Production of recombinant protein hLIF in static and dynamic systems of animal cell culture

Thesis for the Degree of Master of Science in Biotechnology Universidade Nova de Lisboa, Faculdade de Ciências e TecnologiaThe present work focuses the optimization of the production of human Leukemia Inhibitory Factor (hLIF) by human embryonic kidney 293 (HEK293) - EBNA1 cell line cultured as suspension aggregates in spinner flask. The effects of initial cell density and feeding-regime in cellular growth and productivity were evaluated. A first phase occurs until the end of exponential growth in medium containing serum, being followed by puromycin selection of cells containing hLIF plasmid. A second phase, the production phase, is developed under serum-free conditions. Three initial cell densities (2´104 cells.mL-1, 2´105 cells.mL-1 and 2´106 cells.mL-1) were tested and the effect on maximum cell density and cell aggregate size distribution was evaluated. The inoculum of 2´105 cells.mL-1 led to final higher cellular densities around 7´106 cells.mL-1 and homogeneous aggregates around 278 μm were observed. The effect of the feeding-regime was then studied for the production of recombinant hLIF with an initial cell density of 2´105 cells.mL-1 by performing metabolite analysis Metabolite analysis revealed the occurrence of glucose and glutamine starvation upon 9 and 7 days, respectively, in 25% FR. Therefore, glutamine acted as an alternative carbon, energy and aminoacid source in HEK293-EBNA1 growth. Observed lactate levels were bellow the inhibition limit concentrations (30 mM 1).HEK293-EBNA1 did not seem to be affected by the produced levels of ammonia. In conclusion, the 25% feeding regime at 2´105 cells.mL-1 initial cell density lead to the higher viable cell densities, either along culture time at growth and at production phase, with aggregates in suspension below necrotic diameters. This experiment conditions also allowed achieving the higher LIF volumetric productivity, 125 ± 2.2 ng.mL-1

Doença de Marchiafava‑Bignami: A Importância do Diagnóstico e Tratamento Precoces por uma Equipa Multidisciplinar

Marchiafava‑Bignami disease is a rare condition associated with chronic alcohol consumption and/or malnutrition, characterized by demyelination of the corpus callosum, generally attributed to a deficiency in B complex vitamins. We report the case of a 34‑year‑old male with a 10‑year history of alcohol dependence who was admitted to the hospital, after having been found lying on the floor of his house, malnourished and with pressure ulcers on his chest and knees. On clinical observation he was found to be alert but mute. He followed some simple orders. Generalized spastic hypertonia was present. Magnetic resonance imaging showed demyelination of the corpus callosum, suggesting the diagnosis of Marchiafava‑Bignami disease. He was admitted to the Psychiatry Inpatient Unit and evaluated by a multidisciplinary team. He received thiamine, corticosteroids and rehabilitation. After a week, his speech was slurred but comprehensible and he could walk with aid; magnetic resonance imaging findings had improved. After three months in a Convalescence Unit, he was discharged with total autonomy.A doença de Marchiafava‑Bignami é uma condição rara associada ao consumo crónico e abusivo de álcool e/ou desnutrição, caracterizada por desmielinização do corpo caloso, sendo geralmente atribuída a uma deficiência de vitaminas do complexo B. Apresentamos o caso de um homem de 34 anos com 10 anos de dependência de álcool que foi trazido ao hospital, após ter sido encontrado caído no chão de sua casa em decúbito ventral, desnutrido, com úlceras de pressão no tórax e joelhos. Estava vígil, não emitia discurso, cumpria ordens simples e apresentava hipertonia espástica generalizada. Na ressonância magnética identificou‑se desmielinização do corpo caloso, sendo diagnosticado com doença de Marchiafava‑Bignami. Foi internado no serviço de psiquiatria e avaliado por uma equipa multidisciplinar, iniciando terapêutica com tiamina e corticosteróides e um programa de reabilitação. Após uma semana, apresentava melhoria clínica e imagiológica. Após um período de 3 meses numa Unidade de Convalescença, teve alta totalmente autónomo

Aplicação de queuing models para planeamento de capacidade na recepção da Clínica Universitária Egas Moniz

Dissertação para obtenção do grau de Mestre no Instituto Superior de Ciências da Saúde Egas MonizObjectivos: Avaliar os tempos de espera dos pacientes para o atendimento na recepção da Clínica Universitária Egas Moniz e planear a capacidade ideal da estrutura de recursos humanos.Materiais e métodos: Durante 3 semanas, englobando todos os horários de funcionamento da Clínica Universitária Egas Moniz, foram registados os tempos de espera dos pacientes na recepção. Após esse registo foram aplicados os princípios de fila de espera M/M/S de modo a poder verificar a eficiência do sistema e a capacidade ideal da estrutura de recursos humanos.Resultados: Verificou-se que para os tempos de espera registados, o ponto óptimo de eficiência do sistema é de 2 funcionários.Conclusões: Não se verificaram necessidades de alteração da estrutura actual de recursos humanos, ou seja, o número de funcionários no presente é o adequado

Bleb morphology of glaucoma drainage devices on magnetic resonance imaging

© 2023 The Authors. Acta Ophthalmologica published by John Wiley & Sons Ltd on behalf of Acta Ophthalmologica Scandinavica Foundation. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.Purpose: To evaluate bleb morphology features of different glaucoma drainage devices (GDD) using magnetic resonance imaging (MRI). Materials and methods: Prospective cohort study of GDD and bleb morphology in consecutive glaucoma patients implanted with Ahmed Glaucoma Valve (AGV), Paul Glaucoma Implant (PGI), Baerveldt Glaucoma Implant (BGI) and Ahmed ClearPath (ACP) devices. Thirty-six eyes from 30 consecutive patients underwent standardized GDD implantation followed by MRI at least 1 month after surgery. Main outcomes included bleb volume and endplate position relative to the optic nerve. Secondary outcomes included intraocular pressure (IOP), medication and surgical complications during a 12-month follow-up. Results: Seven eyes were implanted with the AGV (19%), 5 with BGI (14%), 16 with PGI (44%) and 8 with ACP (22%). MRI scans were obtained 85 ± 66 days after surgery. Mean total bleb volume was 563 ± 390 mm3 . This bleb volume was inversely correlated with early post-operative IOP (day 7; rs = -0.3326, p = 0.0475) but positively correlated with IOP at 12 months (rs = 0.3592, p = 0.0341). No significant difference in total bleb volume was found between GDD types (p = 0.1223). A double-layered bleb was observed in 34 eyes (94%). The inferior bleb volume was significantly larger in PGI devices versus other GDD types (380 ± 205 vs. 193 ± 161 mm3 ; p = 0.0043). Distance from the endplate to the optic nerve was 9.5 ± 4.0 mm, similar across GDDs (p = 0.2519). Conclusions: Double-layered blebs are a common finding with GDDs. Bleb volume showed different correlations with IOP at distinct timepoints and the PGI device formed larger blebs. A standardized GDD implantation technique ensures a safe distance from the GDD endplate to the optic nerve.info:eu-repo/semantics/publishedVersio

Red Flags Not To Be Missed

BACKGROUND: Urinary sediment is a noninvasive laboratory test that can be performed by an automated analyzer or manually by trained personnel. Manual examination remains the diagnostic standard because it excels at differentiating isomorphic from dysmorphic red blood cells and identifying other urinary particles such as renal tubular epithelial cells (RTECs), lipids, crystals, and the composition of casts. This study aimed to investigate the prevalence of a complete profile of urinary sediment particles and its associations with histologic lesions on kidney biopsy, regardless of diagnosis. METHODS: This was a single-center, observational retrospective study of 131 patients who had contemporary manual urinary sediment evaluation and kidney biopsy. A comprehensive set of urinary particles and histologic lesions were quantified, and their associations were analyzed. RESULTS: In our samples, we found an elevated frequency of findings suggestive of proliferative kidney disease and a low frequency of particles evoking urologic damage. The association of histologic lesions and urinary particles was explored with a multivariate model. We identified urinary sediment characteristics that independently correlated with the presence of some histologic lesions: urinary lipids with mesangial expansion (OR=2.86; 95% confidence interval [95% CI], 1.3 to 6.3), mesangial hypercellularity (OR=2.44; 95% CI, 1.06 to 5.58), and wire loops and/or hyaline deposits (OR=2.89; 95% CI, 1.13 to 7.73); Urinary renal tubular epithelial cells with endocapillary hypercellularity (OR=3.17; 95% CI, 1.36 to 7.39), neutrophils and/or karyorrhexis (OR=4.51; 95% CI, 1.61 to 12.61), fibrinoid necrosis (OR=4.35; 95% CI, 1.48 to 12.74), cellular/fibrocellular crescents (OR=5.27; 95% CI, 1.95 to 14.26), and acute tubular necrosis (OR=2.31; 95% CI, 1.08 to 4.97). CONCLUSIONS: In a population of patients submitted to kidney biopsy, we found that the presence of some urinary particles (renal tubular epithelial cells, lipids, and dysmorphic erythrocytes), which are seldom reported by automated analyzers, is associated with active proliferative histologic lesions. In this regard, manual urinary sediment evaluation may help to shape the indications for performing a kidney biopsy.publishersversionpublishe