Dried Blood Spot (DBS) sampling is a microsampling technique used throughout the World for neonatal screening. The work set out in this thesis shows the development and implementation of DBS in the area of preclinical and clinical pharmaceutical drug development, specifically in support of Toxicokinetics and Pharmacokinetics. The advantages of the technique are explored along with the issues faced. The papers discussed in this commentary, include in papers 1 and 2, the concept of supporting both Toxicokinetics and Pharmacokinetics studies and the validation of bioanalytical assays utilising DBS. Commentary paper 3 further explores the practicalities of DBS in the Clinical environment and commentary paper 4 the transferability of DBS technology between laboratories. Commentary paper 5 uses Incurred Sample Reanalysis data to answer questions around specific DBS issues and commentary paper 6 looks at indicating papers for Dried Plasma Spots. Commentary papers 7 and 8 explore the use of consortia to investigate hematocrit and homogeneity when using DBS and finally commentary paper 9 explores the training required to ensure quality DBS samples. The impact and contributions to this field of research are demonstrated through discussion and critical examination of selected examples of the author’s peerreviewed publications in this area. Developments of scientific practices, where the author has contributed intellectual, leadership and practical insight to achieve significant improvements in the generation of knowledge, are highlighted throughout the commentary.

Photoisomerization of azobenzene can be used to reversibly photo-control peptide and protein structure, thereby offering the potential to probe peptide and protein function directly in living systems. Most azobenzene photo-switches to date however require the use of UV light, which suffers from poor tissue penetration and can lead to cell damage. In addition, the unknown stability of azobenzene photo-switches within the reducing intracellular environment has limited these switches to extracellular applications. The ability to red-shift the photo-switching wavelength and tune the thermal cis-to-trans rate independently from one another has been a central challenge. Incorporating a piperazine-like moiety at the 4,4′-para positions red-shifts the π-π* band of an azobenzene-based cross-linker such that trans-to-cis photoisomerization can be triggered with blue light. Accompanying this red-shift was a faster cis-to-trans thermal rate, which allowed for fast secondary structural changes of the attached peptides (τ½ ~2s). Piperazine-like moieties at the 2,2′-ortho positions of an azobenzene-based cross-linker resulted in a similar red-shift in the π-π* band but a longer-lived cis isomer relative to its para-counterpart (τ½ ~minutes). These results suggest that the thermal rate could be tuned independently from the photo-switching wavelength by appropriate para or ortho substitution. The effect of 2,2′,6,6′-ortho-tetramethoxysubstitution provided an alternative approach to red-shifting the photo-switching wavelength. These groups caused an unconventional red-shift in the n-π* band of the trans isomer allowing for trans-to-cis and cis-to-trans photoisomerization to occur with green and blue light, respectively. In this case, the half-life of the cis isomer was not shortened, but rather extended relative to its parent compound (τ½ ~days versus minutes). These results provide progress in tuning the photo-switching wavelength independently from the thermal rate. A fluorescent reporter was developed to determine the stability of a commonly employed 4,4′-diamido derivative in vivo. Photoisomerization was found to cause time-dependent changes in fluorescein emission intensity. The reporter was microinjected in zebrafish embryos and photo-switching could be imaged for at least two days. This work provides the first direct evidence of azobenzene photo-switching in vivo, and indicates that it will be possible, in general, to photo-control peptide and protein function in living systems.

Photoisomerization of azobenzene can be used to reversibly photo-control peptide and protein structure, thereby offering the potential to probe peptide and protein function directly in living systems. Most azobenzene photo-switches to date however require the use of UV light, which suffers from poor tissue penetration and can lead to cell damage. In addition, the unknown stability of azobenzene photo-switches within the reducing intracellular environment has limited these switches to extracellular applications. The ability to red-shift the photo-switching wavelength and tune the thermal cis-to-trans rate independently from one another has been a central challenge. Incorporating a piperazine-like moiety at the 4,4′-para positions red-shifts the π-π* band of an azobenzene-based cross-linker such that trans-to-cis photoisomerization can be triggered with blue light. Accompanying this red-shift was a faster cis-to-trans thermal rate, which allowed for fast secondary structural changes of the attached peptides (τ½ ~2s). Piperazine-like moieties at the 2,2′-ortho positions of an azobenzene-based cross-linker resulted in a similar red-shift in the π-π* band but a longer-lived cis isomer relative to its para-counterpart (τ½ ~minutes). These results suggest that the thermal rate could be tuned independently from the photo-switching wavelength by appropriate para or ortho substitution. The effect of 2,2′,6,6′-ortho-tetramethoxysubstitution provided an alternative approach to red-shifting the photo-switching wavelength. These groups caused an unconventional red-shift in the n-π* band of the trans isomer allowing for trans-to-cis and cis-to-trans photoisomerization to occur with green and blue light, respectively. In this case, the half-life of the cis isomer was not shortened, but rather extended relative to its parent compound (τ½ ~days versus minutes). These results provide progress in tuning the photo-switching wavelength independently from the thermal rate. A fluorescent reporter was developed to determine the stability of a commonly employed 4,4′-diamido derivative in vivo. Photoisomerization was found to cause time-dependent changes in fluorescein emission intensity. The reporter was microinjected in zebrafish embryos and photo-switching could be imaged for at least two days. This work provides the first direct evidence of azobenzene photo-switching in vivo, and indicates that it will be possible, in general, to photo-control peptide and protein function in living systems.

A preorganised amino acid derivative containing a cyclopropyl constraint was designed to orient an amino acid into its bound conformation. This constrained mimic was determined by ITC to be equally potent to the native Phe derivative. It was found that a more favorable enthalpy of binding was compensated by an equally unfavorable entropy compared to the native ligand. In order to properly ascertain the effects of the cyclopropane constraint, a flexible control containing the same number of heavy atoms was synthesized and tested, and it was found to be at least 200 fold less potent than the constrained analog. However, without structural data of the flexible control, it is difficult to infer if the differences in ligand binding affinity arose from the ligand constraint or some other unknown complexity to binding. We studied the thermodynamic and structural effects of modifying alkyl chains of n-alka(e)nol and phenylalka(e)nol binders to MUP-I by both the removal of a rotor via deletion of a methylene unit and restriction of a rotor via the installation of an internal olefin. In general, we observed that a similar thermodynamic signature accompanies modifications for both the n-alka(e)nol and phenylalka(e)nol ligands: A favorable T[delta][delta]Sºo̳b̳s̳ is compensated by an unfavorable T[delta][delta]Hºo̳b̳s̳ such that T[delta][delta]Gºo̳b̳s̳ for both removal of a methylene and insertion of an internal olefin are unfavorable and equipotent, respectively. The insertion of an internal olefin into an alkyl chain led to significantly more favorable entropies than does methylene removal, yet enthalpy-entropy compensation leads to nearly equipotent binding energetics. However, we did find a strong correlation between [delta]Ho̳b̳s̳° and buried apolar Connolly Surface Area (CSA). The intrinsic free energies of introducing an internal olefin into the n-alkanols and phenylalkanols differ markedly from the observed data. It was observed that intrinsic affinities are more favorable than the observable because a favorable T[delta][delta]S⁰i̳n̳t̳ dominates an unfavorable [delta][delta]Hºi̳n̳t̳. Also, we discovered that the intrinsic entropies of inserting an internal olefin are nearly double that of removing a methylene group, suggesting that the insertion of an internal olefin results in the restriction of more C-C rotors. We have shown through ITC analysis that the added substituents probed in this study provided binding increases to our Grb2 SH2 ligands as expected, but that the thermodynamic driving force of binding affinities depended greatly upon the specific nature and flexible mobility of the ligands in the binding pocket. Through a combination of X-ray and ITC studies it was shown that ligands containing rigid and aromatic functional groups bound with a higher [delta]H° than the more flexible alkyl ligands, and that this effect correlates well with more direct vdW contacts made in the pocket. Finally, we described a case study where a strict adherence to the Topliss operational schemes led to an expedient development of novel MUP-I binding analogs. The validity of the schemes was also depicted through the synthesis and testing of ligands that were correctly predicted to be weaker/equipotent to the starting ligand. Of important note is that the degree to which the schemes led to affinity boost depended greatly on the starting potency of the initial compound. / text

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reinhardtii, a unicellular green alga, is a model organism to study the circadian clock. Cryptochromes are the blue light photoreceptors that entrain the clock in some organisms. The CPH1 protein of C. reinhardtii resembles the cryptochromes of the plant model Arabidopsis, but whether CPH1 entrains the circadian clock in C. reinhardtii is not yet known. Recent reports have suggested the existence of one more cryptochrome in C. reinhardtii, which resembles the cryptochromes of animals. However, the amino acid sequence of this protein shows even higher sequence similarity with the 6-4 DNA photolyase of Arabidopsis. DNA photolyases are involved in the repair of UV light-induced DNA damage using the energy of blue light. In order to determine, if the “animal cryptochrome” gene of C. reinhardtii actually encodes a 6-4 DNA photolyase rather than a photoreceptor, an experimental design was developed to test whether the protein product is able to rescue an E. coli mutant defective in its DNA photolyase gene. The design is as follows: In a first step, the coding region of the “animal cryptochrome” cDNA is cloned. In a second step, the cDNA is inserted in-frame into an E. coli expression vector. In a third step, the construct is transformed into an E. coli photolyase mutant, its expression induced, and the strain tested for better survival after UV light exposure. To accomplish the first step, the cloning of “animal cryptochrome” cDNA, total RNA was successfully extracted from C. reinhardtii 4 hrs into the light phase of a 12 h light/12 h dark cycle and reverse transcribed into cDNA using oligo(dT) primers. After initially unsuccessful attempts at amplifying animal cryptochrome from cDNA or genomic template with a variety of primers and conditions, a short fragment with the expected size of 186 bp was amplifiable with both templates. However, even this fragment was not reliably obtained in every PCR assay. Because of this difficulty, real-time PCR was finally performed in the presence of DMSO (Dimethylsulfoxide) and Betaine. These two adjuvants were reported to improve amplifications particularly for GC-rich templates. C. reinhardtii DNA is especially GC-rich with an average of 64% Gs and Cs. The improved conditions allowed the reliable amplification of the 186 bp fragment from genomic template. It also enabled the amplification of a larger fragment of 528 bp from the same template. The results suggest that a combination of 5% DMSO and 1M Betaine is optimal for the amplification of C. reinhardtii DNA and thus can serve as the basis for successful amplification of the entire 1788 bp coding region of the animal cryptochrome cDNA.

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Indiana University-Purdue University Indianapolis (IUPUI) / Polyacetylenic natural products, compounds containing multiple carbon-carbon triple bonds, have been found in a large collection of organisms. Radiochemical tracer studies have indicated that these bioactive metabolites are synthesized from fatty acid precursors through a series of uncharacterized desaturation and acetylenation steps. To date, there are three main pathways believed to be involved in acetylenic natural product biosynthesis. However, it is apparent that the crepenynic acid pathway is the origin of a vast majority of the known plant and fungal acetylenic products. This investigation provides concrete evidence that the polyacetylenic natural products found in the fungus Fistulina hepatica and the medicinal plant species Echinacea purpurea are biosynthesized from crepenynic acid. Through heterologous expression in Yarrowia lipolytica, two acetylenases capable of producing crepenynic acid were identified from E. purpurea. Furthermore, heterologous expression of two diverged desaturases isolated from F. hepatica, uncovered a ∆12-acetylenase and the first multifunctional enzyme capable of ∆14-/∆16- desaturation and ∆14-acetylenation.

Depuis l’émergence du concept de cellules souches cancéreuses (CSC), de telles cellules ont été isolées à partir de diverses tumeurs solides, dont les glioblastomes. Les CSC et les propriétés qui les caractérisent permettent de mieux comprendre l’hétérogénéité tumorale, ainsi que l’agressivité de certaines tumeurs et les récidives après traitement. Avec la mise en évidence des CSC, un nouveau paradigme est apparu dans le domaine de la thérapie anticancéreuse visant à cibler non seulement les cellules de la masse tumorale, mais également les CSC, plus résistantes aux chimio- et radiothérapies, mais aussi capables d’entrer en quiescence et de reformer la tumeur d’origine. L’isolement de CSC à partir des tumeurs, leur physiopathologie et la recherche de molécules capables de les détruire ou de les différencier afin de les rendre plus sensibles aux traitements mobilisent un nombre croissant d’équipes de recherche et certaines industries pharmaceutiques. Cette thèse présente un travail sur des CSC isolées de glioblastomes humains et s’inscrit dans la démarche énoncée ci-dessus. / Since the emergence of the concept of cancer stem cells (CSC), such cells were isolated from various solid tumors including glioblastomas. The CSC and the properties that characterize them allow a better understanding of tumor heterogeneity and aggressiveness of certain tumors and recurrences after treatment. With the highlighting of CSC, a new paradigm has emerged in the field of cancer therapy. New strategies aim at targeting not only the cells of the tumor mass, but also the CSC, more resistant to chemo- and radiotherapy, but also capable of enter into a quiescent state and to reform the original tumor. The isolation of CSC from solid tumors, their pathogenesis and the search for molecules capable of triggering their death or differentiate them to make them more sensitive to treatment, mobilize a growing number of research teams and some pharmaceutical industries.This thesis presents a work on CSC isolated from human glioblastomas and is framed inside the approach set out above.