer therapy. Here, we discuss the knowledge of anthracyclines as Topoisomerase II poisons, their molecular and cellular effects and toxicity along with current efforts to improve the therapeutic index. Then, we discuss the contribution of the immune system in the anticancer activity of anthracyclines, and the need to increase our knowledge of molecular mechanisms connecting the drug targets to the immune stimulatory pathways in cancer cells. We propose that the complete definition of the molecular interaction of anthracyclines with the immune system may open up more effective and safer ways to treat patients with these drugs
Discovery of new antisense transcripts induced by camptothecin at CpG island promoters of human cells.
No full textType I DNA Topoisomerases
No full textDNA topoisomerases constitute a large family of enzymes that are essential for all domains of life. Although they share general reaction chemistry and the capacity to govern DNA topology and resolve strand entanglements during fundamental molecular processes, they are characterized by differences in their structural organization, modes of enzymatic catalysis, and biological functions. Moreover, hundreds of compounds interfere with bacterial and/or eukaryotic enzymes, some of which are effective drugs for the treatment of infectious diseases and cancers. Research over the past decade has focused on the biological functions of DNA topoisomerases, and several findings have revealed unexpected roles of type I DNA topoisomerases, a subclass of these enzymes, in regulating gene expression and DNA and chromatin conformations. These new findings highlight that type I topoisomerases are still interesting targets for drug discovery for the treatment of several human diseases, including multidrug-resistant infections and genetic disorders
Characterization of novel antisense HIF-1α transcripts in human cancers
No full textWhole transcriptome analyses have revealed new classes of long ncRNA (lncRNA), the functions of which are however largely unknown. Recently, we showed that the antitumor DNA topoisomerase I (Top1) inhibitor camptothecin (CPT) increases the cellular levels of two antisense lncRNAs at the 5' (5'aHIF-1α) and 3' (3'aHIF-1α) ends of the human HIF-1α gene. To gain insights into their functions, we have here determined structural and functional aspects of the two antisense RNAs in human cancer cell lines and kidney tumor specimen. We found that the antisense transcripts are activated in response to partially different kinds of stress, and that the 5'aHIF-1α has a 5'Cap and a poly(A+) tail, while the 3'aHIF-1α is known to lack both modifications. Cell fractionation experiments showed that 5' and 3' antisense RNAs are nuclear transcripts. Further analyses by RNA-FISH showed that the 5'aHIF-1α accumulates at the perinuclear cellular compartment and co-localizes with the nuclear pore complex Nup62 protein, suggesting a role in nuclear membrane trafficking. Finally, we provide evidence that the studied antisense lncRNAs are expressed in human kidney cancer tissues, highlighting their possible roles in cancer development. Altogether, our findings may suggest a novel function of 5'aHIF-1α in membrane transport that may regulate the cancer-relevant HIF-1α pathway
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
- …
