Effects of sugars and lactic acid isomers on Chlamydia trachomatis infectivity, an in vitro study

The thesis investigates two different in vitro aspects of Chlamydia trachomatis (CT). The thesis analyzes the effect of different sugars on CT infectivity. which is investigated on HeLa cells after 2 hour-incubation of elementary bodies (EBs) with glucose, sucrose or mannitol. Sugars effect on EB membrane fluidity is investigated by fluorescence anisotropy measurement, whereas changes in lipopolysaccharide exposure are examined by cytofluorimetric analysis. By Western blot experiments, the phosphorylation state of Focal Adhesion Kinase in cells infected with EBs pre-incubated with sugars it’s explored. Sugar significantly increase infectivity, acting on the EB structure. Sugars induce an increase of EB membrane fluidity, leading to changes in LPS exposure. After incubation with sucrose and mannitol, EBs lead to higher FAK phosphorylation, enhancing activation of anti-apoptotic and proliferative signals in the host. Secondly, the thesis explores the protective effect of different Lactobacilli against CT infection: Lactobacillus crispatus and Lactobacillus reuteri. CT infectivity is evaluated after host cells were treated for 1 hour with diluted supernatant cell-free fraction or with the bacterial cells. Assessed that L.crispatus is more protective than L.reuteri, lactic acid production is evaluated by HPLC. Subsequently Lactate dehydrogenases activity is evaluated by resazurin assay and by LC-MS. Then, D-lactate dehydrogenase specific activity has been investigated by measuring NADH formation. Afterwards, addition of D or L-lactic acid to L.reuteri supernatant has been performed and their effect in promoting protection in the host cells assessed. Then a metabolic analysis has been carried out by real-time measurement of mitochondrial respiration after treatment. Finally, histone acetylation and lactylation, and gene and protein expression of relevant targets, have been investigated. It is shown that the D isomer is more efficient in conferring protection, causing a shift in the host cell metabolic profile and a pattern of histone modifications that changes the expression of important targets.

Protein-based nanomaterials as protein heterogeneous nucleants and structural studies on enzymes from two photosynthetic organisms

Proteins, the most essential biological macromolecules, are involved in nearly every aspect of life. The elucidation of their three-dimensional structures through X-ray analysis has significantly contributed to our understanding of fundamental mechanisms in life processes. However, the obstacle of obtaining high-resolution protein crystals remains significant. Thus, searching for materials that can effectively induce nucleation of crystals is a promising and active field. This thesis work characterizes and prepares albumin nanoparticles as heterogeneous nucleants for protein crystallization. These stable Bovine Serum Albumin nanoparticles were synthesized via the desolvation method, purified efficiently, and characterized in terms of dimension, morphology, and secondary structure. The ability of BSA-NPs to induce macromolecule nucleation was tested on three model proteins, exhibiting significant results, with larger NPs inducing more nucleation. The second part of this work focuses on the structural study, mainly through X-ray crystallography, of five chloroplast and cytosolic enzymes involved in the fundamental cellular processes of two photosynthetic organisms, Chlamydomonas reinhardtii and Arabidopsis thaliana. The structures of three enzymes involved in the Calvin-Benson-Bassham Cycle, phosphoribulokinase, troseposphatisomerase, and ribulosiophosphate epimerase from Chlamydomonas reinhardtii, were solved to investigate their catalytic and regulatory mechanisms. Additionally, the structure of nitrosylated-CrTPI made it possible to identify Cys14 as a target for nitrosylation, and the crystallographic structure of CrRPE was solved for the first time, providing insights into its catalytic and regulatory properties. Finally, the structure of S-nitrosoglutathione reductase, AtGSNOR, was compared with that of AtADH1, revealing differences in their catalytic sites. Overall, seven crystallographic structures, including partially oxidized CrPRK, CrPRK/ATP, CrPRK/ADP/Ru5P, CrTPI-nitrosylated, apo-CrRPE, apo-AtGSNOR, and AtADH1-NADH, were solved and are yet to be deposited in the PDB.

Effetto dell' ormone tiroideo (T3) durante ex vivo lung perfusion (EVLP) sul polmone di ratto: valutazione dell'azione protettiva nel danno da ischemia riperfusione.

La perfusione polmonare extracorporea (EVLP) è una tecnica utilizzata dal 2010 per valutare e migliorare la qualità dell'organo da trapiantare e il danno da ischemia-ripefusione (IRI). Tale perfusione utilizza la soluzione di Steen, la cui composizione è solo parzialmente nota. Lo scopo è quello di identificare gli effetti di T3 su IRI polmonare ex vivo, in un modello di ratto di donatore a cuore non battente. Animali (40) randomizzati in otto gruppi e il protocollo EVLP sono stati standardizzati nel nostro centro. Sono state valutate la funzione polmonare, PEEP, la resistenza vascolare polmonare totale a 45, 60, 120 e 180 minuti di EVLP per eseguire analisi di gas, dosaggio del mediatore di infiammazione, mitocondriale libero DNA, freeT3 e freeT4. Alla fine dei campioni di tessuto polmonare sono stati congelati dal dosaggio ATP, espressione genica, DNA mitocondriale, T3. Non date le concentrazioni del produttore, abbiamo analizzato gli acidi grassi liberi, vitamine, ormoni e composizione della soluzione Steen. Risultati La soluzione di steen contiene albumina umana x2 nel siero umano (7,5-8 g/dl): le concentrazioni di ft4 e ft3 sono x2 quelle nel siero umano e vengono rilasciati dall'albumina. La concentrazione di ft4 e ft3 non è cambiata durante l'EVLP. La Steen ha alta fluorescenza per l'alta concentrazione delle molecole aromatiche (ormoni) mai descritto in precedenza. NADH e mtDNA nel perfused aumenta con danno ischemico e nel gruppo trattato con T3 Conclusione Il modello EVLP è già convalidato nella perfusione nel trapianto polmonare, ma è necessario approfondire l'effetto della Steen in termini di ormoni e analiti. L'effetto sull'IRI dell'EVLP sembra essere influenzato negativamente da un T3 troppo alto in Steen, cosa che descriviamo per la prima volta. L'ulteriore aggiunta di T3 provoca disfunzione mitocondriale e infiammazione.