Recent studies suggest that factor (F) XII, which is dispensable for hemostasis, is important for thrombus stabilization and growth. Therefore, FXIIa inhibition may attenuate thrombosis without disrupting hemostasis. FXII activation is stimulated by polyanions such as polyphosphates released from activated platelets, and nucleic acids released by cells. Previously, we showed that histidine-rich glycoprotein (HRG) binds FXIIa with high affinity, inhibits FXII autoactivation and FXIIa-mediated activation of FXI, and attenuates ferric chloride-induced arterial thrombosis in mice. Thus, HRG has the capacity to downregulate the contact pathway in vitro and in vivo. This thesis aimed to identify the complementary binding domains of HRG and FXIIa, and to further explore the anticoagulants effects of HRG on FXIIa-mediated contact activation. We hypothesized that FXIIa binds to the zinc-binding histidine-rich region (HRR) of HRG and that HRG binds to the non-catalytic heavy chain of FXIIa to exert its anticoagulant activities on FXIIa-mediated contact activation. We have localized the complementary binding sites of HRG and FXIIa to be within the HRR domain of HRG and NH2-FNII-EGF1 (NFE) domains of FXIIa. Moreover, we show that the HRR binds to short chain polyphosphate with high affinity, suggesting a dynamic complex between HRG, FXIIa, and polyphosphate (polyP) on activated platelets. We provide evidence for two potential mechanisms through which HRG modulates the contact system. These include by 1) inhibiting FXIIa activity and 2) attenuating the procoagulant effect of polyanions, such as polyP on FXIIa-mediated reactions. Indeed, we show that the interaction of HRG with FXIIa and polyphosphate is predominantly mediated by the HRR domain and that HRR analogs have the capacity to recapitulate the anticoagulant effects of HRG in purified and plasma systems. Therefore, by modulating FXIIa-mediated contact pathway reactions, like HRG, HRR analogs may attenuate thrombosis without disrupting hemostasis. / Thesis / Doctor of Philosophy (Medical Science)

An unexpected biological removal of phosphorous in IVL Hammarby Sjöstadsverket pilot membrane bioreactor (MBR) wastewater treatment process was investigated by three distinct methods. a) Investigation and quantification of the biological phosphorous uptake (bio-P) capacity by phosphate release tests (PRT), b) validation of bio-P occurrence by granular polyphosphate (poly-P) staining and microscopy and c) metagenomic community structure analysis to map the sludge habitat. Validation of phosphate accumulating organisms (PAO) was successfully performed using the Neisser Methylene Blue Metachromatic staining protocol. Quantification by a novel staining protocol gave indications of the relative activity of bio-P in the bioreactor process line. The bio-P activity by PRT showed high capacity of phosphate accumulation in the treatment process. Furthermore, the use of ethylenediaminetetraacetic acid (EDTA) to induce stressful conditions was successfully shown to increase the release rate and depletion of intracellular poly-P of PAO. Finally, the impact of temperature dependency in PRT was investigated.A metagenomic community structure analysis by 16S rRNA successfully prepared 28 samples for sequencing and analysis. The project has successfully validated and quantified bio-P with an improved methodology as a foundation for upcoming studies of the treatment process. / En oväntad biologisk fosforrening (bio-P) i IVL Hammarby Sjöstadsverkets membranreaktorpilot undersöktes med tre distinkta metoder. Kvantifiering av det biologiska fosforupptaget undersöktes med fosforsläppstest (PRT). Validering av förekomsten av fosfatackumulerande organismer (PAO) via infärgning av polyfosfatgranuler med Neissers protokoll för infärgning med metylenblå. Ett nytt protokoll för kvantifiering av polyfosfatgranulkluster gav indikationer på relativ aktivitet av bio-P i reningsprocessens bioreaktorer. Bio-P aktiviteten som uppmättes med PRT visade på hög kapacitet av biologisk fosforackumulation. Vidare visades framgångsrikt att stresspåslag av PAO med etylendiamintetraättiksyra (EDTA) ökade fosfatsläppshastigheten och uttömningen av intracellulär poly-P. Slutligen undersöktes temperaturberoendet i PRT. En 16S rRNA metagenomikstudie gav 28 st prover redo att sekvensieras och analyseras. Projektet har framgångsrikt validerat och kvantifierat bio-P med en utvecklad metodologi som kan utgöra grunden för kommande studier vid reningsverket.

The phosphatidylinositol signaling pathway utilizes inositol-containing second messengers to mediate signaling events. The enzymes that metabolize phosphoinositides can in some cases serve to terminate the signaling actions of phosphoinositides. The inositol polyphosphate 5-phosphatases (5PTases) comprise a large protein family that hydrolyzes 5-phosphates from a variety of inositol phosphate and phosphoinositide substrates. I have examined the substrate specificity of the At5PTase11 protein from the model plant, Arabidopsis thaliana. The At5PTase11 gene (At1g47510) encodes an active 5PTase enzyme that can dephosphorylate the phosphoinositide substrates phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2], and phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3]. In addition, the At5PTase11 gene is regulated by abscisic acid, jasmonic acid, and auxin, suggesting a role for phosphoinositide action in these signal transduction pathways. To further delineate the function of At5PTase11 in Arabidopsis thaliana, two independent T-DNA insertion mutant lines were isolated (At5ptase11-1 and At5ptase11-2). Analysis of At5ptase11 mutant lines revealed that At5ptase11 mutant seeds germinate slower compared to wild-type seeds. Moreover, At5ptase11 mutant seedlings demonstrated less hypocotyl growth when grown in the dark. These results indicate that At5PTase11 is required for the early stages of seed germination and seedling growth. Since there are 15 predicted 5PTases in Arabidopsis thaliana, a group of 5PTases have been analyzed to identify the 5PTases with similar substrate selectivity. At5PTase1 (At1g34120), At5PTase2 (At4g18010) and At5PTase3 (At1g71710) have been found to hydrolyze all four potential substrates, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4], PtdIns(4,5)P2, and PtdIns(3,4,5)P3. At5PTase7 (At2g32010) hydrolyzed PtdIns(4,5)P2, and PtdIns(3,4,5)P3 which is similar to the substrate selectivity of At5PTase11. In addition, At5PTase4 (At3g63240), and At5PTase9 (At2g01900) hydrolyzed only PtdIns(4,5)P2. These results indicate that there are different groups of Arabidopsis thaliana 5PTases based on the substrate selectivity. These results suggest that Arabidopsis thaliana 5PTases with similar substrate selectivity may have overlapping functions. In summary, the findings that At5PTase11 is a phospholipid-specific 5PTase and At5PTase11 functions in the early stages of seed germination and seedling growth indicate that 5PTases play important roles in plant growth and development. / Ph. D.

Die Inositol-Pyrophosphate (PP-InsPs) sind eine ubiquitäre Gruppe hochphosphorylierter eukaryotischer Signalmoleküle. Sie werden mit einer Vielzahl zentraler zellulärer Prozesse in Verbindung gebracht, doch fehlt oft ein detailliertes Verständnis der einzelnen Signalereignisse, was zum Teil auf einen Mangel an chemischen Werkzeugen zurückzuführen ist. Diese Arbeit beschreibt die chemische Synthese, Validierung und Anwendung von PP-InsP-Affinitätsreagenzien zur Identifizierung von Proteinbindungspartnern von Inositolhexakisphosphat (InsP6) und 5-Diphosphoinositol-Pentakisphosphat (5PP-InsP5), zwei wichtigen eukaryotischen Metaboliten. Die Affinitätsreagenzien wurden entwickelt, um InsP6 und ein metabolisch stabiles 5PP-InsP5-Analogon auf drei verschiedene Arten darzustellen. Die Anwendung dieser triplexierten Reagenzien auf Säugetier-Lysate lieferte einen ersten umfassenden Datensatz in HCT116- und HEK293T-Zellen. Die Interaktome wurden mittels quantitativer Proteomik annotiert und enthüllten Hunderte von potenziellen Proteinbindungspartnern. Die quantitative Analyse der InsP6- und 5PP-InsP5-bindenden Proteine zeigte Beispiele für hochspezifische Protein-Ligand-Interaktionen auf. Biochemische Untersuchungen ergaben, dass Inositol-5-Phosphatasen, PRPS1 und spezifische Phosphatidyl-Inositolphosphat-Kinasen potenziell unentdeckte Zielproteine von PP-InsPs sind. Darüber hinaus wurde durch die Entwicklung einer neuen Strategie der Myo-Inositol-Desymmetrisierung erstmals die Synthese eines Affinitätsreagens auf der Basis von 1,5-Bisdiphosphoinositol-Tetrakisphosphat (1,5(PP)2-InsP4) beschrieben. Die Affinitätsreagenzien und die proteomischen Datensätze stellen für die Gemeinschaft leistungsstarke Ressourcen dar, um künftige Untersuchungen zu den vielfältigen Signalmodalitäten von Inositolpyrophosphaten einzuleiten. / Inositol pyrophosphates (PP-InsPs) are a ubiquitous group of highly phosphorylated eukaryotic messengers. They have been linked to a panoply of central cellular processes, but a detailed understanding of the discrete signaling events is often missing, which can partially be attributed to a lack of chemical tools. This thesis describes the chemical synthesis, validation and application of PP-InsP affinity reagents to identify protein binding partners of inositol hexakisphosphate (InsP6) and 5-diphosphoinositol pentakisphosphate (5PP-InsP5), two important eukaryotic metabolites. The affinity reagents were developed to display InsP6 and a metabolically stable 5PP-InsP5 analog in three different ways. Application of these triplexed reagents to mammalian lysates provided a first comprehensive data set in HCT116 and HEK293T cells. The interactomes were annotated using quantitative proteomics and uncovered hundreds of potential protein binding partners. Quantitative analysis of InsP6 versus 5PP-InsP5 binding proteins highlighted examples of highly specific protein-ligand interactions. Biochemical studies primed inositol 5-phosphatases, PRPS1 and specific phosphatidyl inositol phosphate kinases as potentially undiscovered targets of PP-InsPs. Moreover, by developing a novel strategy of myo-inositol desymmetrization, the synthesis of an affinity reagent based on 1,5-bisdiphosphoinositol tetrakisphosphate (1,5(PP)2-InsP4) was described for the first time. The affinity reagents and the proteomic data sets constitute powerful resources for the community, to help launching future investigations into the multiple signaling modalities of inositol pyrophosphates.

Inositol phosphates (IPs) are important signalling molecules with various biological roles in a cell. One such role it is often associated with is DNA repair. The DNA repair process following DNA insult is considered crucial for the genomic integrity and stability. Failure to perform this task will result in mutations and possibly disease. Thus, it is important that we expand our knowledge on how these repair processes occur and identify the key factors involved in its regulation. The aim of this project was to investigate whether DNA repair was mediated by inositol diphosphates (IDPs). Using a family of yeast knockout mutants with modulated levels of IPs, it was found that IDPs are crucial in repair of DNA following insult with bleomycin and 5-fluorouracil. The observed sensitivity of the mutants was thought to be due to lack of functional repair protein, UDG-like or APE-like, in the absence of essential cofactor such as IDPs. Experiments conducted revealed that the hypersensitive kcs1Δ contain both the repair proteins required to process the DNA lesions. However, extreme extraction methods were required to access these proteins, suggesting that the proteins are mislocalised and unavailable to access the damage site and perform DNA repair. GFP-tagging the proteins Ung1, Apn1 and Rad52 in kcs1Δ proved to be of little use as it failed to show exact localisation, movement and functionality status of these proteins following bleomycin insult. The enzymes accountable for the dephosphorylation of the IDPs in vivo are the diphosphoinositol polyphosphate phosphohydrolases (DIPPs). Little is known regarding the Michaelis-Menten kinetics parameters for Ddp1p/DIPPs. In this study, using improved methods for the enzymatic synthesis and electrophoretic purification of 1-InsP7, 5-InsP7 and InsP8, the DIPP family has been kinetically characterised. Each DIPP was found to ii display similar Km values for every substrate tested (range: 35-148 nM). The rank order of Kcat values (1-InsP7 > 5-InsP7 = InsP8) was identical for each enzyme, although DIPP-1 activity was observed to be 10- to 60-fold more than DIPP-2α/β and DIPP-3α/β, irrespective of the substrate. This study reveals that Ddp1, the yeast DIPP, is capable of hydrolysing not only 5-InsP7 but also 1-InsP7 and InsP8 to a single product, InsP6. The HPLC data found InsP7 accumulation to be relatively little during InsP8 breakdown by DIPPs. Such low build-up was found to be due to rapid conversion of InsP7 to InsP6. Through this study it is also clear that InsP8 prefers to dephosphorylate through 1-InsP7. In contrary, metabolically and functionally significant steady-state route of InsP8 synthesis was observed to be via 5-InsP7. Oesophageal cancer is considered as one of the deadliest cancers worldwide because of its aggressive nature and low survival rate. Epidemiologic studies have shown that low-dose daily intake of aspirin can decrease the incidence of oesophageal cancer. The data presented in this study show the effects of a number of in-house synthesized novel aspirin analogues on oesophageal cancer cell lines, squamous cell carcinoma (SSC) and adenocarcinoma (ADC). The aspirin analogues, fumaryldiaspirin (PN517) and benzoylsalicylates (PN524, PN528 and PN529), were observed to be more potent against the oesophageal cell lines than aspirin itself. Both, quantitative and qualitative apoptosis experiments conducted revealed that these compounds largely induced apoptosis, although some necrosis was evident with PN528 and PN529. Failure to recover following the treatment with these analogues emphasized that these drugs are largely cytotoxic in nature. The SSC cells (oe21) displayed increased sensitivity to the aspirin analogues compared to the ADC cell lines (flo-1 and oe33). The anticancer properties of these novel aspirin compounds appear to not involve the COX-enzymes at the tested concentrations. These initial findings support further studies into the potential of these aspirin analogues as chemotherapeutic agents against oesophageal cancer.

The purpose of the present study is to develop techniques for manufacturing anatomically-shaped substrates of implants made from calcium polyphosphate (CPP) ceramic. These substrates have tissue-engineered cartilage growing on their top surfaces and can be used as implants for osteochondral focal defect repair. While many research groups have been fabricating such substrates using standard material shapes, e.g., rectangles and circular discs, it is considered beneficial to develop methods that can be integrated in the substrate fabrication process to produce an implant that is specific to a patient’s own anatomy (as obtained from computer tomography data) to avoid uneven and/or elevated stress distribution that can affect the survival of cartilage. The custom-made, porous CPP substrates were fabricated with three-dimensional printing (3DP) and computer numerically controlled (CNC) machining for the first time to the best of the author’s knowledge. The 3DP technique was employed in two routines: indirect- and direct-3DP. In the former, 3DP was used to fabricate molds for pre-shaping of the CPP substrates from two different powder size ranges (<75 μm and 106-150 μm). In the latter, CPP substrates were produced directly from the retrofitted 3DP apparatus in a layer-by-layer fashion from 45-75 μm CPP powder with a polymeric binder. The prototyped samples were then sintered to obtain the required porosity and mechanical properties. These substrates were characterized in terms of their dimensional shrinkage and density. Also, SEM images were used to assess the particle distribution and neck and bond formations. The substrates produced using the indirect-3DP method yielded densities (<75 μm: 66.28 ± 11.62% and 106-150 μm: 65.87 ± 6.12%), which were comparable to the substrates used currently and with some success in animal studies. Geometric adjustment factors were devised to compensate for the slight expansion inherent in the 3DP mold fabricating process. These equations were used to bring the plaster molds into true dimension. The direct-3DP method has proven to be the ultimate choice due to its ability to produce complex anatomically-shaped substrates without the use of a chemical solvent. In addition, it allows for precise control of both pore size and internal architectures of the substrates. Thus, the direct-3DP was considered to be superior than the indirect-3DP as a fabrication method. In the alternative CNC machining approach to fabrication, the ability to machine the CPP ceramic was feasible and by careful selection of the machining conditions, anatomically-shaped CPP substrates were produced. To develop strategies for optimizing the machining process, a mechanistic model was developed based on curve fitting the average cutting forces to determine the cutting coefficients for CPP. These cutting coefficients were functions of workpiece material, axial depth of cut, chip width, and cutter geometry. To explore the utility of this modelling approach, cutting forces were predicted for a helical ball-end mill and compared with experimental results. The cutting force simulation exhibits good agreement in predicting the fundamental force magnitude and general shape of the actual forces. However, there were some discrepancies between the predicted and measured forces. These differences were attributed to internal microstructure defects, density gradients, and the use of a shear plane model in force prediction that was not entirely appropriate for brittle materials such as CPP. The present study successfully developed 3DP and CNC fabrication methods for manufacturing anatomically-shaped CPP substrates. Future studies were recommended to explore further optimization of these fabrication methods and to demonstrate the utility of accurate substrates shapes to the clinical application of focal defect repair implants.

No description available.

The purpose of the present study is to develop techniques for manufacturing anatomically-shaped substrates of implants made from calcium polyphosphate (CPP) ceramic. These substrates have tissue-engineered cartilage growing on their top surfaces and can be used as implants for osteochondral focal defect repair. While many research groups have been fabricating such substrates using standard material shapes, e.g., rectangles and circular discs, it is considered beneficial to develop methods that can be integrated in the substrate fabrication process to produce an implant that is specific to a patient’s own anatomy (as obtained from computer tomography data) to avoid uneven and/or elevated stress distribution that can affect the survival of cartilage. The custom-made, porous CPP substrates were fabricated with three-dimensional printing (3DP) and computer numerically controlled (CNC) machining for the first time to the best of the author’s knowledge. The 3DP technique was employed in two routines: indirect- and direct-3DP. In the former, 3DP was used to fabricate molds for pre-shaping of the CPP substrates from two different powder size ranges (<75 μm and 106-150 μm). In the latter, CPP substrates were produced directly from the retrofitted 3DP apparatus in a layer-by-layer fashion from 45-75 μm CPP powder with a polymeric binder. The prototyped samples were then sintered to obtain the required porosity and mechanical properties. These substrates were characterized in terms of their dimensional shrinkage and density. Also, SEM images were used to assess the particle distribution and neck and bond formations. The substrates produced using the indirect-3DP method yielded densities (<75 μm: 66.28 ± 11.62% and 106-150 μm: 65.87 ± 6.12%), which were comparable to the substrates used currently and with some success in animal studies. Geometric adjustment factors were devised to compensate for the slight expansion inherent in the 3DP mold fabricating process. These equations were used to bring the plaster molds into true dimension. The direct-3DP method has proven to be the ultimate choice due to its ability to produce complex anatomically-shaped substrates without the use of a chemical solvent. In addition, it allows for precise control of both pore size and internal architectures of the substrates. Thus, the direct-3DP was considered to be superior than the indirect-3DP as a fabrication method. In the alternative CNC machining approach to fabrication, the ability to machine the CPP ceramic was feasible and by careful selection of the machining conditions, anatomically-shaped CPP substrates were produced. To develop strategies for optimizing the machining process, a mechanistic model was developed based on curve fitting the average cutting forces to determine the cutting coefficients for CPP. These cutting coefficients were functions of workpiece material, axial depth of cut, chip width, and cutter geometry. To explore the utility of this modelling approach, cutting forces were predicted for a helical ball-end mill and compared with experimental results. The cutting force simulation exhibits good agreement in predicting the fundamental force magnitude and general shape of the actual forces. However, there were some discrepancies between the predicted and measured forces. These differences were attributed to internal microstructure defects, density gradients, and the use of a shear plane model in force prediction that was not entirely appropriate for brittle materials such as CPP. The present study successfully developed 3DP and CNC fabrication methods for manufacturing anatomically-shaped CPP substrates. Future studies were recommended to explore further optimization of these fabrication methods and to demonstrate the utility of accurate substrates shapes to the clinical application of focal defect repair implants.

Polyphosphat-speichernde Bakterien entfernen das im Abwasser enthaltene Phosphat durch Speicherung in Form von Granula, die dann mit einem Teil des Belebtschlammes aus dem Abwasser entfernt werden können. Dies ist wichtig um negative Einflüsse auf Oberflächengewässer wie Flüsse und Seen so gering wie möglich zu halten. Trotz intensiver Forschung ist der Prozess der sogenannten biologischen Phosphatelimination oft uneffektiv und im Jahresverlauf instabil, da über die im Belebtschlamm aktiven Polyphosphat-speichernden Bakterien nur wenig bekannt ist. Hauptproblem ist hierbei die geringe Kultivierbarkeit der Bakterien unter definierten Bedingungen (nur etwa 10-15 % der Mikroorganismen im Belebtschlamm sind kultivierbar). Aus diesem Grund war das Ziel der Arbeit die aktiven, Polyphosphat-speichernden Bakterien durchflusszytometrisch zu bestimmen und deren Dynamiken im Belebtschlamm kultivierungsunabhängig zu messen. Zunächst wurde ein Fixierungsprotokoll für die durchflusszytometrische Untersuchung der Polyphosphat-speichernden Bakterien erarbeitet, welches die größtmögliche Stabilität der hochdiversen mikrobiellen Gemeinschaft in Belebtschlammproben gewährleistet. Eine Mischung aus den Metallen Barium und Nickel (jeweils 5 mM) in einer 10%igen Natriumazidlösung erwies sich als bestes Fixierungsmittel mit einer Belebtschlamm-Stabilität von mindestens 9 Tagen. Um sowohl den DNA-als auch den Polyphosphat-Gehalt der Zellen messen zu können wurde weiterhin eine neue und sehr spezifische Polyphosphatfärbung auf Basis des fluoreszierenden Antibiotikums Tetrazyklin etabliert. Tetrazyklin bindet divalente Kationen, die auch in großer Menge in Polyphosphatgranula enthalten sind und fluoresziert gelblich grün. Die entwickelten Methoden zur Fixierung und Polyphosphatfärbung wurden an Belebtschlamm einer kommunalen Kläranlage getestet. Neben DNA- und Polyphosphat-Gehalt der Bakterienzellen wurde eine Vielzahl abiotischer Parameter (pH, Temperatur, Leitfähigkeit, …) gemessen. Diese wurden zusammen mit den durchflusszytometrischen Daten mittels Korrelationsanalyse ausgewertet. Hieraus ergaben sich wichtige Hinweise auf die Art der Polyphosphat-speichernden Bakterien, fördernde und störende Einflüsse des in der Kläranalage behandelten Abwassers auf die biologische Phosphatelimination und die Abhängigkeiten der mikrobiellen Gemeinschaft von Faktoren wie Temperatur, pH oder der anfallenden Regenmenge. Diese Erkenntnisse können genutzt werden um die biologische Phosphatelimination aus dem Abwasser zu verbessern und damit den Weg zu einer Ressourcen- und Umweltschonenden Phosphatrückgewinnung zu bereiten. Außerdem ist es, bei Kenntnis des kläranlagenspezifischen Prozesses, möglich anhand der durchflusszytometrischen Daten schnell die aktuelle Situation zu erfassen und gegebenenfalls rechtzeitig auf Änderungen zu reagieren, bevor es zu einer massiven Störung kommt. Eine Kombination von Durchflusszytometrie und der Erfassung abiotischer Daten ist nicht nur auf die biologische Phosphateliminierung anwendbar, sondern auch auf viele andere wissenschaftliche Fragestellungen.

Phosphorus (P) is a requirement for biological growth, but this vital nutrient is present at low or limiting concentrations across vast areas of the global surface ocean. Inorganic polyphosphate (poly-P), a linear polymer of at least three orthophosphate units, is one component of the marine P cycle that has been relatively overlooked as compared to other P species, owing in part to a lack of routine analytical techniques that cleanly evaluate it within samples. This thesis demonstrates that inorganic poly-P is a quantitatively significant and dynamic component of the global marine P cycle while also establishing two new techniques for its analysis in biological and environmental samples. In Chapter 2, experiments using the freshwater algae Chlamydomonas sp. and Chlorella sp. illustrate X-ray fluorescence spectromicroscopy as a powerful tool for the sub-micron scale assessment of poly-P composition in organisms. This method enabled the discovery, detailed in Chapter 3, of a mechanism for the long-term sequestration of the vital nutrient P from marine systems via the initial formation of poly-P in surface waters and its eventual transformation into the mineral apatite within sediments. The importance of marine poly-P is furthermore established in Chapter 3 by observations showing that naturally-occurring poly-P represents 7-11% of total P in particles and dissolved matter in Effingham Inlet, a eutrophic fjord located on Vancouver Island, British Columbia. In Chapter 4, a new fluorometric protocol based on the interaction of inorganic poly-P with 4',6-diamidino-2-phenylindole (DAPI) is established as a technique for the direct quantification of poly-P in environmental samples. Chapter 5 presents work from Effingham Inlet utilizing this method that show that inorganic poly-P plays a significant role in the redox-sensitive cycling of P in natural systems.