Zugl.: Hohenheim, Univ., Diss., 2009

published_or_final_version / Architecture / Master / Master of Landscape Architecture

Ubiquitin proteasome pathway is an important cellular pathway that affects the fate of almost all intracellular proteins. Misregulation of this pathway has been found to be associated with a broad range of human diseases, such as cancer, neurodegenerative diseases, as well as viral infections. Ubiquitin recognition by the proteasome is of central importance to this pathway. So far, two proteasome subunits, Rpn10 and Rpn13, have been identified as ubiquitin receptors. An alternative pathway is mediated by shuttling factors. In yeast, three shuttling factors, known as UBL-UBA proteins, have been found. A UBL receptor activity of the proteasome has been attributed to Rpn1. However, yeast cell mutated all five proteasomal ubiquitin receptors is still viable. To identify the additional proteasomal ubiquitin receptor in cells, I first obtained and characterized a new Rpn13 mutant allele. This Rpn13 mutant completely abolished its ubiquitin binding activity, and functionally resembles a null allele. Rpn13 substrate pool has also been sought in this mutant cells. In the second part of this dissertation, I reported a novel ubiquitin binding site on proteasomal subunit Rpn1. With the help of NMR analysis, Rpn1's ubiquitin and UBL binding surfaces were resolved at high resolution and found to substantially overlap. A specific Rpn1 mutation that disrupts both ubiquitin and UBL binding while not compromising the folding of Rpn1 was obtained. This mutant allele shows a pleiotropic proteasomal defect in vivo. Moreover, I found that the dual ubiquitin/UBL binding activity is not unique in Rpn1, but a common feature in all three proteasomal ubiquitin receptors. In summary, the proteasome adopts a multilayer ubiquitin/UBL binding surface to ensure flexible substrate recognition.

Besides collagen, elastin is the most common connective tissue structural protein in vertebrates and similar to collagen relatively resistant to non-specific degradation. Typical elastolytic proteases are the serine-dependent pancreatic and leukocyte elastases, the Zn-dependent matrix metalloproteinase 12, and several lysosomal cysteine proteases. Among the cysteine cathepsins, cathepsins S, K and V are highly potent elastases with cathepsin V displaying the highest activity among all known mammalian elastases. Despite a shared amino acid sequence identity of over 80% between cathepsins V and L and very similar subsite specificities, only cathepsin V has a potent elastase activity whereas cathepsin L lacks it. A series of chimera mutants containing various proportions of cathepsin V and cathepsin L were constructed in an attempt to define a specific region needed for elastin degradation. It was found that retaining the peptide sequence region from amino acids 89 to 119 of cathepsin V preserves the mutant’s elastolytic activity against elastin-Rhodamine conjugates whereas the region FTVVAPGK (amino acids 112-119) contributes approximately 60% of activity retention. Several additional mutant proteins involving mutual swapping of residues VDIPK (amino acids 113-117) of cathepsin L with residues TVVAPGK (amino acids 113-119) of cathepsin V, deletion of Glyl 18 from cathepsin V, and insertion of Gly between Prol 16 and Lysi 17 in cathepsin L were constructed and evaluated for their elastolytic activities. The results obtained with those mutant cathepsin proteins support the importance of the amino acid region spanning the residues from 112 to 119 in cathepsin V. Based on the 3-D structure of cathepsin V, this peptide region is located below subsite binding pocket S2 and forms a wall-like barrier which may act as an exosite for the productive binding of cross-linked elastin.

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The research presented focuses on the use of Carbon-dioxide (CO₂), Nitrogen (N₂) and Argon (Ar) as purge gases for torrefaction. Torrefaction using CO₂ as a purge gas may further improve the fuel characteristics of the torrefied fuel when compared to N₂ and Ar (which are entirely inert), making it better suited for use as a fuel for co-firing with coal or gasification. Three different biomasses were investigated: Juniper wood chips, Mesquite wood chips, and forage Sorghum. Experiments were conducted using a thermo-gravimetric analyzer (TGA, TA Instruments Model Q-600) to determine the effect of the purge gas over a wide range of torrefaction temperatures (200-300°C). TGA weight traces (thermograms) showed an increased mass loss when using CO2 as a purge gas when compared to N₂. The increased mass loss when CO₂ was used is attributed to a hypothesized reaction between the CO₂ and fixed Carbon contained within the biomass. Torrefaction of biomass, using Ar as the purge gas, produced results similar to torrefaction using N₂. Derivative Thermo-Gravimetric analysis (DTG) was done to determine the temperature ranges over which the three main components of biomass (hemicellulose, cellulose, and lignin) decomposed. The DTG results are in agreement with previously published research. From TGA thermograms and DTG analysis it was determined that torrefaction at higher temperatures (>260°C) likely result in the breakdown of cellulose during torrefaction, an undesired outcome. Proximate, ultimate, and heat value analysis was done on all three biomasses. All three contain a relatively high Oxygen content, which serves to decrease the higher heating value (HHV) of the biomass. The HHV of Juniper, Mesquite, and Sorghum on a dry ash-free (DAF) basis were 20,584 kJ/kg, 20,128 kJ/kg, and 19,389 kJ/kg respectively. The HHV of the three biomasses were relatively constant as expected for agricultural biomass. From TGA analysis (thermograms and DTG), an optimal torrefaction temperature was determined (240°C) based upon the amount of mass lost during torrefaction and estimates of energy retained. Batch torrefaction of all three biomasses at the optimal torrefaction temperature was completed using a laboratory oven. All three biomasses were torrefied using CO₂, N₂, and Ar as a purge gas. Proximate, ultimate, and heat value analysis was done for each of the torrefied fuels and compared. Results of the fuel property analysis showed torrefaction reduced the moisture content and oxygen percentage of the fuel resulting in the torrefied biomass having a larger HHV when compared to raw biomass. Due to inherent mass lost during torrefaction, the amount of energy retained in the torrefied biomass was calculated to determine the percentage of the virgin biomass energy content that remained. Torrefaction using CO2 resulted in the lowest amount of energy retention of all three purge gases tested (78.86% for Juniper); conversely, Nitrogen resulted in the highest amount of energy retention (91.81% for Sorghum.) Torrefaction of the biomass also increased the fixed carbon (FC) content of the fuel. The grindability of the torrefied biomass was investigated via size distribution analysis of the raw and ground biomass. Initial size distribution analysis showed that torrefaction of Mesquite and Juniper resulted in smaller particle sizes; with a greater fraction of the torrefied biomass passing through smaller meshes. Analysis of the ground biomass samples showed that torrefaction improved the grindability of the fuel. The percent of torrefied biomass that passed through an 840 micrometer mesh increased by over 20% for both Mesquite and Juniper when ground. Sorghum exhibited similar increases; however, the amount of increase is less apparent due to the smaller particle size distribution of the raw Sorghum.

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