Biological, molecular, immunological and thermostability characterization of Newcastle disease vaccine strains and isolateds from migratory and industrially raised birds in Brazil

Orientadores: Clarice Weis Arns, Fernando Rosado SpilkiTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: O vírus da doença de Newcastle (VDN) é o agente causador de uma das mais importantes doenças em aves e representa uma ameaça para a indústria avícola. O VDN é um membro da família Paramyxoviridae, subfamília Paramyxovirinae, gênero Avulavirus. São vírus envelopados, não segmentados dotados de genoma RNA de fita simples sentido negativo, associado à doença do trato respiratório, digestivo e nervoso das aves. O Controle da DN se baseia em biossegurança, uso de vacinas e detecção precoce de lotes infectados. No presente estudo, examinamos dez vacinas vivas comercializadas no Brasil quanto à sua estabilidade térmica (ET), virulência e imunogenicidade. Em outra etapa do trabalho, investigou-se a soroprevalência perante o VDN em regiões de produção avícola voltada à exportação ou não de produtos aviários, seguido da detecção do VDN em aves migratórias. Os estudos são complementados pela análise filogenética de VDN isolado de aves comerciais, dos resultados alcançados, cumpre ressaltar: i) os testes de ET revelam elevada estabilidade para as vacinas utilizadas no país, mesmo após dois anos de sua fabricação; ii) o grau de proteção conferido por vacinas vivas contra a DN não depende da virulência residual conforme testes de inoculação intracerebral; iii) a soroprevalência contra VDN em aves nas regiões produtoras e exportadoras, foi de 39,1% e foram isoladas 77 amostras virais, sempre com perfil não-patogênico; iv) sorologia realizada em uma segunda oportunidade detectou-se uma soroprevalência de 28,8% e isolamento de 15 amostras virais que também foram caracterizadas como não-patogênicas; v) observou-se numa soropositividade de 41,7 a 84,3% dependendo da região e isolamento de 12 VDN na região Nordeste, caracterizados como não-patogênicas, indicando que nas áreas não exportadoras circulam vírus de baixa patogenicidade; vi) o genoma dos vírus isolados e das vacinas vivas, atesta que os vírus circulantes em aves comerciais são de provável origem vacinal e pertencem à classe II sendo 71,8% do genótipo II ou La Sota-like e 28,2% do genótipo I ou Ulster-like; vii) por último, a caracterização biológica dos isolados de aves migratórias mostram que há circulação de vírus de baixa e alta patogenicidade em nosso território. Portanto, este conjunto de trabalhos evidencia o status do Brasil como país livre da Doença de Newcastle em aves comerciaisAbstract: Newcastle disease virus (NDV) is the agent that causes one of the most important diseases in birds and represents a threat to industrial aviculture. NDV is a member of the Paramyxoviridae family, Paramyxovirinae subfamily, Avulavirus genus. In the present study, live vaccines commercialized in Brazil were examined in regard to their thermostability (TS), virulence and immunogenicity. In another stage of this work, soroprevalence was investigated, with viral isolation and characterization carried out in poultry production regions focused on production for exportation or domestic commercialization, followed by the detection of the virus in migratory birds. The studies were complemented by phylogenic analysis carried out on the isolates from the commercial birds. From the results obtained, it is important to underscore that i) the tests of TS revealed a high stability of the vaccines used in Brazil, even two years after their manufacture; ii) the level of protection given by live vaccines against NDV does not depend on residual virulence, as confirmed by tests of intracerebral inoculation iii) soroprevalence against NDV in regions with production for exportation was 39.1% and it was possible to isolate 77 samples, always with a non-pathogenic profile; iv) on a second opportunity, a soroprevalence of 28.8% was detected, with isolation of 15 samples, also classified as non-pathogenic; v) in the Brazilian Northeast, a seropositivity of 41.7 to 84.3% was observed depend of region, with isolation of 12 NDVs, characterized as non-pathogenic, indicating that virus of low pathogenicity circulates in those areas that do not export; vi) the genome of the isolated virus and vaccine implies that the circulating virus in commercial birds probably originates from the vaccine and belongs to class II, being 71.8% from genotype II or La Sota-like and 28.2% from genotype I or Ulster-like; vii) finally, the biological characterization of isolates from migratory birds showed that there is circulation of virus of low and high pathogenicity in Brazil. However, this set of studies agrees with the status of Brazil as being a country free of Newcastle disease in commercial birdsDoutoradoCiencias BasicasDoutor em Clínica Médic

Legionella control in the water system of antiquated hospital buildings by shock and continuous hyperchlorination: 5 years experience

To control the presence of Legionella in an old hospital water system, an integrated strategy of water disinfection-filtration was implemented in the university hospital Umberto I in Rome. Due to antiquated buildings, hospital water system design and hospital extension (38 buildings), shock hyperchlorination (sodium hypochlorite, 20-50 ppm of free chlorine at distal points for 1-2 h) followed by continuous hyperchlorination (0.5-1.0 mg/L at distal points) were adopted, and microbiological and chemical monitoring of the water supply was carried out in the university hospital (December 2006-December 2011). Overall, 1308 samples of cold 45°C (17.8%) water were collected, determining residual free chlorine (0.43 ± 0.44 mg/L), pH (7.43 ± 0.29) and trihalomethanes (8.97 ± 18.56 μg/L). Legionella was isolated in 102 (9.8%) out of 1.041 water samples without filters (L. pneumophila sg 1 17.6%, L. pneumophila sg 2-14 28.4%, L. non pneumophila 53.9%), and in none of the 267 samples with filters. Legionella was recovered in 23 buildings out of 38 and 29 samples (28.4%) exceeded 103 cfu/L. When considering the disinfection treatment Legionella was isolated: before shock hyperchlorination (21.1%), 15 days after shock hyperchlorination (7.8%), 30 days after shock hyperchlorination (3.5%), during continuous hyperchlorination (5.5%) and without continuous hyperchlorination (27.3%). Continuous hyperchlorination following the shock treatment achieved >70% reduction of positive samples, whereas no continuous hyperchlorination after shock treatment was more frequently associated to Legionella isolation (OR 6.41; 95% CI 3.10-13.26; p 0.5 < 1.0 mg/L) deteriorated water quality (organoleptic and chemical). However, shock and continuous hyperchlorination remains a valid-term option in old buildings with no water system rational design, managing problems due to hospital extension and absence of a proper hot water recirculation system

Expression of FBXW11 in normal and disease-associated osteogenic cells

The ubiquitin-proteasome system (UPS) plays an important role in maintaining cellular homeostasis by degrading a multitude of key regulatory proteins. FBXW11, also known as b-TrCP2, belongs to the F-box family, which targets the proteins to be degraded by UPS. Transcription factors or proteins associated with cell cycle can be modulated by FBXW11, which may stimulate or inhibit cellular proliferation. Although FBXW11 has been investigated in embryogenesis and cancer, its expression has not been evaluated in osteogenic cells. With the aim to explore FBXW11gene expression modulation in the osteogenic lineage we performed molecular investigations in mesenchymal stem cells (MSCs) and osteogenic cells in normal and pathological conditions. In vitro experiments as well as ex vivo investigations have been performed. In particular, we explored the FBXW11 expression in normal osteogenic cells as well as in cells of cleidocranial dysplasia (CCD) patients or osteosarcoma cells. Our data showed that FBXW11 expression is modulated during osteogenesis and overexpressed in circulating MSCs and in osteogenically stimulated cells of CCD patients. In addition, FBXW11 is post-transcriptionally regulated in osteosarcoma cells leading to increased levels of beta-catenin. In conclusion, our findings show the modulation of FBXW11 in osteogenic lineage and its dysregulation in impaired osteogenic cells

A study of neural networks for natural language processing

L’idée initiale de ce travail de recherche était de fournir aux professeurs, que ce soit de hautes écoles ou non, un outil qui leur permettrait de pouvoir corriger automatiquement les examens de leurs étudiants. Pour cela, le prototype aurait besoin d’une liste des questions, d’une liste des réponses justes et des copies des étudiants. Pour pouvoir mettre une idée pareille en pratique, nous avions comme idée d’utiliser un réseau de neurones artificiels sans que je n’y connaisse rien, hormis le nom. Ce travail va donc être découpé en deux parties : - Recherche, compréhension et choix - Prototypage et synthèse Ainsi, ce travail a donc pour but d’explorer quelques types de réseaux de neurones artificiels existants pour le traitement de texte, en s’appropriant leur fonctionnement et d’en choisir un qui va nous servir pour construire notre réseau de neurones artificiel

Pingu virus : a new picornavirus in penguins from Antarctica

Picornaviridae family comprises single-stranded, positive-sense RNA viruses distributed into forty-seven genera. Picornaviruses have a broad host range and geographic distribution in all continents. In this study, we applied a high-throughput sequencing approach to examine the presence of picornaviruses in penguins from King George Island, Antarctica. We discovered and characterized a novel picornavirus from cloacal swab samples of gentoo penguins (Pygoscelis papua), which we tentatively named Pingu virus. Also, using RT-PCR we detected this virus in 12.9 per cent of cloacal swabs derived from P. papua, but not in samples from adelie penguins (Pygoscelis adeliae) or chinstrap penguins (Pygoscelis antarcticus). Attempts to isolate the virus in a chicken cell line and in embryonated chicken eggs were unsuccessful. Our results expand the viral diversity, host range, and geographical distribution of the Picornaviridae52FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP13/14929-1; 17/13981-0; 12/24150-9; 15/05778-5; 14/20851-8, 16/01414-1; 06/00572-0This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil (Grant no. 13/14929-1, and Scholarships nos. 17/13981-0; 12/24150-9; 15/05778-5; 14/20851-8; 16/01414-1; 06/00572-0). P.R.M. was supported by the Medical Research Council of the UK (Grant no. MC_UU_120/14/9

Evaluation of humoral and cellular response to four vaccines against COVID-19 in different age groups: A longitudinal study

To date there has been limited head-to-head evaluation of immune responses to different types of COVID-19 vaccines. A real-world population-based longitudinal study was designed with the aim to define the magnitude and duration of immunity induced by each of four different COVID-19 vaccines available in Italy at the time of this study. Overall, 2497 individuals were enrolled at time of their first vaccination (T0). Vaccine-specific antibody responses induced over time by Comirnaty, Spikevax, Vaxzevria, Janssen Ad26.COV2.S and heterologous vaccination were compared up to six months after immunization. On a subset of Comirnaty vaccinees, serology data were correlated with the ability to neutralize a reference SARS-CoV-2 B strain, as well as Delta AY.4 and Omicron BA.1. The frequency of SARS-CoV-2-specific CD4+ T cells, CD8+ T cells, and memory B cells induced by the four different vaccines was assessed six months after the immunization. We found that mRNA vaccines are stronger inducer of anti-Spike IgG and B-memory cell responses. Humoral immune responses are lower in frail elderly subjects. Neutralization of the Delta AY.4 and Omicron BA.1 variants is severely impaired, especially in older individuals. Most vaccinees display a vaccine-specific T-cell memory six months after the vaccination. By describing the immunological response during the first phase of COVID-19 vaccination campaign in different cohorts and considering several aspects of the immunological response, this study allowed to collect key information that could facilitate the implementation of effective prevention and control measures against SARS-CoV-2

Role of continuous glucose monitoring in diabetic patients at high cardiovascular risk. an expert-based multidisciplinary delphi consensus

Background: Continuous glucose monitoring (CGM) shows in more detail the glycaemic pattern of diabetic subjects and provides several new parameters (“glucometrics”) to assess patients’ glycaemia and consensually guide treatment. A better control of glucose levels might result in improvement of clinical outcome and reduce disease complications. This study aimed to gather an expert consensus on the clinical and prognostic use of CGM in diabetic patients at high cardiovascular risk or with heart disease. Methods: A list of 22 statements concerning type of patients who can benefit from CGM, prognostic impact of CGM in diabetic patients with heart disease, CGM use during acute cardiovascular events and educational issues of CGM were developed. Using a two-round Delphi methodology, the survey was distributed online to 42 Italian experts (21 diabetologists and 21 cardiologists) who rated their level of agreement with each statement on a 5-point Likert scale. Consensus was predefined as more than 66% of the panel agreeing/disagreeing with any given statement. Results: Forty experts (95%) answered the survey. Every statement achieved a positive consensus. In particular, the panel expressed the feeling that CGM can be prognostically relevant for every diabetic patient (70%) and that is clinically useful also in the management of those with type 2 diabetes not treated with insulin (87.5%). The assessment of time in range (TIR), glycaemic variability (GV) and hypoglycaemic/hyperglycaemic episodes were considered relevant in the management of diabetic patients with heart disease (92.5% for TIR, 95% for GV, 97.5% for time spent in hypoglycaemia) and can improve the prognosis of those with ischaemic heart disease (100% for hypoglycaemia, 90% for hyperglycaemia) or with heart failure (87.5% for hypoglycaemia, 85% for TIR, 87.5% for GV). The experts retained that CGM can be used and can impact the short- and long-term prognosis during an acute cardiovascular event. Lastly, CGM has a recognized educational role for diabetic subjects. Conclusions: According to this Delphi consensus, the clinical and prognostic use of CGM in diabetic patients at high cardiovascular risk is promising and deserves dedicated studies to confirm the experts’ feeling

Albiglutide and cardiovascular outcomes in patients with type 2 diabetes and cardiovascular disease (Harmony Outcomes): a double-blind, randomised placebo-controlled trial

Background: Glucagon-like peptide 1 receptor agonists differ in chemical structure, duration of action, and in their effects on clinical outcomes. The cardiovascular effects of once-weekly albiglutide in type 2 diabetes are unknown. We aimed to determine the safety and efficacy of albiglutide in preventing cardiovascular death, myocardial infarction, or stroke. Methods: We did a double-blind, randomised, placebo-controlled trial in 610 sites across 28 countries. We randomly assigned patients aged 40 years and older with type 2 diabetes and cardiovascular disease (at a 1:1 ratio) to groups that either received a subcutaneous injection of albiglutide (30–50 mg, based on glycaemic response and tolerability) or of a matched volume of placebo once a week, in addition to their standard care. Investigators used an interactive voice or web response system to obtain treatment assignment, and patients and all study investigators were masked to their treatment allocation. We hypothesised that albiglutide would be non-inferior to placebo for the primary outcome of the first occurrence of cardiovascular death, myocardial infarction, or stroke, which was assessed in the intention-to-treat population. If non-inferiority was confirmed by an upper limit of the 95% CI for a hazard ratio of less than 1·30, closed testing for superiority was prespecified. This study is registered with ClinicalTrials.gov, number NCT02465515. Findings: Patients were screened between July 1, 2015, and Nov 24, 2016. 10 793 patients were screened and 9463 participants were enrolled and randomly assigned to groups: 4731 patients were assigned to receive albiglutide and 4732 patients to receive placebo. On Nov 8, 2017, it was determined that 611 primary endpoints and a median follow-up of at least 1·5 years had accrued, and participants returned for a final visit and discontinuation from study treatment; the last patient visit was on March 12, 2018. These 9463 patients, the intention-to-treat population, were evaluated for a median duration of 1·6 years and were assessed for the primary outcome. The primary composite outcome occurred in 338 (7%) of 4731 patients at an incidence rate of 4·6 events per 100 person-years in the albiglutide group and in 428 (9%) of 4732 patients at an incidence rate of 5·9 events per 100 person-years in the placebo group (hazard ratio 0·78, 95% CI 0·68–0·90), which indicated that albiglutide was superior to placebo (p&lt;0·0001 for non-inferiority; p=0·0006 for superiority). The incidence of acute pancreatitis (ten patients in the albiglutide group and seven patients in the placebo group), pancreatic cancer (six patients in the albiglutide group and five patients in the placebo group), medullary thyroid carcinoma (zero patients in both groups), and other serious adverse events did not differ between the two groups. There were three (&lt;1%) deaths in the placebo group that were assessed by investigators, who were masked to study drug assignment, to be treatment-related and two (&lt;1%) deaths in the albiglutide group. Interpretation: In patients with type 2 diabetes and cardiovascular disease, albiglutide was superior to placebo with respect to major adverse cardiovascular events. Evidence-based glucagon-like peptide 1 receptor agonists should therefore be considered as part of a comprehensive strategy to reduce the risk of cardiovascular events in patients with type 2 diabetes. Funding: GlaxoSmithKline

Estudo das manifestações de varias proteinases do Trypanossoma cruzi

Orientador: Julia Keiko SakuradaDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Os estudos sobre as protease do Trypanosoma cruzi foram realizados utilizando-se as formas epimastigota do parasita. Embora a enzima SH-dependente tenha sido detectada nas diferentes formas evolutivas do parasita (RANGEL et alli,1981 b). No entanto existem poucos estudos das enzimas presentes nas formas tripomastigota e amastigota. Neste trabalho, procuramos investigar através da eletroforese em gel de poliacrilamida, a expressão da proteinase nas formas tripomastigota e amastigota com capacidade de degradara gelatina e obtenção de anticorpos monoclonais reati vos com aproteinase de epimastigota. Os resultados obtidos, no presente trabalho mostram que as formas tripomastigota e amastigota expressam o mesmo pOlipeptideo de 48 kDa com atividade enzimática em pH ácido. Entretanto em pH neutro foi detectada uma banda de 35 kDa, e esse polipeptideo não apresentou nenhuma correlação antigênica com aproteinase de epimastigota.O efeito dos anticorpos monoclonais com especificidade para a proteinase na invasão e multiplicação do T. cruzi nas células hospedeiras mostrou um aumento da infectividade, sugerindo que as proteinase do T.cruzi interferem negativamente na penetração e multiplicação do parasita nas células hospedeirasMestradoImunologiaMestre em Ciências Biológica