Protein Phosphatase 2A (PP2A) is a holoenzyme consisting of three subunits – a scaffold subunit (A), a catalytic subunit (C), and a regulatory subunit (B). One of its functions is to oppose hyperphosphorylation of pocket proteins in the Rb family, including p107, allowing them to form a suppressor complex with E2F that silences E2F-dependent genes required for cell cycle progression. B55α, a regulatory subunit of PP2A, binds p107 mediating its dephosphorylation by the trimeric B55α /PP2A holoenzyme. Our lab has previously shown that binding of B55α/PP2A is dependent on two regions in the intrinsically disordered spacer region of p107, which contain three potential CDK phosphorylated sites. However, it is not known which of these sites are direct B55α/PP2A substrates and how individual phosphorylation sites are presented to the catalytic active site. In order to perform these studies, we first need to purify the B55α/PP2A holoenzyme. We tested various conditions for purification, and found that the most efficient method for purification of complete holoenzymes is to use stably transfected HEK293T cells expressing flag-B55α, which can be pulled down using α-flag beads and eluted using a high concentration of flag peptide which competes with the flag tag for binding to the beads. Once the enzyme had been successfully purified we tested enzymatic activity, and found that purified enzyme was able to dephosphorylate PP2A substrates at a rate equal to or higher commercially available enzyme. We also showed that it was responsive to PP2A inhibitors. Our lab has previously created several GST-p107 fusion proteins allowing the identification of the p107 spacer binding sites for B55α/PP2A complexes using pull downs from cellular lysates. Our results using purified enzyme show that the B55a/PP2A holoenzyme binds p107 directly, and confirms the binding properties previously determined using crude lysates. We plan to use a series of p107 mutants to determine whether the CDK phosphorylated sites in these mutants are direct targets of this holoenzyme. / Biomedical Sciences

Insufficient supply and pathogen safety concerns regarding plasma-derived therapeutic proteins, such as fibrinogen and immunoglobulins, have been the impetus for the development of genetic engineering techniques and separations methods for the economical and safe production of these proteins. This study is concerned with the isolation of these important therapeutics from complex media. Immunoaffinity chromatography has been an important method in the isolation of these products, typically being implemented as the final cleanup step yielding an extremely pure, homogenous final product. However, the use of immunoaffinity chromatography in large-scale purification processes have been precluded due to high capital costs and the inherent lability of immunosorbents. Peptide-based affinity sorbents are being developed in order to surmount the inherent limitations posed by monoclonal antibodies that are used as ligands in immunosorbents. The objective of this research is to quantitatively assess the impact of affinity ligand orientation, local density and transport phenomena on peptide-based affnity sorbent performance. The peptides under study herein can form high-affinity complexes with their protein targets, thus these ligands are one of the newest technologies arising from combinatorial chemistry with applications to the difficult problem of purifying high-molecular weight proteins from complex mixtures. Two types of structural motifs which are common to small peptide affinity ligands derived from combinatorial chemistry are studied here: a linear peptide which is comprised of the affinity recognition sequence in its entirety and a chain structure which displays multiple branches of the recognition sequence emanating from a central lysinic core structure. Two recognition sequences are studied here which bind plasma proteins. One peptide recognition sequence forms a high affinity complex with fibrinogen. Another peptide recognition sequence binds the Fc region of immunoglobulins. Immunglobulins are plasma proteins which range in molecular weight from 155 to 900-kDa and are valuable for therapeutic uses for imparting passive immunity. This study seeks to identify factors analogous to those manifested in immunosorbent performance that may also be important in the optimal design of peptide-based affinity sorbents. In general, previous research with the design of immunosorbents have found that immunosorbent performance, i.e., target-binding efficiency or activity, is substantially dependent upon several factors which include effects associated with ligand orientation, and local density as related to steric incumbrance of target binding sites, and transport phenomena as related to under utilization of intra matrix volume. In summary, this study asks the questions: (1) What factors regarding ligand orientation, local ligand density, and intraparticle transport phenomena, are important in the optimal design of peptide affinity sorbents?; and (2) Are these effects analogous to those manifested in immunosorbent performance? This study seeks to investigate the use of techniques used to mitigate the effects associated with these negative factors upon immunosorbent performance in order to elucidate the nature of these same effects upon peptide-based affinity sorbents. For example, oriented ligand immobilization can be facilitated through selective coupling chemistries and the premasking of ligand binding domains prior to immobilization. In addition, the manipulation of local ligand density using novel spatially controlled matrix activation and ligand immobilization methods can be assessed and implemented for the optimization of the performance and design of peptide-based affinity sorbents. This study has found that enhanced transport phenomena into the matrix interior volume can be achieved by using low solids content cellulose matrices having a low extent of crosslinking. This study demonstrates the effective use of these large-particle diameter, low-solids content cellulose hydrogel matrices in immunoaffinity, peptide-based affinity and ion exchange chromatography in the separation of high-molecular weight therapeutic proteins. / Ph. D. / This report presents an evaluation of the design of low-solids content, large-particle diameter beaded cellulose supports for column-mode protein purification. The study presented here optimizes the molecular accessibility of the cellulose support to high molecular weight proteins relative to the mechanical stability of the support at high operating linear velocities by the manipulation of bead particle diameter and solids content. A novel epoxidegradient activation method (epoxy-GAM) is developed for creating a gradient of support activation for support crosslinking and affinity ligand installation in the preparation of DEAE hydrogel matrices. These cellulose hydrogel supports were evaluated with regards to structure, dynamic and static binding capacity, pressure-flow stability, chemical stability and intraparticle transport phenomena. The utility of the low-solids content, large-particle diameter DEAE hydrogel matrices was demonstrated in a column-mode protein purification using albumin and fibrinogen mixtures.

Lake restoration procedures are reviewed with the objective of making recommendations toward future lake restorations for central Florida lakes. The progress of drawdown for restoration is examined. A total phosphorus model is developed for Lake Eola to predict the success of the drawdown. No one restoration procedure should be applied to every lake in central Florida. Each situation must be studied separately. It appears that lake drawdown was the best technique to use for Lake Eola. However, the stormwater inputs and free-flowing drains must be purified to insure the success of the drawdown or the Lake will return within the immediate future to its previous eutrophic state.

This thesis discusses the causes and methods for removing taste and odor compounds from water supplies.

Call number: LD2668 .T4 1986 B46 / Master of Landscape Architecture / Landscape Architecture/Regional and Community Planning

Thesis (PhD)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: Microorganisms show a tendency to accumulate on surfaces in aqueous environments to form biofilms. Microbial biofilms represent a significant problem in public health microbiology as the development of these microbial communities, especially in water distribution systems, may lead to (i) the enhanced growth of opportunistic pathogens, (ii) the development of organoleptic problems, (iii) the reduction in the flow rate and (iv) the regrowth of microorganisms. In this project, biofilm monitors were installed in a large water distribution system to study biofilm phenomena in drinking water systems, and to deduce the biological stability and quality of the potable water. Measurements of biofilm formation potential showed that biofilms did not reach a steady state after 100 to 150 days. The microbial cells in these biofilms were mostly non-culturable. The contribution of the heterotrophic colony count to active biomass, as determined with cell numbers based on ATP measurements were often < 1%, while the ratio of heterotrophic plate counts and direct acridine orange counts were also <1%. The ratio between cell numbers based on ATP measurements and direct acridine orange counts were often < 100%. Results also showed that under certain conditions, such as those investigated in the present study, 1 pg of ATP may not be equal to approximately 104 active bacteria/cells, as stipulated by previous investigations, and that the average ATP content per active bacterial cell is indeed less than 10-16 - 10-15 g. It was calculated that threshold values for assimilable, and dissolved organic carbon below -5 IJg Gil and -0.5 mg Gil, respectively, should be target values for the control of biofilm formation in this system. It was shown that polyethylene, polyvinylchloride, teflon, plexiglass, copper, zinc-coated steel and aluminium provide favourable attachment surfaces that allowed primary colonisation and subsequent biofilm formation. Significant (p < 0.05) differences in surface colonisation on the materials were observed, indicating that the composition of the material has a direct influence on microbial colonisation. The two grades of stainless steel evaluated in this study were the least favourable materials for biofilm formation. It was further demonstrated that the nature of the surface of these materials, flow conditions and water type all had a direct influence on biofilm formation. While modification of the attachment surface did not result in significant differences (p > 0.05) in disinfection efficiency of two commonly used biocides, the concentration of the biocide, as well as the material to which the biofilm is attached, greatly influenced biocidal efficiency. The results show that biofilm monitoring needs to be implemented at the water treatment plants in addition to common biostability measurements. / AFRIKAANSE OPSOMMING: Mikro-organismes neig om te akkumuleer aan oppervlaktes in akwatiese omgewings om biofilms te vorm. Mikrobiese biofilms verteenwoordig In betekenisvolle probleem in publieke gesondheidsmikrobiologie omdat die ontwikkeling van hierdie mikrobiese gemeenskappe in waterverspreidingsisteme mag lei tot (i) die verhoogde groei van opportunistiese patogene, (ii) ontwikkeling van organoleptiese probleme, (iii) die vermindering in die vloeitempo en (iv) die hergroei van mikro-organismes. In hierdie projek was biofilm monitors geïnstalleer in In groot waterverspreidingsisteem om biofilm fenomene in drinkwatersisteme to bestudeer, en om die biologiese stabiliteit en kwaliteit van drinkwater af te lei. Bepalings van biofilmvormingspotensiaal het aangetoon dat biofilms nie In stabiele stadium na 100 tot 150 dae bereik nie. Die mikrobiese selle in hierdie biofilms was meestal niekweekbaar. Die bydrae van die heterotrofiese kolonie tellings tot aktiewe biomassa, soos bepaal deur seltellings gebaseer op ATP metings was dikwels < 1%, terwyl die verhouding van die heterotrofiese plaatteIIings en direkte akridien oranje tellings ook < 1% was. Die verhouding tussen seltellings, gebaseer op ATP metings en direkte akridien oranje tellings was dikwels < 100%. Resultate het ook aangetoon dat onder sekere omstandighede, soos dié wat ondersoek was in die huidige studie, 1 pg ATP nie gelyk is aan min of meer 104 aktiewe bakterieë/selle soos gestipuleer deur vorige ondersoeke nie, en dat die gemiddelde ATP inhoud per aktiewe bakteriële sel inderdaad minder as 10-16 tot 10-15 g is. Dit was bereken dat die drempelwaardes vir assimileerbare en opgeloste organiese koolstof onder -51-1g C/l en -0.5 mg C/l, onderskeidelik, teikens moet wees vir die beheer van biofilmvorming in hierdie sisteem. Dit was aangetoon dat polyetileen, polyvinielchlroried, teflon, plexiglas, koper, sink-bedekte staal en aluminium gunstige aanhegtings oppervlaktes voorsien wat primêre kolonisering en daaropvolgende biofilmvorming toelaat. Betekinisvolle (p <0.05) verskille in oppervlak kolinisering op die materiale was waargeneem, wat aandui dat die samestelling van die materiaal In direkte invloed op mikrobiese kolonisering het. Die twee tipes vlekvryestaal wat geëvalueer was in hierdie studie, was die minder gunstige materiale vir biofilmvorming. Dit was verder gedemonstreer dat die aard van die oppervlak van hierdie materiale, vloeitoestande, en water tipe almal In direkte invloed het op biofilmvorming. Terwyl die aanpassing van aanhegtingsoppervlak nie die ontsrnettinqsdoeltreffendheid resultaat van die twee algemeen-gebruikte biosiede betekinisvol (p > 0.05) beïnvloed het nie, het die konsentrasie van die biosiede doeltreffendheid grootliks beïnvloed. asook die aanhegtings-materiaal, biosied Die resultate het aangetoon dat biofilm monitering geïmplementeer moet word by waterbehandelingsaanlegte as In alternatief vir algemene biostabiliteit metings.

published_or_final_version / abstract / Civil Engineering / Master / Master of Philosophy

Dissertation submitted in compliance with the requirements for the Master's Degree in Technology: Biotechnology, Technikon Natal, 2001. / Up until now extensive work has been done to develop kinetic models and related software that can be used successfully to simulate and design nitrification denitrification (ND) and nitrification denitrification biological excess phosphorus removal (NDBEPR) systems for efficient nitrogen removal. The denitrification kinetics of these systems have primarily been determined and attributed to the ordinary heterotrophic bacteria, now also known as the OHO fraction, otherwise not involved in biological excess phosphorus removal. However, denitrification kinetics determined for ND systems have been found to vary considerably at times when applied to NDBEPR systems because of varying OHO active fraction estimates and the unexplained occurrence of anoxic phosphorus removal and anysuccess achieved to date has been some what fortuitous. Ultimately variations in process performance and kinetics are attributable to inadequate control and lack of understanding of the ecological, physiological and biochemical activities of constituent microorganisms. There is growing concern and movement towards a better understanding of the microbial community within activated sludge in order to gain optimal control of the process. / M

Dissertations submitted in compliance with the requirements for the Master's Degree in technology: Biotechnology, Technikon Natal, 2001. / Hexavalent chromium [Cr(VI)] or chromate, is a toxic, water-soluble contaminant present in many soils and industrial eflluents. As a result of contaminated discharges from industrial applications, and inappropriate wastedisposal practices, significant amounts of chromate have found their way into the environment. This poses a health risk to man as well as animals and plants due to the carcinogenicity, mutagenicity, and teratogenicity of chromate. In man, acute, high level exposures to Cr(VI) can result in ulceration of the skin, eyes, and mucous membranes. Exposure of plants to Cr(VI) can result in reduced biomass production, and in extreme cases, death. Upon reduction ofCr(VI) to trivalent chromium [Cr(III], the toxic effects are significantly decreased because of a decrease in the solubility and bioavailability of Cr(III). Traditionally, Cr(VI) has been recovered from aqueous systems using processes exploiting the differential solubility properties described above. The use of chromate reducing bacteria represents a potential mechanism for the development of an efficacious, cost effective alternative to traditional chemical/physical processes for Cr(VI) recovery from the environment. Therefore, the aim of this research was to isolate and identify chromate reducing bacteria from soil, and characterise the chromate reductase enzyme in order to determine the potential of bacteria to detoxify chromate by reduction. Bacteria from soils and wastewater were examined for chromate reducing potential and identified on the basis of biochemical tests and API 20E. Organisms were isolated by the spread plate technique. Species of Pseudomonas maltophilia, Bacillus subtilis, Acinetobacter calcoaceticus, and Cellumonas cellasea were capable of catalyzing the reduction ofCr(VI) to Cr(IlI) in batch experiments. Reduction capability as high as 99% by the isolates was detected from an initial Cr(VI) concentration of 150 mg.L' in batch cultures. Chromate reduction was determined by means of the diphenylcarbazide method and total chromium was measured by atomic absorption spectroscopy. Pseudomonas maltophilia was observed to be the most suited organism for the efficient detoxification ofCr(VI) due to its wide temperature and pH requirements, low substrate utilization, and tolerance to heavy metal ions of'Cu', Cd2+,Zn2+,and Ni2+which commonly appear in industrial eflluents along with Cr(VI). Reduction rate in a batch reactor for this organism was calculated to be 1.75 mg.g+h'. Comparison of the rates of chromate reduction by Cr(VI) grown cells and cells grown without chromate indicated that the chromate reductase activity is constitutive. Reductase activity was detected by means of the lysozyme-EDTA method in aerobically grown cells, with highest specific activity in the cytoplasmic fraction of the cell. The Cr(VI)-reductase was found to be NAD(p)H-dependent and yielded an activity of 3.24 ml.I.mg' of protein in the cytoplasmic fraction. Once optimization of the parameters in the batch reactor was achieved, cells of Ps. maltophilia was immobilized into polyacrylamide gel and packed in a column. Mass balance studies indicated that ca 147 mg.L' chromate passing through the column undergoes reduction with an initial Cr(VI) concentration of 150 mg.L' resulting in a Cr(VI) reduction efficiency of98%. An amount of 0.11 mg.L' remained in the cells, 0.11 mg.L' in the cell wash water, and 1.65 mg.L' was unaccounted for in the mass balance. Chromate reduction rate in the continuous-upflow reactor system was calculated to be 5.34 mg.g'l.h', which was 3-fold higher than that calculated for the batch reactor. Chromium-contaminated industrial eflluent obtained from Sheffield, Natal, and Saayman Danks Electroplaters was pumped into the continuous-upflow reactor containing immobilized cells of Ps. maltophilia to determine the industrial applicability of the reactor to treat chromate-containing effluents. Complete Cr(VI) reduction / M

No description available.