Our research revolves around in situ hybridization employing nucleic acid mimics. Hybridization processes, where a nucleic acid strand recognizes and binds to another strand, occur naturally inside cells and are crucial for their development and replication. Our interests range from the elucidation of the mechanistics of these biotechnological processes to the development and application of novel hybridization-based techniques to localize and treat microorganisms.
Mechanistics of biotechnological processes
Methods such as fluorescence in situ hybridization (FISH) are commonly used to track specific nucleic acid sequences inside the cells. Like everything in nature, these molecular methods still obey the “laws of science”. For instance, in FISH, a fluorescently-labeled nucleic acid has to diffuse through the extracellular space, cell envelope and cytoplasm before reaching its target, in what is essentially a mass transfer process. When in contact with the target, hybridization will only occur if the attractive forces outweigh the repulsive forces, in a process mainly driven by thermodynamics and, consequently, by temperature. In this topic we try to elucidate these processes combining experimental, mathematical and computational approaches1,2,3.
In vivo treatment
Development of hybridization-based techniques
As part of an Engineering faculty, we aim at developing novel techniques based on NAMs that can be of use for both research and the society. As examples of work being carried out so far, we have the detection of microorganisms directly within the human body (FIVH), the differentiation between more than 8 species simultaneously (CLASI-FISH), or the combination of FISH and microfluidics for the detection of microorganisms in the food industry without the need for pre-enrichment7,8,9.
Spatial location of microorganisms in biofilms
Biofilms are known as the cities of microbes due to the intricate mechanisms that govern their structure and activity. In order to better understand how biofilms behave, we employ FISH methods for the spatial location of individual cells in multispecies biofilms. Examples of studied multispecies biofilms include urinary catheters and cystic fibrosis10,11. In addition, we also develop minimum information guidelines to improve the reproducibility of biofilm experiments12.
1. Fontenete S., Guimarães N., Wengel J. and N.F. Azevedo (2016). “Prediction of melting temperatures in fluorescence in situ hybridization (FISH) procedures using thermodynamic models”. Critical Reviews in Biotechnology, 36(3):566-77.
2. Rocha, R., Almeida C. and N.F. Azevedo (2018). “Influence of the fixation/permeabilization step on peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) for the detection of bacteria”. Plos One, 13(5): e0196522.
3. Pérez Rodríguez G., Gameiro D., Pérez-Pérez M., Lourenço A. and N.F. Azevedo (2016). “Single Molecule Simulation of Diffusion and Enzyme Kinetics”. The Journal of Physical Chemistry B, 120(16):3809-20.
4. Lopes S.P., Azevedo N.F. and M.O. Pereira (2018). “Quantitative assessment of individual populations within polymicrobial biofilms”. Scientific Reports, 8, Article number: 9494.
5. Azevedo A.S., Almeida C., Melo L.F. and N.F. Azevedo (2017). “Impact of polymicrobial biofilms in catheter-associated urinary tract infections”. Critical Reviews in Microbiology, 43(4):423-439.
6. Lourenço A., Coenye T., Goeres D., Donelli G., Azevedo A.S., Ceri H., Coelho F.L., Flemming H.-C., Juhna T., Lopes S.P., Oliveira R., Oliver A., Shirtliff M.E., Sousa A.M., Stoodley P., Pereira M.O. and N.F. Azevedo (2014). “Minimum information about a biofilm experiment (MIABiE): standards for reporting experiments and data on sessile microbial communities living at interfaces”. Pathogens and Disease, 70(3):250-256.
7. Santos R.S., Dakwar G.R., Zagato E., Brans T., Figueiredo C., Raemdonck K., Azevedo N.F., De Smedt S.C., and K. Braeckmans (2017). “Intracellular delivery of oligonucleotides in Helicobacter pylori by fusogenic liposomes in the presence of gastric mucus”. Biomaterials, 138:1-12.
8. Lima J.F., Carvalho J., Ribeiro I.P., Almeida C., Wengel J., Cerqueira L., Figueiredo C., Oliveira C. and N.F. Azevedo (2018). “Targeting miR-9 in gastric cancer cells using Locked Nucleic Acid Oligonucleotides”. BMC Molecular Biology, 19(1):6.
9. Santos R.S., Figueiredo C., Azevedo N.F., Braeckmans K. and S.C. De Smedt (2018). “Nanomaterials and molecular transporters to overcome the bacterial envelope barrier: towards advanced delivery of antibiotics”. Advanced Drug Delivery Reviews, 136-137:28-48.
10. Müller V., Sousa J.M., Koydemir H.C., Veli M., Tseng D., Cerqueira L., Ozcan A., Azevedo N. F. and F. Westerlund (2018). “Identification of pathogenic bacteria in complex samples using a smartphone based fluorescence microscope”, RSC Advances, 8(64):36493-36502.
11. Fontenete S., Leite M., Cappoen D., Santos R., Figueiredo C., Wengel J., Cos P. and N.F. Azevedo (2016). “Fluorescence in vivo hybridization (FIVH) for detection of Helicobacter pylori infection in a C57BL/6 mouse model”. PLoS ONE, 11(2):e0148353.
12. Ferreira A.M., Cruz-Moreira D., Cerqueira L., Miranda J.M. and N.F. Azevedo (2017). “Yeasts identification in microfluidic devices using peptide nucleic acid fluorescence in situ hybridization (PNA-FISH)”. Biomedical Microdevices, 19(1):art11.