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LabRMN-CNC – Nuclear Magnetic Resonance Laboratory
Center of Neurosciences and Cell Biology, University of Coimbra

The laboratory is currently part of the Area E “Biophysics and Biomedical NMR” of the CNC. It is also part of the Portuguese National Network of Nuclear Magnetic Resonance Spectroscopy (PTNMR), under the supervision of the Foundation for Science and Technology (FCT), Portugal.

The main objectives of the LabRMN-CNC are the provision of Nuclear Magnetic Resonance to the scientific community, the disclosure and promotion of NMR and training of users. In addition of developing its own research projects, the LabRMN-CNC aims to support work of scientists of the national academic community as business environment, from public or private institutions, thru collaboration in research projects or provision of services.

NMR Unit Coordinator:
Prof. Carlos F.G.C. Geraldes, PhD
Contact: geraldes@bioq.uc.pt

NMR Unit Manager:
Dr. Emeric Wasielewski, PhD
Contact: emeric@cnc.uc.pt
Voice: +351 304 502 926
Fax: +351 239 853 409

NB: Meeting with the NMR Unit Coordinator or the NMR Unit Manager are with appointment only. Appointments can be requested per E-mail at least 24 hours in advance.

NMR Time schedule:

NMR time schedules are planned on a monthly basis, beginning the first Monday of the month.

Online calendars	Periods
Red: NMR events (as planning days)	M => morning (9:30 AM to 1:45 PM)
Mustard: VNMRS 600 MHz schedule	A => afternoon (1:45 PM to 6 PM)
Aluminium: Unity 500 MHz schedule	D => daytime (9:30 AM to 6 PM)
Cyan: Open time (for both spectrometer)	N => night (6 PM to 9:30 AM)
Blue: Minispec mq20	F => full day (start at 9:30 AM)
Violet: Portuguese holidays.

Additional information about session is available by clicking on the name.

NB: For more information about the operating of the LabRMN-CNC, please read the "Guide for NMR access and use" below.

Research Interests:

A. Inorganic Complexes for Medical Diagnostics: MRI and Molecular Imaging:
Studies focus on inorganic compounds for medical diagnostic imaging, in particular contrast agents for magnetic resonance imaging (MRI CAs). They include design, development and physico-chemical evaluation of metal based nanoparticles and small metal chelate agents for multimodal targeted medical imaging agents, followed by in vitro and animal model evaluation using MRI and nuclear imaging techniques.

Contact person:
Prof. Carlos F.G.C. Geraldes
geraldes@bioq.uc.pt

Representative Publications:

M.F. Ferreira, A.F. Martins, C.I.O. Martins, É. Tóth, T.B. Rodrigues, D. Calle, S. Cerdan, P. López-Larrubia, J.A. Martins, C.F.G.C. Geraldes, “Amide conjugates of the DO3A-N-(-amino)propionate ligand: leads for stable, high relaxivity Contrast Agents for MRI?”, Contrast Media Mol. Imaging, (in press).

A.C. Mendonça, A.F. Martins, A. Melchior, S.M. Marques, S. Chaves, S. Villette, S. Petoud, P.L. Zanonato, M. Tolazzi, C.S. Bonnet, É. Tóth, P. Di Bernardo, C.F.G.C. Geraldes, M.A. Santos, “New Tris-3,4-HOPO lanthanide complexes as potential imaging probes: complex stability and magnetic properties”, Dalton Trans., (in press).

A. de Sá, C.S. Bonnet, C.F.G.C. Geraldes, É. Tóth, P.M.T. Ferreira, J.P. André, “Thermodynamic stability and relaxation studies of small, triaza-macrocylic Mn(II) chelates, Dalton Trans., (in press).

H.S. Leitão, S. Doblas, G. d’Assignies, P. Garteiser, J.L. Daire, V. Paradis, C.F.G.C. Geraldes, V. Vilgrain, B.E. Van Beers, “Fat deposition decreases diffusion parameters at MRI: a study in phantoms and patients with liver steatosis”, European Radiology, (in press).

A.F. Martins, M.I.M. Prata, S.P.J. Rodrigues, C.F.G.C. Geraldes, P.J. Riss, A.A. Coarasa, C. Burchardt, C. Kroll, Frank Roesch, “Spectroscopic, radiochemical, and theoretical studies of the Ga³⁺-N-2-hydroxyethyl piperazine-N’-2-ethanesulfonic acid (HEPES buffer) system: Evidence for the formation of Ga³⁺- HEPES complexes in ⁶⁸Ga labeling reactions”, Contrast Media Mol. Imaging, (in press).

S. Figueiredo, J.C. Cutrin, S. Rizzitelli, E. De Luca, J.N. Moreira, C.F.G.C. Geraldes, S. Aime and E. Terreno, “MRI Tracking of Macrophages using Glucan Particles Entrapping a Paramagnetic Agent”, Molecular Imaging and Biology, (in press).

S.L.C. Pinho, H. Faneca, C.F.G.C. Geraldes, M.H. Delville, L.D. Carlos, J. Rocha, “Lanthanide-DTPA Grafted Silica Nanoparticles as Bimodal-Imaging Contrast Agents”, Biomaterials, 33, 925-935 (2012).

S.L.C. Pinho, S. Laurent, J. Rocha, A. Roch, M.H. Delville, L.D. Carlos, L. Vander Elst, R.N. Muller, C.F.G.C. Geraldes. “Relaxometric studies of γ-Fe₂O₃@SiO₂ core shell nanoparticles: when the coating matters”, J. Phys. Chem. C, 116, 2285-2291 (2012).

M.F. Ferreira, B. Mousavi, P.M. Ferreira, C.I.O. Martins, L. Helm, J.A. Martins and C.F.G.C. Geraldes, “Gold nanoparticles functionalised with stable, fast water exchanging Gd³⁺ chelates as high relaxivity Contrast Agents for MRI”, Dalton Trans., 41, 5472 – 5475 (2012).

L.M.P. Lima, R. Delgado, P. Hermann, R. Ševčík, P. Lubal, H.F. Carvalho, A.F. Martins, É. Tóth and C.F.G.C. Geraldes, “Tris(phosphonomethyl) cyclen derivatives: 2) Thermodynamic stability, kinetics, solution structure, and relaxivity of Ln³⁺ complexes”, Eur. J. Inorg. Chem., 2548-2559 (2012).

S.L.C. Pinho, H. Faneca, C.F.G.C. Geraldes, J. Rocha, L.D. Carlos and M.H. Delville, “Silica Nanoparticles for Bimodal MRI-Optical Imaging via Grafting of Gd³⁺ and Eu³⁺/Tb³⁺ Complexes”, Eur. J. Inorg. Chem., 2828-2837 (2012).

F.M. Coreta-Gomes, W.L.C. Vaz, E. Wasielewski , C.F.G.C. Geraldes and M.J. Moreno, “Quantification of Cholesterol Solubilized in Bile Salt Micellar Aqueous Solutions by ¹³C NMR”, Anal. Biochem., 427, 41-48 (2012).

M.L. Debasu, D. Ananias, S.L.C. Pinho, C.F.G.C. Geraldes, L.D. Carlos, J. Rocha, “(Gd,Yb,Tb)PO4 Up-Conversion Nanocrystals for Bimodal Luminescence-MR Imaging”, Nanoscale, 4, 5154-5162 (2012).

I. Bertini, V. Calderone, L. Cerofolini, M. Fragai, C.F.G.C. Geraldes, P. Hermann, C. Luchinat, G. Parigi, J.M.C. Teixeira, “The catalytic domain of MMP-1 studied through tagged lanthanides”, FEBS Lett., 586, 557-567 (2012)

G.A. Pereira, J.A. Peters, E. Terreno, D. Delli Castelli, S. Aime, S. Laurent, L. Vander Elst, R.N. Muller and C.F.G.C. Geraldes, “Supramolecular Adducts of Negatively Charged Lanthanide(III) DOTP Chelates and Cyclodextrins Functionalized with Ammonium Groups : Mass Spectrometry and Nuclear Magnetic Resonance Studies”, Eur. J. Inorg. Chem., 2087-2098 (2012).

S. Figueiredo, J.N. Moreira, S. Rizzitelli, S. Aime, E. Terreno, C.F.G.C. Geraldes, “Nature-inspired particles as carriers for multimodal molecular imaging applications”, 2^nd IEEE-EAS Portuguese Meeting in Bioengineering (ENBENG), Coimbra, Portugal, IEEE Conference Publications, pg. 1 – 4, 2012.

J.P. Monteiro, A.F. Martins, M. Lúcio, S. Reis, T.J. Pinheiro, C.F.G.C Geraldes, P.J. Oliveira and A.S. Jurado, "Nimesulide interaction with membrane model systems: are membrane physical effects involved in nimesulide mitochondrial activity?", Toxicology in Vitro, 25, 1215-1223 (2011).

D.M. Dias, J.M.C. Teixeira, I. Kuprov, E.J. New, D. Parker and C.F.G.C. Geraldes, "Enantioselective binding of a lanthanide(III) complex to human serum albumin studied by ¹H STD NMR techniques", Org. Biomol. Chem., 9, 5047-5050 (2011).

J.P. Monteiro, A.F. Martins, M. Lúcio, S. Reis, C.F.G.C Geraldes, P.J. Oliveira and A.S. Jurado, "Interaction of carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) with lipid membrane systems: a biophysical approach with relevance to mitochondrial uncoupling", J. Bioenerg. Biomembranes, 43, 287–298 (2011).

J.M.C. Teixeira, D.M. Dias, F.J. Cañada, J.A. Martins, J.P. André, J. Jiménez-Barbero and C.F.G.C. Geraldes, "The Interaction of La³⁺ Complexes of DOTA/DTPA-Glycoconjugates with the RCA₁₂₀ lectin: A Saturation Transfer Difference (STD) NMR Spectroscopic Study", J. Biol. Inorg. Chem., 16, 725-734 (2011).

S. Figueiredo, J.N. Moreira, C.F.G.C. Geraldes, S. Aime and E. Terreno, "Supramolecular Protamine/Gd-loaded Liposomes Adducts as Relaxometric Protease Responsive Probes", Bioorg. Medicinal Chem., 19, 1131-1135 (2011).

S.L.C. Pinho, G.A. Pereira, P. Voisin, J. Kassem, V. Bouchaud, L. Etienne, J.A. Peters, L. Carlos, S. Mornet, C.F.G.C. Geraldes, J. Rocha and M.H. Delville, "Fine tuning of the relaxometry of γ-Fe2O3 nanoparticles by tweaking the thickness of the silica coating", ACS Nano, 4, 5339–5349 (2010).

M.P.C. Campello, S. Lacerda, I.C. Santos, G.A. Pereira, C.F.G.C. Geraldes, J. Kotek, P. Hermann, J. Vaněk, P. Lubal, V. Kubíček, E. Tóth and I. Santos, "Lanthanide(iii) Complexes of 4,10-Bis(phosphonomethyl)-1,4,7,10-tetraazacyclo-dodecane-1,7-diacetic acid (trans-H₆do2a2p) in Solution and in the Solid State: Structural Studies Along the Series", Chem. Eur. J., 28, 8446-8465 (2010).

G.A. Pereira, J.A. Peters, F.A. Paz, J. Rocha and C.F.G.C. Geraldes, "Evaluation of [Ln(H2cmp)(H2O)] Metal Organic Framework Materials for Potential Application as MRI Contrast Agents", Inorganic Chemistry, 49, 2969–2974 (2010).

M. Polášek, P. Hermann, J.A. Peters, C.F.G.C. Geraldes and I. Lukeš, "PAMAM dendrimers conjugated with an uncharged gadolinium(iii) chelate with a fast water exchange: the influence of chelate charge on rotational dynamics", Bioconj. Chem., 20, 2142–2153 (2009).

M.F. Ferreira, A.F. Martins, J.A. Martins, P.M. Ferreira, E. Tóth and C.F.G.C. Geraldes, "Gd(DO3A-N-α-aminopropionate): a versatile and easily available synthon with optimized water exchange for the synthesis of high relaxivity, targeted MRI contrast agents", Chem. Commun., 6475-6477 (2009).

NMR expertise:

	Design, development and physico-chemical characterization of paramagnetic chelates through relaxometric (T₁, T₂, T₂*) and chemical shift studies using multinuclear NMR (¹⁷O, ¹H, ²H), NMRD techniques, EPR and simultaneous fitting of paramagnetic data.
	Relaxometric (T₁, T₂, T₂*) characterization of paramagnetic nanoparticulate systems.
	In vitro (cells) and in vivo (small animals) MRI characterization of paramagnetic nanoparticulate systems.
	Paramagnetic shifts (¹H, ¹³C, ³¹P...) study to obtain solution structure and dynamics of lanthanide chelates and, together with residual dipolar couplings (RDCs), of covalently tagged oriented proteins.
	Interaction study of ligands and complexes with biomacromolecules using STD technique.
	Structural characterization of small molecules using 1D and 2D NMR techniques.

B: Inorganic Biochemistry: Inorganic Complexes and Species for Therapy and Environment:
We are interested on the design and study of inorganic compounds as drugs for medical therapy (lithium Li⁺ and bipolar disorder; vanadium V(IV/V) complexes as oral insulin-mimetic agents). We focus on their mechanisms of action using cell and animal models.

Contact person:
Dra. M. Margarida C.A. Castro (gcastro@bioq.uc.pt)

Representative Publications:

A.M. Metelo, R. Pérez-Carro, M.M.C.A. Castro and P. López-Larrubia, "Pharmacological effects of VO(dmpp)2 as assessed by in vivo Magnetic Resonance Imaging and Spectroscopy", J. Inorg. Biochem., 115, 44-49 (2012).

A.M. Metelo, R. Pérez-Carro, M.M.C.A. Castro and P. López-Larrubia, "Pharmacological effects of VO(dmpp)₂ as assessed by in vivo Magnetic Resonance Imaging and Spectroscopy", Magn. Reson. Med., (submitted, July 2011).

M. Passadouro, A.M. Metelo, A.S. Melão, J.R. Pedro, H. Faneca, E. Carvalho, M.M.C.A. Castro, "Study of the antidiabetic capacity of the VO(dmpp)₂ complex", J. Inorg. Biochem., 104, 987-992, (2010).

T.B. Rodrigues, C.P. Fonseca, M.M.C.A. Castro, S. Cerdán and C.F.G.C. Geraldes, "¹³C NMR tracers in neurochemistry: implications for molecular imaging", Quart. J. Nucl. Med. Mol. Imaging, 53, 631-645, (2009).

H. Faneca, V.A. Figueiredo, I. Tomaz, G. Gonçalves, F. Avecilla, M.C.P. de Lima, C.F.G.C. Geraldes, J.C. Pessoa and M.M.C.A. Castro, "Vanadium compounds as therapeutic agents: some chemical and biochemical studies", J. Inorg. Biochem., 103, 601-608, (2009).

C.P. Fonseca, A. Sierra, C.F.G.C. Geraldes, M.M.C.A. Castro and S.Cerdán, "Mechanisms underlying Li⁺ effects in glutamatergic and GABAergic neurotransmissions in the adult rat brain and in primary cultures of neural cells as revealed by ¹³C NMR", J. Neurosci. Res., 87, 1046-1055, (2009).

T.C. Delgado, D. Pinheiro, M. Caldeira, M.M.C.A. Castro, C.F.G.C. Geraldes, P. López-Larrubia, S. Cerdán and J. G. Jones, "Sources of hepatic triglyceride accumulation during high-fat feeding in the healthy rat", NMR in Biomedicine, 22, 310-317, (2009).

L.P. Montezinho, A. Mork, C.B. Duarte, S.Penschuck, C.F.G.C.Geraldes and M.M.C.A. Castro, "Effect of mood stabilizers on dopamine D2-like receptors-mediated inhibition of adenylate cyclase", Bipolar Disorders, 9, 290-297, (2007).

D.M. De Freitas, M.M.C.A. Castro and C.F.G.C. Geraldes, "Is Competition Between Li⁺ and Mg²⁺ the Underlying Theme in the Proposed Mechanisms for the Pharmacological Action of Lithium Salts in Bipolar Disorder?", Acc. Chem. Res., 39, 283-291 (2006).

L.P. Montezinho, M.M.C.A. Castro, C.B. Duarte, S. Penschuck, C.F.G.C. Geraldes and A. Mørk, ", The interaction between dopamine D₂-like and beta-adrenergic receptors in the prefrontal cortex is altered by mood stabilizing agents", J. Neurochem., 96, 1336-1348 (2006).

NMR expertise:

	Characterization of the structure, speciation and cell interactions of complexes in solution and cell suspensions using multinuclear NMR and EPR.
	Study of the effects of inorganic compounds on cell transport metabolism using metal (eg. ⁷Li, ²³Na) through ³¹P and ¹³C NMR (with isotopomer analysis of ¹³C-enriched metabolites).

C. Polymers and their complexes:
Synthesis and structural studies of metal complexes with biologically relevant molecules using NMR spectroscopy, DFT computational methods and luminescence studies, with reference to their potential ability to chelate metals in vivo. Related studies on the interaction of tri- and divalent metal ions, such as aluminium(III), gallium(III), zinc(II) and copper(II) with hydroxyaromatic ligands, such as 8-hydroxyquinoline-5-sulfonate and with anionic synthetic polyelectrolytes using multinuclear NMR coupled with various other techniques, with the objective of the development of luminescent sensors for the analytical determination of these metals at low concentrations. Extension of these studies to biopolymers, such as nucleic acids, and other small molecules of biological interest, and the relevance to their potential ability to chelate hazardous metals in vivo.

Study on the structure and interaction of conjugated polymers and polyelectrolytes, with particular reference to their applications in biosensors of nucleic acids, sugars, proteins and in optoelectronic devices.

Analysis of the electronic structure and aggregation behaviour of conjugated polymers, studied as models for DNA, using nuclear magnetic resonance spectroscopy (NMR), computational methods based on density functional theory (DFT), small angle neutron scattering (SANS), UV/vis absorption and fluorescence and other photophysical techniques.

Contact person:
Dra. M. Luisa Ramos (mlramos@ci.uc.pt)

Representative Publications:

M.L. Ramos, A.R.E. de Sousa, L.L.G. Justino, S.M. Fonseca, C.F.G.C. Geraldes, H.D. Burrows, “Structural and photophysical studies on Gallium(III) 8-hydroxyquiniline-5-sulphonates. Does excited state decay involve ligand photolabilization?”, Dalton Trans., (in press).

T.M.R. Maria, R.A.E. Castro, M. Ramos Silva, M.L. Ramos, L.L.G. Justino, H.D. Burrows, J. Canotilho, M.E.S. Eusébio, “Polymorphism and melt recrystallisation of racemic betaxolol, a beta-adrenergic antagonist drug”, J. Therm. Anal. Calorim. (in press).

M.L. Ramos, L.L.G. Justino, A. Branco, S.M. Fonseca, H.D. Burrows, “Theoretical and experimental insights into the complexation of 8-hydroxyquinoline-5-sulfonate with divalent ions of Group 12 metals", Polyhedron, (in press).

A.T. Marques, H.D. Burrows, J.S. Seixas de Melo, A.J.M. Valente, L.L.G. Justino,U. Scherf, E. Fron, S. Rocha, J. Hofkens, E. W. Snedden and A.P. Monkman, "Spectroscopic properties, excitation and electron transfer in an anionic water-soluble-poly(fluorinealtphenylene)-perylenediimide copolymer”, J. Phys. Chem. B., 116 7548-7559 (2012).

H.D. Burrows, D. Costa, M.L. Ramos, M. da G. Miguel, M.H. Teixeira, A.A.C.C. Pais, A.J.M. Valente, M. Bastos and G. Baib, “Does cation dehydration drive the binding of metal ions to polyelectrolytes in water? What we can learn from the behaviour of aluminium(III) and chromium(III)”, Phys. Chem. Chem. Phys., 14, 7950-7953 (2012).

M.L. Ramos, L.L.G. Justino, A.I.N. Salvador, A.R.E. de Sousa, P.E. Abreu, S.M. Fonseca and H.D. Burrows, “NMR, DFT and luminescence studies of the complexation of Al(III) with 8-hydroxyquinoline-5-sulfonate”, Dalton Trans., 41, 12478–12489 (2012).

M.L. Ramos, L.L.G. Justino, A. Branco, C.M.G. Duarte, P.E. Abreu, S.M. Fonseca, H.D. Burrows, “NMR, DFT and Luminescence Studies of the Complexation of Zn(II) with 8-Hydroxyquinoline-5-Sulfonate”, Dalton Trans. (in press).

M.L. Ramos, L.L.G. Justino and H.D. Burrows, "Structural considerations and reactivity of peroxocomplexes of V(V), Mo(VI) and W(VI)", Dalton Trans., 40, 4374-4383, (2011).

L.L.G. Justino, M.L. Ramos, M. Knaapila, A.T. Marques, C.J. Kudla, U. Scherf, L. Almásy, R. Schweins, H.D. Burrows and A.P. Monkman, "Formation and Interpolymer Alkyl Chain Interactions with Poly(9,9-dioctylfluorene-2,7-diyl) (PFO) in Toluene Solution: Results from NMR, SANS, DFT, and Semiempirical Calculations and Their Implications for PFO beta-Phase Formation", Macromolecules, 44, 334-343 (2011).

H.D. Burrows, T.O. Chimamkpam, T. Encarnação, S.M. Fonseca, R.F.P. Pereira, M.L. Ramos and A.J.M. Valente, "Trivalent Metal Ion Binding to Surfactants and Polyelectrolytes – A Review", J. of Surf. Sc. and Technolog., 26, 1-16 (2010).

H.D. Burrows, M. J. Tapia, S.M. Fonseca, A.J.M. Valente, V.M.M. Lobo, L.L.G. Justino, S. Qiu, S. Pradhan, U. Scherf, N. Chattopadhyay, M. Knaapila and V.M. Garamus, "Aqueous Solution Behaviour of Anionic Fluorene-co-thiophene Based Conjugated Polyelectrolytes", ACS Applied Materials & Interfaces, 1, 864-874, (2009).

L.L.G. Justino, M.L. Ramos, M. Kaupp, H.D. Burrows, C. Fiolhais and V.M.S. Gil, "Density functional theory study of the oxoperoxo vanadium(V) complexes of glycolic acid. Structural correlations with NMR chemical shifts", Dalton Trans., 9735-9745, (2009).

M.L. Ramos, L.L.G. Justino, V.M.S. Gil and H.D. Burrows, "NMR and DFT studies of the complexation of W(VI) and Mo(VI) with 3-phospho-D-glyceric and 2- phospho-D-glyceric acids", Dalton Trans., 9616–9624, (2009).

L.L.G. Justino, M.L. Ramos, P.E. Abreu, R.A. Carvalho, A.J.F.N. Sobral, U. Scherf and H.D. Burrows, "Conformational Studies of Poly(9,9-dialkylfluorene)s in solution using NMR Spectroscopy and Density Functional Theory Calculations", J. Phys.Chem.B, 113, 11808–11821 (2009).

R.F.P. Pereira, A.J.M. Valente, H.D. Burrows, M.L. Ramos, A.C.F. Ribeiro and V.M.M. Lobo, "Flocculation and micellization of sodium dodecyl sulfate solutions in the presence of aluminium nitrate: effect of concentration and temperature", Acta Chimica Slovenica, 56, 45–52 (2009).

NMR expertise:

	NMR characterization of metal complexes in solution, combined with DFT calculations.
	NMR characterization of polymers in solution, combined with DFT calculations.

D. NMR studies of metabolic fluxes:
We study intermediary metabolism using ²H, ¹³C and ¹⁵N stable isotope tracers. Glucose, protein and lipid homeostasis are studied in human subjects, in animal models of human diseases, and in isolated perfused animal organs.

Contact person:
Dr. John G. Jones (jones@cnc.uc.pt)

Representative Publications:

T.C. Delgado, F.O. Martins, F. Carvalho, A. Gonçalves, D.K. Scott, R.M. O’Doherty, M.P. Macedo and J.G. Jones, “²H-enrichment distribution of hepatic glycogen from ²H₂O reveals the contribution of dietary fructose to glycogen synthesis”, Am. J. Physiol. (in press).

R. Basu, C. Barosa, B. Norby, B., Dicke, J.G. Jones, A. Basu, A. & R.A. Rizza, “Pathogenesis of prediabetes: Role of the liver in isolated fasting hyperglycemia and combined fasting and postprandial hyperglycemia”, J. Clin. Endo. & Metab. (in press).

C. Barosa, J.G. Jones, R.A. Rizza and R. Basu, “Acetaminophen glucuronide and plasma glucose report identical estimates of gluconeogenesis and glycogenolysis for healthy and pre-diabetic subjects using the deuterated water method”, Magn. Res. Med. (in press).

T.C. Delgado, C. Barosa, P.M. Nunes, S. Cerdán, C.F.G.C. Geraldes, J.G. Jones, “Resolving the Sources of Plasma Glucose Excursions in the Rat following a Glucose Tolerance Test with Deuterated Water and [U-13C]Glucose using NMR Spectroscopy analysis”, PLoSONE, 7, e34042 (2012).

T.C. Delgado, C. Barosa, P.M. Nunes, D.K. Scott, R.M. O’Doherty, S. Cerdán, C.F.G.C. Geraldes and J.G. Jones, “Effect of cyclosporine A on hepatic carbohydrate and lipid metabolism in rat: implications for post-transplant Diabetes Mellitus”, Expert Opinion on Drug Metabolism and Toxicology, 8, 1223-1230 (2012).

A.F. Soares, R.A. Carvalho, F.J. Veiga, M.G. Alves, F.O. Martins, I. Viegas, J.D. Gonzalez, I. Meton, I.V. Baanante, J.G. Jones, “Restoration of direct pathway glycogen synthesis flux in the STZ-diabetes rat model by insulin administration”, Amer. J. Physiol.- Endocrinology and Metabolism, 303, E875-E885 (2012).

I. Viegas, J. Rito, I. Jarak, S. Leston, R.A. Carvalho, I. Meton, M.A. Pardal, I.V. Baanante, J.G. Jones, “Hepatic glycogen synthesis in farmed European seabass (Dicentrarchus labrax L.) is dominated by indirect pathway fluxes”, Comparative Biochem. and Physiol. A – Molecular and Integrative Physiology, 163, 22-29 (2012).

C. Reis, R.J. Neufeld, F. Veiga, I. Figueiredo, J.G. Jones, A.F. Soares, P. Nunes, C. Damge and R.A. Carvalho, “Effects of an oral insulin nanoparticle administration on hepatic glucose metabolism assessed by ¹³C and ²H isotopomer analysis”, J. Microencapsulation, 29, 167-176 (2012).

J.D. González, A. Caballero, I. Viegas, I. Metón, J.G. Jones, J. Barra, F. Fernández, F. and I.V.Baanante, “Effects of ALT inhibition on the intermediary metabolism in Sparus aurata through dietary amino-oxyacetate supplementation”, Br. J. Nutr. 107, 1747-1756 (2012).

C. Barosa, C. Silva, A. Fagulha, L. Barros, M.M. Caldeira, M. Carvalheiro and J.G. Jones, “Sources of hepatic glycogen synthesis following a milk-containing breakfast meal in healthy subjects”, Met. Clin & Exp. 61, 250-254 (2012).

C. Barosa, C. Silva, A. Fagulha, L. Barros, M.M. Caldeira, M. Carvalheiro and J.G. Jones, "Sources of hepatic glycogen synthesis following a milk-containing breakfast meal in healthy subjects", Met. Clin & Exp. 61, 250-254 (2012)

C. Barosa, C. Silva, A. Fagulha, L. Barros, M.M. Caldeira, M. Carvalheiro and J.G. Jones, "Sources of hepatic glycogen synthesis following a milk-containing breakfast meal in healthy subjects", Met. Clin & Exp. (in press 2011).

I. Viegas, V.M. Mendes, S. Leston, I. Jarak, R.A. Carvalho, M.A. Pardal, B. Manadas and J.G. Jones, "Analysis of glucose metabolism in farmed european sea bass (Dicentrarchus labrax L.) using deuterated water", Comp. Biochem & Physiol. A. (in press 2011).

M. Kacerovsky, J.G. Jones, A.I. Schmid, C. Barosa, A. Lettner, G. Kacerovsky-Bielesz, J. Szendroedi, M. Chmelik, P. Nowotny, V. Chandramouli, M. Wolzt and M. Roden, "Postprandial and fasting hepatic glucose fluxes in longstanding type 1 diabetes", Diabetes, 60,1752-1758, (2011).

R. Basu, C. Barosa, A. Basu, V. Pattan, A. Saad, J.G. Jones and R. Rizza, "Transaldolase exchange and its effects on measurements of gluconeogenesis in humans", Am. J. Physiol., 300, 296-303, (2011).

C. Barosa, M. Almeida, M.M. Caldeira, F. Gomes and J.G. Jones, "Contribution of proteolytic and metabolic sources to hepatic glutamine by ²H NMR analysis of urinary phenylacetylglutamine ²H enrichment from ²H₂O", Metabolic Engineering, 12, 53-61, (2010).

A.F. Soares, F.J. Veiga, R.A. Carvalho and J.G. Jones, "Effects of galactose on direct and indirect pathway estimates of hepatic glycogen synthesis", Metabolic Engineering, 12, 552-560, (2010).

P.N. Nunes and J.G. Jones, "Quantifying endogenous glucose production and contributing source fluxes from a single ²H NMR Spectrum", Magn. Res. Med., 62, 802-807, (2009).

T.C. Delgado, C. Silva, I. Fernandes, M.M. Caldeira, M. Bastos, C. Baptista, M. Carvalheiro, C.F.G.C Geraldes and J.G. Jones, "Sources of hepatic glycogen synthesis during an oral glucose tolerance test: effect of transaldolase exchange on flux estimates", Magn. Res. Med., 62, 1120-1128, (2009).

T.C. Delgado, D. Pinheiro, M.M. Caldeira, M.M.C.A. Castro, C.F.G.C. Geraldes, P. Lopez-Larrubia, S. Cérdan and J.G. Jones, "Mechanisms of hepatic triglyceride accumulation during high-fat feeding in the healthy rat", NMR in Biomedicine, 22, 310-317, (2009).

J.G. Jones, P. Garcia, C. Barosa, T.C. Delgado and L. Diogo, "Hepatic anaplerotic outflow fluxes are redirected from gluconeogenesis to lactate synthesis in patients with Type 1a Glycogen Storage Disease", Metabolic Engineering, 11, 155-162, (2009).

NMR expertise:

	Analysis of metabolic flux.
	¹³C NMR isotopomer analysis of ¹³C-enriched metabolites from perfused organs, plasma glucose and urinary metabolites by direct and indirect detection methods.
	2H NMR analysis of metabolite positional, ²H-enrichments from ²H₂O in perfused organs, plasma glucose and urinary metabolites.

Equipment:

Varian VNMR 600 System with 3 observe channels (V600)

Probes:
3-mm PFG Indirect Detection (optimal sample volume = 180-200 μl)
3-mm PFG Broadband (optimal sample volume = 180-200 μl)
5-mm PFG triple resonance Indirect Detection (optimal sample volume = 600-700 μl)
5-mm PFG Broadband (optimal sample volume = 600-700 μl)
4-mm PFG nanoprobe (HR-MAS)

The Varian VNMRS 600 MHz NMR spectrometer is an equipment part of the Portuguese National Network of Nuclear Magnetic Resonance Spectroscopy (PTNMR) as type A spectrometer (i.e. high ﬁeld spectrometer). As such 70% of its time of operation is dedicated in priority to the PTNMR Users (i.e. registered Users with a project approved by PTNMR referees). Remaining time is allocated to registrered CNC Users. Its conditions of use are the ones of a PTNMR type A spectrometer.

Note on the use of 3-mm microprobes: The 3-mm probes offer a ~40% increase in sensitivity over their 5-mm counterparts provided that the molecule of interest is soluble in the smaller volume and that the signal line widths are preserved. For many biological samples that contain salt, e.g. protein solutions or tissue extracts, the smaller volumes result in higher salt concentrations leading to longer pulse widths, less efficient ¹H-broadband decoupling, and increased sample heating. This later effect may result in a drifting lock signal particularly if the lock solvent is D₂O.

Varian UNITY 500 System (U500)

Probes:
5-mm PFG Broadband (optimal sample volume = 600-700 μl)
5-mm Broadband Low frequency (optimal sample volume = 600-700 μl)
5-mm Indirect Detection (optimal sample volume = 600-700 μl)
10-mm Broadband (optimal sample volume = 1ml)

The Varian Unity 500 MHz NMR spectrometer depends on the LabRMN-CNC. Its conditions of use and access are following the ones of a PTNMR type B spectrometer (i.e. low field NMR spectrometer).

Data Backup Service

The LabRMN-CNC does not currently provide archive or backup service: Users are strongly recommended to backup their data during their NMR session.

Guide for NMR access and use:

1) Location:

The LabRMN-CNC is located in the ground floor of the Faculty of Medicine at the University of Coimbra, Pólo I (bus stop: Universidade). The local opens directly into the courtyard of the Medicine Building aside the principal staff entrance into the courtyard, opposite to the entrance for vehicles.

Parking inside the courtyard is reserved for Faculty cardholders. There is no visitor car park. The street parking in and around the University fills up quickly after 8:30 AM. Please be aware that cars either parked improperly or in designated sites for any length of time will almost certainly be clamped.

There are regular bus services connecting the University with coach and train stations.

The LabRMN-CNC facility is open to every User from 9:30 AM to 6:00 PM, from Monday to Friday, with a pause between ~ 1:00 PM - 2:00 PM. Access to the lab during the night or the weekend is restraint only to LabRMN-CNC Users with personal required access cards (Medicine Faculty and LabRMN-CNC). If doors are closed, Users are pleased to ring at the entrance (right of the door).

2) Spectrometer availability:

Equipment of LabRMN-CNC is not free access. The use of both NMR spectrometers is limited to CNC Users registered in the LabRMN-CNC Users database and to PTNMR Users with appropriate approved PTNMR projects.

New Users planning to access to service provided by the LabRMN-CNC are pleased to contact the NMR Unit Manager first or the NMR Unit Coordinator to discuss about their projects and their needs. They are also directly informed about the specific operating of the NMR Unit and the occasional costs of the services. After the meeting, Users are registered in the LabRMN-CNC Users database as in the [CNC NMR] mailing list. They receive the NMR time request forms and are allowed to request NMR time.

The organization and NMR time allocation in the LabRMN-CNC agree with standards of the National Network for Nuclear Magnetic Resonance (PTNMR) and the Foundation for Science and Technology (FCT). Both NMR spectrometers are following PTNMR access rules. NMR time not allocated to PTNMR Users is shared between CNC Users (members and outsiders of LabRMN-CNC).

NMR Service:
Along of its own research projects the LabRMN-CNC aims to support work of scientists of the national academic community as business environment, from public or private institutions, by providing a regular weekly service (called NMR Service), usually performed during the Saturday. This Service provides NMR experiments (mono and multidimensional experiments acquisition, analysis) for a large panel of nuclei. This NMR Service is exclusively performed by LabRMN-CNC staff. Users interested by the NMR Service are please to contact first the NMR Unit Manager for more informations.

3) Scheduling:

NMR time schedules of both spectrometers are planned on a monthly basis, generally starting the first Monday of the month.

NMR schedule is usually planned the Thursday before the coming month. Users wanting to request some NMR time must fully fill a PDF NMR time request form, specific to the spectrometer they want to use. Filled NMR time request forms must be send to the NMR Unit Manager (emeric@cnc.uc.pt) before the Wednesday 8:00 PM of the “planning week”. Collection of requests starts the Monday of the “planning week”. (Please consult the online calendar to check the dates)

A preview of the new month NMR time schedule will be available the Thursday evening on the LabRMN-CNC online calendar. Any request for modification will be processed during the following Friday and weekend if necessary.

Possible OPEN TIME (free NMR time not distributed) will be distributed on demand during the same period. All request for OPEN TIME must be done with a new fully filled NMR time request form.

The definitive NMR time schedule will be posted on the online calendar and send by e-mail through the [CNC-NMR] mailing-list before the Sunday evening (usually the Friday evening).

Cancellations of NMR time must be immediately notified to the NMR Unit Manager, at least a day before. Non usage of time (without reasonable notification of cancellation) will result in a two weeks suspension.

4) Materials and Sample Preparation:

The LabRMN-CNC, as the PTNMR, does not provide any consumable. Users have to supply their own tubes and NMR solvents. For gHX Nano NMR probe Users, rotors from the LabRMN-CNC are available for use and have to be requested to the Unit Manager during NMR time booking.

For External Users: NMR samples may be prepared in the wet-lab next to the NMR room belonging to the LabRMN-CNC teams. The wet-lab is equipped with pipettes, a balance, a thermostated bath and micro-centrifuge. There is also a fridge / freezer to temporary store perishable samples. Please notice the wet-lab does not have fume hood or solvent evaporator.

Users are pleased to consider the following recommendations:
- use a deuterated solvent.
- use an intact and good quality NMR tube.
- avoid too big sticker on the tube (2 cm max glue on top of the tube) All samples must be labelled.
- use constantly the recommended amount of sample in the tube: 200 µL for 3 mm tubes and 650 µL for 5 mm tubes.

Pricing informations for the use of the LabNMR spectrometers (starting September 1st 2011)

I – Costs of the service for Academics (Non-Profit)/PTNMR Users

Spectrometer	Spectral Acquisition (€ / hour)
Spectrometer	With operator*	Without operator**
VNMRS 600	10,00	7,00
Unity 500	8,00	5,50

* Experiments preparation time with operator charged.
** Cost for acquisition extra time after preparation.

Costs include the legal taxe IVA of 23%.

II – Costs of the services for companies (For-Profit)^a

Spectrometer	VNMRS 600	Unity 500
Spectrometer	Cost (€)	Cost (€)
¹H	65,00	40,00
¹³C	190,00	120,00
³¹P	190,00	120,00
¹⁹F	190,00	120,00
¹³C-DEPT	190,00	120,00
COSY	190,00	120,00
HSQC	320,00	200,00
HMBC	570,00	360,00
NOESY	680,00	420,00
TOCSY	190,00	120,00
Package 1D*	390,00	240,00
Package 1D+2D**	1180,00	730,00

*includes ¹H, ¹³C, ¹³C-DEPT
**includes ¹H, ¹³C, ¹³C-DEPT, COSY, HSQC, HMBC

Costs include the legal taxe IVA of 23%.

^a Costs are based on an average time of preparation and acquisition of the experiments. They include the use of the equipments as the preparation of sample and standard deutered solvants (CDCl3, D2O, DMSO, acetone).

Related with the sample quantity (MM), costs are valid for:

¹H and 2D > 10 mg; ¹³C and ¹³C-DEPT > 200 mg; ³¹P > 10 mg; ¹⁹F > 10 mg

In case more acquisition time is needed than established for some experiments additionnal hours are charged (600 MHz 90€/h and 500 MHz 70€/h). Analysis of sample in relatively small quantity (concentration between 2 and 5 mg) is possible under conditions and a budget will be given after consultation of the service.

Processed spectra are send in electronic format (PDF) and acquisition files are available.

Elaboration of reports of spectra interpretations is possible. But due to projects specificities reports are charged in accordance with the time of their execution (110 € / hour).

III - Bio-NMR

Spectrum	VNMRS 600
Spectrum	Cost (€)^a
¹⁵N-HSQC	190,00
¹³C-HSQC	590,00
3D-HNCO	1490,00
3D-HN(CA)CO	1490,00
3D-HN(CO)CACB	1490,00
3D-HNCACB	1490,00
3D-15N-NOESY	2900,00
3D-¹⁵N-TOCSY	1490,00
3D-hCCH-TOCSY	2900,00
3D-HNHA	1490,00
3D ¹³C-NOESY	2900,00

Costs include the legal IVA tax of 23%.

^a Costs are based on an average time of preparation and acquisition of the experiments for labelled samples (¹³C and ¹⁵N) with a standard concentration of 0.5 mM and an approximative molecular mass of 20 kDa.

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