Silvery threadmoss is a problematic weed of golf putting greens, growing interspersed with turf, decreasing aesthetic quality and playability.  Moss is typically controlled postemergence and currently only one herbicide, carfentrazone, is registered for silvery threadmoss control on greens.  Carfentrazone controls moss up to 75% applied at a three week interval throughout the growing season.  Alternatives providing longer residual or more effective control are desirable.  Studies were conducted to examine the growth of moss gametophytes from spores and bulbils and to evaluate turf protection products for pre and postemergence moss control.  Moss gametophytes develop best from spores at 30"aC and from bulbils at 23"aC.  Products which control moss equivalent to carfentrazone (>70%) both pre and postemergent include sulfentrazone, saflufenacil, flumioxazin, oxadiazon, and oxyfluorfen.  Fosamine and fosetyl-Al alone controlled moss equivalent to carfentrazone post-, but not preemergent.  14C glyphosate absorption and translocation through moss colonies was examined from 12 to 192 hours after treatment (HAT) to understand how herbicides are absorbed by silvery threadmoss.  It appears that 14C reaches equilibrium by 24 HAT in capillary water of the moss colony and inside moss tissues.  Subsequently, 14C is lost to the system presumably through microorganism degradation of 14C glyphosate in capillary water.  The final objective of this work was to identify and evaluate two fungal organisms observed to cause disease of silvery threadmoss on putting greens in efforts to develop a biological control.  The organisms were identified by morphology and ITS sequence as Alternaria sp. and Sclerotium rolfsii.  Alternaria sp. causes a leaf disease of silvery threadmoss and Sclerotium rolfsii causes Southern blight of silvery threadmoss.  Host specificity testing demonstrated moderate pathogenicity of S. rolfsii to annual bluegrass but not to "¥Penn A4"" creeping bentgrass.  Both organisms have potential to be effective biological controls for silvery threadmoss; however, host specificity indicates Alternaria sp. may be a better choice.  Data from these experiments suggest herbicides in two chemical classes control mosses both pre and postemergence, and sulfentrazone, fosetyl-Al, and Alternaria sp. may be new alternatives to carfentrazone for use on golf putting greens. / Ph. D.

Τα ραδιοπεπτίδια γνώρισαν μεγάλη εξέλιξη τα τελευταία 30 χρόνια και συνέβαλαν σημαντικά στην πειραματική και κλινική ογκολογία. Η στοχευμένη απεικονιστική διάγνωση και ραδιονουκλιδική θεραπεία του καρκίνου με διαμεσολάβηση υποδοχέων πεπτιδίων (Peptide Receptor Imaging and Radionuclide Therapy, PRΙ και PRRT) βρίσκει ολοένα μεγαλύτερη εφαρμογή. Βασική προϋπόθεση για την αποτελεσματικότητα της στρατηγικής αυτής είναι η υπερέκφραση των αντίστοιχων υποδοχέων στα καρκινικά κύτταρα-στόχους, σε σχέση με τους παρακείμενους υγιείς ιστούς. Η φυσική πεπτιδορμόνη σωματοστατίνη (SS14) ασκεί τη βιολογική της δράση μετά από αλληλεπίδραση με πέντε υποδοχείς, sst1-5. Λόγω ταχείας αποικοδόμησης της SS14 στο πλάσμα του αίματος, έχουν αναπτυχθεί μεταβολικά σταθεροποιημένα ανάλογα με στόχο την εφαρμογή στην κλινική πράξη. Το OctreoScan® είναι ένα μεταβολικά σταθεροποιημένο ραδιοπεπτίδιο της SS με υψηλή συγγένεια δέσμευσης για τον sst2, διαμέσου του οποίου και εσωτερικεύεται. Σήμερα αποτελεί το ραδιοφάρμακο επιλογής στη διάγνωση των NETs, οι οποίοι εκφράζουν τον sst2 σε υψηλή πυκνότητα. Όμως, δεδομένου ότι οι sst1-5 εκφράζονται συχνά μαζί και σε διάφορους συνδυασμούς σε πολλούς ανθρώπινους όγκους, αναμένεται ότι σταθεροποιημένα ανάλογα με ιδιότητες πανσωματοστατίνης (μιμητές σωματοστατίνης) θα απεικονίζουν ή θα θεραπεύουν μεγαλύτερο φάσμα ανθρώπινων όγκων. Επιπλέον, λόγω αλληλεπίδρασης με πολλαπλούς υποδοχείς (συνέκφραση των sst) θα επιτυγχάνεται αύξηση του διαγνωστικού σήματος και του θεραπευτικού αποτελέσματος. Σκοπός της παρούσας διατριβής ήταν η ανάπτυξη ραδιοπεπτιδίων SS με υψηλή συγγένεια δέσμευσης για τους sst1-5 και υψηλή ικανότητα εσωτερίκευσης διαμέσου των υποτύπων αυτών, ώστε να στοχεύουν αποτελεσματικότερα διευρυμένο φάσμα όγκων με πολλαπλή έκφραση sst. Για το σκοπό αυτό αναπτύξαμε δώδεκα νέα πεπτιδικά ανάλογα (ΑΤΧΣ) στα οποία διατηρήθηκε ο αριθμός των αμινοξέων της SS14. Επιπλέον, σε όλα τα πεπτιδικά ανάλογα έχει προσδεθεί ομοιοπολικά στο Ν-τελικό άκρο ο καθολικός υποκαταστάτης DOTA για τη δέσμευση του 111In και άλλων δισθενών ή τρισθενών μεταλλικών ραδιονουκλιδίων με κλινική εφαρμογή, ενώ παράλληλα επιτυγχάνεται προστασία του Ν-τελικού άκρου. Για την αύξηση της μεταβολικής σταθερότητας των νέων αναλόγων πραγματοποιήθηκαν επιπλέον τροποποιήσεις, όπως σταδιακή μείωση του αριθμού αμινοξέων στο δακτύλιο, αντικατάσταση L-αμινοξέων από D- ή μη φυσικά αμινοξέα ή από κατάλοιπα PEGΧ και τέλος εισαγωγή δεύτερης δισουλφιδικής γέφυρας. Η σύνθεση των πεπτιδικών αναλόγων (ΑΤΧΣ) σε στερεή φάση ήταν επιτυχής καθώς παραλήφθηκαν υψηλής καθαρότητας (92-99%) τελικά προϊόντα με την αναμενόμενη δομή. Η ικανότητα δέσμευσης στους sst1-5 διατηρείται στα ανάλογα με δωδεκαμελή δακτύλιο. Όμως, όσο μειώνεται ο αριθμός των αμινοξέων του δακτυλίου (από δώδεκα σε έξι) μειώνεται και η συγγένεια δέσμευσης των αναλόγων στους περισσότερους sst1-5. Παρατηρείται ακόμα και πλήρης απώλεια δέσμευσης σε συγκεκριμένους υπότυπους και ιδιαίτερα στον sst1. Η επισήμανση των ATΧΣ με 111In πραγματοποιήθηκε με δέσμευση του στον υποκαταστάτη DOTA. Τα ραδιοπεπτίδια [111In]ΑΤΧΣ παρελήφθησαν με ειδική ραδιενέργεια 0.1-0.2 mCi/nmol (επαρκή για in vivo στόχευση υποδοχέων), με υψηλή απόδοση (> 94%) και ραδιοχημική καθαρότητα (> 95%), όπως καταδεικνύεται με ανάλυση RP-HPLC. Η ικανότητα των αναλόγων [111In]ΑΤΧΣ να εσωτερικεύονται διαμέσου του sst2 μειώνεται όσο μειώνεται και ο αριθμός των αμινοξέων στο δακτύλιο. Επιπλέον, τα [111In]ΑΤ1Σ και [111In]ΑΤ2Σ (δωδεκαμελής δακτύλιος) εσωτερικεύονται διαμέσου του sst3 και λιγότερο διαμέσου του sst5. Το δικυκλικό [111In]ΑΤ6Σ παρουσίασε υψηλότερη ικανότητα εσωτερίκευσης διαμέσου του sst3 σε σύγκριση με τον sst2. Η μελέτη της in vivo σταθερότητας των [111In]ΑΤΧΣ καταδεικνύει ότι η σταδιακή μείωση του αριθμού αμινοξέων από δώδεκα σε έξι στο δακτύλιο έχει σαν αποτέλεσμα την προστιθέμενη αύξηση της μεταβολικής σταθερότητας των νέων αναλόγων. Μερική αύξηση της μεταβολικής σταθερότητας των αναλόγων παρατηρήθηκε με αντικατάσταση επιλεγμένων L-αμινοξέων από D- αμινοξέα. Η εισαγωγή δεύτερης δισουλφιδικής γέφυρας στο [111In]ΑΤ6Σ προκάλεσε την πλήρη σταθεροποίηση του. Η φαρμακοκινητική συμπεριφορά των [111In]ΑΤΧΣ αξιολογήθηκε με μελέτες βιοκατανομής σε υγιή και παθολογικά πρότυπα πειραματοζώων. Η πρόσληψη των [111In]ΑΤΧΣ σε πειραματικούς όγκους AR4-2J ή/και HEK293-hsst2/ -hsst3/ -hsst5 αξιολογήθηκε με πειράματα βιοκατανομής σε ποντίκια SCID. Το μεταβολικά σταθερό δικυκλικό [111In]AT6Σ είχε την υψηλότερη πρόσληψη (1.9% ID/g) στους πειραματικούς όγκους AR4-2J, ενώ παρόμοια πρόσληψη (1.8% ID/g) είχε και το [111In]AT2Σ (δωδεκαμελής δακτύλιος). Για τα υπόλοιπα ανάλογα παρατηρήθηκε μείωση της πρόσληψης στους πειραματικούς όγκους AR4-2J με τη μείωση του αριθμού αμινοξέων στο δακτύλιο. Όπως αναλύθηκε πριν, η μείωση του δακτυλίου είχε σαν αποτέλεσμα την μειωμένη ικανότητα δέσμευσης των αναλόγων στους sst1-5 και στην μειωμένη ικανότητα εσωτερίκευσης διαμέσου του sst2 με την παρατηρούμενη άμεση επίπτωση στην πρόσληψη στους πειραματικούς όγκους AR4-2J. Βιοκατανομή των [111In]AT1Σ και [111In]AT2Σ σε ποντίκια με πειραματικούς όγκους HEK293-hsst2/ -hsst3/ -hsst5 έδειξε ότι το πιο σταθερό [111In]AT2Σ είχε υψηλότερη πρόσληψη στους όγκους απότι το [111In]AT1Σ. Το δικυκλικό [111In]AT6Σ έδειξε πολύ μεγαλύτερη πρόσληψη (3.7 % ID/g) στους πειραματικούς όγκους HEK293-hsst3 σε σχέση με το [111In]AT2Σ (1.2 % ID/g). Φαίνεται ότι στo [111In]AT6Σ η πρόσληψη στους πειραματικούς όγκους sst2+ και sst3+ ευνοείται λόγω της υψηλής μεταβολικής του σταθερότητας, ενώ στο [111In]AT2Σ, το οποίο έχει υψηλότερη συγγένεια δέσμευσης για τους sst1-5 και μεγαλύτερη ικανότητα εσωτερίκευσης διαμέσου των sst2 και sst3, η ικανότητα στόχευσης των πειραματικών όγκων sst2+ και sst3+ μετριάζεται λόγω της χαμηλής μεταβολικής σταθερότητας. Συμπερασματικά, για τη διατήρηση της συγγένειας δέσμευσης για τους πέντε sst1-5 αλλά και της πλήρους φαρμακολογικής δράσης της ενδογενούς SS14 σε ένα ανάλογο (ιδιαίτερα τη διατήρηση της εσωτερίκευσης διαμέσου του sst2) αναδεικνύεται ουσιαστική η ύπαρξη του δωδεκαμελή δακτυλίου της SS14. Δεδομένης όμως της χαμηλής μεταβολικής σταθερότητας των αναλόγων SS14 με δωδεκαμελή δακτύλιο συνεχίζονται οι προσπάθειες για περαιτέρω σταθεροποίηση τους. Η μελέτη αυτή κατέστησε επιπλέον σαφές ότι τα εξωκυκλικά αμινοξέα των αναλόγων και ιδιαίτερα αυτών με εξαμελή δακτύλιο επιδρούν δραματικά στην συγγένεια για τους sst1-5. Επομένως, καθίσταται σημαντική περαιτέρω αξιολόγηση τους με διαμορφωσιακές μελέτες, η οποία θα ενισχυθεί με τη σύνθεση και αξιολόγηση αναλόγων με σταδιακά μειούμενο αριθμό εξωκυκλικών αμινοξέων. / Over the past 30 years great progress has been made in the field of radiopeptides, with major impact in experimental and clinical oncology. Peptide receptor imaging and radionuclide therapy is a promising new approach in nuclear medicine. An essential prerequisite for the effectiveness of this strategy is the overexpression of the corresponding peptide receptors on tumor cells, as opposed to their minimal or lack of expression in healthy surrounding tissues. Native somatostatin (SS14) exerts its inhibitory actions after binding to five receptor subtypes, sst1-5, on target cells. Due to the rapid in vivo degradation of SS14, metabolically stabilized analogs have been developed for clinical application. Octreoscan® is a metabolically stabilized radiopeptide with high affinity to sst2 and high sst2-mediated internalization into target-cells. Today this radiopharmaceutical is the agent of choice for the diagnostic imaging of NETs expressing the sst2 in high density. The above five sst1-5 are expressed concomitantly and in various combinations in several human tumors. Consequently, radiolabeled stabilized analogs with pansomatostatin-like properties are expected to detect or treat a wider range of human tumors. Furthermore, due to their multiple-receptor interaction on tumors, an increased diagnostic sensitivity and a higher therapeutic efficacy are expected by their use. The aim of the present Thesis was to develop radiopeptides which are true mimics of native SS14. In else, they will preserve a high affinity to all five sst1-5 and the pharmacological traits of SS14, especially its sst-mediated internalization capacity which will eventually lead to effective targeting of multi-sst expressing tumors. For this purpose, twelve new cyclic peptide analogs (ATXS) were developed, all containing 14 amino acids similarly to SS14. Furthermore, the universal DOTA chelator was attached to their N-terminus for stable binding of 111In and several other divalent or trivalent radionumetals used in clinical oncology. This modification has advertently led to N-terminal capping. Furthermore, ATXS underwent additional modifications to increase metabolic stability, such as progressive reduction of ring-size (from 12 to 6 amino acids), replacement of L- by D- or by unnatural amino acids or PEGx residues and eventually, introduction of a second disulfide bridge in the peptide backbone. The synthesis ATXS conducted on the solid support was successful and the final products were obtained in high purity (92-99%), as verified by analytical methods. All analogs containing a 12 amino acid ring displayed high binding affinity to all sst1-5. However, reducing the number of amino acids in the ring (from twelve to six) caused reduction of affinity to most sst1-5 subtypes. Some analogs even lost their affinity to certain subtypes, particularly to the sst1. Labeling of ATXS with 111In was mediated by the chelator DOTA attached to their N-terminus according to published protocols. The resulting radiopeptides, [111In]ATXS, were obtained at a typical specific activity of 0.1-0.2 mCi/nmol (sufficient for receptor targeting purposes) and in high yield (>94%) and radiochemical purity (>95%), as verified by RP-HPLC analysis. Internalization of [111In]ΑΤΧS into sst2-expressing cells declined as the size of the ring decreased. Furthermore, [111In]ΑΤ1S and [111In]ΑΤ2S (12-member ring) showed higher sst3- than sst5-mediated internalization in transfected HEK293 cells. On the other hand, [111In]ΑΤ6S (bi-cyclic 8,12-ring) showed higher sst3- than sst2-mediated internalization. The in vivo stability of [111In]ATXS progressively increased as the number of amino acids in the ring decreased from twelve to six. Additional increase of metabolic stability was observed in analogs undergone replacement of selected L-amino acids by D-amino acids. The most striking effect on metabolic stability was observed by the introduction of a second disulfide bridge in [111In]AT6S. The pharmacokinetic behavior of [111In]ΑΤΧS was evaluated by biodistribution studies in healthy and SCID mice. The uptake of [111In]ΑΤΧS in the AR4-2J or HEK293-hsst2 / -hsst3 / -hsst5 / experimental tumors was determined during biodistribution experiments in SCID mice. The metabolically stable bicyclic [111In]AT6S showed the highest uptake (1.9%ID/g) in the AR4-2J tumors, while [111In]AT2S displayed similar uptake (1.8%ID/g). For the other analogs the decrease of ring-size resulted in reduced uptake in the AR4-2J tumors. This effect may be assigned to the lower binding affinity to hsst1-5 and the lower sst2-mediated internalization observed in these analogs. The more stable [111In]AT2S showed higher uptake in all HEK293-hsst2 / -hsst3 / -hsst5 experimental tumors as compared to [111In]AT1S. On the other hand, the bicyclic [111In]AT6S showed much higher uptake (3.7%ID/g) in the HEK293-hsst3 tumors as compared to [111In]AT2S (1.2%ID/g). It can be assumed, that due to its higher metabolic stability, [111In]AT6S targets more effectively both the sst2+ and the sst3+ tumors in comparison to [111In]AT2S, although the latter displayed a higher affinity to sst1-5 and a faster sst2- and sst3-mediated internalization. It can be concluded, that the twelve-member ring of SS14 seems to be essential for maintaining the affinity to all five sst1-5 and the pharmacological profile of the mother hormone, especially in regards to the sst2-mediated internalization, to eventually make available clinically useful pansomatostatin-like radiopeptides. The sub-optimal metabolic stability displayed by the 12-member ring ATXS analogs of the present study indicate the need for further stabilization by innovative structural interventions. Another conclusion of this study, is the negative effect of the exo-cyclic amino acids of the 6-member ring analogs leading to dramatic loss of affinity to all sst1-5. This finding warrants further investigation with conformational studies and will be greatly assisted by the availability and evaluation of analogs with gradually declining number of exo-cyclic amino acid residues.

Platinum based chemotherapy is amongst the mainstream DNA-damaging agents used in clinical cancer therapy today. Agents such as cisplatin, carboplatin are clinically prescribed for the treatment of solid tumors either as single agents, in combination, or as part of multi-modality treatment strategy. Despite the potent anti-tumor activity of these drugs, overall effectiveness is still hampered by inadequate delivery and retention of drug in tumor and unwanted normal tissue toxicity, induced by non-selective accumulation of drug in normal cells and tissues. Utilizing molecular imaging and nanoparticle technologies, this thesis aims to contribute to better understanding of how to improve the profile of platinum based therapy. By developing a novel fluorinated derivative of carboplatin, incorporating a Flourine-18 (18F) moiety as an inherent part of the molecule, quantitative measures of drug concentration in tumors and normal tissues can be directly determined in vivo and within the intact individual environment. A potential impact of this knowledge will be helpful in predicting the overall response of individual patients to the treatment. Specifically, the aim of this project, therefore, is the development of a fluorinated carboplatin drug derivative with an inherent positron emission tomography (PET) imaging capability, so that the accumulation of the drug in the tumor and normal organs can be studied during the course of therapy . A secondary objective of this research is to develop a proof of concept for simultaneous imaging of a PET radiolabeled drug with a SPECT radiolabeled liposomal formulation, enabling thereby bi-modal imaging of drug and delivery vehicle in vivo. The approach is challenging because it involves development in PET radiochemistry, PET and SPECT imaging, drug liposomal encapsulation, and a dual-modal imaging of radiolabeled drug and radiolabeled vehicle. The principal development is the synthesis of fluorinated carboplatin 19F-FCP using 2-(5-fluoro-pentyl)-2-methyl malonic acid as the labeling agent to coordinate with the cisplatin aqua complex. It was then used to treat various cell lines and compared with cisplatin and carboplatin at different concentrations ranging from 0.001 µM to 100 µM for 72 hrs and 96 hrs. IC50 values calculated from cell viability indicated that 19F-FCP is a more potent drug than Carboplatin. Manual radiosynthesis and characterization of [18F]-FCP was performed using [18F]-2-(5-fluoro-pentyl)-2-methyl malonic acid with coordination with cisplatin aqua complex. Automated radiosynthesis of [18F]-FCP was optimized using the manual synthetic procedures and using them as macros for the radiosynthesizer. [18F]-FCP was evaluated in vivo with detailed biodistribution studies and PET imaging in normal and KB 3-1 and KB 8-5 tumor xenograft bearing nude mice. The biodistribution studies and PET imaging of [18F]-FCP showed major uptake in kidneys which attributes to the renal clearance of radiotracer. In vivo plasma and urine stability demonstrated intact [18F]-FCP. [111In]-Labeled Liposomes was synthesized and physiochemical properties were assessed with DLS. [111In]-Labeled Liposome was evaluated in vivo with detailed pharmacokinetic studies and SPECT imaging. The biodistribution and ROI analysis from SPECT imaging showed the spleen and liver uptake of [111In]-Labeled Liposome and subsequent clearance of activity with time. [18F]-FCP encapsulated [111In]-Labeled Liposome was developed and physiochemical properties were characterized with DLS. [18F]-FCP encapsulated [111In]-Labeled Liposome was used for in vivo dual tracer PET and SPECT imaging from the same nanoconstruct in KB 3-1 (sensitive) and COLO 205 (resistant) tumor xenograft bearing nude mice. PET imaging of [18F]-FCP in KB 3-1 (sensitive) and COLO 205 (resistant) tumor xenograft bearing nude mice was performed. Naked [18F]-FCP and [18F]-FCP encapsulated [111In]-Labeled Liposome showed different pharmacokinetic profiles. PET imaging of [18F]-FCP showed major uptake in kidneys and bladder. However, [18F]-FCP encapsulated [111In]-Labeled Liposome showed major uptake in RES in both PET and SPECT images. ROI analysis of SPECT image enabled by 111In corresponded with PET image enabled by 18F demonstrating the feasibility of dual tracer imaging from the single nanoconstruct. Future work involves the intensive in vitro characterization of [18F]-FCP encapsulated [111In]-Labeled Liposome and detailed in vivo evaluation of [18F]-FCP encapsulated [111In]-Labeled Liposome in various tumor models.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2018-09-13T17:48:46Z No. of bitstreams: 1 2017 - Camila da Costa Barros de Souza.pdf: 3294573 bytes, checksum: 19a6a94e300565df61e362d626a65cf1 (MD5) / Made available in DSpace on 2018-09-13T17:48:46Z (GMT). No. of bitstreams: 1 2017 - Camila da Costa Barros de Souza.pdf: 3294573 bytes, checksum: 19a6a94e300565df61e362d626a65cf1 (MD5) Previous issue date: 2017-02-17 / FAPERJ / Residual herbicides, in some cases, can affect subsequente crops. In these situations, The use of phytoremediation species may be an alternative in the degradation of these molecules, minimizing the carryover risks. The first step in establishing if some species can be used as a phytoremediation, is check that it has tolerance to the product, for later verify its remedial effect. Against the foregoing, The present master's thesis aimed to identify plant species capable of phytoremediation the diclosulam herbicide, elucidating the biological mechanism of phytoremediation used by plants. For this, 3 different experiments were performed. In the first experiment, conducted at the Dow Agrosciences Experiment Station, it was selected, among the species Arachis pintoi, Brachiaria brizantha, Brachiaria decumbens, Canavalia ensiformis, Cajanus cajan e Crotalaria juncea, those that shows tolerance to the diclosulam herbicide. In the second experiment, conducted in the Universidade Federal Rural do Rio de Janeiro, it was evaluated the efficiency of the previously selected plant species to remediate soil contaminated with the diclosulam herbicide, using cucumber as a bioindicator plant. In the third experiment, conducted at Centro de Energia Nuclear na Agricultura (CENA/USP), it was verified if the tolerance mechanism of the two species that presented the greatest phytoremediation potential occurs due to the phenomena of absorption and / or translocation of the herbicide by the plant. The doses of diclosulam herbicide tested in the first and second experiments were 21, 42, 63, e 84 g ha-1 + control (without presence of the herbicide); And in the third experiment, the doses that were tested was 42 g ha-1 + control (without presence of the herbicide). The species Arachis pintoi, Canavalia ensiformis, Cajanus cajan e Crotalaria juncea showed a tolerance to the diclosulam herbicide at the doses tested, and the species Canavalia ensiformis, Cajanus cajan e Crotalaria juncea were efficient in the dissipation/degradation of these molecules in the soil, it is pointed out that the last two species were those that presented the greatest remedial effect. Using the 14C radiolabeled herbicide in its moleculares structure, could be inferred through the mass balance and observation of the radioimagens of the species Crotalaria juncea e Cajanus cajan that the phytoremediative action performed by these species is, probably, through phytostimulation. In addition, there is an anatomic / metabolic barrier of diclosulam translocation in the collar of these species, which gives them tolerance to this herbicidal molecule. / Herbicidas residuais, em alguns casos, podem afetar culturas subsequentes. Nestas situa??es, o uso de esp?cies fitorremediadoras pode ser uma alternativa na degrada??o destas mol?culas, minimizando o risco de carryover. O primeiro passo para estabelecer se uma esp?cie pode ser utilizada como fitorremediadora, ? verificar se a mesma apresenta toler?ncia ao produto, para posteriormente verificar o seu efeito remediador. Diante do exposto, a presente disserta??o de mestrado teve por objetivo identificar esp?cies vegetais capazes de fitorremediar o herbicida diclosulam, elucidando o mecanismo biol?gico de fitorremedia??o empregado pelas plantas. Para isso foram realizados 3 experimentos distintos. No primeiro experimento, realizado na Esta??o Experimental da Empresa Dow Agrosciences, foi selecionado, dentre as esp?cies Arachis pintoi, Brachiaria brizantha, Brachiaria decumbens, Canavalia ensiformis, Cajanus cajan e Crotalaria juncea, aquelas que apresentem toler?ncia ao herbicida diclosulam. No segundo experimento, realizado na Universidade Federal Rural do Rio de Janeiro, avaliou-se a efici?ncia das esp?cies vegetais, previamente selecionadas, em remediar solo contaminado com o herbicida diclosulam, utilizando o pepino como planta bioindicadora. No terceiro experimento, realizado no Centro de Energia Nuclear na Agricultura (CENA/USP), foi verificado se o mecanismo de toler?ncia das duas esp?cies que apresentaram maior potencial fitorremediador ocorre devido aos fen?menos de absor??o e/ou transloca??o do herbicida pela planta. As doses do herbicida diclosulam testadas no primeiro e segundo experimento foram 21, 42, 63, e 84 g ha-1 + controle (sem presen?a do herbicida); E no terceiro experimento foram testadas as doses 42 g ha-1 + controle (sem presen?a do herbicida). As esp?cies Arachis pintoi, Canavalia ensiformis, Cajanus cajan e Crotalaria juncea apresentaram toler?ncia ao herbicida diclosulam nas doses testadas, sendo as esp?cies Canavalia ensiformis, Cajanus cajan e Crotalaria juncea eficientes na dissipa??o/degrada??o dessas mol?culas no solo, posto que as duas ?ltimas esp?cies foram as que apresentaram maior efeito remediador. Utilizando o herbicida radiomarcado com 14C em sua estrutura molecular, p?de-se inferir atrav?s do balan?o de massa e observa??o das radioimagens das esp?cies Crotalaria juncea e Cajanus cajan que a a??o fitorremediadora exercida por essas esp?cies ?, provavelmente, atrav?s da fitoestimula??o. Ademais, existe uma barreira anat?mica/metab?lica de transloca??o do diclosulam no coleto dessas esp?cies, o que lhes confere toler?ncia ? essa mol?cula herbicida.

Two-dimensional (2-D) micro-autoradiography is typically used to identify the location of a radio-labeled ligand bound to a cellular target in tissue sections. Data, such as a histological image, combined with the autoradiographic data provide a spatial relationship of the radiolabel to cellular structures. However, the disadvantage of 2-D imaging is that it only provides a local distribution of the radiolabel within a tissue slice, and not a volumetric regional distribution in the structure of interest. The development of 3-D autoradiographic/histological visualizations would provide important information not otherwise apparent, such as the ability to visualize the distribution of the labeled agents in subcutaneous tissue. We plan to obtain digital micro-autoradiographic images and fuse them to their corresponding histological images using commercially available software. We plan to create a series of 2-D fused images. This series of 2-D fused images will then form a basis for creating 3-D visualization of autoradiographic/histological images using another commercially available software. These type of fused 3-D images, which we will refer to as micro-autoradiographic fusion tomography (MAFT), are not currently available. We will illustrate the use of MAFT with the distribution of vascular endothelial growth factor (VEGF) in subcutaneous tissue. [14C]-VEGF will be injected into rat subcutaneous tissue. VEGF has been found to stimulate angiogenesis, or the growth of new blood vessels, which could prove beneficial by aiding the function of an implantable blood glucose sensor. The diffusion coefficient for VEGF in subcutaneous tissue has not yet been characterized. MAFT would be an ideal technique to use for this type of study. My thesis will address the following specific aims: 1) To label the nicotine receptors in adult and adolescent rat brains, and to obtain digital micro-autoradiographic images and histological images; 2) To fuse a 2-D digital micro-autoradiographic image with a 2-D histological image; 3) To create a 3-D image from a series of 2-D fusion images; and 4) To assess the increased information value obtained using MAFT.

Master of Science / Department of Kinesiology / Timothy I. Musch / Dietary supplementation with omega-3 polyunsaturated fatty acids (PUFAs) containing docosahexaenoic (DHA) and eicosapentaenoic acid (EPA) has been demonstrated to produce advantageous effects on vascular function. Specifically, PUFA supplementation has resulted in enhanced brachial artery blood flow (Q), dilation, and vascular conductance (VC) during rhythmic handgrip exercise. The effects of fish oils (FO) on skeletal muscle blood flow (Qm) during dynamic whole body exercise, however, remain unknown. PURPOSE: To test our hypothesis that 6 weeks of dietary FO supplementation with DHA and EPA enhances regional Qm and VC to the hindlimb musculature during submaximal treadmill exercise. METHODS: Following 6 weeks of dietary supplementation with safflower oil (SO) (control; n = 9) or FO (n = 8), heart rate (HR), mean arterial pressure (MAP), and Q[subscript]m to the hindlimb were measured at rest and during submaximal treadmill exercise (20 m/min, 10%, ~65% VO[subscript]2max) via radiolabeled microspheres in adult male Sprague-Dawley rats. RESULTS: HR and MAP were not different between SO and FO at rest or exercise (P<0.05). Q[subscript]m and VC were not different between SO and FO at rest. During exercise, FO exhibited greater Q[subscript]m in 8 of the 28 muscle parts measured as well as greater VC in 11 of the 28 muscle parts measured. Additionally, FO exhibited greater (Q)[subscript] m (158[plus or minus]9) and VC (1.156[plus or minus]0.066) to the total hindlimb musculature than SO (128[plus or minus]10 ml/min/100g, 0.918[plus or minus]0.077 ml/min/100g/mmHg) (P<0.05). CONCLUSION: These results demonstrate that 6 weeks of dietary FO supplementation with DHA and EPA results in enhanced Q[subscript]m and VC to the hindlimb during submaximal exercise. Thus, supplementation with DHA and EPA may have therapeutic effects on oxygen delivery and vascular function in patients with impaired vascular function and exercise tolerance (i.e., congestive heart failure, diabetes).

Epoxy fatty acids (EXA) are valuable to industry as they are used in synthesizing plasticizers such as of poly vinyl chloride, resins, adhesives, coating materials such as paint, lubricant, lubricant additives, insecticides, insect repellants, crop oil concentrates and formulations of carriers for slow release pesticides and herbicides. There is interest in developing commercial oilseeds accumulating epoxy fatty acids to at least 50% of the seed oil. Soybeans are the most widely cultivated oilseed and its oil has high levels of linoleic acid which can be a substrate for epoxygenase enzymes. Cahoon et al., expressed a cytochrome P450 enzyme (CYP726A1) from Euphorbia lagascae in soybean somatic embryos and found that the epoxy fatty acid, vernolic acid, reached ~8% of the total fatty acids in transgenic somatic embryos. Rabbit Livers possess a cytochrome P450, CYP2C2, which catalyzes the same epoxidation reaction as the E. lagascae enzyme but might be less likely to be influenced by regulatory machinery in plant cells. This CYP2C2 gene was placed in a plant expression vector under a seed-specific promoter and used to transform soybean, Glycine max, somatic embryos. The ten putative transgenic clones observed after 4-5 weeks were separated and proliferated under selection. glucuronidase (GUS) assays and PCR analyses performed on selected clones were positive. However vernolic acid in total lipids and specific lipid classes was not detected as analyzed by GC. In vitro enzyme assay performed on microsomes isolated from mature somatic embryos at three weeks of maturation using [14C] 18:2 PC as substrate showed presence of [14C] methyl vernoleate. Preliminary analyses on toxicity of epoxy fatty acids and corresponding diols in bacteria, yeast and caco-2 cells showed that leukotoxin diol (LD) most toxic.

Plusieurs maladies rénales et cardiovasculaires présentent une altération de l’expression des récepteurs de type 1 de l'angiotensine (AT1R) et sont traitées par le candésartan ou le losartan. Le radiomarquage de cette famille de molécules développé au sein de notre laboratoire a démontré une liaison spécifique pour les AT1R rénaux chez les rats et les cochons. Le [11C]méthyl-candésartan présente une cinétique supérieure à celle du [11C]méthyl-losartan. Néanmoins, la présence d’un radiométabolite hydrophobe, interférant avec le signal TEP du traceur d’origine, rend la quantification des AT1R complexe. Le [18F]fluoropyridine-losartan est un antagoniste qui possède une affinité de liaison élevée ainsi que peu de métabolites radiomarqués dans les reins de rats. Cependant, sa faible activité molaire ne permet pas de visualiser les niveaux d’AT1R myocardiques présents en faible densité. Le but de cette recherche est de développer les radiosynthèses automatisées d'analogues 18F-fluoropyridinés du candésartan et du losartan en très hautes puretés et activités molaires et d'évaluer l'efficacité du [18F]fluoropyridine-candésartan comme traceur TEP sélectif aux AT1R. Le [18F]fluoropyridine-candésartan et le [18F]fluoropyridine-losartan ont été synthétisés via la cycloaddition de Huisgen catalysée au cuivre(I) entre le 2-[18F]fluoro-3-(pent-4-yn-1-yloxy)pyridine ([18F]FPyKYNE) et un dérivé azidé du candésartan ou du losartan. Le [18F]FPyKYNE est préalablement obtenu par radiofluoration de NO2PyKYNE puis purifié par HPLC, empêchant ainsi la formation de sous-produits nitropyridinés. Après optimisation, les traceurs sont obtenus avec un rendement radiochimique de 11% et des activités molaires (>380 GBq/μmol), des puretés chimiques et radiochimiques (>97%) très élevées. Les tests de contrôle de qualité complet du [18F]fluoropyridine-losartan permettant son utilisation dans des études cliniques futures ont aussi été développés. Le fluoropyridine-candésartan a révélé une affinité élevée (Ki=5,9 nM) dans des membranes exprimant les AT1R, comparable au fluoropyridine-losartan (Ki=5,6 nM) mais inférieure à celle du candésartan (Ki=0,4 nM). Des études d’ultrafiltration ont démontré que le [18F]fluoropyridine-candésartan se lie fortement aux protéines plasmatiques (99,3%). La liaison spécifique aux AT1Rs a été confirmée ex vivo par biodistribution. Une réduction significative (-86%) de la captation dans le cortex rénal de rats prétraités (candésartan ou losartan) est constatée. Des résultats similaires sont obtenus in vitro par autoradiographie avec co-incubation en présence de losartan. La présence de métabolites radiomarqués a été évaluée ex vivo dans du plasma et dans les reins d’animaux témoins ou prétraités (candésartan ou losartan) par HPLC. Le [18F]fluoropyridine-candésartan a été métabolisé en composés radiomarqués hydrophiles, affichant une interférence minimale sur la liaison rénale aux AT1R avec 82% de traceur inchangé dans les reins 20 min après injection. Les images TEP/TC dynamiques du [18F]fluoropyridine-candésartan, acquises pendant 60 min, ont révélé des accumulations élevées et éliminations lentes dans le cortex rénal et le foie avec des rapports tissu-sang élevés. La spécificité de liaison aux AT1R a été démontrée par le blocage des sites de liaison avec du losartan 20 min après l'injection (réductions des rapports tissu-sang dans le cortex rénal (-84%) et dans le foie (-93%)). Aucun changement n'a été observé dans les reins de rats prétraités avec l'antagoniste des AT2R PD123,319, confirmant la sélectivité de liaison pour les AT1R. Les radiosynthèses reproductibles automatisées, les propriétés de liaison ainsi que la stabilité in vivo font du [18F]fluoropyridine-candésartan et du [18F]fluoropyridine-losartan des radiotraceurs potentiels permettant l’imagerie TEP des AT1R dans plusieurs maladies cardiovasculaires ou rénales, offrant ainsi la possibilité de suivre la progression de ces maladies dans des études longitudinales mais aussi de mieux guider la thérapie. / Numerous renal and cardiovascular diseases exhibit alterations in the Angiotensin type 1 receptor (AT1R) levels that are treated with candesartan or losartan. Our previous radiolabeled derivatives of these drugs displayed specific binding for renal AT1R in rats and pigs. [11C]Methyl-candesartan exhibited superior kinetics than [11C]methyl-losartan, but a hydrophobic radiometabolite interfered with the PET signal and AT1R quantification. High binding affinity, full antagonistic efficacy and minimal interference of labeled metabolites in rat kidneys were observed with [18F]fluoropyridine-losartan. However, the tracer’s low molar activity prevented visualization of low-density myocardial AT1R. The aim of this research was to develop automated radiosyntheses of 18F-fluoropyridinated analogs of candesartan and losartan in very high purities and molar activities and to evaluate [18F]fluoropyridine-candesartan efficacy as a selective AT1R PET tracer. [18F]Fluoropyridine-candesartan and [18F]fluoropyridine-losartan were synthesized via the copper(I)-catalyzed Huisgen cycloaddition between 2-[18F]fluoro-3-(pent-4-yn-1-yloxy)pyridine ([18F]FPyKYNE) and an azido-derivative of candesartan or losartan, respectively, followed by acid deprotection and C18-HPLC purification. [18F]FPyKYNE was obtained by radiofluorination of NO2PyKYNE and purified by silica-gel HPLC, preventing the formation of nitropyridinated by-products in the second step. The reaction parameters and purifications were optimized to provide tracers in 11% radiochemical yield with very high molar activities (>380 GBq/μmol), chemical and radiochemical purities (>97%). Full quality control testing of [18F]fluoropyridine-losartan was performed to validate its safe use in future clinical studies. The binding and pharmacokinetic properties of the novel [18F]fluoropyridine-candesartan were evaluated in vitro and ex/in vivo in male Sprague-Dawley rats. Competition binding assays in AT1R-expressing membranes revealed a high affinity of fluoropyridine-candesartan (Ki=5.9 nM), which is similar to fluoropyridine-losartan (Ki=5.6 nM) but lower than candesartan (Ki=0.4 nM). [18F]Fluoropyridine-candesartan bound strongly to plasma proteins (99.3%) as assessed by ultrafiltration. Specific binding was confirmed by ex vivo biodistribution 20 min after tracer injection, with a significant uptake reduction (-86%) in the AT1R-rich kidney cortex of pretreated (candesartan or losartan) rats and by in vitro autoradiography with losartan co-incubation. Radiolabeled metabolites in plasma and kidneys of control and pretreated (candesartan or losartan) animals were analyzed by column-switch HPLC. [18F]Fluoropyridine-candesartan was mainly metabolized to radiolabeled hydrophilic compounds, displaying minimal interference on renal AT1R binding with 82% of unchanged tracer in the kidneys at 20 min post-injection. Dynamic PET/CT images of [18F]fluoropyridine-candesartan, acquired for 60 min at baseline, revealed high accumulation and slow clearance from the kidney cortex and liver with high tissue-to-blood ratios. Binding specificity for AT1R was demonstrated with marked reductions in kidney-cortex (-84%) and liver (-93%) tissue-to-blood ratios at 20 min post-injection, when blocking with losartan. No change was observed in kidneys of rats pre-treated with the AT2R antagonist PD123,319, confirming binding selectivity for AT1 over AT2 receptors. The favorable binding properties and in vivo stability of [18F]fluoropyridine-candesartan support further studies to validate its potential as AT1R PET radioligand. The reproducible automated radiosyntheses developed in my research will allow innovative in-vivo PET imaging of AT1R-expression in several diseases, offering the possibility to follow disease progression in longitudinal studies and guide therapy.