Modelling Immunological Memory

Accurate immunological models offer the possibility of performing highthroughput experiments in silico that can predict, or at least suggest, in vivo phenomena. In this chapter, we compare various models of immunological memory. We first validate an experimental immunological simulator, developed by the authors, by simulating several theories of immunological memory with known results. We then use the same system to evaluate the predicted effects of a theory of immunological memory. The resulting model has not been explored before in artificial immune systems research, and we compare the simulated in silico output with in vivo measurements. Although the theory appears valid, we suggest that there are a common set of reasons why immunological memory models are a useful support tool; not conclusive in themselves.

Multi-body simulation of a canine hind limb: model development, experimental validation and calculation of ground reaction forces

Background: Among other causes the long-term result of hip prostheses in dogs is determined by aseptic loosening. A prevention of prosthesis complications can be achieved by an optimization of the tribological system which finally results in improved implant duration. In this context a computerized model for the calculation of hip joint loadings during different motions would be of benefit. In a first step in the development of such an inverse dynamic multi-body simulation (MBS-) model we here present the setup of a canine hind limb model applicable for the calculation of ground reaction forces. Methods: The anatomical geometries of the MBS-model have been established using computer tomography- (CT-) and magnetic resonance imaging- (MRI-) data. The CT-data were collected from the pelvis, femora, tibiae and pads of a mixed-breed adult dog. Geometric information about 22 muscles of the pelvic extremity of 4 mixed-breed adult dogs was determined using MRI. Kinematic and kinetic data obtained by motion analysis of a clinically healthy dog during a gait cycle (1 m/s) on an instrumented treadmill were used to drive the model in the multi-body simulation. Results and Discussion: As a result the vertical ground reaction forces (z-direction) calculated by the MBS-system show a maximum deviation of 1.75%BW for the left and 4.65%BW for the right hind limb from the treadmill measurements. The calculated peak ground reaction forces in z- and y-direction were found to be comparable to the treadmill measurements, whereas the curve characteristics of the forces in y-direction were not in complete alignment. Conclusion: In conclusion, it could be demonstrated that the developed MBS-model is suitable for simulating ground reaction forces of dogs during walking. In forthcoming investigations the model will be developed further for the calculation of forces and moments acting on the hip joint during different movements, which can be of help in context with the in silico development and testing of hip prostheses.

"Mutações no gene OCT4 em células eritroleucêmicas humanas: implicações sobre o fenótipo de resistência a múltiplas drogas (MDR)”

O fator de transcrição OCT4 é um importante marcador de células tronco e tem sido relacionado com o conceito de células tronco tumorais (CTTs). Recentemente, ele tem sido também relacionado ao fenótipo de resistência a múltiplas drogas (MDR). O objetivo deste trabalho foi testar se o OCT4 está ligado ao fenótipo MDR em células eritroleucêmicas derivadas da linhagem LMC-K562. Para isso, foi realizada a análise de expressão de genes associados à superfamília de transportadores ABC (ATP Binding Cassette) e, também, de fatores de transcrição relacionados a células tronco. Os primeiros resultados apontaram uma relação direta entre OCT4 e transportadores ABC na linhagem MDR derivada de K562 (Lucena). O sequenciamento de promotores ABC não revelou qualquer mutação que pudesse explicar a expressão diferenciada do OCT4 na linhagem MDR. Posteriormente, o sequenciamento da região contendo o domínio homeobox do gene OCT4 de ambas as linhagens celulares evidenciou, pela primeira vez, que este fator de transcrição é alvo de mutações que podem estar relacionados com o fenótipo MDR. As mutações encontradas implicam substituições de vários aminoácidos em ambas as linhagens celulares. K562 teve sete substituições (três exclusivas), enquanto Lucena teve 13 (nove exclusivas). Além disso, um busca in silico por motivos de fosforilação dentro da sequência de aminoácidos comparada, revelou que o OCT4 humano normal possui sete motivos potenciais de fosforilação. Entretanto, K562 perdeu um motivo e Lucena dois deles. Moléculas com diferentes padrões de fosforilação podem ter sua função modificada. Estes resultados indicam o OCT4 com uma alvo potencial para o tratamento do câncer, especialmente aqueles resistentes à quimioterapia.

Control HSMC para regulación de glucosa sanguínea

Este artículo propone una nueva estrategia de control basada en medidas continuas de glucosa y un controlador por modo deslizante que se habitúa (HSMC). El HSMC es desarrollado, combinando la ley de control por modo deslizante y los principios de control por habituación. El HSMC aplicado a la regulación de glucosa sanguínea en la unidad de cuidados intensivos, incluye tanto entrada de glucosa, como de infusión de insulina intravasculares a fin de proveer el suministro de nutrición y mejorar el rechazo a la perturbación. El estudio basado en simulaciones (in silico), usando un modelo fisiológico de la dinámica glucosa-insulina, muestra que la estrategia de control propuesta funciona apropiadamente. Finalmente, se compara el desempeño del controlador propuesto con respecto a un controlador PID estándar.

Caracterização e comparação de genes expressos em ápices meristemáticos de cana-de-açúcar cultivados em SP e RN

In this work, we used sugarcane as a model due to its importance for sugar and ethanol production. Unlike the current plant models, sugarcane presents a complex genetics and an enormous allelic variation. Here, we report the analysis of SAGE libraries produced using the shoot apical meristem from contrasted genotypes by flowering induction (non-flowering vs. early-flowering varieties) grown under São Paulo state conditions. The expression pattern was analyzed using samples from São Paulo (SP) and Rio Grande do Norte (RN) states. These results showed that cDNAs identified by SAGE libraries had differential expression only in São Paulo state samples. Furthermore, the cDNA identified CYP (Citocrome P450) was chosen for in silico and genome characterization because it was found in SAGE libraries and subtractive libraries from samples from RN. Phylogenetic trees showed the relationship for these sequences. Furthermore, the qRT-PCR for CYP showed a potential role as flowering indutor for RN samples considering different isophorms. Considering the results present here, it can be consider that CYP gene may be used as molecular marker

Polimorfismos nos genes ERBB2 e PARK2/PACRG e sua associação com a hanseníase em populações do Rio Grande do Norte e Pará

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Caracterização funcional de dois cDNAs homólogos à ciclofilina e à proteína reprimida por auxina (ARP) identificados em bibliotecas subtrativas a para floração em tomateiro

Flowering is a fundamental process in the life cycle for plant. This process is marked by vegetative to reproductive apical meristem conversion, due to interactions between several factors, both internal and external to plant. Therefore, eight subtractive libraries were constructed using apical meristem induced or not induced for two contrasting species: Solanum lycopersicum cv. Micro-Tom and Solanum pimpinellifolium. Several cDNAs were identified and among these, were selected two cDNAs: one homologous cDNA to cyclophilin (LeCYP1) and the other to Auxin repressed protein (ARP). It has observed that LeCYP1 and ARP genes are important in the developmental process to plants. In silico analysis, were used several databases with the exclusion criterion E-value <1.0x10-15. As a result, conservation was observed for proteins analyzed by means of multiple alignments and the presence of functional domains. Then, overexpression cassettes were constructed for the ARP cDNA in sense and antisense orientations. For this step, it was used the CaMV35S promoter. The cDNA orientation (sense or antisense) in relation to the promoter was determined by restriction enzymes and sequencing. Then, this cassette was transferred to binary vector pZP211 and these cassettes were transferred into Agrobacterium tumefaciens LBA4404. S. lycopersicum cv. Micro-Tom (MT) and MT-Rg1 plants were transformed. In addition, seedlings were subjected to hormone treatments using a synthetic auxin (- naphthalene acetic acid) and cyclosporin A (cyclophilin inhibitor) treatments and it was found that the hormone treatment there were changes in development of lateral roots pattern, probably related to decreases in auxin signaling caused by reduction of LeCYP1 in MT-dgt plants while cyclosporin A treatments, there was a slight delay in flowering in cv. MT plants. Furthermore, assay with real-time PCR (RT-qPCR) were done for expression level analysis from LeCYP1 and ARP in order to functionally characterize these sequences in tomato plants.

Análise filogenética e funcional de dois genes de reparo homólogos a AP endonuclease em cana-de-açúcar: ScARP1 e ScARP3

The genome of all organisms constantly suffers the influence of mutagenic factors from endogenous and/or exogenous origin, which may result in damage for the genome. In order to keep the genome integrity there are different DNA repair pathway to detect and correct these lesions. In relation to the plants as being sessile organisms, they are exposed to this damage frequently. The Base Excision DNA Repair (BER) is responsible to detect and repair oxidative lesions. Previous work in sugarcane identified two sequences that were homologous to Arabidopsis thaliana: ScARP1 ScARP3. These two sequences were homologous to AP endonuclease from BER pathway. Then, the aim of this work was to characterize these two sequence using different approaches: phylogenetic analysis, in silico protein organelle localization and by Nicotiana tabacum transgenic plants with overexpression cassette. The in silico data obtained showed a duplication of this sequence in sugarcane and Poaceae probably by a WGD event. Furthermore, in silico analysis showed a new localization in nuclei for ScARP1 protein. The data obtained with transgenic plants showed a change in development and morphology. Transgenic plants had slow development when compared to plants not transformed. Then, these results allowed us to understand better the potential role of this sequence in sugarcane and in plants in general. More work is important to be done in order to confirm the protein localization and protein characterization for ScARP1 and ScARP3

Proteômica diferencial da Chromobacterium violaceum em resposta ao estresse oxidativo induzido por peróxido de hidrogênio

The β-proteobacterium Chromobacterium violaceum is a Gram-negative, free-living, saprophytic and opportunistic pathogen that inhabits tropical and subtropical ecosystems among them, in soil and water of the Amazon. It has great biotechnological potential, and because of this potential, its genome was completely sequenced in 2003. Genome analysis showed that this bacterium has several genes with functions related to the ability to survive under different kinds of environmental stresses. In order to understand the physiological response of C. violaceum under oxidative stress, we applied the tool of shotgun proteomics. Thus, colonies of C. violaceum ATCC 12472 were grown in the presence and absence of 8 mM H2O2 for two hours, total proteins were extracted from bacteria, subjected to SDS-PAGE, stained and hydrolysed. The tryptic peptides generated were subjected to a linear-liquid chromatography (LC) followed by mass spectrometer (LTQ-XL-Orbitrap) to obtain quantitative and qualitative data. A shotgun proteomics allows to compare directly in complex samples, differential expression of proteins and found that in C. Violaceum, 131 proteins are expressed exclusively in the control condition, 177 proteins began to be expressed under oxidative stress and 1175 proteins have expression in both conditions. The results showed that, under the condition of oxidative stress, this bacterium changes its metabolism by increasing the expression of proteins capable of combating oxidative stress and decreasing the expression of proteins related processes bacterial growth and catabolism (transcription, translation, carbon metabolism and fatty acids). A tool with of proteomics as an approach of integrative biology provided an overview of the metabolic pathways involved in the response of C. violaceum to oxidative stress, as well as significantly amplified understanding physiological response to environmental stress. Biochemical and "in silico" assays with the hypothetical ORF CV_0868 found that this is part of an operon. Phylogenetic analysis of superoxide dismutase, protein belonging to the operon also showed that the gene is duplicated in genome of C. violaceum and the second copy was acquired through a horizontal transfer event. Possibly, not only the SOD gene but also all genes comprising this operon were obtained in the same manner. It was concluded that C. violaceum has complex, efficient and versatile mechanisms in oxidative stress response

Diseño y evaluación de un vector adenoviral recombinante con un promotor sintético de respuesta a campos electromagnéticos

Los adenovirus recombinantes actualmente representan la opción más utilizada en terapia génica por su eficiencia de transducción, amplio tropismo celular y gran versatilidad en comparación con otros sistemas de transferencia de genes. De igual forma, el uso de un promotor sintético inducible por campos electromagnéticos (CEM) para la expresión del gen reportero de luciferasa ha sido utilizado como vacuna de ADN en ensayos in vitro e in vivo con resultados prometedores al momento de evaluar su respuesta ante la exposición a un campo magnético (CM). En el presente trabajo se sintetizó un fragmento poliA in silico para flanquear el promotor CEM y su gen reportero, aislándolo de la acción promotora e inhibitoria de las regiones ITR (Inverted Terminal Repeat) del genoma adenoviral. Este casete de expresión se introdujo en un plásmido acarreador que comparte regiones de recombinación homóloga con el plásmido poseedor del genoma adenoviral en el sistema comercial AdEasy. Las partículas adenovirales recombinantes AdCEM-Luc generadas fueron utilizadas para medir los niveles de luminiscencia a diferentes tiempos de transducción y de exposición al CM en células HEK 293. Los resultados obtenidos comprobaron la viabilidad del adenovector AdCEM-Luc para transducir células HEK 293, y además revelaron una correlación directa entre el tiempo de incubación y los niveles de luminiscencia. No obstante, no se encontró diferencia significativa en la luminiscencia obtenida ante la exposición o ausencia del CM, lo cual es atribuible a la permisividad en la expresión génica adenoviral de la línea celular HEK 293.

Modelling early transitions toward autonomous protocells.

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Results of a round-robin exercise on read-across

A round-robin exercise was conducted within the CALEIDOSLIFE project. The participants were invited to assess the hazard posed by a substance, applying in silico methods and read-across approaches. The exercise was based on three endpoints: mutagenicity, bioconcentration factor and fish acute toxicity. Nine chemicals were assigned for each endpoint and the participants were invited to complete a specific questionnaire communicating their conclusions.The interesting aspect of this exercise is the justification behind the answers more than the final prediction in itself. Which tools were used? How did the approach selected affect the final answer?

The identification and differentiation of the Candida parapsilosis complex species by polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region of the rDNA

Currently, it is accepted that there are three species that were formerly grouped under Candida parapsilosis : C. parapsilosis sensu stricto, Candida orthopsilosis , and Candida metapsilosis . In fact, the antifungal susceptibility profiles and distinct virulence attributes demonstrate the differences in these nosocomial pathogens. An accurate, fast, and economical identification of fungal species has been the main goal in mycology. In the present study, we searched sequences that were available in the GenBank database in order to identify the complete sequence for the internal transcribed spacer (ITS)1-5.8S-ITS2 region, which is comprised of the forward and reverse primers ITS1 and ITS4. Subsequently, an in silico polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to differentiate the C. parapsilosis complex species. Ninety-eight clinical isolates from patients with fungaemia were submitted for analysis, where 59 isolates were identified as C. parapsilosis sensu stricto, 37 were identified as C. orthopsilosis, and two were identified as C. metapsilosis. PCR-RFLP quickly and accurately identified C. parapsilosis complex species, making this method an alternative and routine identification system for use in clinical mycology laboratories.

Identification of regulatory polymorphisms associated with breast cancer risk

Dissertação de Mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2014

Functional characterisation of putative Cis-Regulatory Risk Loci for breast cancer

Dissertação de Mestrado, Qualidade em Análises - Erasmus Mundus, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015