This work deals with carborane and metallacarborane clusters, in terms of their fundamental chemistry and complexation with cyclodextrins, and in the context of emerging pharmacophores applicable in medicinal chemistry. Arguably, the most important part of this work is the preparation of cobalt bis(dicarbollide) sulfamide derivatives. The sulfamido group is attached to the metallacarborane carbon vertex by an alkyl chain that may be modified in its length. This was accomplished by, firstly, the abstraction of the acidic hydrogen, located on the {CH}-vertex from the metallacarborane, by reaction with lithium base, followed by, secondly, reaction with electrophilic agents (PFA, oxirane and oxetane), which leads to a cascade of reactions to give the desired sulfamide derivatives. These compounds were then tested by collaborators in other institutes for in vitro and in vivo activity towards Carbonic Anhydrase IX (CA IX), which is an enzyme associated with tumour growth. In vivo tests on mice have shown that these types of substances are able to effectively reduce tumour size by 30%. The synthetic research continued with the preparation of sulfonamide compounds of the isomers of the carborane series. The reactions began exclusively with propylhydroxy carborane starting materials, which provide optimum...

Carbonic anhydrase is a ubiquitous zinc-metalloenzyme that catalyzes the interconversion of carbon dioxide and carbonate and has been found to play a wide range of roles in animals, plants and bacteria. Cotton genomic and cDNA libraries were screened for the plastidial isoform of carbonic anhydrase. The nucleotide sequences of two 1.2 Kb partial cDNA clones were determined. These clones exhibit high homology to carbonic anhydrases from other dicot plants and possess all the expected peptide motifs. For example, serine and threonine rich chloroplastic targeting peptide and conserved zinc binding residues are both present. These clones were utilized to isolate two carbonic anhydrase genes that were shown to encode different isoforms by PCR and RFLP analysis.

Concrete is the most widely used construction material in the world and is responsible for 7% of global carbon emissions. It is inherently brittle, and it requires frequent repair or replacement which is economically expensive and further generates large volumes of carbon dioxide. Current methods of repair by agents such as mortar, epoxies, and bacteria result in structures with reduced strength and resiliency. Recent advances in the design of structural composites often mimic natural microstructures. Specifically, the structure of abalone nacre with its high stiffness, tensile strength, and toughness is a source of inspiration from the process of evolution. The inspiration from nacre can lead to design of a new class of architected structural materials with superb mechanical properties. This body of work first presents a method to reinforce concrete with an architected polymer phase. Second is presented how a ubiquitous enzyme, Carbonic anhydrase (CA), can be used to repair and strengthen cracked concrete, and how it can be used as an additive in fresh concrete. The first study presents an experimental and computational study on a set of bioinspired architected composites created using a cement mortar cast with brick-and-mortar and auxetic polymer phases. The impact of this unit-cell architected polymer phase on the flexural and compressive strengths, resilience, and toughness is studied as a function of microstructural geometry. All mechanical properties of the architected composite samples are found to be greater than those of control samples due to prevention of localized deformation and failure, resulting in higher strength. The microstructurally designed composites showed more layer shear sliding during fracture, whereas the control samples showed more diagonal shear failure. After initial cracking, the microstructurally designed composites gradually deformed plastically due to interlocking elements and achieved high stresses and strains before failure. Results also show that microstructurally designed composites with the architected polymer phase outperform control samples with equal volume fraction of a randomly oriented polymer fiber phase. Computational studies of the proposed unit cells are also performed, and the results suggest that the orientation of cells during loading is critical to achieve maximum performance of a cementitious composite. The implications of these results are immense for future development of high performing construction materials. The second study outlines methods for repair of concrete and lays the groundwork to develop a self-healing concrete that uses trace amounts of the CA enzyme. The CA catalyzes the reaction between calcium ions and carbon dioxide to create calcium carbonate that naturally incorporates into concrete structures with similar thermomechanical properties as concrete. The reaction is safe, actively consumes carbon dioxide, generates low amounts of heat, and avoids using unhealthy reagents, resulting in a strong structure. This repair method results in concrete samples with similar strength and water permeability as the intact materials. These results offer an inexpensive, safe, and efficient method to create self-healing concrete structures. The science underlying the creation of self-healing concrete is described, producing a material intrinsically identical to the original using the CA enzyme. Using this strategy, a preliminary self-healing concrete mix is able to self-repair fractures via hydration. This body of work addresses a major issue: Is there an efficient and ecological repair for decaying concrete infrastructure? These methods propose alternative reinforcement, alleviates high monetary and energy costs associated with concrete replacement, and consume the greenhouse gas, carbon dioxide.

Gammaproteobacterium Thiomicrospira crunogena thrives at deep-sea vents despite extreme oscillations in the environmental supply of dissolved inorganic carbon (DIC; =CO2 + HCO3- + CO3-2). Survival in this habitat is likely aided by the presence of a carbon concentrating mechanism (CCM). Though CCMs are well-documented in cyanobacteria, based on this study T. crunogena is the first chemolithoautotroph to have a physiologically characterized CCM. T. crunogena is capable of rapid growth in the presence of 20 micrometers DIC, has the ability to use both extracellular HCO3- and CO2, and generates intracellular DIC concentrations 100-fold greater than extracellular, all of which are consistent with a CCM analogous to those present in cyanobacteria. Interestingly, however, the T.crunogena genome lacks apparent orthologs of many of the components of the cyanobacteria CCM (e.g., HCO3- transporters). However, despite this lack, several candidate genes were identified during genome annotation as likely to play a role in DIC uptake and fixation (three carbonic anhydrase genes: alpha-CA, beta-CA, and csoSCA, as well as genes encoding three RubisCO enzymes: cbbLS, CScbbLS, and cbbM, which encode a cytoplasmic form I RubisCO, a carboxysomal form I RubisCO, and a form II RubisCO, respectively). In order to clarify their possible roles in DIC uptake and fixation, alpha-CA, beta-CA and csoSCA transcription by low-DIC and high-DIC T. crunogena were assayed by qRT PCR, heterologous expression in E. coli, and potentiometric assays of low-DIC and high-DIC T. crunogena. Transcription of alpha-CA and beta-CA were not sensitive to the DIC concentration available during growth. When overexpressed in E.coli, carbonic anhydrase activity was detectable, and it was possible to measure the effects of the classical carbonic anhydrase inhibitors ethoxyzolamide and acetazolamide, as well as dithiothreitol (DTT; recently determined to be a carboxysomal CA inhibitor). The alpha-CA was sensitive to both of the classical inhibitors, but not DTT. Beta-CA was insensitive to all inhibitors tested, and the carboxysomal carbonic anhydrase was sensitive to both ethoxyzolamide and DTT. The observation that the CA activity measureable potentiometrically with intact T. crunogena cells is sensitive to classical inhibitors, but not DTT, strongly suggests the alpha-CA is extracellular. The presence of carbonic anhydrase activity in crude extracts of high-DIC cells that was resistant to classical inhibitors suggests that beta-CA may be more active in high-DIC cells. Incubating cells with ethoxyzolamide (which permeates cells rapidly) resulted in inhibition of carbon fixation, but not DIC uptake, while incubation with acetazolamide (which does not permeate cells rapidly) had no apparent effect on either carbon fixation or DIC uptake. The observations that inhibition of alpha-CA has no effect on DIC uptake and fixation, and that the beta-CA is not transcribed more frequently under low-DIC conditions, make it unlikely that either play a role in DIC uptake and fixation in low-DIC cells. Further studies are underway to determine the roles of alpha-CA and beta-CA in T. crunogena. To assay the entire genome for genes transcribed more frequently under low-DIC conditions, and therefore likely to play a role in the T. crunogena CCM, oligonucleotide arrays were fabricated using the T. crunogena genome sequence. RNA was isolated from cultures grown in the presence of both high (50 mM) and low (0.05 mM) concentrations of DIC, directly labeled with cy5 fluorophore, and hybridized to microarrays. Genes encoding the three RubisCO enzymes present in this organism demonstrated differential patterns of transcription consistent with what had been observed previously in Hydrogenovibrio marinus. Genes encoding two conserved hypothetical proteins were also found to be transcribed more frequently under low-DIC conditions, and this transcription pattern was verified by qRT-PCR. Knockout mutants are currently being generated to determine whether either gene is necessary for growth under low-DIC conditions. Identifying CCM genes and function in autotrophs beyond cyanobacteria will serve as a window into the physiology required to flourish in microbiallydominated ecosystems where noncyanobacterial primary producers dominate.

Indiana University-Purdue University Indianapolis (IUPUI) / Pancreatic ductal adenocarcinoma (PDAC) is an extremely deadly disease characterized by aggressive metastasis and therapeutic resistance. Reactive stroma in pancreatic tumors contributes to tumor signaling, fibrosis, inflammation, and hypoxia. Hypoxia signaling creates a more aggressive phenotype with increased potential for metastasis and decreased therapeutic efficacy. Carbonic anhydrase IX (CA9) functions as part of the cellular response to hypoxia by regulating intracellular pH to promote cell survival. Apurinic/Apyrimidinic Endonuclease-1-Reduction/oxidation Effector Factor 1 (APE1/Ref-1) is a multi-functional protein with two major activities: endonuclease activity in DNA base excision repair and a redox signaling activity that reduces oxidized transcription factors, enabling them to bind target sequences in DNA. APE1/Ref-1 is a central node in redox signaling, contributing to the activation of transcription factors involved in tumor survival, growth, and hypoxia signaling. This work evaluates the mechanisms underlying PDAC cell responses to hypoxia and APE1/Ref-1 redox signaling control of hypoxia inducible factor 1 alpha (HIF1a), a critical factor in hypoxia-induced CA9 transcription. We hypothesized that obstructing the HIF-CA9 axis at two points via APE1/Ref-1 inhibition and CA9 inhibition results in enhanced PDAC cell killing under hypoxic conditions. We found that HIF1a-mediated induction of CA9 is significantly attenuated following APE1/Ref-1 knock-down or redox signaling inhibition in patient-derived PDAC cells and pancreatic cancer-associated fibroblast cells. Additionally, dual-targeting of APE1/Ref-1 redox signaling activity and CA9 activity results in enhanced acidification and cytotoxicity of PDAC cells under hypoxic conditions as well as decreased tumor growth in an ex-vivo 3-dimensional tumor co-culture model. Further experiments characterized novel analogs of clinically relevant drugs targeting the key enzymes in this pathway, resulting in improved potency. These results underscore the notion that combination therapy is essential and demonstrate the potential clinical utility of blocking APE1/Ref-1 and CA9 function for novel PDAC therapeutic treatment.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20614号 / 医博第4263号 / 新制||医||1023(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 高田 穣, 教授 平井 豊博, 教授 岩井 一宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

<p>Renal cell carcinoma (RCC) is the most frequently lethal of urological cancers. It arises in the lining of the proximal convoluted tubule of the kidney and is most common in men ages 50–70. Often, partial or radical nephrectomy is needed to effectively treat the disease, leaving patients with reduced kidney function. RCC frequently displays significant radiation resistance, limiting the usefulness of traditional radiation therapy which might spare patients’ normal tissue. The enzyme carbonic anhydrase 9 (CA9), a product of the hypoxia pathway, is found upregulated in the majority of RCC, particularly the clear cell type. It catalyses the dissolution of carbon dioxide into water as bicarbonate and has been linked to increased invasion and migration in RCC tumour cells. The radiation resistance of two RCC cell lines 786-O (human CCRCC) and RAG (murine renal adenocarcinoma) was investigated by the clonogenic assay in the presence of a CA9 inhibitor or silencing RNA. The interference with CA9 by either of these methods significantly sensitizes 786-O cells to the effects of ionizing radiation <em>in vitro</em>. Moreover, fractionation of the dose delivered can increase this sensitization effect. It is hoped that current targeting of CA9 can make radiation therapy a more feasible option in the treatment of RCC.</p> / Master of Science (MSc)

Symbiosis has been an important factor in evolution, and continues to drive speciation and allows organisms to fill new ecological niches. Symbiotic relationships in which both partners benefit from the association, or mutualisms, are ubiquitous in both terrestrial and aquatic ecosystems. Many of the symbionts in these associations are photosynthetic algae or cyanobacteria that fix carbon through photosynthesis and translocate a portion of this energy to their hosts. Host organisms utilize this fixed carbon for a variety of physiological processes, including growth and development, thus, photosynthetically-fixed carbon is vital for many hosts. The following chapters will describe carbon fixation and translocation in two algal symbioses: the freshwater association between the alga Oophila and the eggs of Ambystoma maculatum salmanders, and the relationship between the dinoflagellate Symbiodinium and marine zoanthids. These chapters will discuss carbon flux in symbiosis, and reveal some of the ways in which environmental factors alter photosynthesis in algal mutualisms. / Biology

Cancer is the second most common cause of death in the world. One of the objectives of this thesis is to biologically evaluate a series of anti-cancer polymeric aromatic/heterocyclic bis-sulfonamides and pyridinium sulfonamides which were synthesized from three established aminosulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitor pharmacophores. Testing of these novel inhibitors and their precursors against a panel of membrane-bound CA isoforms, including tumor-overexpressed CA IX and XII and cytosolic isozymes, identified nanomolar-potent inhibitors against both classes and several compounds with medium isoform selectivity. In the case of pyridinium sulfonamides we used complexes of the inhibitors with cyclodextrins or sulfocalixarene to enhance aqueous solubility for biological testing. The ability of CA inhibitors to kill tumor cells overexpressing CA IX and XII was tested under normoxic and hypoxic conditions, using 2D and 3D in vitro cellular models. The study identified a nanomolar potent PEGylated bis-sulfonamide CA inhibitor (25), as well as cyclodextrin and sulfocalixarenes complexes, which were able to significantly reduce the viability of colon HT-29, breast MDA-MB231, and ovarian SKOV-3 cancer cell lines, thus revealing the potential of polymer conjugates in CA inhibition and cancer treatment. As a different disease state yet still a concern, cognitive dysfunction markedly impacts patients with a host of psychiatric conditions including attention deficit hyperactivity disorder, autism spectrum disorder, drug addiction, schizophrenia, depression, bipolar disorder, obsessive-compulsive disorder, and of course, Parkinson’s and Alzheimer’s diseases and other types of dementia. Another objective of this thesis was to profile several series of bis-imidazoles for physicochemical, in-vitro and in-vivo properties as potential memory and learning enhancers (nootropics). Biological testing on eight isozymes of carbonic anhydrase (CA) present in the human brain revealed compounds with nanomolar potency against at least one membrane bound, cytosolic or mitochondrial CA isozymes, combined with good physicochemical properties. We also identified lead compounds with the ability to rescue experimental animals from drug-induced memory deficits, using an optimized Novel Object Recognition Task (NORT) procedure. / Pharmaceutical Sciences