INPhINIT Fellowship @ ITQB NOVA 2022

Electrochemical investigations of biomolecular interactions under controlled conditions (Felipe Conzuelo)

HOST ORGANIZATION
MOSTMICRO-ITQB Nova - Microbiologia Molecular, Estrutural e Celular

AREA OF KNOWLEDGE
Physical Sciences, Mathematics and Engineering Panel

GROUP OF DISCIPLINE
Chemistry and Chemical Engineering

GROUP LEADER
Dr. Felipe Conzuelo Fernandez
felipe.conzuelo@itqb.unl.pt

RESEARCH PRODUCT / RESEARCH GROUP
The Bioelectrochemistry and Electrobiotechnology Lab led by Dr. Felipe Conzuelo focuses on the integration of biomolecules and microorganisms with electrode materials to solve fundamental questions in the study of biological redox processes, as well as the implementation of various biotechnological devices, ranging from energy conversion and storage to biosensing applications.
https://www.itqb.unl.pt/research/biological-chemistry/bioelectrochemistry-and-electrobiotechnology

POSITION DESCRIPTION
Research Project / Research Group Description

To date, a great number of isolated enzymes have been exploited for catalyzing various important reactions, finding applications in a wide range of fields expanding from the synthesis of pharmaceuticals and commodity products to the development of biosensors for healthcare applications. In resemblance to the natural processes occurring at the cellular level, several reports have shown the great advantage of conversion systems integrating a number of enzymes, which are coupled on demand to perform sequential conversion steps in so-called catalytic cascade reactions. The confinement of biocatalysts in such systems can greatly promote the rates of conversion, the stability of the biomolecules, and the selectivity of the overall conversion process, leading to more efficient reactions. However, important questions remain open in this field encouraging a deeper examination and optimization of multi-step biocatalytic conversion processes in vitro toward the development of advanced systems with a potential impact on new and improved practical applications. This research project aims at the investigation of intricate biomolecular interactions coupled with electrochemical manipulations. The approach will enable the exploitation of several electrochemical techniques for a detailed characterization of the biocatalytic processes. At the same time, redox reactions can be used as a means of control and stimulation of the investigated reactions. Particular emphasis will be given to the study of microenvironments established at the biotic/abiotic interfaces and their influence on the conversion performance with regard to the possibility of tuning the conversion kinetics of the individual steps and adjusting the accumulation and transfer of reaction intermediates. Model catalytic cascades will be established for the planned investigations with potential applications in biosensing as well as biocatalysis for energy conversion and valorization of waste residues.

Job position description

The successful candidate will work on the previously described project, acquiring solid expertise in the fields of bioelectrochemistry, biocatalytic conversions, and the fabrication of biotechnological devices for practical applications. The tasks to be performed include research on the implementation of hierarchically structured electrodes for hosting the isolated enzymes to be coupled, the establishment of microfluidic systems for assessing the influence of accumulation and transfer rates of reaction intermediates, and the investigation and tuning of relative reaction kinetics of the different biocatalysts involved in the catalytic reactions. The unique opportunity to control and optimize the nature of the established interfaces together with the possibility for electrochemical control will make it possible to understand the mechanisms of biocatalytic interactions between isolated enzymes coupled on demand for the establishment of specific catalytic cascades. The results obtained in the realization of the project will also be important to provide a deeper knowledge of the importance of adequate microenvironments established near the immobilized biomolecules and their influence on the performance of catalytic conversions. In consequence, the project will set new milestones and provide the foundations for advanced technologies utilizing bioelectrochemical cascades. In particular, the work will contribute to the development of modern electrochemical biosensors for human health assessment, aiming at simpler and more reliable strategies. In addition, the development of more efficient catalytic cascade reactions under electrochemical control will be applied to the utilization of biomass and abundant waste residues as starting materials for conversion into more valuable products, contributing to circular economies and a sustainable society.

Establishing the interactions between a Type II diabetes drug and the gut microbiome / PhD on drug-gut microbiome interactions

HOST ORGANIZATION
MOSTMICRO-ITQB Nova - Microbiologia Molecular, Estrutural e Celular

AREA OF KNOWLEDGE
Life Sciences Panel

GROUP OF DISCIPLINE
Human Biology, Microbiology, Molecular Biology, Genetics, Cell Biology, Genomics and Proteomics, Biochemistry, Basic Neuroscience

GROUP LEADER
Dr. Sarela Garcia-Santamarina
sarela.santamarina@itqb.unl.pt

RESEARCH PRODUCT / RESEARCH GROUP
Webpage of the Human Microbiota – Xenobiotics Interactions Lab, which contains a brief description of the Lab’s Research Interests and a list of selected publications
https://www.itqb.unl.pt/research/biology/human-microbiota-xenobiotics-interactions

POSITION DESCRIPTION
Research Project / Research Group Description

The Human Microbiota-Xenobiotic Interactions Laboratory belongs to the Mostmicro Unit of the Instituto de Tecnologia Quimica e Biologica (ITQB) from the University Nova of Lisbon. We aim to investigate the effects of xenobiotics (eg, drugs, contaminants, nutrients) on the composition and function of the gut microbiota and their effects on the host.

Our research answers questions such as how xenobiotics affect the structure of gut bacterial communities, gut bacterial metabolism, or nutrient exchanges at the host-microbe interface. The proposed PhD project is framed within the context of understanding how a drug used to treat diabetes influences the composition of the intestinal microbiota, and how this influence affects the efficacy of the drug for the treatment of this disease.

To carry out this research we have state-of-the-art equipment that consists of an anaerobic chamber equipped with technology that allows high-throughput phenotypic characterizations of the intestinal microbiota, and that we can couple to 'omics' readings (metagenomics, metaproteomics, and metabolomics).

Job position description

The gut microbiota may be a key factor contributing to the great problem of interpersonal variations in drug responses. Many studies have correlated the baseline microbiota, or changes in microbiota composition after drug treatment with drug efficacies in diabetes, cardiovascular disease, psychiatric diseases, and others. However, few studies have mechanistically addressed why. By understanding these mechanisms, personalized treatments can be designed that allow better pharmacological management of different diseases.

Based on data from clinical studies and our data, we hypothesize that the gut microbiota, and their nutritional status influence the mode of action of a drug to treat diabetes. To study this, the PhD student will develop a bottom-up systematic research pipeline with the goals of:

(1) establishing the direct interactions between drug and gut bacteria,

(2) identifying how these interactions affect gut bacterial communities and the underlying mechanisms,

(3) identifying which drug’s effects on gut bacterial communities influence drug mode of action and gut microbiota-host interactions.

The doctoral student will be enrolled in the Molecular Biosciences doctoral program at ITQB NOVA. To carry out this research, the doctoral student is expected to acquire interdisciplinary skills and competencies in high-throughput phenotypic screening, systematic assembly of gut microbial consortia, characterization of phenotypic responses to stressors of gut microbial communities by amplicon sequencing, use of genome-wide mutant libraries, genetic manipulation of non-model organisms, use of ‘omics’ technologies, and data analysis and integration among others. By undertaking this research, the doctoral student will also gain transferable skills including project management, problem solving, teamwork, creativity, public speaking, scientific writing, research ethics, and scientific communication to non-scientific audiences.

Peptide-based supramolecular catalytic systems 

HOST ORGANIZATION
MOSTMICRO-ITQB Nova - Microbiologia Molecular, Estrutural e Celular

AREA OF KNOWLEDGE
Physical Sciences, Mathematics and Engineering Panel

GROUP OF DISCIPLINE
Chemistry and Chemical Engineering

GROUP LEADER
Dr. Ana Sofia Pina
ana.pina@itqb.unl.pt

RESEARCH PRODUCT / RESEARCH GROUP
The lab works at the interface of chemistry and biology with a focus on the use of supramolecular peptide chemistry to understand the complexity of biological systems. This complexity arises from chemical reactions networks often involving molecular interactions between proteins, and phase transition towards compartmentalization.
https://www.itqb.unl.pt/research/chemistry/bioinspired-peptide-systems

POSITION DESCRIPTION
Research Project / Research Group Description

Nature has a vast repertoire of enzyme functions with high catalytic efficiency and a degree of specificity. Enzymes evolved from cooperative interactions among peptides, where functionality emerged from the fusion and diversification of short peptide sequences identified today as highly conserved regions responsible for the enzyme’s functionality (10.1021/acs.chemrev.9b00664). Peptides are successful chiral catalysts in synthetically relevant reactions (10.1021/ja994280y). However, the efficiency of peptide catalysts in aqueous media is still modest and has met considerable skepticism due to their conformational heterogeneity.

Nevertheless, simpler molecules such as small peptides and amino acids can be advantageous because they are easily synthesized, modular, tunable, and act at non-standard pH and temperature.

Both de novo design and screening for peptide sequence-encoded function, with a particular focus on catalysis, are still challenging. However, in vitro screening techniques like phage display can screen for billions of sequences and multiple peptide combinations toward a rationally engineered target. Depending on the target, phage display can identify peptide sequences with interesting frameworks and encoded functionality. Using targets with multivalence and supramolecular order can simultaneously aid in discovering peptides with defined frameworks improving the catalytic via an induced-fit model (DOI: 10.1039/D1SC04420F).  

In this research project, we aim to develop short-constrained peptide catalysts by exploring molecular in vitro display technologies with stable supramolecular targets and thus drive the process of molecular recognition by conformational selection.

Our lab aims to comprehend the dynamics of the peptide motifs’ interactions in the living systems with the intention to control/create self-assembly and function (e.g., catalysis and supramolecular recognition), to program complexity in peptide systems with simplified emergent behaviours.

Job position description

Screening methodologies linking sequence to function can contribute to gain fundamental understanding of the natural mechanisms of living systems in terms of their molecular cooperativity and reactivity.  This work uses phage display with synthetic supramolecular targets (amphiphile-based molecules) to conformationally select peptide sequences with ability for phosphate hydrolysis. The functionality and secondary structures of the peptides-will be extensively studied by resorting to biophysical methodologies.

The enhancement of catalytic functionality on the discovered peptide sequences will further explore self-assembly strategies triggered by environmental conditions (pH, temperature, ionic strength), with respective characterization by microscopy techniques. This project sits at the interface between peptide chemical biology, synthetic and supramolecular chemistry and materials science.

Enzyme-like functional peptides can provide solutions to overcome biotechnological barriers (e.g., engineer peptide catalysts to control reactions under environmentally friendly conditions). Moreover, this minimalistic peptide sequences encompassed dynamic features of the living systems and will be a step forward to create artificial life-inspired nanosystems and for future technological applications in materials science and biomedicine.

The research activities leading to obtaining the academic degree of doctor of the selected fellow will be developed at the Bioinspired Peptide Systems Lab at The Instituto de Tecnologia Química e Biológica António Xavier (ITQB NOVA) of the Universidade Nova de Lisboa, and under the Doctoral Programme in Molecular Biosciences of ITQB-NOVA. The student to whom the scholarship is awarded will be automatically admitted to the doctoral programme in Molecular Biosciences of the Universidade Nova de Lisboa.

OTHER RELEVANT WEBSITES
Google Scholar- Ana Pina
https://scholar.google.com/citations?user=97tcx90AAAAJ&hl=en&oi=ao

The Life electric: structural and functional characterization of cytochromes that enable bacteria to live by producing electricity

HOST ORGANIZATION
MOSTMICRO-ITQB Nova - Microbiologia Molecular, Estrutural e Celular

AREA OF KNOWLEDGE
Life Sciences Panel

GROUP OF DISCIPLINE
Human Biology, Microbiology, Molecular Biology, Genetics, Cell Biology, Genomics and Proteomics, Biochemistry, Basic Neuroscience

GROUP LEADER
Prof. Ricardo O. Louro
louro@itqb.unl.pt

RESEARCH PRODUCT / RESEARCH GROUP
Inorganic Biochemistry and NMR lab website
https://www.itqb.unl.pt/labs/inorganic-biochemistry-and-nmr/home

POSITION DESCRIPTION
Research Project / Research Group Description

In line with the notion expressed by the Nobel Prize winner Albert Szent-Györgyi that  ‘Life is nothing but an electron looking for a place to rest’, some bacteria are capable of living by delivering electrons to electrical circuits. This enabled the development of BioElectrochemical Technologies that are powered by the microbial metabolism, and that operate in conditions of low ecological footprint. Despite great strides in the development of these technologies, decades of effort have failed to produce a structure of the redox enzymes at the beginning of these novel bioenergetics metabolic chains. These are the key biochemical players where the metabolism diverges from the traditional aerobic respiration with oxygen or anaerobic respiration of numerous compounds. These key enzymes interface the quinone pool of the cytoplasmic membrane with the specific electron transfer chains that deliver the electrons to the electrical circuit outside of the cell. In the model electricity generating bacteria of the genus Shewanella, the key enzyme is CymA. It is a cytochrome of ~20kDa containing four hemes with a N-terminal alpha helix inserted in the cytoplasmic membrane. NMR spectroscopy is uniquely suited for the detailed structural and functional characterization of a membrnane associated enzyme of this size. Its characterization will conclude the structural and functional characterization of this unique electron transfer chain allowing, for the first time, to have a complete functional and structural picture of how “Life Electric” operates.

The hosting laboratory is composed by a mature research team headed by the PI and an assistant researcher that are recognized leaders in the international panorama of biochemical studies in BioElectrochemical Technologies. We are currently funded by one national research project, one European project, and engaged in two COST action, all relevant for this proposal.

Job position description

This PhD research work comprises

i) Protein expression and purification. The candidate will follow-up on methodologies developed in the lab for the expression of the target proteins and develop strategies for their efficient expression and purification from cells grown in minimal medium. This will enable the preparation of isotopically labelled protein for NMR spectroscopy studies.

ii) NMR data collection. The candidate will prepare samples with protein in the oxidized and in the reduced state. Cyanide will be used as strong-field ligand to convert the high-spin heme into its low spin form to facilitate spectral assignment. Standard methods will be used for data collection of the diamagnetic reduced samples, whereas non-standard methods recently developed by the host lab will be used for data collection in the oxidized sample which is paramagnetic.

iii) Signal assignment and characterization of interactions with physiological partners. Computer-aided resonance assignment will be performed iteratively with additional data collection that may prove to be necessary to complete the task. The assignment will provide the opportunity to identify the binding region of physiological redox partners.

iv) Structural calculation. Nuclear overhauser effect distance constrains will be used for structural calculation of   the reduced diamagnetic protein whereas these data will be complemented by paramagnetic constrains (pseudocontact shifts and paramagnetic relaxation enhancements) to calculate the structure in the oxidized paramagnetic state. T1, T2 and heteronuclear NOE will be used to assess the amplitude and timescale of the motions of the protein, and how these change with redox state and upon binding with physiological partners.

This workplan provides advanced training on the whole pipeline of modern structural biology workflow using the top-rated know-how and facilities of the host lab and privileged access to European research infrastructure.

Study of the traits that define the inheritance of the microbiota in Solanaceae

HOST ORGANIZATION
GREEN-IT - Biorecursos para a Sustentabilidade
https://www.itqb.unl.pt/green-it

AREA OF KNOWLEDGE
Life Sciences Panel

GROUP OF DISCIPLINE
Agriculture, Veterinary Studies, Animal Production, Forest Sciences

GROUP LEADER
Dr. Juan Ignacio Vílchez
nacho.vilchez@itqb.unl.pt

RESEARCH PRODUCT / RESEARCH GROUP
Online site that includes all information about group members, objectives, former projects and most relevant publications
https://www.itqb.unl.pt/research/plant-sciences/Plant-Microbiome-Interaction/iplantmicro

POSITION DESCRIPTION
Research Project / Research Group Description

In the last decade we have observed an increasing in climatic and productive challenges for agriculture. Facing a growing global population, these conditionings will suppose a serious problem. In this way, it is time to look for more sustainable models for agricultural management that will ensure its production and general supply in the future.

Solanaceae are among the most common crop plants. The food production together with industrial use (textile, pharmaceutical or horticulture), make this family of plants very important. However, this family has a high range of pathogens that condition their develop and production. Thus, they are capable of infecting reproductive organs, making plants susceptible to transferring them to the next generation (seedborne). This phenomenon is also reported for beneficial strains, suggesting some filtering system. We hypothesize these traits are mostly linked to Solanaceae characteristic chemical compounds (alkaloids, terpenes). Moreover, the presence of microbiota in seeds seems to depend on a series of skills as mobility, scavenging of reactive oxygen species, detoxifying or buffering compounds, cellulolytic capacity, chemotactic sensitivity, resistance stage (cystic, sporulation), or molecular camouflage.

Unrevealing these microbiota-plant traits in seedborne population would represent a revolution in terms of agricultural management: we propose to use these traits through to actively select a beneficial microbiota in next generation (seedborne), which will allow the application of biocontrol models, facing stress conditions or prepare a preventive hardening. This pathway would be essential in the fight against the effects of climate change, as well as against the soil degradation that condition the production of food and products of industrial interest, aligning our bioengineering project with the UN 2030 objectives providing a sustainable alternative and guaranteeing food production worldwide.

Job position description

The PhD student will participate in 4 main tasks:

• Task 1: Selection of species from Solanaceae family, their propagation and adjustment of conditions to carry out comparative studies.
• Task 2: Greenhouse tests to determine the relationship with the microbiota: isolation of strains, molecular pathways and phenotyping.
• Task 3: Analysis of the effects of the selected compounds on the microorganisms.
• Task 4: Testing of compounds to influence the selection of strains within the natural microbiota in the next generation (seedborne).

Among others, this student will be involved in the use of devices and software:

• HPLC and GC-MS.
• MultiskanTM FC Microplate Photometer.
• Scanning Electron Microscope, bright field and confocal fluorescence microscopes.
• Phenotyping devices as MultispeQ, FluorPen FP 110 or ThermaCAM7.
• Cytoscape v3.0.
• EZRhizo and RhizoV (root architecture analysis).

At the end of the project, we expect the student will be able to develop independently technics as:

• Culture-dependent microbiota studies
• In vitro/in planta tests
• Greenhouse and field tests
• Microscopy techniques
• Molecular biology
• Chemical treatments
• HQ-sequencing and analysis

Moreover, our research group together with all groups in our research center (ITQB) and research unit (GREEN-IT), we have an internal collaboration network. Since we have multiple common programs and projects, our students have access to specific equipment, facilities, services, techniques and resources, as well as periodical training and assistance in their use. They will be as well guided in paper writing and conferences presentations. Finally, iPlantMicro Lab has an extensive network of collaborators, including high-impact multidisciplinary groups in Spain, Ireland, France, UK, Germany, Turkey, Brazil, Argentina, India, Korea and China. The student would have access to international training, to specialized research technologies, and to the advice of some of the highest-level experts in the world.

OTHER RELEVANT WEBSITES

Website of iPlantMicro Lab at GREEN-IT Unit
https://www.itqb.unl.pt/green-it/groups/iplantmicro

Research Gate of PI
https://www.researchgate.net/profile/Juan-Ignacio-Vilchez

PI’s ORCID
https://orcid.org/0000-0003-4524-7384