Influenza virus susceptibility to antiviral drugs : drug susceptibility profiling, whole-genome mutational landscape and selective pressure footprints

Tese de doutoramento, Ciências e Tecnologias da Saúde (Microbiologia), Universidade de Lisboa, Faculdade de Medicina, 2018Antivirals play an important and decisive role in the clinical management of influenza and in the underlying reduction of related morbidity and mortality. The emergence of antiviral resistance, and particularly of transmissible resistance, poses a serious threat to public health as it could render influenza antivirals useless against circulating viruses. This is even more worrying when considering the current paucity of alternative antiviral therapy choices. This PhD research project aimed at disclosing the susceptibility of human influenza viruses circulating in Portugal to nationally approved antivirals, and at improving the knowledge on the evolutionary dynamics underlying the emergence and/or spread of influenza variants resistant or with decreased susceptibility to neuraminidase inhibitor (NAI) drugs. To this end, the project focused on three main areas: antiviral susceptibility testing; whole-genome sequencing; and selective pressure (SP) footprints on human influenza neuraminidase (NA)(NAI target). Antiviral susceptibility testing was performed on human influenza viruses circulating in both community and hospital settings from 2004/2005 to 2012/2013, after establishing a technological platform for comprehensive evaluation of virus susceptibility to M2 protein inhibitors and the NAIs oseltamivir (OS) and zanamivir (ZA) (objective 1). Important findings were made on: the circulation of drug-resistant A(H3N2) (M2 inhibitors) and former seasonal (H1N1) (OS) viruses; the cut-off for potentially clinically relevant sub-populations of drug-resistant virus; a potential novel amino acid substitution conferring slightly decreased susceptibility to ZA (N2 NA) and a novel source for a variant with decreased susceptibility; and, the virus type or subtype specificity of two amino acid substitutions conferring reduced susceptibility to the drug. Overall susceptibility data contributed at a better understanding of the relationship between virus NAI susceptibility phenotype and genotype and of the natural variations in the in vitro NAI susceptibility of circulating viruses over time. The emergence of new drift variants (former seasonal A(H1N1), A(H3N2)), the co-circulation of distinct virus lineages (influenza B) and the increase in OS drug use (A(H1N1)pdm09) were found to potentially play a role in this latter. Influenza viruses exhibiting resistance or decreased susceptibility to OS and/or ZA were further evaluated through whole-genome sequencing to identify and characterize the amino acid substitutions specific of their genome (objective 2). No genetic support was found for the fitter NA H275Y OS resistant former seasonal A(H1N1) viruses, but mutations known to or that based on its structural location or functional impact may play a role in the overall viral fitness, were identified in the genome of single or few viruses resistant or with decreased susceptibility to the drug. Large datasets of full-length NA gene sequences of worldwide circulating viruses were created to estimate the global and site-specific SP acting on influenza NA, particularly on the sites associated with NAI resistance or reduced susceptibility and/or contacting with the drug (objective 3a). Further temporal splitting of NA gene sequences allowed to investigate for the first time the impact of NAI introduction into clinic (1999) and/or its increased use during 2009 A(H1N1) pandemic on the SP acting on NA (objective 3b). Major findings include: the potential role of positive SP (PSP) in the low-level and locally variable spread of NA H275Y OS-resistant A(H1N1)pdm09 viruses that has been observed in the community; a potential risk of spread of a synergistic drug-resistant (H275Y/S247N) or a RI (S247G) variant in A(H1N1)pdm09 subtype and a RI variant (A395E) in B/Victoria lineage (positive diversifying selection); and the potential lack of impact of both NAI introduction into clinic and its increased use during 2009 A(H1N1) pandemic on the global and site-specific SP acting on influenza NA, with the single exception of site 154 of B/YAM-lineage NA (framework active site residue). Overall mapping of site-specific SP across the different NA subtypes or lineages allowed for further identify 7 potential new regions for drug targeting. This project marked the beginning of influenza antiviral susceptibility testing and monitoring activities in Portugal. It not only established the technological capacity and capability required to perform such activities but also generated comprehensive information on the susceptibility of circulating human influenza viruses, essential to contribute to both global and European influenza surveillance on antiviral susceptibility. The project also contributed at finding potential determinants of viral fitness in the genome of influenza virus resistant or with decreased susceptibility to NAIs, based on its location onto the protein structure; and at elucidating the role of PSP in the evolutionary pathways to NAI resistance or reduced susceptibility.Fundação Calouste Gulbenkian, projetos FCG 76676 e SDH49; Administração Central do Sistema de Saúde, I.P. (ACSS), projeto SDH4

Green synthesis of 2-Oxazoline-based polymers with antimicrobial activity using scCO2

Using a green methodology, 17 different poly(2-oxazolines) were synthesized starting from four different oxazoline monomers. The polymerization reactions were conducted in supercritical carbon dioxide under a cationic ring-opening polymerization (CROP) mechanism using boron trifluoride diethyl etherate as the catalyst. The obtained living polymers were then end-capped with different types of amines, in order to confer them antimicrobial activity. For comparison, four polyoxazolines were end-capped with water, and by their hydrolysis the linear poly(ethyleneimine) (LPEI) was also produced. After functionalization the obtained polymers were isolated, purified and characterized by standard techniques (FT-IR, NMR, MALDI-TOF and GPC). The synthesized poly(2-oxazolines) revealed an unusual intrinsic blue photoluminescence. High concentration of carbonyl groups in the polymer backbone is appointed as a key structural factor for the presence of fluorescence and enlarges polyoxazolines’ potential applications. Microbiological assays were also performed in order to evaluate their antimicrobial profile against gram-positive Staphylococcus aureus NCTC8325-4 and gram-negative Escherichia coli AB1157 strains, two well known and difficult to control pathogens. The minimum inhibitory concentrations (MIC)s and killing rates of three synthesized polymers against both strains were determined. The end-capping with N,N-dimethyldodecylamine of living poly(2- methyl-2-oxazoline) and poly(bisoxazoline) led to materials with higher MIC values but fast killing rates (less than 5 minutes to achieve 100% killing for both bacterial species) than LPEI, a polymer which had a lower MIC value, but took a longer time to kill both E.coli and S.aureus cells. LPEI achieved 100% killing after 45 minutes in contact with E. coli and after 4 hours in contact with S.aureus. Such huge differences in the biocidal behavior of the different polymers can possibly underlie different mechanisms of action. In the future, studies to elucidate the obtained data will be performed to better understand the killing mechanisms of the polymers through the use of microbial cell biology techniques

Bank runs : suspension of convertibility and deposit insurance

Dissertação de mestrado em EconomiaFrom the very beginning, the banking system has been vulnerable to bank runs. A bank run occurs when a large number of depositors attempt to withdraw their funds simultaneously because they are afraid that the bank will not be able to repay the deposits in full and on time. The view that financial crises are costly may be based primarily on the Wall Street crash of the early 1930s. This was one of the most extreme crises which had significant impact on the banking system of the United States. History does not end and the long lines outside Northern Rock branches in Britain, on September 2007, as well as the bankruptcy of Lemman Brother Holdings Inc., in 2008, brought back memories from the worldwide monetary history. The financial crisis of 2007 and 2008 is reminiscent of a bank run and it seems obvious that research on bank runs and financial instability has taken on a new urgency. The model presented in this master thesis is consistent both with the sunspots and business cycle view of the origins of banking panics. The main motivation of this work is to compare, from a welfare point of view, two different banking regulations that try to avoid or mitigate the effects of bank runs - suspension of convertibility and government deposit insurance. With the first mechanism, payments are suspended at a certain level and with the second, deposits are always guaranteed when the bank fails. We show that if the level of risk aversion is high enough, suspension of convertibility dominates deposit insurance, however we also show, numerically, that the relation is not monotone for low values of risk aversion.Desde o seu início que o sistema financeiro enfrenta o problema de corridas e pânicos bancários. Diz-se que há uma corrida aos bancos quando, simultaneamente, um número elevado de indivíduos tenta retirar os seus depósitos do banco, devido ao receio de que este não seja capaz de cumprir os seus compromissos de liquidez. As crises financeiras são, em geral, caracterizadas por períodos de grande instabilidade e acarretam graves custos para a sociedade. Um exemplo presente na memória de todos nós é o da Grande Depressão dos anos 30 que teve lugar nos Estados Unidos da América. Recentemente, as longas filas à entrada do banco britânico Northern Rock em Setembro de 2007, assim como, a falência do banco de investimento Lemman Brother Holdings Inc., em 2008, desencadearam uma nova vaga de falências bancárias. O modelo presente nesta tese de mestrado considera que corridas e pânicos bancários ocorrem devido a variáveis não correlacionadas com fundamentos económicos, como por exemplo sunspots, e devido a informação existente na economia sobre o retorno futuro dos activos bancários. Este estudo pretende avaliar, do ponto de vista do bem-estar, dois contractos que atenuam e previnem os efeitos de corridas e pânicos bancários – suspensão de convertibilidade e seguros de depósitos. No quadro do nosso modelo demonstramos que quando o nível de aversão ao risco é elevado, a suspensão de convertibilidade é preferível ao seguro de depósito. No entanto, demonstramos também que para níveis de aversão ao risco suficientemente baixos, esta relação não é monótona

Private equity challenge - assessment of Calavo growers´ suitability as an Lbo target

The following work project was developed by a group of Masters in Finance students and consists on the development of an Investment Committee Paper on Calavo Growers, an American fresh fruit and health yon-the-go fresh food it ems company, in an academical context. The company operates through three segments: Fresh Products–which deals with fresh fruit, mostly avocados; Calavo Foods–that transforms avocados in to processed products, such as guac a mole; and RFG–which hand lesa portfolio of fresh and healthy foods, including fresh-cut fruits. In this segment, the value creation strategy and business plan are presented, which intend to create value for investors during a 7-yeart imehorizon through organic and inorganic growth, consisting of revenue growth, operational improvements, add-on acquisitions and international expansion. This plan is supported by extensive research on the company and on the markets in which it operates

Harm Reduction for Corporations

When corporations set out to do good for the environment and society, they usually do so under the banner of corporate social responsibility. This approach has become commonplace among the public, in business schools, and in issues of academic journals. However, corporate social responsibility has a few flaws. First, some corporations may never be socially responsible because of their core business. Second, there are corporations who pursue corporate social responsibility campaigns that are highly removed from their core business and these campaigns seem inappropriate. Finally, there are corporations for whom corporate social responsibility is unattainable because it requires too many resources. This thesis offers another tool for social responsibility: harm reduction for corporations. My harm reduction framework fills the gaps left by corporate social responsibility and encourages every corporation to set and meet goals that reduce the harms that they do to the environment and society. My harm reduction framework offers a low threshold for engagement, where corporations can reduce harms for any reason they choose and to any extent that they choose. This low threshold approach makes room for every corporation to contribute to reducing harms done to the environment and society

Responses presented by adult patients with COVID-19, based on the formulated nursing diagnoses: a scoping review

(1) Background: this review aims to identify the human responses exhibited by adult patients with COVID-19, by listing the corresponding nursing diagnoses. Nursing diagnosis it's a clinical analysis of human responses to a person, family, or community. Therefore, it is possible to state that nursing diagnoses represent human responses. (2) Methods: a scoping review was conducted following recommendations provided by the Joanna Briggs Institute (JBI) and the research was carried out between December 2020 and 15 January, 2021, via CINAHL Complete, Complementary Index, MEDLINE, Science Direct, Academic Search Complete, Science Citation Index, Directory of Open Access Journals, Scopus, Social Sciences Citation Index, Business Source Complete, eBook Index (by B-on), and the Cochrane Database of Systematic Reviews (by Cochrane Library). (3) Results: with respect to studies using the NANDA-I taxonomy, the findings have shown that "impaired gas exchange" was the most highlighted nursing diagnosis. ICNP taxonomy, the relevant nursing diagnosis is "cough present". (4) Conclusions: concurrently, as suggested by the human responses documented in this review, throughout the pandemic, the requirements for adequate care provision have been constantly updated, to improve the quality of life of those patients, as much as possible.info:eu-repo/semantics/publishedVersio

Hemin ameliorates the inflammatory activity in the inflammatory bowel disease: a non-clinical study in rodents

FCT_UIDB/05608/2020. FCT_UIDP/05608/2020.Background: Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract. Currently, there is no cure, and pharmacological treatment aims to induce and maintain remission in patients, so it is essential to investigate new possible treatments. Hemin is a heme-oxygenase inducer that can confer anti-inflammatory, cytoprotective, and antiapoptotic effects; therefore, it can be considered an asset for different gastrointestinal pathologies, namely for IBD. Aim: This experiment aims to evaluate the efficacy and safety of hemin, in a chronic 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis model in rodents. Methods: The induction of chronic colitis consisted of five weekly intrarectal administrations of 1% TNBS. Then, the mice were treated daily with 5 mg/kg/day or 10 mg/kg/day of hemin, through intraperitoneal injections, for 14 days. Results: Hemin demonstrated an anti-inflammatory effect through the reduction in tumor necrosis factor (TNF)-α levels, fecal calprotectin, and fecal hemoglobin. It was also found to be safe in terms of extraintestinal manifestations since hemin did not promote renal and/or hepatic changes. Conclusions: Hemin could become an interesting tool for new possible pharmacological approaches in the management of IBD.info:eu-repo/semantics/publishedVersio

Nesting material enrichment reduces severity of overgrooming-related self-injury in individually housed rats

Individual or singly-housing laboratory rats is common in many animal facilities, but has an adverse impact on the welfare of this social species. It has previously been shown that a small proportion of individually housed mice (∼5%) engage in pathological overgrooming behaviour, but this has not been assessed in rats. We performed an observational study to determine the prevalence of overgrooming-related self-injury and whether providing nesting material enrichment throughout an animal’s life would affect the prevalence or severity of overgrooming-related self-injury. Due to protocol differences between projects in our behavioural neuroscience lab, unenriched rats received a nylabone and a shelter (n = 167), while baseline-enriched rats received a nylabone, shelter and shredded paper nesting material throughout experiments (n = 238). Unenriched rats received nesting material enrichment after the onset of overgrooming-related self-injury. Over 18 months, rats were monitored by their experimenters on a daily basis (5–7 days/week over 2–3 months/project) and any cases of overgrooming-related self-injury were recorded. Replicating the findings of previous studies in mice, we observed 20 cases of overgrooming-related self-injury (∼5%) with no difference in prevalence between rats on the basis of supplier, cage position, experimental procedure (behavioural only or involving surgical procedures), reinforcer (ethanol or sugar) or level of baseline-enrichment. While there was no difference in onset severity between rats that were unenriched at baseline and baseline-enriched rats, baseline-enriched rats had lower self-injury severity scores at one-, two- and four-week follow-ups. These results suggest that nesting material enrichment provided throughout an animal’s life may reduce overgrooming-related self-injury