INTERACTION BETWEEN THE HYPOXIA INDUCIBLE FACTOR 1 ALPHA AND THE HUMAN POLYOMAVIRUS BK: A RISK FACTOR FOR THE DEVELOPMENT OF POLYOMAVIRUS ASSOCIATED NEPHROPATHY

Polyomaviruses are nonenveloped viruses with an icosahedral capsid of about 40-45 nm in diameter. The human polyomavirus BK (BKV) is a member of the Polyomaviridae family detected in 1971 in the urine of an immunosuppressed renal transplant recipient who developed ureteric stenosis. BKV has a worldwide seroprevalence of about 90%. After the primary infection, BKV establishes a life-long latency within the urogenital tract. The severe immunological impairment occurring in transplant kidney recipients leads to BKV reactivation that may result in the polyomavirus associated nephropathy (PVAN). During transplantation, kidney is subjected to hypoxic conditions, driven by the action of Hypoxia Inducible Factor (HIF). It has been proved that HIF isoform-1\u3b1 (HIF-1\u3b1) may interact with several viruses, but till now there are no evidences regarding the interaction between BKV and HIF-1\u3b1. In the present study, we aimed to achieve a better understanding about the relation between BKV infection and hypoxia conditions in kidney cells in case of transplantation. Firstly, 17 kidney paraffin-embedded tissue samples were collected from kidney transplant patients, who developed or not PVAN (PVAN and NOT PVAN group) and from a control group in order to evaluate HIF-1\u3b1 expression levels in vivo. Total RNA was extracted from paraffin embedded tissues, reverse transcripted into cDNA and HIF-1\u3b1 expression was measured by means of a qualitative Real Time PCR. Then, in vitro experiments were conducted using the VERO cell line to evaluate the possible interaction between the BKV promoter and HIF-1\u3b1. Luciferase and Chromatin Immunoprecipitation (ChIP) assays were performed on BKV transfected VERO cells, to verify the interaction between the viral promoter and HIF-1\u3b1. In parallel, to clarify the nature of the interaction between HIF-1\u3b1 and the BKV promoter, the sequences were in silico analyzed using BLASTN 2.2.32+ software to find whether one or more hypoxia response elements (HRE) core sequences were present in the BKV promoters. Finally, the effect of hypoxia on BKV replication was assessed by evaluating BKV replication in BKV infected VERO cells, treated and not treated with the hypoxia mimic Cobalt Chloride (CoCl2). The HIF-1\u3b1 expression level resulted 13.6 folds higher in PVAN tissues than in the control group while no differences were observed between the NOT PVAN tissues and the control group. Luciferase assay showed that the presence of HIF-1\u3b1 stabilized the BKV promoter, increasing its activity from 2-folds to 6-folds (p<0.05) in transfected cells. ChIP assay showed a physical interaction between HIF-1\u3b1 and the BKV promoter. BLASTN analysis showed no match between HRE sequence and the BKV promoter sequences, confirming that no binding sites for HIF-1\u3b1 are present in the viral promoter. Finally, data obtained from BKV infected VERO cells revealed that BKV viral load was 5-fold increased in CoCl2 treated cells compared to not treated infected cells. These data, taken together, are significant to define the role of hypoxic stress in BKV replication after renal transplantation. In particular, it can be concluded that the replication of this opportunistic virus, mainly due to immunosuppressive therapy, is furthermore stimulated and favorite by HIF-1\u3b1 activation, driven by hypoxic conditions during transplantation. The experimental results suggested a hypothetical molecular mechanism underling this thesis, which can be outlined as follows: cellular response to hypoxic environment prevents HIF-1\u3b1 proteasomal degradation, allowing the creation of a HIF-1 active complex. This complex translocates to the nucleus where it binds to the BKV promoter, stimulating the transcription of viral genes and promoting the BKV replication. If confirmed by further experiments, this scenario may have important clinical implications: exposition to hypoxia during renal transplantation process should be considered as a crucial risk factor for the development of PVAN. These findings could be translated into clinical practice, replacing modulation of HIF system with ex-vivo organ preservation technologies, such as extra-corporeal membrane oxygenation, or considering HIF-1\u3b1 as a target to be inhibited, perhaps using RNA interference technology

Permeation of Ternary Mixture Containing H2S, CO2 and CH4 in Aquivion® Perfluorosulfonic Acid (PFSA) Ionomer Membranes

Aquivion (R) E87-12S Perfluorosulfonated acid ionomer material (PFSA) has been studied as a membrane technology for natural gas sweetening from CO2 , H2S due to its interesting chemical and mechanical stability and good separation performance for polar compounds in humid environments. In the present work, permeation of the H2S/CO2/CH4 ternary mixture in this short-side PFSA chain was investigated at pressures up to 10 bar, temperatures up to 50 degrees C, and in a range of relative humidity (RH) from 20% to 90%. The results obtain confirm the strong dependence of Aquivion (R) on water activity and temperature, and its ability to separate gases based on their water solubility without substantial differences between pure and mixed gas experiments. Indeed, even when tested in ternary mixture, the permeation behavior remains similar to that observed for pure components and binary mixtures. In particular, the permeability of H2S is higher than that of CO(2 )and methane CH4, reaching values of 500 Barrer at 50 degrees C and 80% RH, against 450 and 23 Barrer for the other two gases respectively. Additionally, when tested at higher pressures of up to 10 bar under humid conditions, the membrane properties remained largely unchanged, thus confirming the overall stability and durability of Aquivion (R) E87-12S in acid environments

Avaliação in vitro da atividade anti-leishmania da ivermectina e dipropionato de imidocarb sobre Leishmania braziliensis e Leishmania infantum

Orientadora: Profa. Dra. Iara José de Messias-Reason.Coorientador: Prof. Dr. Fabiano Borges Figueiredo e Dr. Gustavo GonçalvesDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências da Saúde, Programa de Pós-Graduação em Medicina Interna e Ciências da Saúde. Defesa : Curitiba, 20/12/2022Inclui referências: p.58-62Resumo: As leishmanioses compõem um grupo de doenças tropicais infecciosas, endêmicas e prioritárias globalmente. Frente à indisponibilidade vacinal, baixa disponibilidade, alto custo e toxicidade de fármacos, o uso de drogas comercializadas para tratamento de outras doenças, que não àquelas tradicionalmente propostas pelo fabricante, é desejável. Neste contexto destacam-se os fármacos Ivermectina (IVM) e Dipropionato de Imidocarb (DPI), cuja potencial atividade anti-leishmania pode ser investigada. O objetivo geral é avaliar a atividade anti-leishmania dos fármacos veterinários IVM e DPI. Os materiais e métodos compreendem experimentação in vitro, realizada por meio de teste de viabilidade das promastigotas (obtido pelo teste MTT) e amastigotas (obtido por leitura no equipamento Operetta®) de L. braziliensis e L. infantum frente aos fármacos, teste de citotoxicidade das linhagens celulares VERO e THP-1 frente aos fármacos. Para as formas amastigotas foi empregada a linhagem THP-1 como células hospedeiras. A curva de crescimento das promastigotas de L. braziliensis e L. infantum frente aos fármacos IVM e DPI foi realizada ao longo de oito dias consecutivos. Os resultados foram analisados com auxílio de testes estatísticos; p<0,05 representou diferença estatística significativa entre o teste e o grupo controle. A análise da viabilidade da IVM frente à promastigota de L. braziliensis e de L. infantum apresentou diferença estatística em todas as concentrações. A viabilidade do DPI frente à promastigota de L. braziliensis apresentou diferença estatística entre as concentrações de 100 e 1,23 ug/mL; e entre 100 a 3,7 ug/mL frente à promastigota de L. infantum. Quanto à citotoxicidade da IVM frente às células VERO, houve diferença em todas as concentrações; quanto ao DPI houve diferença estatística apenas na concentração de 333,33ug/mL. No teste de citotoxicidade da IVM frente às células THP-1 houve diferença estatística nas duas maiores concentrações, 100 e 33,33ug/mL; para DPI a análise mostrou não haver diferença nas diversas concentrações. Na análise da viabilidade das amastigotas de L. braziliensis com IVM, não houve diferença entre as concentrações testadas, diferentemente dos testes com amastigotas de L. infantum, em que houve diferença estatística das concentrações mais altas, 17 e 3,4ug/mL. Na viabilidade das amastigotas de L. braziliensis com DPI, houve diferença estatística nas concentrações 25, 5 e 1ug/mL; para amastigotas de L. infantum houve diferença nas concentrações de 25 e 5 ug/mL. A IVM foi eficaz contra os parasitos, porém tóxica contra as células; o DPI foi eficaz contra os parasitos e apresentou baixa toxicidade para as células. Conclui-se que o reposicionamento de DPI para o tratamento de leishmanioses causadas por L. braziliensis e L. infantum é promissor considerando a atividade anti-leishmania com baixa toxicidade demonstrada in vitro. Resultado esse inédito e promissor para futuros ensaios in vivo.Abstract: Leishmaniases make up a group of infectious, endemic and priority tropical diseases globally. Faced with vaccine unavailability, low availability, high cost and drug toxicity, the use of drugs marketed for the treatment of other diseases, other than those traditionally proposed by the manufacturer, is desirable. In this context, the drugs Ivermectin (IVM) and Imidocarb Dipropionate (DPI) stand out, whose potential anti-leishmania activity can be investigated. The general objective is to evaluate the anti-leishmanial activity of the veterinary drugs IVM and DPI. The materials and methods include in vitro experimentation, carried out by means of a viability test of promastigotes (obtained by the MTT test) and amastigotes (obtained by reading in the Operetta® equipment) of L. braziliensis and L. infantum against drugs, cytotoxicity test of VERO and THP-1 cell lines against drugs. For amastigotes, the THP-1 cell line was used as host cells. The growth curve of L. braziliensis and L. infantum promastigotes against the drugs IVM and DPI was carried out over eight consecutive days. The results were analyzed with the aid of statistical tests; p<0.05 represented a statistically significant difference between the test and the control group. The viability analysis of IVM against the promastigotes of L. braziliensis and L. infantum showed statistical difference in all concentrations. The viability of DPI against the promastigote of L. braziliensis showed statistical difference between the concentrations of 100 and 1.23 ug/mL; and between 100 and 3.7 ug/mL against the promastigotes of L. infantum. As for the cytotoxicity of IVM against VERO cells, there was difference in all concentrations; as for the DPI, there was a statistical difference only in the concentration of 333.33ug/mL. In the IVM cytotoxicity test against THP-1 cells, there was a statistical difference in the two highest concentrations, 100 and 33.33ug/mL; for DPI the analysis showed no difference in the different concentrations. In the viability analysis of L. braziliensis amastigotes with IVM, there was no difference between the tested concentrations, unlike the tests with L. infantum amastigotes, in which there was a statistical difference in the highest concentrations, 17 and 3.4ug/mL. In the viability of L. braziliensis amastigotes with DPI, there was statistical difference in the concentrations 25, 5 and 1ug/mL; for amastigotes of L. infantum there was a difference in the concentrations of 25 and 5 ug/mL. IVM was effective against parasites, but toxic against cells; DPI was effective against parasites and showed low toxicity to cells. It is concluded that the repositioning of DPI for the treatment of leishmaniasis caused by L. braziliensis and L. infantum is promising considering the anti-leishmanial activity with low toxicity demonstrated in vitro. This unprecedented and promising result for future in vivo tests

Impact of a multimodal rehabilitative intervention on demented patients and their caregivers

Alzheimer's disease is becoming a social, political, and economic issue as a result of both the growing number of people affected and the enormous economic, social, and emotional costs involved in caring for Alzheimer's patients. The aim of this study is to evaluate the effects of a multimodal intervention program for patients with Alzheimer's disease and their caregivers. The study was conducted on a sample of 32 subjects: 16 Alzheimer's patients and their caregivers. The results obtained after the multimodal rehabilitation program showed that the Alzheimer's patients had a more stable cognitive status and improved mood. Regarding the psychoeducational program, the results demonstrate the efficacy of such interventions in terms of increasing and preserving the caregivers' coping skills and enhancing their perception of the value of support groups

Patient- and parent-reported outcome measures of developmental adaptive abilities in visually impaired children: The Visual Impairment Developmental Autonomy (VIDA) scale

In the pediatric context, parents’ and patients’ engagement in the care process is strongly recommended and could be pursued using patient-reported outcome measures (PROMs), which therefore become useful for planning and monitoring treatments. Nevertheless, few data are available from families of children with neurodevelopmental disorders such as visual impairment (VI). The Visual Impairment Developmental Autonomy (VIDA) project aims to develop and validate a patient- and parent-reported tool to measure the most relevant aspects concerning everyday adaptive abilities in children and adolescents with visual impairment: the VIDA scale. The present paper illustrates the Delphi process of item generation engaging parents and patients and presents a protocol for the validation of this new co-designed tool in an Italian visually impaired pediatric population. Twenty-three families and five adolescents provided a list of 192 items and assessed their relevance. Items were categorized in 5 areas of adaptive abilities (i.e., table manners, clothing, personal hygiene, orientation and mobility, and socio-affectivity) and into three age ranges based on the patient's age. The final 102-item Vida Scale will be administered to a minimum of 300 visually impaired children together with measures of quality of life and child adjustment to investigate its psychometric properties

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Molecular dynamics calculations have been used to study structural relaxation and dynamical correlations near the glass transition in the system [Ca(N0 3 h]o.4 [KN0 3 ]o.6&apos; As in a typical molten salt, the overall structure is determined by charge ordering. However, the radial distribution function for Ca 2 + ions is unusual in that even at high temperatures it shows a split first peak due to specific spatial correlations of the cations with the nitrate anions. Structural relaxation that accompanies cooling of the system has been characterized with the aid of the van Hove real-space correlation functions G s (r,t) for the constituent atoms (Ca, K, N, 0). The relaxation of the incoherent structure factor Fs (k,t), with a wave vector k near the peak of the static structure factor, has been investigated as a function of temperature. The results clearly reveal both the a and /3 relaxation processes; the former can be well represented by a master curve with a stretched exponential shape. An analysis of the susceptibility, which agrees qualitatively with neutron spin-echo data, suggests that the glass transition for the model occurs around 400 K. The relatively small discrepancy with the experimental transition temperature derived from neutron scattering data (366 K) is likely related to inadequacies in the model employed for the interionic interactions. The functions C1(t) and C 2 (t) , which describe the reorientational relaxation of the threefold symmetry axes of the nitrate ions, are shown to exhibit a scaling behavior analogous to that of the structure factor. In the region of the glass transition, where translational diffusion has essentially stopped, the nitrate ions continue to flip predominantly about their twofold axes