Interrogating Cancer Metabolism using Novel Imaging Techniques

Altered metabolism is one of the hallmarks of cancer and is known to enable the proliferation and survival of cancer cells in changing microenvironments. Metabolic changes can be interrogated using non-invasive metabolic imaging techniques, which enable the investigation of tumour metabolism in vivo and the identification of imaging biomarkers. Hyperpolarised 13C Magnetic Resonance Spectroscopic Imaging (MRSI) is a metabolic imaging technique that enables dynamic imaging of isotopically labelled metabolites in vivo, with [1-13C]pyruvate being the most widely used substrate in pre-clinical studies and the only substrate used so far in clinical studies. In the first part of this thesis, hyperpolarised 13C pyruvate MRSI was directly compared to mass spectrometry imaging in a murine lymphoma model, cross-validating both methods for providing information on the relative spatial distribution of 13C-labelled lactate. In the second part, the potential of hyperpolarised [1-13C]Ketoisocaproic Acid as a substrate for hyperpolarised imaging was investigated. Ketoisocaproic acid is a substrate for Branched Chain Aminotransferase 1 (BCAT1), the first enzyme in the catabolic pathway of Branched Chain Amino Acids. This enzyme is upregulated in many cancers and is linked to cancer progression. In this project, the role of BCAT1 in cell proliferation was confirmed in glioblastoma patient derived cells and tumours and BCAT1 was shown to regulate the cells’ metabolic and transcriptional profiles through regulation of alpha-ketoglutarate metabolism. In vivo imaging of BCAT1 activity is therefore suggested as a potential technique for patient stratification and potentially treatment monitoring. [1-13C]Ketoisocaproic acid was successfully hyperpolarised and showed fast uptake into subcutaneous EL4 lymphoma tumours, healthy rat brain and orthotopic gliomas in rat brain, following intravenous injection. Sufficient 13C leucine signal was generated in the lymphoma tumour models enabling in vivo imaging of BCAT activity but the reaction was found to be significantly slower in the healthy rat brain and in the glioma tumours. This was explained by low levels of glutamate, a co-substrate for the reaction, in the glioma tumours, which is a potential limiting factor for this reaction.Cancer Research UK Rosetrees Trus

A multi spin echo pulse sequence with optimized excitation pulses and a 3D cone readout for hyperpolarized 13 C imaging.

PURPOSE: Imaging tumor metabolism in vivo using hyperpolarized [1-13 C]pyruvate is a promising technique for detecting disease, monitoring disease progression, and assessing treatment response. However, the transient nature of the hyperpolarization and its depletion following excitation limits the available time for imaging. We describe here a single-shot multi spin echo sequence, which improves on previously reported sequences, with a shorter readout time, isotropic point spread function (PSF), and better signal-to-noise ratio. METHODS: The sequence uses numerically optimized spectrally selective excitation pulses set to the resonant frequencies of pyruvate and lactate and a hyperbolic secant adiabatic refocusing pulse, all applied in the absence of slice selection gradients. The excitation pulses were designed to be resistant to the effects of B0 and B1 field inhomogeneity. The gradient readout uses a 3D cone trajectory composed of 13 cones, all fully refocused and distributed among 7 spin echoes. The maximal gradient amplitude and slew rate were set to 4 G/cm and 20 G/cm/ms, respectively, to demonstrate the feasibility of clinical translation. RESULTS: The pulse sequence gave an isotropic PSF of 2.8 mm. The excitation profiles of the optimized pulses closely matched simulations and a 46.10 ± 0.04% gain in image SNR was observed compared to a conventional Shinnar-Le Roux excitation pulse. The sequence was demonstrated with dynamic imaging of hyperpolarized [1-13 C]pyruvate and [1-13 C]lactate in vivo. CONCLUSION: The pulse sequence was capable of dynamic imaging of hyperpolarized 13 C labeled metabolites in vivo with relatively high spatial and temporal resolution and immunity to system imperfections

Dual-light antibacterial photodynamic therapy in peri-implant mucositis treatment

Introducere. Relația existentă între acumularea biofilmului în jurul implanturilor dentare și dezvoltarea unei afecțiuni inflamatorii a fost demonstrată în diverse studii pre-clinice și clinice.În cazul pacienților cu mucozită peri-implantară, ghidul de practică clinică de nivel S3 al EFP sugerează să nu se utilizeze agenți administrați local (antiseptice, postbiotice, gel desicant) sau terapia fotodinamică, ca metode adjuvante la îndepărtarea mecanică profesională a plăcii bacteriene (PMPR). În schimb, se poate lua în considerare utilizarea unei autoadministrări limitate în timp a antisepticelor prin clătituri orale (pe bază de clorhexidină și pe bază de plante) sau autoadministrarea probioticelor ghidată de un medic. În studiile anterioare, unele antiseptice au fost asociate cu efecte secundare nedorite, cum ar fi senzația de anestezie tranzitorie la nivelul mucoasei orale sau niveluri sporite de colorare a dinților sau a limbii. Anterior, terapia fotodinamică antibacteriană (aPDT) a fost utilizată în principal în cabinetele stomatologice, iar utilizările rare, atunci când a fost aplicată rar (1-4 ori pe an), de obicei nu a determinat o îmbunătățire a indicilor clinici. Recent, aPDT pe bază de LED a fost dezvoltată, facilitând utilizarea repetată și practică la domiciliu.Scopul lucrării. Acest studiu a avut ca scop determinarea rezultatelor clinice obținute cu terapia fotodinamică antibacteriană regulată în tratamentul mucozitei peri-implantare. Material și metode. După aplicarea criteriilor de includere și excludere, 29 de pacienți au fost incluși în studiu. Fiecare dintre ei avea cel puțin 1 implant diagnosticat cu mucozită peri-implantară. Pacienții au fost repartizați aleatoriu în 2 grupuri: Grupul de studiu (GS) - 14 pacienți, și Grupul de control (GC) - 15 pacienți. Pacienții din GS, pe lângă instrumentarea submarginală, au primit tratament repetat cu aPDT. În cadrul studiului s-a folosit un dispozitiv medical care conținea apă de gură Lumorinse® activată cu ajutorul luminii și un activator luminos Lumoral®. Variabila de rezultat primar a fost reducerea adâncimii de sondare periimplantară (PiPD), în timp ce variabilele de rezultat secundare au fost modificările indicelui de sângerare la sondare (mBOP) și ale indicelui de placă modificat (mPI). Indicii clinici au fost evaluați inițial și la 2 luni. Rezultate. Toți cei 29 de pacienți incluși în studiu au finalizat studiul. În GS și, respectiv, GC, rezultatele au fost următoarele: PiPD de 5,3±0,6 mm și 5,4±0,5 mm inițial, iar după 2 luni de 4,1±0,5 mm și respectiv 4,2±0,7 mm (p >0,05), mPI a fost mai redus în grupul GS decât în GC (p <0,05), iar mBoP a fost mai mic în GS comparativ cu GC (p <0,05). Concluzii. Pacienții din grupul de control au prezentat rezultate bune ale indicilor mPI și mBoP. Tratamentul repetat cu aPDT la domiciliu poate îmbunătăți igiena orală și rezultatele tratamentului mucozitei peri-implantare. Sunt necesare studii suplimentare pentru a valida rezultatele actuale.Background. The link between biofilm accumulation around dental implants and the development of an inflammatory disease around them has been demonstrated in classical pre-clinical and clinical studies. In patients with peri-implant mucositis, the EFP S3 level clinical practice guideline suggest not to use locally administered agents (antiseptics, postbiotics, desiccant gel) or photodynamic therapy, as adjunct methods to professional mechanical plaque removal (PMPR). Instead, the use of a time limited self-administration of oral rinse antiseptics (chlorhexidine and herbal-based) or the professionally guided self-administration of probiotics may be considered. In previous studies, some antiseptics have been associated with undesirable side effects, such as transient anaesthetic sensation in the oral mucosa or higher levels of staining of the teeth or tongue. Previously, antibacterial photodynamic therapy (aPDT) has been used mainly in dental offices practice and rare aplications (1-4 times per year ) usually did not improve the results. Recently, LED based aPDT has been developed allowing repeated and practical use at home.Objective of the study. This study aimed to determine the clinical outcomes obtained with regular antibacterial photodynamic therapy in peri-implant mucositis treatment. Materials and methods. After applying inclusion and exclusion criteria, 29 patients were enrolled in the study. Each of them had at least 1 implant diagnosed with mucositis. Patients were randomized into 2 groups: Study Group (GS) – 14 patients, and Control Group (GC) – 15 patients. Patients from GS, besides submarginal instrumentation, received repeated aPDT treatment. The study used a medical device containing a light-activated Lumorinse® mouthwash and a Lumoral® light activator. The primary outcome variable was peri-implant probing depth reduction (PiPD), while the secondary outcome variables were changes in bleeding on probing (mBOP) and plaque index (mPI). Clinical parameters were assessed at the beginning and after 2 months. Results. All 29 patients included in the study finalized the study. In GS and GC respectively, the results were: PiPD of 5.3±0.6 mm and 5.4±0.5 mm initially, and after 2 months 4.1±0.5 mm and 4.2±0.7 mm respectively (p >0.05), mPI was more reduced in GS group than GC (p < 0.05), and mBoP was lower in GS comparing to GC (p < 0.05). Conclusion. Patients from the control group showed better results on mPI and mBoP indices. Repeated aPDT treatment at home can improve oral hygiene and the results of peri-implant mucositis treatment. Additional studies are required to validate the current results

Mechanical modification of the implant surface as an adjunctive measure during surgical treatment of peri-implantitis

Introducere. Peri-implantita a fost definită ca o condiție patologică asociată cu biofilmul, care afectează țesuturile din jurul implanturilor dentare și se caracterizează prin inflamație în mucoasa peri-implantară și prin resorbția progresivă a țesutului osos de suport. Numeroase abordări chirurgicale, inclusiv chirurgia cu lambou de acces și tehnici de rezecție sau de augmentare, au fost propuse anterior în tratamentul peri-implantitei. Modificarea mecanică a suprafeței implantului a fost propusă ca o măsură adjuvantă în timpul tratamentului chirurgical al periimplantitei. Implantoplastia este o procedură bazată pe îndepărtarea mecanică a firelor implantului pentru a crea o suprafață netedă, mai puțin susceptibilă la acumularea plăcii și la reinfecție.Scopul lucrării. Scopul acestui studiu a fost de a evalua eficacitatea clinică a modificării mecanice a suprafeței implantului ca măsură adjuvantă în timpul tratamentului chirurgical rezectiv al peri-implantitei. Material și metode. Peri-implantita a fost definită la implanturile cu o resorbție osoasă radiologică ≥3 mm și/sau adâncimea pungii ≥6mm, împreună cu sângerarea la sondare.12 pacienți cu peri-implantită au fost incluși în studiu. Grupul de control (CG, n=6) a beneficiat de tratament chirurgical rezectiv cu implantoplastie, în timp ce grupul de testare (TG, n=6) a fost supus aceluiași tratament folosind perii rotative din titan pentru decontaminare. Instrumentele din carbură de tungsten au fost utilizate pentru implantoplastie în timpul tratamentului chirurgical al periimplantitei. Debridarea mecanică a suprafețelor implanturilor a fost precedată de aplicarea de agenți antibacterieni, cum ar fi gluconatul de clorhexidină 0,05 % și peroxidul de hidrogen 3% Au fost analizate următoarele variabile: adâncimea de sondare peri-implantară (PiPD), indicele desângerarea la sondare modificat (mBoP), indicele de placă modificat (mPI), supurația (SUP) și recesiunea (REC). Rezultate. Toți subiecții au fost supuși anterior tratamentului non-chirurgical. La controlul de 3 luni, ambele grupuri au prezentat ameliorări ale variabilelor clinice. Reducerea indicelui clinic mBoP a fost similară între grupul de studiu și grupul de control (p > 0,05). Cu toate acestea, grupul GC a demonstrat o reducere mai mare, semnificativă din punct de vedere statistic, a indicilor mPI și PiPD (p < 0,05). Nu a existat nicio diferență semnificativă statistic în ceea ce privește indicele de supurație între cele două grupuri (p > 0,05). Situsurile tratate prin implantoplastie au evidențiat o recesiune semnificativ mai mare a țesuturilor moi (CG: 1,9±0,4 mm; TG: 1,1±0,35 mm; p < 0,05) și, în consecință, o expunere accentuată a suprafeței implantului. Concluzii. Pacienții din grupul de control au înregistrat rezultate mai bune ale indicilor mPI și PiPD. Ambele metode au dus la obținerea unor condiții stabile, cu o ameliorare semnificativă a indicilor clinici. Implantoplastia este mai potrivită pentru zonele laterale. Șlefuirea suprafeței implantului este indicată în situsurile peri-implantare la care nu se preconizează regenerarea osoasă.Background. Peri-implantitis has been defined as a biofilm-associated pathological condition, occurring in tissues around dental implants, and characterized by inflammation in the peri-implant mucosa and subsequent progressive loss of supporting bone. Numerous surgical approaches, including access flap surgery, and resective or augmentative techniques, have been proposed previously in order to treat peri-implantitis. The mechanical modification of the implant surface has been proposed as an adjunctive measure during surgical treatment of peri-implantitis. Implantoplasty is a procedure based on the mechanical removal of implant threads to create a smooth surface that is less predisposed to plaque accumulation and reinfection.Objective of the study. This study aims to evaluate the clinical efficacy of mechanical modification of the implant surface as an adjuvant measure during resective surgical treatment of peri-implantitis. Material and Methods: Peri-implantitis was defined as radiographic bone loss ≥ 3mm and/or probing depths ≥ 6mm, followed by profuse bleeding. 12 patients with peri-implantitis were included in the study. The control group (CG, n=6) received resective surgical treatment with implantoplasty, while the test group (TG, n=6) received the same treatment using rotating titanium brushes for decontamination. Tungsten carbide instruments were used for implantoplasty on titanium during the surgical treatment of peri-implantitis. Mechanical debridement of implant surfaces was preceded by the application of antibacterial and chemical agents, such as 0,05 % chlorhexidine gluconate and 3% hydrogen peroxide The following outcome variables were assessed: peri-implant probing depth (PiPD), modified bleeding on probing (mBoP), modified plaque index (mPI), suppuration (SUP) and recession (REC). Results. All subjects had previously received submarginal instrumentation. At the 3-month follow-up, both groups showed clinical improvements. The reduction in clinical index, mBoP were quite similar between the study and control groups (p > 0.05). However, the GC group demonstrated a statistically significant greater reduction of the indices mPI and PiPD (p < 0.05). There was no statistically significant difference in suppuration index between the two groups (p > 0.05). The sites treated with implantoplasty revealed significantly more soft-tissue recession (CG: 1.9±0.4 mm; TG: 1.1±0.35 mm; p < 0.05) and consequently marked exposure of the implant surface. Conclusions. Patients from the control group showed better results on mPI and PiPD indices. Both methods resulted in stable conditions, with high improvement in clinical indices. Implantoplasty is more suitable for lateral areas. Implant surface polishing is indicated at implant sites where no bone regeneration is expected

Review of the scientific literature to analyse the cariogenic potential of the diet of military personnel on active duty

Introducere. În 2021, Organizația Mondială a Sănătății a adoptat o rezoluție privind sănătatea orală ca răspuns la prevalența ridicată a bolilor orale la nivel global. Se estimează că 3,5 miliarde de persoane suferă de boli orale, cele mai frecvente fiind caria dentară și boala parodontală. Rezoluția solicită statelor membre ale ONU să ia măsuri pentru a atenua factorii de risc pentru bolile orale. În timp ce în rândul populației generale preocuparea comunității științifice se reflectă într-o disponibilitate crescută a datelor privind sănătatea orală, în rândul militarilor numărul de studii științifice este limitat. Sănătatea orală este considerată a fi o componentă importantă a sănătății generale, cu impact asupra capacității personalului de a-și îndeplini sarcinile în mod corespunzător. Tiparele alimentare care cresc riscul de carii dentare includ consumul constant și pe termen lung de alimente care conțin zaharuri, alimente lipicioase și băuturi zaharoase. Revizuirea literaturii de specialitate a arătat că nu există date disponibile cu privire la starea de sănătate orală a personalului militar activ din Republica Moldova.Scop. Analiza literaturii științifice disponibile pentru a determina influența dietei cu potențial cariogen asupra sănătății orale a personalului militar. Materiale și metode. Datele au fost colectate folosind termenii de căutare (MESH) despre potențialul cariogenic al dietei la personalul militar - Pubmed, MEDLINE. Rezultate. Analiza literaturii științifice a constatat o prevalență crescută a cariilor dentare la personalul militar care a consumat în mod constant produse bogate în carbohidrați. Conform studiului propus de Tudoronion et al pe 2020 dieta bogată în carbohidrați a dus la un procent mai mare de subiecți diagnosticați cu carii dentare 70,9%, comparativ cu cei care nu prezentau semne clinice de carie dentară 29,1% . Concluzii și strategii de atenuare. Diferite studii au arătat că o creștere a procentului de consum de carbohidrați în raport cu energia zilnică totală crește prevalența cariei dentare, ceea ce este în concordanță cu rezultatele multor studii anterioare. OMS a recomandat un consum de < 10% de carbohidrați pentru a preveni cariile dentare.Introduction. In 2021 the World Health Organization adopted a resolution on oral health in response to the high prevalence of oral diseases globally. It is estimated that 3.5 billion people suffering from oral diseases, the most common being dental caries and periodontal diseases. It requires UN member states to take action to mitigate the risk factors for oral diseases. While in the general population the concern of the scientific community is reflected in an increased availability of oral health data, in the military the number of scientific studies is limited. Oral health is considered to be an important component of general health, with an impact on the ability of personnel to perform their duties properly. Dietary patterns that increase the risk of dental caries include consistent and long-term consumption of foods containing sugars, sticky foods, and sugary drinks. The literature review revealed that there is no data available regarding oral health status of active military personnel in the Republic of Moldova.Objective of the study. Analysis of available scientific literature to determine the influence of diet with cariogenic potential on oral health of military personnel. Materials and methods. Data was collected using search terms (MESH) about the cariogenic potential of diet in military personnel – Pubmed, MEDLINE. Results. A review of the scientific literature found an increased prevalence of dental caries in military personnel who consistently consumed high carbohydrate products. According to the study proposed by Tudoronion et al on 2020 high carbohydrate diet resulted in a higher percentage of subjects diagnosed with dental caries 70.9%, compared to those who did not show clinical signs of dental caries 29.1% . Conclusion and mitigation strategies. Different studies showed that an increase in the percentage of carbohydrate consumption relative to the total daily energy increases the prevalence of dental caries, which is consistent with the results of many previous studies. The WHO has recommended < 10% consumption of sugars to prevent dental caries. In contrast to many other studies indicating the anti-caries properties of fruits, increasing fruit consumption during the day increased susceptibility to caries

Magnetic Resonance Imaging Is More Sensitive Than PET for Detecting Treatment-Induced Cell Death-Dependent Changes in Glycolysis.

Metabolic imaging has been widely used to measure the early responses of tumors to treatment. Here, we assess the abilities of PET measurement of [18F]FDG uptake and MRI measurement of hyperpolarized [1-13C]pyruvate metabolism to detect early changes in glycolysis following treatment-induced cell death in human colorectal (Colo205) and breast adenocarcinoma (MDA-MB-231) xenografts in mice. A TRAIL agonist that binds to human but not mouse cells induced tumor-selective cell death. Tumor glycolysis was assessed by injecting [1,6-13C2]glucose and measuring 13C-labeled metabolites in tumor extracts. Injection of hyperpolarized [1-13C]pyruvate induced rapid reduction in lactate labeling. This decrease, which correlated with an increase in histologic markers of cell death and preceded decrease in tumor volume, reflected reduced flux from glucose to lactate and decreased lactate concentration. However, [18F]FDG uptake and phosphorylation were maintained following treatment, which has been attributed previously to increased [18F]FDG uptake by infiltrating immune cells. Quantification of [18F]FDG uptake in flow-sorted tumor and immune cells from disaggregated tumors identified CD11b+/CD45+ macrophages as the most [18F]FDG-avid cell type present, yet they represented <5% of the cells present in the tumors and could not explain the failure of [18F]FDG-PET to detect treatment response. MRI measurement of hyperpolarized [1-13C]pyruvate metabolism is therefore a more sensitive marker of the early decreases in glycolytic flux that occur following cell death than PET measurements of [18F]FDG uptake. SIGNIFICANCE: These findings demonstrate superior sensitivity of MRI measurement of hyperpolarized [1-13C]pyruvate metabolism versus PET measurement of 18F-FDG uptake for detecting early changes in glycolysis following treatment-induced tumor cell death

Heterologous expression and purification of the hypoxia-induced factor HIF-1 human aiming structural and biochemical studies and structural studies of prolyl-hydroxylases (PHDs) human, isoforms 1 and 3, in complex with inhibitors

Orientador: Andre Luís Berteli AmbrósioDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: A adaptação das células cancerosas ao microambiente é o ponto central que leva ao fenótipo invasivo e metastático, e é garantida principalmente através do controle preciso da expressão gênica. A resposta às necessidades energéticas e biossintéticas e principalmente à disponibilidade de oxigênio intracelular, por exemplo, é em grande parte mediada pelo fator de transcrição induzido por hipóxia 1 (HIF-1). HIF-1 é um heterodímero composto pelas subunidades ? e ?, que respondem a sequência consenso (5'-RCGTG-3') e ativam a transcrição de mais de 100 genes envolvidos em diversos aspectos cruciais da biologia tumoral, incluindo angiogênese, metabolismo de glicose, diferenciação celular, apoptose e resistência a radio e quimioterapias. São conhecidas três isoformas da subunidade ? (1 a 3) e todas se heterodimerizam com a subunidade ?. No geral, HIFs são constituídas de diferentes domínios funcionais, como de ligação ao DNA, de heterodimerização, transativação e degradação. Atualmente, pouco se sabe sobre os mecanismos estruturais e funcionais dos domínios da HIF- 1, deste modo este trabalho objetivou o estudo estrutural destes domínios. Os domínios bHLH, Pas-1 e Pac de HIF-1? e HIF-? em diferentes combinações entre si e o domínio Pac da HIF-3? foram clonados, as proteínas foram expressas em sistema bacteriano e purificadas por diferentes técnicas cromatográficas. Diversas destas construções se mostraram insolúveis ou suscetíveis a degradação, enquanto outras foram purificadas com sucesso. As construções Pac, um exemplo de sucesso na produção, foram submetidas a ensaios de anisotropia de fluorescência e ressonância magnética nuclear, o que nos permitiu a caracterização dos perfis de interação entre as várias combinações de heterodimerização. Neste contexto, os resultados mostram que o equilíbrio dinâmico da interação entre Pac-1? com a subunidade -1? é alcançado imediatamente, enquanto que para a interação entre Pac-3? e -1?, são necessários pelo menos 30 horas de incubação. O mesmo pode ser extraído da caracterização da interação direta entre Pac-1? e Pac-3?. Nos experimentos de RMN, foi possível identificar a região de interação entre as subunidades -1? e -3? com a subunidade ?, separadamente. Ambas as subunidades ? interagem com a Pac-1? na região das fitas-beta 1 e 5 e no loop entre as fitas 4 e 5. Em conjunto, estes resultados impactam no mecanismo de antagonização de HIF-3? na atividade transcricional de HIF-1?. Houve ainda a formação de monocristais da subunidade Pac-3?, que foram submetidos a experimentos preliminares de difração de raios X, que apesar de resultar em dados anisotrópicos e insuficientes para resolução estrutural, permitiram a caracterização dos parâmetros cristalinos, incluindo a presença de um alto conteúdo de solvente. Adicionalmente, são também apresentados os resultados obtidos visando a expressão e cristalização das Prolilhidroxilases (PHDs) isoformas 1 a 4, durante estágio de seis meses no Structural Genomics Consortium (SGC), da Universidade de Oxford, na Inglaterra. Foram expressas de maneira solúvel e purificadas, diversas construções das isoformas 1 e 3 das PHDs humanas. Cristais foram obtidos, porém estes foram determinados como sendo de compostos inorgânicos presentes na condição de cristalização. Como resultado final, está sendo estabelecida uma colaboração entre o nosso grupo e o SGC para que os estudos estruturais com PHDs se estendam e sejam realizados em nosso laboratório aqui no BrasilAbstract: The adaptation process of cancer cells to the microenvironment is the central point leading to the invasive and metastatic phenotypes, and is guaranteed mainly through the precise control of gene expression. The cell response to the energetic and biosynthetic needs and especially to the availability of intracellular oxygen is mediated by the hypoxia inducible transcription factor 1, or HIF-1. HIF-1 functions as a heterodimer composed by subunits ? and ?, binding to responsive elements with the consensus sequence 5'-RCGTG-3 ', thus activating the transcription of more than 100 genes involved in many crucial aspects of tumor biology, including angiogenesis, metabolism glucose, cell differentiation, apoptosis, and resistance to radiotherapy and chemotherapy. There are three known isoforms of the ? subunit (1, 2 and 3) and all heterodimerize with the ? subunit. HIFs are composed of different functional domains, such as the DNA binding domain, the heterodimerization, transactivation and the oxygen-dependent degradation domains. Currently, little is known about the mechanisms of structural and functional domains of HIF-1, thus this work was to study these structural domains. The domain (bHLH, Pas-1 and Pac) of HIF-1? and HIF-? in different combinations with each other and Pac domain of HIF-3? were cloned, the proteins were expressed in bacterial system and purified by various chromatographic techniques. Several of these constructs proved insoluble or susceptible to degradation, while others were purified successfully. The constructs Pac, an example of success in production, were tested for fluorescence anisotropy and nuclear magnetic resonance, which allowed us to characterize the profiles of the interaction between the various combinations of heterodimers. In this context, the results show that the dynamic equilibrium of the interaction between the Pac-1? and -1? subunits is achieved immediately, whereas for the interaction between Pac-3? and -1?, it takes at least 30 hours of incubation. The same can be observed from the characterization of direct interaction between Pac-1? and -3?. From the NMR experiments, it was possible to identify the region of interaction between the subunits -1? and -3? with the -1? subunit. Both ? subunits interact with Pac-1? via betastrands 1 and 5 and the loop between the strands 4 and 5. Overall, these results impact in the mechanism of HIF-3? antagonizing the transcriptional activity of HIF-1?. We also obtained single crystals for Pac-3? subunit, which were subjected to preliminary experiments of X-ray diffraction. Although resulting in anisotropic, insufficient data for and structural resolution, it has allowed the characterization of crystalline parameters, including the presence of a high solvent content. Additionally, we also present the results targeting the expression and crystallization of Prolyl-hydroxylases (PHDs) human isoforms 1-4, during the six-month period at the Structural Genomics Consortium (SGC), the University of Oxford in England. Several construct from of isoforms 1 and 3 were successfully expressed and purified in the soluble form. Likewise, crystals were obtained, but these were determined to be composed by inorganic compounds present in the crystallization conditions. At the end, a collaboration was established between our and the SGC group for the structural studies with the PHDs to extend and carry out the experiments in our lab here in BrazilMestradoClinica MedicaMestra em Clínica Médic

HIF-3alpha : structural studies, identification and characterization of a high-affinity ligand of hypoxia inducible factor (HIF) and structural studies of prolil-hydroxilases (PHDs) from non usual organisms

Orientador: Andre Luis Berteli AmbrosioTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: HIFs (Hypoxia inducible factors) são fatores de transcrição multidomínio, formados pelo heterodímero entre as subunidades alfa e beta. São conhecidas três isoformas para a subunidade alfa (HIF-1alfa, HIF-2alfa e HIF-3alfa), sob condições de normóxia todas as isoformas alfa sofrem regulação pós traducional sendo reguladas por uma família de hidroxilases, as PHDs (Prolil hidroxilases), as quais hidroxilam dois resíduos de prolina presente no domínio ODDD (domínio dependente de oxigênio), e subsequente levando a poli ubiquitinação destinando as subunidades alfa para degradação via proteossomo. As PHDs são hidroxilases dependentes de oxigênio, das quais são conhecidas quatro isoformas, e todas são capazes de hidroxilar as subunidades alfa das HIF e são, portanto, seu principal regulador. O complexo HIF-alfa e HIF-beta podem regular a transcrição de mais de 100 genes, dos quais muitos estão envolvidos no desenvolvimento e progressão do câncer, como a regulação de genes envolvidos em angiogênese, eritropoiese, proliferação celular e apoptose. A HIF-3alfa foi a última isoforma a ser descoberta entre as subunidades ?, e ela é distinta de várias maneiras em comparação às demais. Primeiro, o gene de codificação para HIF-3alfa (HIF3A) é regulado pela HIF-1alfa e é exclusivamente sujeito a splicing alternativo, com seis isoformas confirmadas em níveis proteicos, das quais o único domínio comum entre todas elas é o domínio Per-ARNT-Sim (PASb), que é responsável pela heterodimerização com a HIF-beta. Além disso, a ausência completa do domínio C-terminal (CTAD) leva a isoforma a ser a menor entre as isoformas alfa. Dada a importância das HIFs na biologia e progressão do câncer os objetivos deste trabalho foram estudar o domínio PASb da HIF-3alfa humana e as prolil hidroxilases de organismos não usuais. Objetivamos ainda produzir e realizar ensaios estruturais com a isoforma PASb-3alfa4 da HIF-3alfa, porém após diversas tentativas de expressão e purificação, a proteína continuou na forma insolúvel o que impossibilitou a continuidade dos estudos. Desta forma, apresentamos a estrutura cristalográfica do domínio PASb de HIF-3alfa na resolução de 1,15 Å, na qual identificamos uma cavidade hidrofóbica volumosa exclusiva com 510Å3 ligada ao ácido graxo insaturado cis-vaccenico de 18 carbonos como um inesperado e novo cofator intracelular para HIF-3alfa. Ensaios in vitro mostraram que a proteína é capaz de se ligar a diferentes tipos de lipídeos, os quais foram submetidos à análise por espectrometria de massas através da extração da fase orgânica da amostra. Foram identificados dois fosfolípideos como sendo uma liso-fosfoetanolamina 1-(11Z-octadecenol)-2-hidroxil-sn-glicero-3-fosfoetanolamina (18: 1-LPE) e Liso-fosfoglicerol 1-(11Z-octadecenoil) -2-hidroxil-sn-glicerol-3-fosfo- (1'-sn-glicerol) (18:1-LPG). De modo a explorar a cavidade hidrofóbica e identificar outros lipídeos que possam se ligar a proteína, realizamos ensaio de delipidação e titulação de ácidos graxos livres com diferentes comprimentos da cadeia carbônica e níveis de insaturação, que pudessem estar presentes no organismo humano. Como resultado observamos uma alta afinidade com ácidos graxos oleico (18:1) e linoleico (18: 2) com kd de 39±1nM e 0,8±0,2 nM respectivamente. Estes resultados evidenciam que esta é a primeira vez que um domínio PAS eucariótico pode servir como um sensor para os níveis intracelulares de lipídeos, possivelmente ditando a expressão do gene dependente de HIF-3alfa em resposta a resultados biossintéticos e bioenergéticos, tais como acumulação ou depleção de lipídeos, em exposição à hipóxia. Apresentamos também os resultados da colaboração com o Dr. Christopher Schofield e seu grupo de trabalho (Chemistry Department-Oxford University), através de estudos estruturais das prolil hidroxilases (PHD1, 2 e 3) para os quais foram utilizados os genes presentes em organismos não convencionais, como morsa, ornitorrinco e python que apresentaram similaridade relativa em relação às hidroxilases humanas. Foram selecionados oito organismos, dos quais obtivemos proteínas solúveis para seis deles. Duas isoformas para a PHD3 (genes de ornitorrinco e python) se mostraram mais estáveis e foram purificadas com alto grau de pureza. Ambas as proteínas foram submetidas a teste de atividade enzimática utilizando peptídeos sintéticos da HIF-1?, onde observamos que as enzimas são ativas e hidroxilam preferencialmente à prolina contida no domínio CODD da HIF. Também foram realizados ensaios de biologia estrutural através da cristalização da PHD3 de python, o qual inicialmente apresentou a formação de esferulitas e após algumas otimizações, não obtivemos cristais únicos. Para as demais PHDs que foram obtidas na forma solúvel diversas otimizações nos protocolos de purificação tiveram de ser realizadas a fim de obter amostras mais estáveis e promissoras para os ensaios de cristalização e atividade enzimáticaAbstract: HIFs (Hypoxia-inducible-factors) are heterodimers formed by subunits alpha and beta that regulate the transcription of more than 100 genes, of which are involved in the development and progression of cancer. Three isoforms for the alpha-subunit are known, and under normoxia conditions, they are regulated by PHDs (Prolyl hydroxylases) which lead to the hydroxylation of two residues of proline and subsequent assignment for degradation via the proteosome. PHDs are dioxygenases and the master regulator for HIFs. There are known four isoforms and all of them can hydroxylate the alpha subunits of HIF under normoxia. The complex of HIF-alpha:HIF-beta can regulate the transcription of more than 100 genes, many of them are involved in the development and progression of cancer, such as the regulation of genes involved in angiogenesis, erythropoiesis, cell proliferation and apoptosis. The subunit -3alpha is the last addition to the HIF-alpha class subunits (HIF-3alpha) and a distinctive one in a number of ways. Firstly, the gene encoding for HIF-3alpha (HIF3A) is itself under regulation of HIF-1alpha and exclusively subject to alternative splicing, with six isoforms confirmed by protein levels so far. Besides the complete absence of C-terminal (CTAD), HIF-3alpha is considered the short isoform in comparison with HIF-1alpha and -2alpha. Secondly, the only common domain between all -3alpha isoforms is the C-terminal Per-ARNT-Sim sensor domain (PASb), which is responsible for heterodimerization with HIF-beta. The aims of this project were to study the PASb domain of human HIF-3alpha and the prolyl hydroxylases of unusual organisms. We also intend to produce and perform structural assays with the PASb-3alpha4 isoform of HIF-3alpha, but after several attempts at expression and purification, the protein continued in the insoluble form, which made it impossible to continue the studies. Here we report the crystal structure of the PASb domain of HIF-3alpha at 1.15 Å maximum resolution and we identify an exclusive bulky hydrophobic cavity with 510Å3, bound to the unsaturated 18-carbon-long fatty acids as an unexpected and novel high-affinity intracellular cofactors of HIF-3alpha. The lipids were extracted from the samples and two phospholipids were identified by mass spectrometry as a Lysophosphoethanolamine 1-(11Z-octadecenoul)-2-hydroxil-sn-glycero-3-phosphoethanolamine (18:1-LPE) and Lysophosphoglycerol 1-(11Z-octadecenoyl)-2-hydroxil-sn-glycerol-3-phospho-(1'-sn-glycerol) (18:1 LPG). To explore the hydrophobic cavity and identify other lipids that could be interacting with the protein, we performed delipidation assays and titration of free fatty acids with different sizes and insaturation that could be present in the human organism. The results showed a higher affinity with oleic and linoleic fatty acids (18:1 and 18:2 carbons) with kd of 39±1nM and 0,8±0,2 nM respectively. Overall, this is the first evidence that an eukaryotic PAS domain may serve as a sensor for the lipid levels of the cell, possibly dictating HIF-3alpha-dependent gene expression in response to biosynthetic and bioenergetic outcomes, such as lipid accumulation or depletion, upon exposure to hypoxia. We also planned to study the three prolyl hydroxylases (PHD1, 2 and 3) as a collaboration with Dr. Christopher Schofield and their group (Chemistry Department-Oxford University). The genes of organisms that contained the predicted sequences for these enzymes, such as walrus, platypus and python. Were selected eight organisms, from which we obtained soluble proteins for six of them. Two isoforms for PHD3 (platypus and python genes) were shown to be more stable and purified with high purity. As result of the enzymatic activity of PHD3 for python and platypus organisms, we observed that the enzymes are active and they preferentially hydroxylate the proline contained in the CODD domain of HIF. Upon crystallization assay of PHD3 from python, we obtained small malformed crystals and after some refinement cycles we did not obtain single crystals that could be taken to diffraction. For the remaining PHDs that were obtained in a soluble form, several optimizations on the purification protocols had to be made to obtain more stable and promising samples, so that crystallization and enzymatic activity assays can be performedDoutoradoFármacos, Medicamentos e Insumos para SaúdeDoutora em Ciências2014/04927-4FAPES