Development of human central nervous system 3D in vitro models for preclinical research

Neurological disorders are a major concern in modern societies, with increasing prevalence mainly related with the higher life expectancy. Most of the current available therapeutic options can only control and ameliorate the patients’ symptoms, often be-coming refractory over time. Therapeutic breakthroughs and advances have been hampered by the lack of accurate central nervous system (CNS) models. The develop-ment of these models allows the study of the disease onset/progression mechanisms and the preclinical evaluation of novel therapeutics. This has traditionally relied on genetically engineered animal models that often diverge considerably from the human phenotype (developmentally, anatomically and physiologically) and 2D in vitro cell models, which fail to recapitulate the characteristics of the target tissue (cell-cell and cell-matrix interactions, cell polarity). The in vitro recapitulation of CNS phenotypic and functional features requires the implementation of advanced culture strategies that enable to mimic the in vivo struc-tural and molecular complexity. Models based on differentiation of human neural stem cells (hNSC) in 3D cultures have great potential as complementary tools in preclinical research, bridging the gap between human clinical studies and animal models. This thesis aimed at the development of novel human 3D in vitro CNS models by integrat-ing agitation-based culture systems and a wide array of characterization tools. Neural differentiation of hNSC as 3D neurospheres was explored in Chapter 2. Here, it was demonstrated that human midbrain-derived neural progenitor cells from fetal origin (hmNPC) can generate complex tissue-like structures containing functional dopaminergic neurons, as well as astrocytes and oligodendrocytes. Chapter 3 focused on the development of cellular characterization assays for cell aggregates based on light-sheet fluorescence imaging systems, which resulted in increased spatial resolu-tion both for fixed samples or live imaging. The applicability of the developed human 3D cell model for preclinical research was explored in Chapter 4, evaluating the poten-tial of a viral vector candidate for gene therapy. The efficacy and safety of helper-dependent CAV-2 (hd-CAV-2) for gene delivery in human neurons was evaluated, demonstrating increased neuronal tropism, efficient transgene expression and minimal toxicity. The potential of human 3D in vitro CNS models to mimic brain functions was further addressed in Chapter 5. Exploring the use of 13C-labeled substrates and Nucle-ar Magnetic Resonance (NMR) spectroscopy tools, neural metabolic signatures were evaluated showing lineage-specific metabolic specialization and establishment of neu-ron-astrocytic shuttles upon differentiation. Chapter 6 focused on transferring the knowledge and strategies described in the previous chapters for the implementation of a scalable and robust process for the 3D differentiation of hNSC derived from human induced pluripotent stem cells (hiPSC). Here, software-controlled perfusion stirred-tank bioreactors were used as technological system to sustain cell aggregation and dif-ferentiation. The work developed in this thesis provides practical and versatile new in vitro ap-proaches to model the human brain. Furthermore, the culture strategies described herein can be further extended to other sources of neural phenotypes, including pa-tient-derived hiPSC. The combination of this 3D culture strategy with the implemented characterization methods represents a powerful complementary tool applicable in the drug discovery, toxicology and disease modeling.

Bacterial cellulose as a novel substrate for retinal pigment epithelium cells transplantation in age-related macular degeneration

Tese de Doutoramento em Engenharia Química e Biológica.

Otimização do perfil farmacocinético e farmacodinâmico de fármacos anticancerígenos através da sua veiculação em nanossistemas lipídicos

Dissertação de mestrado em Biofísica e Bionanossistemas

Protein-based nanodevices for therapeutic applications

Tese de Doutoramento em Biologia Molecular e Ambiental (área de especialização em Biologia Celular e Saúde).

Development of polymeric nanoparticles containing neuroprotective compounds of Hypericum perforatum

Tese de Doutoramento em Biologia das Plantas - MAP BIOPLANT

Development of Monoolein-based lipofection vectors for therapeutic siRNA delivery

Tese de Doutoramento em Biologia Molecular e Ambiental - Especialidade em Biologia Celular e Saúde

New bio-strategies for ochratoxin A detoxification using lactic acid bacteria

The occurrence of mycotoxigenic moulds such as Aspergillus, Penicillium and Fusarium in food and feed has an important impact on public health, by the appearance of acute and chronic mycotoxicoses in humans and animals, which is more severe in the developing countries due to lack of food security, poverty and malnutrition. This mould contamination also constitutes a major economic problem due the lost of crop production. A great variety of filamentous fungi is able to produce highly toxic secondary metabolites known as mycotoxins. Most of the mycotoxins are carcinogenic, mutagenic, neurotoxic and immunosuppressive, being ochratoxin A (OTA) one of the most important. OTA is toxic to animals and humans, mainly due to its nephrotoxic properties. Several approaches have been developed for decontamination of mycotoxins in foods, such as, prevention of contamination, biodegradation of mycotoxins-containing food and feed with microorganisms or enzymes and inhibition or absorption of mycotoxin content of consumed food into the digestive tract. Some group of Gram-positive bacteria named lactic acid bacteria (LAB) are able to release some molecules that can influence the mould growth, improving the shelf life of many fermented products and reducing health risks due to exposure to mycotoxins. Some LAB are capable of mycotoxin detoxification. Recently our group was the first to describe the ability of LAB strains to biodegrade OTA, more specifically, Pediococcus parvulus strains isolated from Douro wines. The pathway of this biodegradation was identified previously in other microorganisms. OTA can be degraded through the hydrolysis of the amide bond that links the L-β-phenylalanine molecule to the ochratoxin alpha (OTα) a non toxic compound. It is known that some peptidases from different origins can mediate the hydrolysis reaction like, carboxypeptidase A an enzyme from the bovine pancreas, a commercial lipase and several commercial proteases. So, we wanted to have a better understanding of this OTA degradation process when LAB are involved and identify which molecules where present in this process. For achieving our aim we used some bioinformatics tools (BLAST, CLUSTALX2, CLC Sequence Viewer 7, Finch TV). We also designed specific primers and realized gene specific PCR. The template DNA used came from LAB strains samples of our previous work, and other DNA LAB strains isolated from elderberry fruit, silage, milk and sausages. Through the employment of bioinformatics tools it was possible to identify several proteins belonging to the carboxypeptidase family that participate in the process of OTA degradation, such as serine type D-Ala-D-Ala carboxypeptidase and membrane carboxypeptidase. In conclusions, this work has identified carboxypeptidase proteins being one of the molecules present in the OTA degradation process when LAB are involved.

Fibroelastoma of the mitral valve as a cause of transient ischemic stroke

A 44-year-old woman had a transient ischemic stroke, fibroelastoma of the mitral valve being the source of the embolus. The patient evolved with neutropenia induced by ticlopidine after 10 days of treatment. We report the major clinical features, therapeutical options, and medicamentous toxicity resulting from the use of antiplatelet drugs.

Does a Role Exist for Tilting-Guided Therapy in the Management of Neurocardiogenic Syncope?

PURPOSE: Upright tilt-table testing (UTT) is an useful method for identifying patients with neurocardiogenic syncope, but its role in the evaluation of therapeutic efficacy is controversial. The aim of this study was to determine the correlation between negative UTT after therapy introduction (acute efficacy) and symptom recurrence during follow-up (chronic efficacy). METHODS: We studied 56 severely symptomatic patients (age 27±19 years) with recurrent (7±12 episodes) neurocardiogenic syncope (positive UTT). Once empirical pharmacological therapy was initiated, all patients underwent another UTT (therapeutic evaluation test - TET). Therapy was not modified after TET results. The probability of symptom recurrence was analyzed with the Kaplan-Meier method and compared by log-rank test in patients with negative and positive TET. RESULTS: Negative UTT after therapy was related to a significantly lower probability of recurrence during follow-up (4.9 versus 52.4% in 12 months, P<0.0001). CONCLUSION: A good correlation exists between acute and long-term efficacy of pharmacological therapy for neurocardiogenic syncope, so that serial UTT may be considered a good method for identifying an effective therapeutic strategy.

Development of nanohydrogels as a vehicle for the release of bioactive compounds in food matrices

PhD in Chemical and Biological Engineering

Effects of PDE type 5 inhibitors on Left Ventricular Diastolic Dysfunction in Resistant Hypertension

Resistant hypertension (RHTN) is a multifactorial disease characterized by blood pressure (BP) levels above goal (140/90 mmHg) in spite of the concurrent use of three or more antihypertensive drugs of different classes. Moreover, it is well known that RHTN subjects have high prevalence of left ventricular diastolic dysfunction (LVDD), which leads to increased risk of heart failure progression. This review gathers data from studies evaluating the effects of phosphodiesterase-5 (PDE-5) inhibitors (administration of acute sildenafil and short-term tadalafil) on diastolic function, biochemical and hemodynamic parameters in patients with RHTN. Acute study with sildenafil treatment found that inhibition of PDE-5 improved hemodynamic parameters and diastolic relaxation. In addition, short-term study with the use of tadalafil demonstrated improvement of LVDD, cGMP and BNP-32 levels, regardless of BP reduction. No endothelial function changes were observed in the studies. The findings of acute and short-term studies revealed potential therapeutic effects of IPDE-5 drugs on LVDD in RHTN patients.

Investigações sobre doenças de Psittacideos

The present paper colligates the notions acquired in previous investigations, already published, and new observations upon diseases of the psittacidae, liable to be confused with psittacosis of parrots. The author calls attention to the indifference with regard to this question shown by investigators, even by those who dealt with the study of this disease on the occasion of the latest outbreak of psittacosis, in flagrant contrast with the researches upon the alterations induced by pathogenic agents of other diseases transmissible to man, when these agents pass through animals or when the latter are depositaries of the virus. This remark considerably enhances the importance of the presence paper from a hygienic and epidemiologic point of view, representing moreover a contribution to general knowledge and to veterinary medicine. The researches carried out since the appearance of the latest outbreak of psittacosis,-which occurred simultaneously with an epizooty in parrots lodged in aviary of the park of Agua Branca (Directory of Animal Industry of the State São Paulo)-led to the verification of the frequent existence in these animals of various diseases liable to be confused with psittacosis. These diseases are due to two kinds of pathogenic agents: virus and bacteria. In the first group there are to be found the diseases occasioned by the virus of human psittacosis, discovered by Western, Bedson and Simpson, and the disease me with in parrots coming from traders in S. Paulo. The infections by bacteria of the genus Salmonella and by those of other genera belong to the second group. As differential characters of the two infections due to virus, delineated on the strength of notions drawn from a detailed experimental study and from the literature on this subject, the following are given: ¹ Samples of our virus were sent, for comparison, to various investigators of psittacosis. Amongst them, Prof. M. Rivers acceded to our request; he found its nature to be different from that of the virus of psittacosis studiedby him. We are very much obliged to him for the attention he paid to this verification. Virus of psittacosis - Infectiousness: man, monkey, rabbit, mouse, hen, canary. Neurotropic affinity. Inclusions: small, protoplasmic. Exsiccation: the virus has good power of preservation. Symptoms: inactivity, drowsiness, frequent diarrhoea, oculo-nasal discharge and cough, coma. Duration: 4 to 5 days. Bodily lesions: congestion of intestines, splenomegaly. Virus of S. Paulo - Infects only psittacidae, particularly those of the genus Amazona. No localization in the nervous system. Large, nuclear. Is rapidly destroyed. Inactivity, inappetency, adynamia (drooping of the wings, indifference, leaning its beak against the bars of the cage in order not to fall down); profuse diarrhoea, of whitish stools, at times enterorrhagia; prolonged coma. 2 to 8 days. Foci of yellowish necrosis in liver, spleen and lung. At times, congestion of intestines. Characteristic features common to the two viruses.-They act in great dilutions, filter through tight candles though being partly retained, are preserved under glycerine or Bedson's solution, are stable at 55°C. heat and are destroyed by physical and chemical agents. Both virus diseases are very seldom met with in psittacidae: only once, amongst numberless sick parrots, the author met with a disease of the virus differring from that of psittacosis. This disease, greatly transmissible to man, ought to be more frequent, if it were common in parrots. On the contrary, bacteria cause diseases in these animals with great frequency, presenting variable characters, from a severe epizootic form, rapidly mortal, to ambulatory or silent forms, for the most part developing towards a cure or assuming a chronic character. Amongst the bacteria which cause the infection of this group the salmonellae predominate and amongst them the bacterium discovered by Nocard, as well as a species which in the course of this study is characterized under the name of Salmonella nocardi. The author believes that in the epizooty from which Nocard isolated his bacterium there was association of the virus-disease inducing the epizooty of that epoch in Paris with the bacterial disease, as must have happened in Argentina, where the disease was transmitted to man, and Santillan, according to Barros, isolated from the sick parrots bacteria of the genus Salmonella. The diseases of the two groups, that due to virus and that due to bacteria, are differentiated: Virus-diseases - Evolution: rapid, nearly always followed by death. Symptoms: sadness, profuse diarrhoea, of whitish stools, at times enterorrhagia, complete inappetency, adynamia, indifference, prolonged coma. Clinical forms: acute and subacute. Lesions: Foci of necrosis in liver and spleen without cellular reaction around the focus, yellow liver, multiple serositis. Presence of protoplasmic or nuclear granulations. Bacteriology: Complete lack or inconstant presence of bacteria in the organs and blood. Infectiousness of the organs and blood after filtration: positive. Bacterial diseases - Varies from one week to a month or more, not always fatal. Sadness, partial inappetency, tremblings, intensive thirst, mucous or mucosanguineous diarrhoea, lack of adynamia (reacts to stimulations and moves well at any time of the disease, though showing little disposition to locomotion), soiling of feathers. Frustrate, acute, subacute and chronic. Hepatic and intestinal cogestion, foci of necrosis in liver, spleen and lung with cellular reaction around the focus. Lack of granulations. Constant presence of bacteria in the organs and blood. Negative. The analysis of the litterature shows that the characteristic features of the diseases in parrots referred to parrot psittacosis, more frequently approach the bacterial diseases here described of these animals, a hypothesis which is reinforced by the observation of the greater frequency of infections...

Trypanosoma cruzi: circulating antigens

Circulating antigens were detected in sera of mice experimentally infected with a high close of Trypanosoma cruzi by reaction with sera from chronically infected mice. The immunodiffusion reaction between homologous acute and chronic sera produced four precipitation lines. By reaction with chronic mouse serum, circulating antingens were detected in sera from heavily infected hamsters, dogs, rabbits and in sera from chagasic patients. A reaction was also found in urine from acutely infected mice and dogs. Trypanosoma cruzi exoantigen was detected in trypanosome culture medium and in the supernatant of infected cell cultures. Attempts to isolate the antigens are described.

Renal response to the angiotensin II receptor subtype 1 antagonist irbesartan versus enalapril in hypertensive patients.

OBJECTIVE: To compare the acute and sustained renal hemodynamic effects on hypertensive patients of 100 mg irbesartan and 20 mg enalapril each once daily. PATIENTS: Twenty patients (aged 35-70 years) with uncomplicated, mild-to-moderate essential hypertension and normal serum creatinine levels completed this study. STUDY DESIGN: After random allocation to treatment (n=10 per group), administration schedule (morning or evening) was determined by further random allocation, with crossover of schedules after 6 weeks' therapy. Treatment and administration assignments were double-blind. Twenty-four-hour ambulatory blood pressure was monitored before and after 6 and 12 weeks of therapy. Renal hemodynamics were determined on the first day of drug administration and 12 and 24 h after the last dose during chronic treatment. RESULTS: Administration of each antihypertensive agent induced a renal vasodilatation with no significant change in glomerular filtration rate. However, the time course appeared to differ: irbesartan had no significant acute effect 4 h after the first dose, but during chronic administration a renal vasodilatory response was found 12 and 24 h after the dose; enalapril was effective acutely and 12 h after administration, but no residual effect was found 24 h after the dose. Both antihypertensive agents lowered mean ambulatory blood pressure effectively, with no significant difference between treatments or between administration schedules (morning versus evening). CONCLUSIONS: Irbesartan and enalapril have comparable effects on blood pressure and renal hemodynamics in hypertensive patients with normal renal functioning. However, the time profiles of the renal effects appear to differ, which might be important for long-term renoprotective effects.

Treatment of central retinal vein occlusion-related macular edema with intravitreal bevacizumab (Avastin): preliminary results.

PURPOSE: To assess the safety and efficacy of treatment of macular edema secondary to central retinal vein occlusion (CRVO) with intravitreal bevacizumab. PATIENTS AND METHOD: The ongoing prospective study included 8 consecutive patients (8 eyes) with macular edema secondary to CRVO (6 non ischemic and 2 ischemic), treated with intravitreal injection of 1.25 mg (0.05 mL) of bevacizumab. Main outcome was best corrected visual acuity (BCVA) and central foveal thickness (CFT) measured by optical coherence tomography monthly during one year. Retreatment criteria include decrease of BCVA, persistence of macular edema on angiograms and increase of CFT. RESULTS: Mean age of the eight patients was 68 years (range: 50-82 years). Mean duration of symptoms before injection was 98 days (range: 3-289). Mean follow-up was 3.25 months. At baseline, mean BCVA was 0.84 logMar and mean baseline CFT was 771 microm. Mean BCVA was 0.36 and mean CFT thickness was 275 microm (n = 8) at month 1, 0.41 and 411 microm at month 2 (n = 7), 0.3 and 344 microm at month 3 (n = 6), 0.3 and 397 microm at month 4 (n = 5), respectively. In 75 % of patients, a single injection was not sufficient, and retreatment needed. No serious adverse events were observed. CONCLUSIONS: Treatment of macular edema secondary to CRVO with intravitreal bevacizumab injection of 1.25 mg was well tolerated and associated with marked macular thickness reduction and BCVA improvement in all patients. A trend towards reduction of foveal thickness and improvement of visual acuity was observed in both acute and chronic CRVO.