Identification of protein-protein interactions between plasmodium falciparum and the human erythrocyte membrane protein 4.1

Student Number : 9605361W - PhD thesis - School of Pathology - Faculty of ScienceMalaria is one of the most debilitating parasitic infections to have afflicted humanity and remains an expanding health risk for many countries. This is attributed largely to the complexity of the parasite’s life cycle and refined ability to evade host immunity. During development within the erythrocyte, Plasmodium falciparum induces a wide array of changes to the ultrastructure, function and antigenic properties of the host membrane. Numerous proteins encoded by the parasite associate with the erythrocyte skeleton and appear to be essential for P. falciparum survival. The elucidation of new protein-protein interactions has therefore formed a key area of malaria research. To circumvent the difficulties provided by conventional protein techniques, a novel application of phage display technology was used in this research. P. falciparum phage display libraries were created and biopanned against human erythrocyte skeletal protein 4.1 (4.1R). DNA sequencing and bioinformatic investigations uncovered a number of parasite proteins with binding specificity toward 4.1R. They included five hypothetical proteins, two invasion proteins, namely erythrocyte binding antigen-175 (EBA-175) and EBA-181, two predicted protein kinases and a putative aminopeptidase. A common binding motif displaying homology to muscle myosin and neurofilament sequences was also identified in four of the ten proteins. The interaction between EBA-181 and 4.1R was characterised further by mapping the domain in 4.1R responsible for binding to the parasite protein. Recombinant proteins were used in blot-overlay and pull-down experiments, which revealed specific interaction between the highly conserved 10kDa domain and the 4.1R binding region in EBA-181. Binding was concentration dependent, as well as saturable and was abolished by heat denaturation of 4.1R. Functions of the 4.1R-specific parasite proteins remain to be determined, however, they are potentially involved in parasite growth and survival during intra-erythrocytic development. Furthermore, these proteins may also participate in the entry and/or exit of parasites from the human erythrocyte. The interaction of EBA-181 with the 10kDa domain of 4.1R provides new insight into the molecular mechanisms utilised by P. falciparum during erythrocyte entry. It also highlights the multifunctional role of malaria invasion proteins, which may contribute to the success of the pathogenic stage of the parasite’s life cycle

Automated Setup to Accurately Calibrate Electrical DC Voltage Generators

At National Institute of Metrological Research (INRIM), an automated setup to calibrate DC Voltage generators, mainly top-level calibrators from 1 mV to 1 kV has been developed. The heart of the setup is an INRIM-built automated fixed ratios DC Voltage divider. The significant achievement of this setup is the possibility to interconnect the divider, a DMM characterized in linearity, a DC Voltage Standard and a DC Voltage generator under calibration and automatically to manage the calibration process. This calibration method allows to save a lot of time, to improve the reliability and to increase the accuracy of the calibration of generators. The relative uncertainties of the system span from 0.6x10-6 to 1.2x10-4 improving the previous capabilities of the INRIM laboratory for calibration of programmable multifunction instruments. In addition, this system allows to avoid the employment of several Standards (some of them still manually operating) carrying out the entire process without changing the setup configuration and without the presence of operators. The concept of this setup can be transferred to secondary high-level electrical calibration Laboratories that could be consider it useful for their calibration activities.Comment: 6 pages 8 figure

Construction and use of Plasmodium falciparum phage display libraries to identify host parasite interactions

BACKGROUND: The development of Plasmodium falciparum within human erythrocytes induces a wide array of changes in the ultrastructure, function and antigenic properties of the host cell. Numerous proteins encoded by the parasite have been shown to interact with the erythrocyte membrane. The identification of new interactions between human erythrocyte and P. falciparum proteins has formed a key area of malaria research. To circumvent the difficulties provided by conventional protein techniques, a novel application of the phage display technology was utilised. METHODS: P. falciparum phage display libraries were created and biopanned against purified erythrocyte membrane proteins. The identification of interacting and in-frame amino acid sequences was achieved by sequencing parasite cDNA inserts and performing bioinformatic analyses in the PlasmoDB database. RESULTS: Following four rounds of biopanning, sequencing and bioinformatic investigations, seven P. falciparum proteins with significant binding specificity toward human erythrocyte spectrin and protein 4.1 were identified. The specificity of these P. falciparum proteins were demonstrated by the marked enrichment of the respective in-frame binding sequences from a fourth round phage display library. CONCLUSION: The construction and biopanning of P. falciparum phage display expression libraries provide a novel approach for the identification of new interactions between the parasite and the erythrocyte membrane

Severity classification for idiopathic pulmonary fibrosis by using fuzzy logic

OBJECTIVE: To set out a severity classification for idiopathic pulmonary fibrosis (IPF) based on the interaction of pulmonary function parameters with high resolution computed tomography (CT) findings. INTRODUCTION: Despite the contribution of functional and radiological methods in the study of IPF, there are few classification proposals for the disease based on these examinations. METHODS: A cross-sectional study was carried out, in which 41 non-smoking patients with IPF were evaluated. The following high resolution CT findings were quantified using a semi-quantitative scoring system: reticular abnormality, honeycombing and ground-glass opacity. The functional variables were measured by spirometry, forced oscillation technique, helium dilution method, as well as the single-breath method of diffusing capacity of carbon monoxide. With the interaction between functional indexes and high resolution CT scores through fuzzy logic, a classification for IPF has been built. RESULTS: Out of 41 patients studied, 26 were male and 15 female, with a mean age of 70.8 years. Volume measurements were the variables which showed the best interaction with the disease extension on high resolution CT, while the forced vital capacity showed the lowest estimative errors in comparison to total lung capacity. A classification for IPF was suggested based on the 95% confidence interval of the forced vital capacity %: mild group (>92.7); moderately mild (76.9-92.6); moderate (64.3-76.8%); moderately severe (47.1-64.2); severe (24.3-47.0); and very severe (<24.3). CONCLUSION: Through fuzzy logic, an IPF classification was built based on forced vital capacity measurement with a simple practical application

Care pathways models and clinical outcomes in disorders of consciousness

Objective: Patients with Disorders of consciousness, are persons with extremely low functioning levels and represent a challenge for health care systems due to their high needs of facilitating environmental factors. Despite a common Italian health care path-way for these patients, no studies have analyzed information on how each region have implemented it in its welfare system correlating data with patients’ clinical outcomes. Materials and Methods: A multicenter observational pilot study was realized. Clinicians collected data on the care pathways of patients with Disorder of consciousness by ask-ing 90 patients’ caregivers to complete an ad hoc questionnaire through a structured phone interview. Questionnaire consisted of three sections: sociodemographic data, description of the care pathway done by the patient, and caregiver evaluation of health services and information received.Results: Seventy- three patients were analyzed. Length of hospital stay was different across the health care models and it was associated with improvement in clinical diag-nosis. In long- term care units, the diagnosis at admission and the number of caregivers available for each patient (median value=3) showed an indirect relationship with worsening probability in clinical outcome. Caregivers reported that communication with professionals (42%) and the answer to the need of information were the most critical points in the acute phase, whereas presence of Non- Governmental Organizations (25%) and availability of psychologists for caregivers (21%) were often missing during long-term care. The 65% of caregivers reported they did not know the UN Convention on the Rights of Persons with Disabilities. Conclusion: This study highlights relevant differences in analyzed models, despite a recommended national pathway of care. Future public health considerations and ac-tions are needed to guarantee equity and standardization of the care process in all European countries

The 10 kDa domain of human erythrocyte protein 4.1 binds the Plasmodium falciparum EBA-181 protein

BACKGROUND: Erythrocyte invasion by Plasmodium falciparum parasites represents a key mechanism during malaria pathogenesis. Erythrocyte binding antigen-181 (EBA-181) is an important invasion protein, which mediates a unique host cell entry pathway. A novel interaction between EBA-181 and human erythrocyte membrane protein 4.1 (4.1R) was recently demonstrated using phage display technology. In the current study, recombinant proteins were utilized to define and characterize the precise molecular interaction between the two proteins. METHODS: 4.1R structural domains (30, 16, 10 and 22 kDa domain) and the 4.1R binding region in EBA-181 were synthesized in specific Escherichia coli strains as recombinant proteins and purified using magnetic bead technology. Recombinant proteins were subsequently used in blot-overlay and histidine pull-down assays to determine the binding domain in 4.1R. RESULTS: Blot overlay and histidine pull-down experiments revealed specific interaction between the 10 kDa domain of 4.1R and EBA-181. Binding was concentration dependent as well as saturable and was abolished by heat denaturation of 4.1R. CONCLUSION: The interaction of EBA-181 with the highly conserved 10 kDa domain of 4.1R provides new insight into the molecular mechanisms utilized by P. falciparum during erythrocyte entry. The results highlight the potential multifunctional role of malaria invasion proteins, which may contribute to the success of the pathogenic stage of the parasite's life cycle

Food biotechnology and South African consumer attitudes: implications for purchase behaviour

Prof. S. Kruge

Analisi del divisore automatico di Tensione continua di precisione a rapporti fissi sviluppato all’INRIM

Presso l'Istituto nazionale di ricerca metrologica (INRIM), è stato realizzato un divisore a rapporto fissi di Tensione continua di precisione automatico che consente di realizzare i rapporti di divisione 10:1 e 100:1. È staton realizzato utilizzando cento resistori selezionati da 10 kΩ a film metallico a basso coefficiente di temperatura. Essendo lo strumento interfacciabile con un multimetro (DMM) caratterizzato dalla linearità sulla portata 10 V e con un calibratore di Tensione continua, può essere rapidamente e automaticamente tarato quando necessario. Il metodo di taratura rapida del divisore è stato validato confrontandolo con un altro metodo che utilizza il campione di rapporto di Tensione continua dell’INRIM. Un altro particolare è che la caratterizzazione della linearità del DMM sulla portata 10 V consente di considerare nel budget di incertezze della taratura del divisore, le incertezze di tale caratterizzazione invece delle specifiche di accuratezza del multimetro molto maggiori. Le incertezze estese di taratura dei rapporti 10:1 e 100:1 sono rispettivamente 4,6×10–7 e 6,6×10–7, adatte per poter utilizzare il divisore in un sistema di misura automatico con un multimetro e un campione di tensione continua per tarare generatori di tensione continua come calibratori di alto livello, ampiamente diffusi nei laboratori elettrici secondari

Automated setup to accurately calibrate electrical dc voltage generators

At National Institute of Metrological Research (INRIM), an automated setup to calibrate dc voltage generators, mainly top-level calibrators, from 1 mV to 1 kV has been developed. The heart of the setup is an INRIM-built automated precision fixed ratios dc voltage divider. It can be interconnected to a DMM characterized in linearity, to a dc voltage standard and to a dc voltage generator under calibration to manage automatically the calibration process. Novelty of the system is the employment of the divider being such an instrument not commercially available. Main results are the improvement of the reliability and the increasing of the accuracy of the calibration process saving a lot of time. In addition, it is possible to avoid several standards (still manually operating) carrying out the whole process without changing the setup configuration and without the presence of operators. The expanded uncertainties of the system span from 6.0 × 10−7 to 1.3 × 10−4 improving the previous capabilities of the INRIM laboratory for calibration of multifunction instruments. The setup concept can be transferred to secondary high-level electrical laboratories to improve and expedite their calibrations