Internships at ITQB NOVA

BACKGROUND

Invasive microbial infections are quickly becoming a very serious health threat due to the growing antimicrobial resistance of common pathogens and also to the scarce antimicrobial drugs pipeline. While such drugs are traditionally based on organic compounds, metal-based compounds have recently found promise owing to their intrinsically tridimensional structures and various possible modes of action. We are currently developing new metal complexes for potential antifungal and/or antibacterial applications. Such complexes are to be based on bioactive azole scaffolds, and will explore a range of suitable transition and post-transition metal cations.

OBJECTIVES

During this internship the student will synthesize organic azole ligands and use them to produce metal complexes with selected metal cations, which will then be screened for their antimicrobial activity. The work planned includes both synthetic chemistry techniques and chemical characterization studies, and will be mostly hands-on.

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Luís Lima (llima@itqb.unl.pt)

BACKGROUND

The use of carbon dioxide (CO₂) as a renewable C1 feedstock to produce value-added products has attracted enormous attention from the scientific community in the last decade. Among the existing processes, the addition of CO₂ to epoxides represents one of the most promising reactions; a process that allows easy access to cyclic carbonates, which are valuable products widely used in industrial applications. In this project we propose the preparation of metal complexes, based on Earth-abundant metals, for their application as catalysts in the synthesis of cyclic carbonates through the reaction of CO₂ with epoxides.

OBJECTIVES

This project will allow students to receive laboratory training in the synthesis of metallic compounds (working under inert atmosphere conditions-Schlenk-line methodology), organic synthesis, and gas handling (CO₂ reactions carried out in depressurized millireactors), as well as in the characterization of chemical compounds (NMR, IR, MS, etc.).

BACKGROUND

The conservation of our natural resources is a fundamental challenge of mankind. New catalytic systems based on Earth-abundant and nontoxic transition metals that allow rapid and selective chemical transformations through atom-economy processes will have a significant impact in our society.

OBJECTIVES

The present project aims to develop of a new generation of highly efficient phosphine-free metal-based catalysts with tunable reactivity for the synthesis of valuable organic molecules through atom-economy methods. This project will allow students to receive laboratory training in the synthesis of organometallic compounds (working under inert atmosphere conditions-Schlenk-line methodology) and organic synthesis, as well as in the characterization of chemical compounds (NMR, IR, MS, etc.).

This project will involve the synthesis of metal complexes (Fe, Mn, Ni, Co, Cu) complexes bearing N-heterocyclic carbene ligands (NHCs) and their application as catalysts for a variety of catalytic reactions. In particular, in Acceptorless Dehydrogenation and Borrowing Hydrogen Processes for the preparation of pharmaceutically relevant targets.

BACKGROUND

It is well known that plastics play an important role in our lives. These materials are used in agricultural industry, automotive, medicine, among other applications. However, plastic production leads to serious environmental problems and for this reason, it is essential to develop new technologies that are able to carry out recycling effectively. The strategy to be developed in the present proposal is the selective depolymerization of waste polymers, breaking down the polymers into valuable monomers or building blocks that could be then reused.

OBJECTIVES

This project will allow students to receive laboratory training in the synthesis of organometallic compounds (working under inert atmosphere conditions-Schlenk-line methodology; microwave equipment), organic synthesis, as well as in the characterization of chemical compounds (NMR, IR, MS, etc.).

The aim of this project is to develop Fe, Mn and Ni-based catalytic systems for the depolymerization of polyesters through reduction reactions (hydrosilylation, hydrogenation, transfer hydrogenation). To meet this goal, we propose the synthesis of robust organometallic complexes containing tethered N-heterocyclic carbene ligands (NHCs). These metal complexes will be applied as catalysts for the depolymerization of polyesters.

BACKGROUND

Antivirulence agents aim to circumvent resistance by disarming the pathogen as opposed to affecting viability. A common strategy consists in interfering with adhesion of the pathogen to the host using multivalent glycoconjugates. We are seeking to explore arylamide foldamers as a new class of multivalent glycoconjugates. These oligomers adopt stable helical conformations in solution, whose predictability, tunability and ease of synthesis, make them suitable to allow precise control of number, nature and orientation of carbohydrate ligands. In addition, they can feature proteinogenic side chains to mimic protein surfaces. Foldamer-based multivalent glycoconjugates can therefore be promising mimics of glycoproteins that decorate host cells, to competitively interfere with adhesion processes.

OBJECTIVES

The project involves the synthesis of monomers featuring proteinogenic side chains and saccharide ligands; their incorporation into foldamer sequences; biophysical characterization of the glycoconjugate/adhesin interaction; evaluation of the inhibition of bacterial adhesion to host cells.

BACKGROUND

Glycan microarrays are powerful tools for investigating the carbohydrate binding specificity of proteins and antibodies, identifying potential drug targets, and detecting the presence of carbohydrate-binding biomarkers of disease states. This research will consist of developing arylamide foldamers as new scaffolds for the well-defined display of carbohydrates and validating their suitability for integration with current microarray technology platforms. This class of molecules adopts helical conformations in solution, whose stability, predictability, tunability, and ease of synthesis make them particularly suitable for precise control of the number, nature, and orientation of glycoligands. Additionally, they can feature proteinogenic side chains. Therefore, arylamide foldamers are expected to allow for the mimicking of natural glycoproteins that decorate host cell surfaces and the creation of a more physiologically authentic platform for probing glycan-binding proteins.

OBJECTIVES

The project involves synthesizing glycofoldamer scaffolds featuring an appropriate functional group, which will enable their covalent immobilization onto glass slides, non-contact robotic printing of the glycofoldamers onto a microarray platform, and examining their spatial specificity against selected lectin targets.

BACKGROUND

The WHO has declared antimicrobial resistance (AMR) as one of the major public health problems of the 21st century. To address this challenge, researchers are seeking antimicrobials with new modes of action and less prone to resistance. In this regard, antimicrobial peptides (AMPs) have been heralded as a potential solution to AMR because they affect the microbial cell membrane in a non-specific manner, as opposed to traditional antibiotics. However, their peptidic nature has hampered their use in the clinic. For that reason, peptidomimetics (small molecules that mimic the AMPs physicochemical properties and biological activity) have received considerable attention. In this project we seek to explore the antimicrobial potential of quinoline-derived foldamers, i.e. folding oligomers containing quinoline units. Their rigid backbone and preference to adopt helical conformations make them interesting candidates for the development of new proteolytically-stable mimics of -helical amphipathic AMPs with improved antimicrobial activities.

OBJECTIVES

The work will consist in the synthesis of amphipathic foldamers by microwave-assisted solid-phase synthesis; in evaluating the antimicrobial performances/potential of the foldamers against bacteria and fungi; assessment of disruption to key microbial cell structures by fluorescence microscopy and evaluation of foldamer hemolytic activity and cytotoxicity in two major epithelial barrier cell lines.

BACKGROUND

Staphylococcus epidermidis include the skin microbiota and contribute to homeostasis and protection against pathogens. However, they are the most frequent cause of medical device-associated infections. Skin isolates belonging to clonal complex 2 (CC2) lineage are the major colonizers and the more frequent strains in infection, but they share their ecological niche with other minor genetic backgrounds (non-CC2). Discovery of biological processes and/or metabolic pathways that discriminate strains from these two lineages will have potential application on the development of additives to supplement general use disinfectants during clinical practice. Comparative genomics and proteomics analyses previously performed by us for two selected S. epidermidis strains belonging to the two lineages, showed that they display different metabolic and phenotypic profiles. Enrichment on the cell wall proteome will complement the data previously obtained from the whole cell with more accessible protein targets.

OBJECTIVES

Identification of proteins and biological processes that can be used as specific targets for antimicrobial activity against S. epidermidis pathogenic strains colonizing the skin.

BACKGROUND

Staphylococcus epidermidis (SE) is the most representative member of coagulase-negative staphylococci (CoNS) in human skin, being ubiquitously present on this natural human barrier. Despite the many benefits to the host as a skin commensal, SE has emerged as an opportunistic pathogen, causing infections associated to indwelling medical devices. The SE population structure on the human skin consists of two main clonal lineages, A/C and B. Each lineage presents distinct genetic and phenotypic features associated to commensalism (lineage B) or pathogenicity (lineage A/C). However, there is still a lack of information on how strains from these clusters interact with the human host. In particular, the response of SE to antimicrobial fatty acids (AFAs), key components of skin innate immunity in humans, is poorly explored. Our data, showed that a strain, belonging to the B cluster, had a lower susceptibility to the tested AFAs than a strain from the A/C cluster. However, it is still unknown what is the molecular basis of such a difference.

OBJECTIVES

Compare two S. epidermidis strains, representatives of both lineages, their membrane lipid composition in the absence and presence of AFAs. The obtained results will a) allow to determine membrane structural differences that can be explored for the design of specific anti-microbials against SE pathogenic strains; b) generate new insights on the AFAs anti-microbial mechanism of action.

BACKGROUND

In Europe, the burden caused by antibiotic resistant bacterial infections is equivalent to influenza, HIV/Aids and Tuberculosis combined. Infections caused by Staphylococcus aureus (S. aureus) strains are the second most relevant in this context. A chief factor suggested to contribute for the high incidence and prevalence of S. aureus infections is its capacity to persist and divide inside host cells, escaping antibiotics and extracellular immune recognition. Formerly regarded as an exclusively extracellular pathogen, S. aureus is in fact a facultative intracellular pathogen. S. aureus can infect immune and non-immune cells and evade autonomous immunity (intracellular recognition of pathogens). This is thought to be a major factor in continuance of carriage, chronicity of infection and dissemination within the host. In an extracellular context the resolving power and live-cell compatibility of super-resolution microscopy has allowed to perform a comprehensive characterization of S. aureus cell division progression (10.1038/ncomms9055). Based on morphology changes (e.g. septum formation at mid-cell), S. aureus cell division can be divided into 3 different phases: phase 1-cells prior to division septum initiation, phase 2-cells undergoing septum synthesis, phase 3-division septum is complete prior to splitting into to two daughter cells. Importantly, these phases are altered as a consequence of different challenges (e.g. antibiotics). Further, it also permitted to understand the subcellular localization and antibiotic induced changes to a multitude of important S. aureus cell division proteins (10.1038/nature25506). 

OBJECTIVES
A wealth of knowledge that needs to be expanded to an infection context in order to truly understand the cell biology of this bacterial pathogen as well as the role of antibiotics in the context of infection. Here we aim to take advantage of this comprehensive characterization as the basis to explore S. aureus cell division inside host cells. We will infect human cells with S. aureus strains and characterize the morphology changes associated with S. aureus cell division progression (TASK 1) as well as the subcellular localization of key cell division proteins (TASK 2). Finally, we will explore the effect antimicrobial molecules have on these processes and organization (TASK 3). This master project will provide training in microbiology, cell biology and microscopy.

BACKGROUND
In Europe, the burden caused by antibiotic resistant bacterial infections is equivalent to influenza, HIV/Aids and Tuberculosis combined. Infections caused by Staphylococcus aureus (S. aureus) strains are the second most relevant in this context. A chief factor suggested to contribute for the high incidence and prevalence of S. aureus infections is its capacity to persist and divide inside host cells, escaping antibiotics and extracellular immune recognition. Formerly regarded as an exclusively extracellular pathogen, S. aureus is in fact a facultative intracellular pathogen. S. aureus can infect immune and non-immune cells and evade autonomous immunity (intracellular recognition of pathogens). This is thought to be a major factor in continuance of carriage, chronicity of infection and dissemination within the host. The cell surface molecular signatures important for host autonomous immunity recognition of facultative intracellular pathogens, such as S. aureus, and the host autonomous immunity factors responsible for identifying them are still not fully understood. In this project we propose to develop an experimental framework that will have far-reaching impact on our understanding of autonomous immunity function and how we approach the mechanisms of intracellular recognition of pathogens. The knowledge gained here will be applicable to other bacterial pathogens and in the development of new chemotherapy options to tackle the challenges of bacterial infections.

OBJECTIVES
To create a high-resolution map of the interplay between S. aureus and host cell components we need a multi-target near-molecular scale resolution strategy. For this purpose we will use super-resolution microscopy (SRM) which seamlessly provides multi-target molecular specific labelling and sub-diffraction spatial resolution. SRM congregates several approaches the lab has expertise on, from classical single-molecular localization microscopy (SMLM) and structured illumination microscopy (SIM) to very recent expansion microscopy (ExM). These approaches permit achieving spatial resolution bellow diffraction (as low as 10 nm) of multiple host and bacterial targets. However, these techniques rely heavily in immunolabelling (the use of antibodies to localize specific biological targets). In S. aureus biology this is not a trivial task as this in this bacteria Protein A is a major cell wall component. Protein A is a virulence factor of Staphylococcus aureus that binds to the Fc region of IgG antibodies. By binding to the Fc portion of antibodies, protein A renders them inaccessible to opsonins, thus impairing phagocytosis of the bacteria via immune cell attack. This helps protect S. aureus from being engulfed and subsequently killed by immune cells. For microscopy purposes this also signifies that most antibodies will bind to S. aureus cells. Hence TASK 1 of this master project is to develop a blocking strategy to “saturate” all protein A binding sites before labelling with the antibody of interest, this way permitting immunolabelling of the target of interest to be specific. In parallel TASK 2 we will map the interaction of S. aureus cells at different times post infection with cytoskeletal and autonomous immune host factors. Task 2 will be performed using antibodies, for which we will use a protein A mutant of S. aureus, albeit not ideal from a biological perspective (as this strain is lacking a virulence factor) it will permit to optimize the analysis pipeline. A major prospect if to analyse this interaction in live cells to understand the spatiotemporal dynamics of the process, however most high-resolution SRM approached are light intensive an often do not allow multi-colour imaging. To overcome this, TASK 3 will entail developing and using image analysis pipelines based on deep learning approaches to correlate low resolution data with high-resolution data. The combination of these approaches will allow constructing a high-resolution image of the multi-component host cell cytoskeleton and S. aureus containing subcellular compartments. This will permit to correlate how endocytosis, autophagy and cytoskeleton markers correlate with S.aureus cell division and how antibiotic chemotherapy influences these processes. This master project will provide training in microbiology, cell biology, microscopy, and deep learning strategies.

About the PI
Pedro Pereira completed his degree in Applied Chemistry in 2006 at FCT NOVA and his PhD in Cell Biology from ITQB NOVA in 2013. He did post-doctoral work at UCL in the UK developing super-resolution approaches and applying them to host-pathogen interaction cell biology questions. In late 2021, he secured an Auxiliary Researcher position at the ITQB NOVA, through a Mostmicro open call. Throughout his career path, he published 31 articles in peer-reviewed international journals and presented his results in several national and international conferences, seminars, and workshops. His research focuses on 1) exploring the dynamic interaction between human cells and bacterial pathogens, 2) the development of imaging approaches to fully understand these interactions and 3) developing strategies to overcome the antimicrobial molecule shortage challenge.
Ciência ID: 2112-9437-36FA
ORCID ID: 0000-0002-1426-9540

BACKGROUND
Staphylococcus aureus is a major human opportunistic pathogen responsible for a wide range of infections from mild- to life threatening conditions, with a high capacity for acquisition of antimicrobial resistance and virulence determinants. While methicillin resistant S. aureus (MRSA) have been a major cause of hospital associated multidrug resistant infections worldwide, methicillin-susceptible S. aureus (MSSA) infections have been increasingly reported, with an estimated of 84% rise in blood infections in Europe between 2005 and 2018. Like the European scenario, Portuguese hospitals have been identifying an increase in MSSA infections associated to serious illness. Previous studies showed that more than 60% of MSSA from hospital and community infections belonged to four clonal lineages (clonal complex (CC)30, CC5, CC45 and CC8) which, with the exception of CC5, are not commonly found among nosocomial MRSA population in the same settings. Thus, continued surveillance of the MSSA population circulating in hospitals is essential to understand the S. aureus epidemiology and infection dynamics. Additionally, knowledge of the clonal relationship and the presence of determinants of resistance to antibiotics and biocides among MSSA isolates responsible for nosocomial infections may contribute to the identification of dissemination routes and contribute to effective and targeted infection prevention guidelines.

OBJECTIVES
The major objectives will be the identification of antibiotic and biocide resistance profiles as well as the virulence potential of invasive MSSA isolates recovered in Portuguese hospitals between October 2022 and June 2023. In addition, molecular characterization, including whole genome analysis of the isolates, will provide information on major MSSA clonal lineages responsible for the increasing trend in invasive infections in Portugal.

BACKGROUND

Methicillin-resistant Staphylococcus aureus (MRSA) is a frequent cause of hospital-, community-, and livestock-associated infections and is listed, as a high priority pathogen by the World Health Organization (WHO). S. aureus is a versatile microbe that can evolve rapidly to adapt to new environmental conditions. Under antibiotic pressure, for instance, S. aureus can escape the killing action of antibiotics by entering in a tolerant, non-dividing physiological state, becoming undetectable to clinicians. This adaptive behavior does not imply a change in the level of resistance. This phenomenon may lead to misclassification of tolerant strains as resistant, or vice versa, resulting in ineffective treatments. Once the antibiotic is removed, tolerant bacteria can resume their growth and lead to recurrent infections. Recent in vitro and in vivo studies showed that intermittent antibiotic exposures, the most common therapy approach used to treat patients, lead to the rapid evolution of tolerance followed by emergence of resistance, stressing the possible threat to human health. In a previous study, we identified, through whole-genome sequencing (WGS), genes that may be involved in the tolerance phenotype in clinical methicillin-susceptible S. aureus isolates recovered from bloodstream infection samples. 

OBJECTIVES

The goal of this project is to produce proof for the concept that the identified genes may be involved on the β-lactam tolerance phenotype in S. aureus.

BACKGROUND

The Latency-Associated Nuclear Antigen (LANA) is a herpesvirus multifunctional protein responsible for tethering the viral DNA to the chromosome, ensuring maintenance and segregation of the viral genome during cell division. Besides its main role in viral maintenance, LANA also physically interacts with several host proteins (USP7 & Elongin BC) to modulate cell functions to maintain viral latency. 

OBJECTIVES

The project will focus on assessing the relationship between USP7 and Elongin BC on binding to LANA, from both KSHV and MHV-68 herpesviruses, using various biophysical techniques.

BACKGROUND

The Latency-Associated Nuclear Antigen (LANA) is a herpesvirus multifunctional protein responsible for tethering the viral DNA to the chromosome, ensuring maintenance and segregation of the viral genome during cell division. Besides its main role in viral maintenance, LANA also physically interacts with several host proteins (USP7 & Elongin BC) to modulate cell functions to maintain viral latency. 

OBJECTIVES

The project will focus on assessing the relationship between USP7 and Elongin BC on binding to LANA, from both KSHV and MHV-68 herpesviruses, using SEC, ITC and EMSA technqiues.

BACKGROUND

Latency-Associated Nuclear Antigen (LANA) is a herpesvirus multifunctional protein responsible for tethering viral DNA to the chromosome ensuring maintenance and segregation of the viral genome during cell division. LANA is a DNA binding protein and localizes to multiple terminal repeats (TR) DNA sites on the viral epigenome that assemble to form supramolecular dot structures. These LANA orchestrated assembles in KSHV-infected cells recruit heterochromatin components to the viral genome that modulate chromatin structure to ensure its persistence in the host.

OBJECTIVES

The project will use an integrative approach to explore and detail the LANA interactions, using recombinant DNA strategies (affinity tags, co-expression, design of protein truncations) and biophysical methods (ITC interaction analysis, structure determination and analysis), to characterise viral LANA protein interactions with host cell chromatin adaptor proteins. 

Suitable for those interested in protein interactions using biophysical methods.

In this project we will characterize silver nano-pillars (AgNPi), custom tailored in collaboration with Nanomaterial and nanotechnology group from Luxembourg Institute of Science and Technology, for construction of novel enzyme based biosensors. We will explore surface enhancement and electrochemical response of structurally and functionally different oxidoreductase enzymes attached to AgNPi, employing surface enhanced vibrational spectroscopy and electrochemistry.

As part of LegumBiome project, we plan to prepare the sampling 2023. During this sampling, we will collect soil, roots and seeds from wild, desert-adapted legumes in order to isolate their associated microbiota as a source of new biotreatments to help commercial legume varieties that are sensitive to drought and heat. The microbiota will be identified and characterized in order to select the most suitable to test them as treatment in greenhouses experiments.

We have to improve crop productivity In the context of climate change, with several stresses affecting plant growth. We work with state-of-the-art devices within our indoor and field phenotyping platforms to identify tolerant plants, high-yielding varieties and the metabolic mechanisms of interest for crop improvement.

We need to increase crop productivity to meet future food demands. This will be made even more difficult by the negative effects of climate change, so we need to understand the regulation of primary metabolism in crops of interest for the Mediterranian agriculture. Through field and greenhouse trials we analyse different metabolites of carbon and nitrogen metabolism, and antioxidant capacity of the plant to contribute to crop improvement.

The skin provides a critical barrier against bacterial infections. Topical antimicrobial treatments are commonly used to treat skin infections. They provide antimicrobial activity specifically against the causative agent, a high and continued drug concentration at the infected site, they have fewer systemic side effects
and reduce the development of antibiotic resistance. Nanostructured lipid carriers (NLCs) have gained significant attention as safe and biodegradable systems for the dermal drug delivery due to their stability, biocompatibility, and ability to improve drug bioavailability. The aim in this project is to characterize the most promising NLCs, to evaluate these systems for their ability to permeate the skin, both in vitro and in vivo; to understand the molecular mechanisms of NlCs permeation through human skin; and to investigate their antimicrobial capacity in vitro and in vivo.