No description available.

Teleost fishes incorporate renal ammonia excretion as part of a greater acid-base regulatory system. However, the transport mechanisms employed by the renal epithelium to excrete ammonia are relatively unknown. I hypothesized that, under metabolic acidosis, increased renal ammonia excretion would be the product of tubular secretion and involve a Na+/NH4+ exchange metabolon mediated through Rhesus (Rh) glycoproteins. To induce metabolic acidosis, goldfish (Carassius auratus) were exposed to a low pH environment (pH 4.0; 48-h). There was a clear signal of metabolic acidosis: a reduction in both plasma [HCO3-] and blood pH with no influence on plasma PCO2. Goldfish demonstrated an elevation in total plasma [ammonia] with a reduction in PNH3 under acidosis. Metabolic acidosis induced higher rates of urinary excretion of acidic equivalents in the form of both NH4+ and titratable acidity-HCO3- (TA-HCO3-) excretion. Urinary Na+ excretion was not affected by acidosis and urine [Na+] did not correlate with urinary [ammonia]. Alanine aminotransferase activity in the kidney was higher in acidotic goldfish. Glomerular filtration rate and urine flow rate were not affected by acidosis. Increased renal NH4+ excretion was due to increased secretion, and not increased filtration, of ammonia. There was a corresponding elevation in Rhcg1b mRNA expression but no change in renal Na+ reabsorption. My data support a secretion-based mechanism of teleost renal ammonia transport. This system is Na+ independent and is likely mediated by Rh glycoproteins and H+ ATPase, involving a parallel H+/NH3 secretion mechanism. To investigate effects of metabolic acidosis on elasmobranch fish, Pacific spiny dogfish (Squalus acanthias suckleyi) were infused with an acidic saline (125 mM HCl/375 mM NaCl; 3 ml/kg/h; 24-h). The results are preliminary, with no marked effects of HCl infusion on plasma acid-base or N-status, but increased branchial NHE2 and lower renal NHE3 protein expressions. These data are summarized in an Appendix. / Thesis / Master of Science (MSc)

本論文では，配列制御ペプチド−ビニルポリマー・ハイブリッドの合目的的な機能設計とその精密合成法の確立を目指し，グラフト型，トリブロック型，マルチブロック型など多様な高分子形状のハイブリッドポリマーの設計およびその合成法について明らかにした．ペプチド−ビニルポリマー・ハイブリッドは，各構成成分の種類 (一次構造) やその組み合わせ方 (高分子形状) により無限の機能・構造設計が可能な新しいスマートバイオマテリアルであり，そのハイブリッド化の組み合わせ方は機能発現の鍵を握る重要な因子である．特に，ペプチド−ビニルポリマー・ハイブリッドによるGFP様の蛍光発現の再現，および全ユニットがアミノ酸で構成されたハイブリッドポリマーによる単鎖フォールディング形成の実現は，合成高分子化学の究極の目標の一つである人工タンパク質開発において大変意義深く，新しい設計・合成指針を提供する． / 博士(工学) / Doctor of Philosophy in Engineering / 同志社大学 / Doshisha University

No description available.

Recombinant proteins have many medical and industrial applications, but their use is complicated by commercial production and stability constraints. These issues are particularly challenging for recombinant proteins used in pharmaceutical therapeutics and clinical diagnostics. Expensive production and distribution limit the accessibility of therapeutics and diagnostics especially in the developing world. Additionally, clinical use of recombinant proteins face further challenges within biological systems including biological degradation and immunogenicity. To increase the accessibility of recombinant proteins, the cost and inefficiencies of protein manufacturing and distribution need to be significantly reduced. A powerful tool to aid in this endeavor is cell-free protein synthesis (CFPS) technology. CFPS is a versatile platform for recombinant protein production due to its open reaction environment, flexible reaction conditions, and rapid protein expression capabilities. These avoid the disadvantages of conventional manufacturing and present the capability of on-demand protein therapeutic production outside of centralized facilities. To improve the efficacy of recombinant proteins for medicinal use, protein engineering techniques such as PEGylation, or the conjugation of PEG polymers to protein surfaces, can be employed. PEGylation is widely used to enhance the pharmacokinetic properties of protein therapeutics. Deciphering optimal PEG conjugation sites is a continuing area of research that can be facilitated by CFPS systems that enable high-throughput, site-specific PEGylation. This dissertation presents advances in CFPS technology to promote increased accessibility and stability of life-saving therapeutics and diagnostics. The work presented here (1) improves on-demand therapeutic production capabilities by creating shelf-stable, endotoxin-free CFPS systems, (2) aids the rational design of next-generation PEGylated protein therapeutics through an in silico-in vitro CFPS screening platform, and (3) advances the development of portable clinical diagnostics for rapid and sustainable deployment at point-of-care through CFPS biosensor technology. The innovations of this dissertation are described in four publications. Specifically, an endotoxin-free CFPS system lyophilized with lyoprotectants is demonstrated that shows improved shelf-stability over standard lyophilized systems. A streamlined procedure for preparing endotoxin-free extract using auto-induction media is presented that significantly reduces CFPS preparation labor and time. A combinatorial screening approach is demonstrated in which coarse-grain molecular simulation informs PEGylation site selection as verified by CFPS experimental results. An inexpensive paper-based, saliva-activated CFPS biosensor platform is developed for the detection of SARS-CoV-2 sequences.

No description available.

No description available.

Applications in paleontological research are far from being limited to taxonomic collection and identification. Nor is such research limited to working solely on fossil data. Actualistic paleontology is the study of modern or recent organisms and processes to better understand those of the past. The bulk of this body of research falls under the category of actualistic paleontology, and examines geochronological methods and error biases in dating biological specimens ranging in age from modern to thousands of years old. Although such methods are arguably not perfect, error rates of ± a few hundred to few thousand years can be extremely important when considering ecological relationships among both Holocene taxa and time-averaged paleocommunities, but quite diminished when considering implications on more traditional dating techniques for ancient strata. Regardless, understanding implications of time resolution is important in analyses of and comparisons between any biological dataset. The following chapters are united by quantitative and statistical management of data with varying levels of temporal resolution, and represent four manuscripts that either are in press or soon to be submitted for publication. / Ph. D.

The emerging evidence of preferential accumulation and long residence time of proteinaceous compounds in soil are counter to the traditional view that their structure is readily broken down through microbial activity. The shift in thinking of their residence time is, however, heavily influenced by physical and chemical protections in soil, representing an important change for understanding global biogeochemical carbon and nitrogen cycling. We investigated the accumulation patterns of proteinogenic amino acids for a long term (thousands of years) related to their sources and sinks. We found clear patterns of change in the amino acids in a 4000 year-chronosequence adjacent to Lake Michigan, USA (Michigan chronosequence) and they were tightly related to the shifts in their biological sources, namely aboveground vegetative community (r2=0.66, p<0.0001) and belowground microbial community (r2=0.71, p<0.0001). Results also showed great variations of approximately 49% between seasons (summer and winter). Moreover, seasonal dynamic patterns (22% variations) of the amino acids in soil mineral associated fraction were rather counter to the conceptual view that it represents a slow soil organic pool with long residence times. The amino acids enriched in the mineral associated fraction, (e.g., positively charged, aromatic, and sulfur containing amino acids), tended to preferentially accumulate in whole soil pool during the 4000 years of ecosystem development. Their interaction with soil minerals, therefore, may play a critical role in the long-term sink and selective accumulation of proteinaceous compounds with some degree of the displacement. This was further confirmed by another chronosequence system near Haast River, New Zealand, which is geologically separated and climatically- and ecologically- different from the Michigan chronosequence. Common trends between two chronosequences suggested that either polar interactions or redox reactions may be relatively more important in the mineral interaction of amino acids than non-polar interactions. The consistency of results at two disparate locations in the southern and northern hemispheres is strong evidence that the processes of pedogenesis and ecosystem development are parsimonious and predictable. Our research demonstrated fundamental understanding of behavior of proteinaceous compounds at the molecular species level, and further provided their partitioning mechanisms associated with soil components. / Ph. D.

No / Drugs which upregulate astrocyte glutamate transport may be useful neuroprotective compounds by preventing excitotoxicity. We set up a new system to identify potential neuroprotective drugs which act through GLT-1. Primary mouse striatal astrocytes grown in the presence of the growth-factor supplement G5 express high levels of the functional glutamate transporter, GLT-1 (also known as EAAT2) as assessed by Western blotting and (3)H-glutamate uptake assay, and levels decline following growth factor withdrawal. The GLT-1 transcriptional enhancer dexamethasone (0.1 or 1 muM) was able to prevent loss of GLT-1 levels and activity following growth factor withdrawal. In contrast, ceftriaxone, a compound previously reported to enhance GLT-1 expression, failed to regulate GLT-1 in this system. The neuroprotective compound riluzole (100 muM) upregulated GLT-1 levels and activity, through a mechanism that was not dependent on blockade of voltage-sensitive ion channels, since zonasimide (1 mM) did not regulate GLT-1. Finally, CDP-choline (10 muM-1 mM), a compound which promotes association of GLT-1/EAAT2 with lipid rafts was unable to prevent GLT-1 loss under these conditions. This observation extends the known pharmacological actions of riluzole, and suggests that this compound may exert its neuroprotective effects through an astrocyte-dependent mechanism.