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Response to Professor E. Lea Johnston, Reconceptualizing Criminal Justice Reform for Offenders with Serious Mental Illness Abstract While Professor Johnston is persuasive that clinical factors such as diagnosis and treatment history are not, in most cases, predictive by themselves of criminal behavior, her concession that those clinical factors are associated with a constellation of risks and needs that are predictive of criminal system involvement complicates her efforts to maintain a clear boundary between the criminalization theory and the normalization thesis. Indeed, Professor Johnston’s article contains a brief section in which she identifies “possible justifications” for the specialized programs that are the target of her critique. These justifications deserve more attention, precisely because they suggest that the normalization thesis, while powerful, may not entirely displace the criminalization theory upon which those specialized programs rest. Moreover, even if the criminalization theory and the normalization theory are at least partially reconcilable, important questions remain regarding the proper allocation of limited resources both within the criminal legal system and the public mental health system. These questions, in turn, press focus on additional questions with respect to the underlying purposes of criminal system coercion more generally. The complex associations between serious mental illness and several of the key risk factors for criminal system involvement—most notably substance misuse—impact the design of effective programs intended to reduce reoffending. Given the practical difficulty of disentangling the treatment of mental disabilities from the provision of effective interventions to interrupt patterns of criminal behavior in offenders who have significant mental illness, some specialized attention to clinical mental health needs is warranted in correctional rehabilitation programs. These integrated programs are resource intensive, howeve

The focus of this thesis has been synthesise and study metal complexes that mimic the structure and function of the active site in two particular metalloenzymes, ribonucleotide reductase (RNR) and manganese catalase (MnCAT). These two metalloenzymes both have two transition metals ions in the cofactor: two manganese ions in MnCAT and either two iron, iron-manganese or two manganese ions in RNR. Three different ligands were synthetized to make model complexes for these cofactors. The first ligand, BPMP, can bind two metal ions and provides two symmetric pockets with two pyridine groups and one amine each, plus a phenolate group that can bridge the two metals. The second ligand DPCPMP had one carboxylate group instead of a pyridine group in one pocket, creating an asymmetric ligand, and the third ligand BPCPMP, had two carboxylate groups, one in each pocket. From the first and the second ligands it was possible to obtain six complexes: low-valent homometallic Mn/Mn and Fe/Fe complexes and a heterometallic complex for each ligand. For the third ligand, only the Fe/Fe complex was synthetized. All seven complexes were characterized by a number of spectroscopic methods. The presence of carboxylate groups in the ligand shifted the redox potential for the metal complexes towards more negative values, particularly in the case of the homometallic Fe/Fe complexes. Surprisingly, for the asymmetric ligand the placement of the metal ions in the two pockets was not dictated by the asymmetry. Additionally, the relative stability of the homometallic complexes versus the heterometallic complexes and the possibility to transform a homometallic complex into a heterometallic complex were investigated. By titrating one metal into a solution containing the other homometallic dimer it was possible to observe that Fe2+ added to a solution of a Mn/Mn complex led to the replacement of one Mn ion in the complex with a Fe ion. The manganese complex of DPCPMP was investigated as a functional mod

Deoxyribonucleoside triphosphates (dNTPs) are precursors for DNA replication and repair. Mammalian cells have two distinct biosynthesis pathways to supply dNTPs: de novo and salvage pathways. These pathways are intimately coordinated to maintain optimal dNTP concentrations throughout different phases of the cell cycle, and perturbations in the production of dNTPs could lead to increased, decreased, or imbalanced dNTP pools. In yeasts, changes in both the level and balance of dNTPs increase mutation rates and genome instability. In mammals, elevated mutation rates and genome instability predispose to numerous diseases, including cancer. However, the correlation of dNTP changes with pathology has not been well established in mammals. In this thesis, I present how we addressed this issue using three separate mouse models – one with an increased dNTP pool, one with a decreased dNTP pool, and one with an imbalanced dNTP pool. To modulate dNTP levels in the mice, we deleted or mutated either sterile alpha motif and histidine-aspartic domain containing protein 1 (SAMHD1) or ribonucleotide reductase (RNR) proteins, which are involved in the salvage and de novo pathways, respectively. In the first model, mouse embryos without the SAMHD1 gene showed a slight increase in dNTP levels. A similar increase in dNTPs conferred moderately elevated mutation rates in cultured cancer cells. In the second model, we created a mouse strain carrying a modified allosteric specificity site in a subunit of RNR. Embryos with a heterozygous mutation had a mildly imbalanced dNTP pool. Heterozygous mutant mice showed a shorter lifespan and increased incidence and earlier onset of cancer. In the third model, the de novo dNTP production was inactivated in cardiac and skeletal muscles through the deletion of a gene encoding RNR. The hearts of knockout pups showed significant depletion of dNTPs, leading to aberrant DNA replication. In addition, knockout pups developed anatomic and histologic cardiac a

The focus of this thesis has been synthesise and study metal complexes that mimic the structure and function of the active site in two particular metalloenzymes, ribonucleotide reductase (RNR) and manganese catalase (MnCAT). These two metalloenzymes both have two transition metals ions in the cofactor: two manganese ions in MnCAT and either two iron, iron-manganese or two manganese ions in RNR. Three different ligands were synthetized to make model complexes for these cofactors. The first ligand, BPMP, can bind two metal ions and provides two symmetric pockets with two pyridine groups and one amine each, plus a phenolate group that can bridge the two metals. The second ligand DPCPMP had one carboxylate group instead of a pyridine group in one pocket, creating an asymmetric ligand, and the third ligand BPCPMP, had two carboxylate groups, one in each pocket. From the first and the second ligands it was possible to obtain six complexes: low-valent homometallic Mn/Mn and Fe/Fe complexes and a heterometallic complex for each ligand. For the third ligand, only the Fe/Fe complex was synthetized. All seven complexes were characterized by a number of spectroscopic methods. The presence of carboxylate groups in the ligand shifted the redox potential for the metal complexes towards more negative values, particularly in the case of the homometallic Fe/Fe complexes. Surprisingly, for the asymmetric ligand the placement of the metal ions in the two pockets was not dictated by the asymmetry. Additionally, the relative stability of the homometallic complexes versus the heterometallic complexes and the possibility to transform a homometallic complex into a heterometallic complex were investigated. By titrating one metal into a solution containing the other homometallic dimer it was possible to observe that Fe2+ added to a solution of a Mn/Mn complex led to the replacement of one Mn ion in the complex with a Fe ion. The manganese complex of DPCPMP was investigated as a functional mod

Deoxyribonucleoside triphosphates (dNTPs) are precursors for DNA replication and repair. Mammalian cells have two distinct biosynthesis pathways to supply dNTPs: de novo and salvage pathways. These pathways are intimately coordinated to maintain optimal dNTP concentrations throughout different phases of the cell cycle, and perturbations in the production of dNTPs could lead to increased, decreased, or imbalanced dNTP pools. In yeasts, changes in both the level and balance of dNTPs increase mutation rates and genome instability. In mammals, elevated mutation rates and genome instability predispose to numerous diseases, including cancer. However, the correlation of dNTP changes with pathology has not been well established in mammals. In this thesis, I present how we addressed this issue using three separate mouse models – one with an increased dNTP pool, one with a decreased dNTP pool, and one with an imbalanced dNTP pool. To modulate dNTP levels in the mice, we deleted or mutated either sterile alpha motif and histidine-aspartic domain containing protein 1 (SAMHD1) or ribonucleotide reductase (RNR) proteins, which are involved in the salvage and de novo pathways, respectively. In the first model, mouse embryos without the SAMHD1 gene showed a slight increase in dNTP levels. A similar increase in dNTPs conferred moderately elevated mutation rates in cultured cancer cells. In the second model, we created a mouse strain carrying a modified allosteric specificity site in a subunit of RNR. Embryos with a heterozygous mutation had a mildly imbalanced dNTP pool. Heterozygous mutant mice showed a shorter lifespan and increased incidence and earlier onset of cancer. In the third model, the de novo dNTP production was inactivated in cardiac and skeletal muscles through the deletion of a gene encoding RNR. The hearts of knockout pups showed significant depletion of dNTPs, leading to aberrant DNA replication. In addition, knockout pups developed anatomic and histologic cardiac a

The focus of this thesis has been synthesise and study metal complexes that mimic the structure and function of the active site in two particular metalloenzymes, ribonucleotide reductase (RNR) and manganese catalase (MnCAT). These two metalloenzymes both have two transition metals ions in the cofactor: two manganese ions in MnCAT and either two iron, iron-manganese or two manganese ions in RNR. Three different ligands were synthetized to make model complexes for these cofactors. The first ligand, BPMP, can bind two metal ions and provides two symmetric pockets with two pyridine groups and one amine each, plus a phenolate group that can bridge the two metals. The second ligand DPCPMP had one carboxylate group instead of a pyridine group in one pocket, creating an asymmetric ligand, and the third ligand BPCPMP, had two carboxylate groups, one in each pocket. From the first and the second ligands it was possible to obtain six complexes: low-valent homometallic Mn/Mn and Fe/Fe complexes and a heterometallic complex for each ligand. For the third ligand, only the Fe/Fe complex was synthetized. All seven complexes were characterized by a number of spectroscopic methods. The presence of carboxylate groups in the ligand shifted the redox potential for the metal complexes towards more negative values, particularly in the case of the homometallic Fe/Fe complexes. Surprisingly, for the asymmetric ligand the placement of the metal ions in the two pockets was not dictated by the asymmetry. Additionally, the relative stability of the homometallic complexes versus the heterometallic complexes and the possibility to transform a homometallic complex into a heterometallic complex were investigated. By titrating one metal into a solution containing the other homometallic dimer it was possible to observe that Fe2+ added to a solution of a Mn/Mn complex led to the replacement of one Mn ion in the complex with a Fe ion. The manganese complex of DPCPMP was investigated as a functional mod

The focus of this thesis has been synthesise and study metal complexes that mimic the structure and function of the active site in two particular metalloenzymes, ribonucleotide reductase (RNR) and manganese catalase (MnCAT). These two metalloenzymes both have two transition metals ions in the cofactor: two manganese ions in MnCAT and either two iron, iron-manganese or two manganese ions in RNR. Three different ligands were synthetized to make model complexes for these cofactors. The first ligand, BPMP, can bind two metal ions and provides two symmetric pockets with two pyridine groups and one amine each, plus a phenolate group that can bridge the two metals. The second ligand DPCPMP had one carboxylate group instead of a pyridine group in one pocket, creating an asymmetric ligand, and the third ligand BPCPMP, had two carboxylate groups, one in each pocket. From the first and the second ligands it was possible to obtain six complexes: low-valent homometallic Mn/Mn and Fe/Fe complexes and a heterometallic complex for each ligand. For the third ligand, only the Fe/Fe complex was synthetized. All seven complexes were characterized by a number of spectroscopic methods. The presence of carboxylate groups in the ligand shifted the redox potential for the metal complexes towards more negative values, particularly in the case of the homometallic Fe/Fe complexes. Surprisingly, for the asymmetric ligand the placement of the metal ions in the two pockets was not dictated by the asymmetry. Additionally, the relative stability of the homometallic complexes versus the heterometallic complexes and the possibility to transform a homometallic complex into a heterometallic complex were investigated. By titrating one metal into a solution containing the other homometallic dimer it was possible to observe that Fe2+ added to a solution of a Mn/Mn complex led to the replacement of one Mn ion in the complex with a Fe ion. The manganese complex of DPCPMP was investigated as a functional mod

Deoxyribonucleoside triphosphates (dNTPs) are precursors for DNA replication and repair. Mammalian cells have two distinct biosynthesis pathways to supply dNTPs: de novo and salvage pathways. These pathways are intimately coordinated to maintain optimal dNTP concentrations throughout different phases of the cell cycle, and perturbations in the production of dNTPs could lead to increased, decreased, or imbalanced dNTP pools. In yeasts, changes in both the level and balance of dNTPs increase mutation rates and genome instability. In mammals, elevated mutation rates and genome instability predispose to numerous diseases, including cancer. However, the correlation of dNTP changes with pathology has not been well established in mammals. In this thesis, I present how we addressed this issue using three separate mouse models – one with an increased dNTP pool, one with a decreased dNTP pool, and one with an imbalanced dNTP pool. To modulate dNTP levels in the mice, we deleted or mutated either sterile alpha motif and histidine-aspartic domain containing protein 1 (SAMHD1) or ribonucleotide reductase (RNR) proteins, which are involved in the salvage and de novo pathways, respectively. In the first model, mouse embryos without the SAMHD1 gene showed a slight increase in dNTP levels. A similar increase in dNTPs conferred moderately elevated mutation rates in cultured cancer cells. In the second model, we created a mouse strain carrying a modified allosteric specificity site in a subunit of RNR. Embryos with a heterozygous mutation had a mildly imbalanced dNTP pool. Heterozygous mutant mice showed a shorter lifespan and increased incidence and earlier onset of cancer. In the third model, the de novo dNTP production was inactivated in cardiac and skeletal muscles through the deletion of a gene encoding RNR. The hearts of knockout pups showed significant depletion of dNTPs, leading to aberrant DNA replication. In addition, knockout pups developed anatomic and histologic cardiac a

Deoxyribonucleoside triphosphates (dNTPs) are precursors for DNA replication and repair. Mammalian cells have two distinct biosynthesis pathways to supply dNTPs: de novo and salvage pathways. These pathways are intimately coordinated to maintain optimal dNTP concentrations throughout different phases of the cell cycle, and perturbations in the production of dNTPs could lead to increased, decreased, or imbalanced dNTP pools. In yeasts, changes in both the level and balance of dNTPs increase mutation rates and genome instability. In mammals, elevated mutation rates and genome instability predispose to numerous diseases, including cancer. However, the correlation of dNTP changes with pathology has not been well established in mammals. In this thesis, I present how we addressed this issue using three separate mouse models – one with an increased dNTP pool, one with a decreased dNTP pool, and one with an imbalanced dNTP pool. To modulate dNTP levels in the mice, we deleted or mutated either sterile alpha motif and histidine-aspartic domain containing protein 1 (SAMHD1) or ribonucleotide reductase (RNR) proteins, which are involved in the salvage and de novo pathways, respectively. In the first model, mouse embryos without the SAMHD1 gene showed a slight increase in dNTP levels. A similar increase in dNTPs conferred moderately elevated mutation rates in cultured cancer cells. In the second model, we created a mouse strain carrying a modified allosteric specificity site in a subunit of RNR. Embryos with a heterozygous mutation had a mildly imbalanced dNTP pool. Heterozygous mutant mice showed a shorter lifespan and increased incidence and earlier onset of cancer. In the third model, the de novo dNTP production was inactivated in cardiac and skeletal muscles through the deletion of a gene encoding RNR. The hearts of knockout pups showed significant depletion of dNTPs, leading to aberrant DNA replication. In addition, knockout pups developed anatomic and histologic cardiac a

The focus of this thesis has been synthesise and study metal complexes that mimic the structure and function of the active site in two particular metalloenzymes, ribonucleotide reductase (RNR) and manganese catalase (MnCAT). These two metalloenzymes both have two transition metals ions in the cofactor: two manganese ions in MnCAT and either two iron, iron-manganese or two manganese ions in RNR. Three different ligands were synthetized to make model complexes for these cofactors. The first ligand, BPMP, can bind two metal ions and provides two symmetric pockets with two pyridine groups and one amine each, plus a phenolate group that can bridge the two metals. The second ligand DPCPMP had one carboxylate group instead of a pyridine group in one pocket, creating an asymmetric ligand, and the third ligand BPCPMP, had two carboxylate groups, one in each pocket. From the first and the second ligands it was possible to obtain six complexes: low-valent homometallic Mn/Mn and Fe/Fe complexes and a heterometallic complex for each ligand. For the third ligand, only the Fe/Fe complex was synthetized. All seven complexes were characterized by a number of spectroscopic methods. The presence of carboxylate groups in the ligand shifted the redox potential for the metal complexes towards more negative values, particularly in the case of the homometallic Fe/Fe complexes. Surprisingly, for the asymmetric ligand the placement of the metal ions in the two pockets was not dictated by the asymmetry. Additionally, the relative stability of the homometallic complexes versus the heterometallic complexes and the possibility to transform a homometallic complex into a heterometallic complex were investigated. By titrating one metal into a solution containing the other homometallic dimer it was possible to observe that Fe2+ added to a solution of a Mn/Mn complex led to the replacement of one Mn ion in the complex with a Fe ion. The manganese complex of DPCPMP was investigated as a functional mod

Deoxyribonucleoside triphosphates (dNTPs) are precursors for DNA replication and repair. Mammalian cells have two distinct biosynthesis pathways to supply dNTPs: de novo and salvage pathways. These pathways are intimately coordinated to maintain optimal dNTP concentrations throughout different phases of the cell cycle, and perturbations in the production of dNTPs could lead to increased, decreased, or imbalanced dNTP pools. In yeasts, changes in both the level and balance of dNTPs increase mutation rates and genome instability. In mammals, elevated mutation rates and genome instability predispose to numerous diseases, including cancer. However, the correlation of dNTP changes with pathology has not been well established in mammals. In this thesis, I present how we addressed this issue using three separate mouse models – one with an increased dNTP pool, one with a decreased dNTP pool, and one with an imbalanced dNTP pool. To modulate dNTP levels in the mice, we deleted or mutated either sterile alpha motif and histidine-aspartic domain containing protein 1 (SAMHD1) or ribonucleotide reductase (RNR) proteins, which are involved in the salvage and de novo pathways, respectively. In the first model, mouse embryos without the SAMHD1 gene showed a slight increase in dNTP levels. A similar increase in dNTPs conferred moderately elevated mutation rates in cultured cancer cells. In the second model, we created a mouse strain carrying a modified allosteric specificity site in a subunit of RNR. Embryos with a heterozygous mutation had a mildly imbalanced dNTP pool. Heterozygous mutant mice showed a shorter lifespan and increased incidence and earlier onset of cancer. In the third model, the de novo dNTP production was inactivated in cardiac and skeletal muscles through the deletion of a gene encoding RNR. The hearts of knockout pups showed significant depletion of dNTPs, leading to aberrant DNA replication. In addition, knockout pups developed anatomic and histologic cardiac a

Det meget velbevarede og stærkt regulerede enzym ribonucleotide reduktase (RNR) forsyner celler med byggesten til DNA syntese, og har en central rolle i vedligeholdelsen af genomets integritet ved at tilvejebringe et passende niveau af deoxyribonukleotider i cellerne. Et studie i Schizosaccharomyces pombe har vist at når disse celler er afhænige af de to humane underenheder af RNR (RRM1 og RRM2), bliver de afhængige af DNA-struktur kontrolpunkterne og ude af stand til at gennemføre meiose. Sammenligninger af sekvenserne viser at de humane og S. pombes underenheder er 70% identiske og at forskellen mellem dem primært ligger i C-terminalen af R1 og N-terminalen af R2. Dette studie omhandler en mulig rolle af N-terminalen af S. pombes R2 (Suc22) som bliver undersøgt ved at skabe et kimært gen, der koder for RRM2 hvor N-terminalen er erstattet af de første 69 aminosyrer fra Suc22 (ChimR2). Når chimR2 bliver udtrykt i stedet for RRM2 i ellers hRNR afhængige celler, er disse celler ikke længere afhængige af DNA-struktur kontrolpunkterne, og sporuleringen genoprettes til et vild-type lignende niveau. Dette studie foreslår en hidtil ukendt rolle af Suc22 N-terminalen i DNA-skaderesponsen og meiose og antyder at ChimR2/RNR reguleres af det lille RNR-hæmmende protein, Spd1, i et omfang lignende det der er set for vildtypen. Yderligere omfatter denne undersøgelse begyndelsen på at fremstille to vektorer der koder for GFP-His-Flag mærkede peptider, der repræsenterer N-terminalerne af Suc22 og RRM2, der skal udtrykkes i S. pombe for coimmunopfældning for at identificere potentielle bindningspartnere, der er specifikke for Suc22 N-terminalen., The highly conserved and tightly regulated enzyme ribonucleotide reductase (RNR) is the provider of DNA building blocks, and has a central role in the maintenance of genome integrity by providing the cells with suitable levels of deoxyribonucleotides. A study has shown that when the genes for the two subunits of RNR (R1 and R2) in Schizosaccharomyces pombe are substituted for the human genes for R1 and R2 the cells are depending on the DNA structure checkpoints and are meiosis deficient. Sequence alignments show that the human and S. pombe RNR subunits share 70% similarity, and that they differ primarly in either the C-terminus of R1 or the N-terminus of R2. This study addresses a possible role of the N-terminus of S. pombe R2 (Suc22) which is being investigated by the creation of a chimeric gene that encodes the human R2 (RRM2) with the sequence encoding the N-terminus of Suc22 (ChimR2). When ChimR2 is expressed in place of RRM2 in otherwise hRNR dependent S. pombe cells, these cells are no longer reliant on the DNA structure checkpoints and sporulation is restored to near wild-type levels. This study suggests a novel role of the Suc22 N-terminal in the DNA damage response and meiosis and suggests that ChimR2/RNR is regulated by the RNR inhibitor, Spd1, in a wild-type like manner. Further, this study includes the preparation of vectors encoding GFP-His-Flag-tagged peptides representing the N-termini of Suc22 and RRM2 to be expressed in S. pombe for co-immunoprecipitation to identify potential binding partners that are specific for the Suc22 N-terminus.

Inactivation of Mec1, the budding yeast ATR, results in a permanent S phase arrest followed by chromosome breakage and cell death during G2/M. The S phase arrest is proposed to stem from a defect in Mec1-mediated degradation of Sml1, a conserved inhibitor of ribonucleotide reductase (RNR), causing a severe depletion in cellular dNTP pools. Here, the casual link between the S phase arrest, Sml1, and dNTP-levels is examined using a temperature sensitive mec1 mutant. In addition to S phase arrest, thermal inactivation of Mec1 leads to constitutively high levels of Sml1 and an S phase arrest. Expression of a novel suppressor, GIS2, a conserved mRNA binding zinc finger protein, rescues the arrest without down-regulating Sml1 levels. The dNTP pool in mec1 is reduced by ∼17% and GIS2 expression restores it, but only partially, to ∼93% of a control. We infer that the permanent S phase block following Mec1 inactivation can be uncoupled from its role in Sml1 down-regulation. Furthermore, unexpectedly modest effects of mec1 and GIS2 on dNTP levels suggest that the S phase arrest is unlikely to result from a severe depletion of dNTP pool as assumed, but a heightened sensitivity to small changes in its availability.

Inactivation of Mec1, the budding yeast ATR, results in a permanent S phase arrest followed by chromosome breakage and cell death during G2/M. The S phase arrest is proposed to stem from a defect in Mec1-mediated degradation of Sml1, a conserved inhibitor of ribonucleotide reductase (RNR), causing a severe depletion in cellular dNTP pools. Here, the casual link between the S phase arrest, Sml1, and dNTP-levels is examined using a temperature sensitive mec1 mutant. In addition to S phase arrest, thermal inactivation of Mec1 leads to constitutively high levels of Sml1 and an S phase arrest. Expression of a novel suppressor, GIS2, a conserved mRNA binding zinc finger protein, rescues the arrest without down-regulating Sml1 levels. The dNTP pool in mec1 is reduced by ∼17% and GIS2 expression restores it, but only partially, to ∼93% of a control. We infer that the permanent S phase block following Mec1 inactivation can be uncoupled from its role in Sml1 down-regulation. Furthermore, unexpectedly modest effects of mec1 and GIS2 on dNTP levels suggest that the S phase arrest is unlikely to result from a severe depletion of dNTP pool as assumed, but a heightened sensitivity to small changes in its availability.

Inactivation of Mec1, the budding yeast ATR, results in a permanent S phase arrest followed by chromosome breakage and cell death during G2/M. The S phase arrest is proposed to stem from a defect in Mec1-mediated degradation of Sml1, a conserved inhibitor of ribonucleotide reductase (RNR), causing a severe depletion in cellular dNTP pools. Here, the casual link between the S phase arrest, Sml1, and dNTP-levels is examined using a temperature sensitive mec1 mutant. In addition to S phase arrest, thermal inactivation of Mec1 leads to constitutively high levels of Sml1 and an S phase arrest. Expression of a novel suppressor, GIS2, a conserved mRNA binding zinc finger protein, rescues the arrest without down-regulating Sml1 levels. The dNTP pool in mec1 is reduced by ∼17% and GIS2 expression restores it, but only partially, to ∼93% of a control. We infer that the permanent S phase block following Mec1 inactivation can be uncoupled from its role in Sml1 down-regulation. Furthermore, unexpectedly modest effects of mec1 and GIS2 on dNTP levels suggest that the S phase arrest is unlikely to result from a severe depletion of dNTP pool as assumed, but a heightened sensitivity to small changes in its availability.

LKAB is a mining company in Sweden, which among other things, is running an iron mine and several ore enrichment plants in Kiruna. The main product produced in Kiruna is iron pellets: finely grinded iron ore and additives sintered together. The ore enrichment process separates iron ore from contaminating materials. Enriched ore is thereafter mixed with additives which combined gives the product the desired quality. The mix between enriched ore and additives is critical to quality and a consistent product quality is important for every competitive company.Product quality is defined by several parameters, one of which is chemical composition. Several measurement systems are used within the enrichment process. Data from these systems are used for monitoring and controlling the process, where of additive/iron ore ratio is one adjustable factor. LKAB is dependent on these measurement systems. LKAB is therefore interested in knowing whether these measurement systems are reliable and how data from these measurement systems is best put to use.This study addresses the reliability of two manual and one automated measurement system at LKAB’s pelletization plant KK2 in Kiruna. The study also investigate how data from the measurement systems can be used together with statistical process control.Some conclusions drawn from the study are:- The measurements system error is large in comparison to the internal quality requirements at LKAB but well within the requirements of the ISO standard 3082.- There is no proven bias between the manual and automatic measurement system controlling enriched iron ore.- The bias between pre and post pelletization is significant.- Grinders and x-ray equipment used in the measurement systems needimprovement.- Data from the automated measurement system is auto correlated andcurrent control charts should be replaced or combined with suggestedARIMA-charts.- The additive mixing process is not in statistical balance and need to bestabilized in order to get, Validerat; 20150125 (global_studentproject_submitter)

In Saccharomyces cerevisiae, absence of the checkpoint kinase Mec1 (ATR) is viable upon mutations that increase the activity of the ribonucleotide reductase (RNR) complex. Whether this pathway is conserved in mammals remains unknown. Here we show that cells from mice carrying extra alleles of the RNR regulatory subunit RRM2 (Rrm2(TG)) present supraphysiological RNR activity and reduced chromosomal breakage at fragile sites. Moreover, increased Rrm2 gene dosage significantly extends the life span of ATR mutant mice. Our study reveals the first genetic condition in mammals that reduces fragile site expression and alleviates the severity of a progeroid disease by increasing RNR activity.

Inactivation of Mec1, the budding yeast ATR, results in a permanent S phase arrest followed by chromosome breakage and cell death during G2/M. The S phase arrest is proposed to stem from a defect in Mec1-mediated degradation of Sml1, a conserved inhibitor of ribonucleotide reductase (RNR), causing a severe depletion in cellular dNTP pools. Here, the casual link between the S phase arrest, Sml1, and dNTP-levels is examined using a temperature sensitive mec1 mutant. In addition to S phase arrest, thermal inactivation of Mec1 leads to constitutively high levels of Sml1 and an S phase arrest. Expression of a novel suppressor, GIS2, a conserved mRNA binding zinc finger protein, rescues the arrest without down-regulating Sml1 levels. The dNTP pool in mec1 is reduced by ∼17% and GIS2 expression restores it, but only partially, to ∼93% of a control. We infer that the permanent S phase block following Mec1 inactivation can be uncoupled from its role in Sml1 down-regulation. Furthermore, unexpectedly modest effects of mec1 and GIS2 on dNTP levels suggest that the S phase arrest is unlikely to result from a severe depletion of dNTP pool as assumed, but a heightened sensitivity to small changes in its availability.

Inactivation of Mec1, the budding yeast ATR, results in a permanent S phase arrest followed by chromosome breakage and cell death during G2/M. The S phase arrest is proposed to stem from a defect in Mec1-mediated degradation of Sml1, a conserved inhibitor of ribonucleotide reductase (RNR), causing a severe depletion in cellular dNTP pools. Here, the casual link between the S phase arrest, Sml1, and dNTP-levels is examined using a temperature sensitive mec1 mutant. In addition to S phase arrest, thermal inactivation of Mec1 leads to constitutively high levels of Sml1 and an S phase arrest. Expression of a novel suppressor, GIS2, a conserved mRNA binding zinc finger protein, rescues the arrest without down-regulating Sml1 levels. The dNTP pool in mec1 is reduced by ∼17% and GIS2 expression restores it, but only partially, to ∼93% of a control. We infer that the permanent S phase block following Mec1 inactivation can be uncoupled from its role in Sml1 down-regulation. Furthermore, unexpectedly modest effects of mec1 and GIS2 on dNTP levels suggest that the S phase arrest is unlikely to result from a severe depletion of dNTP pool as assumed, but a heightened sensitivity to small changes in its availability.

Trypanosoma brucei causes African sleeping sickness in humans and Nagana in cattle. There are no vaccines available against the disease and the current treatment is also not satisfactory because of inefficacy and numerous side effects of the used drugs. T. brucei lacks de novo synthesis of purine nucleosides; hence it depends on the host to make its purine nucleotides. T. brucei has a high affinity adenosine kinase (TbAK), which phosphorylates adenosine, deoxyadenosine (dAdo), inosine and their analogs. RNAi experiments confirmed that TbAK is responsible for the salvage of dAdo and the toxicity of its substrate analogs. Cell growth assays with the dAdo analogs, Ara-A and F-Ara-A, suggested that TbAK could be exploited for drug development against the disease. It has previously been shown that when T. brucei cells were cultivated in the presence of 1 mM deoxyadenosine (dAdo), they showed accumulation of dATP and depletion of ATP nucleotides. The altered nucleotide levels were toxic to the trypanosomes. However the salvage of dAdo in trypanosomes was dramatically reduced below 0.5 mM dAdo. Radiolabeled dAdo experiments showed that it (especially at low concentrations) is cleaved to adenine and converted to ATP. The recombinant methylthioadenosine phosphorylase (TbMTAP) cleaved methylthioadenosine, dAdo and adenosine into adenine and sugar-1-P in a phosphate-dependent manner. The trypanosomes became more sensitive to dAdo when TbMTAP was down-regulated in RNAi experiments. The RNAi experiments confirmed that trypanosomes avoid dATP accumulation by cleaving dAdo. The TbMTAP cleavage-resistant nucleoside analogs, FANA-A and Ara-A, successfully cured T. brucei-infected mice. The DNA building block dTTP can be synthesized either via thymidylate synthase in the de novo pathway or via thymidine kinase (TK) by salvage synthesis. We found that T. brucei and three other parasites contain a tandem TK where the gene sequence was repeated twice or four times in a single open readi, Targeting the nucleotide metabolism of the mammalian pathogen Trypanosoma brucei.

Trypanosoma brucei causes African sleeping sickness in humans and Nagana in cattle. There are no vaccines available against the disease and the current treatment is also not satisfactory because of inefficacy and numerous side effects of the used drugs. T. brucei lacks de novo synthesis of purine nucleosides; hence it depends on the host to make its purine nucleotides. T. brucei has a high affinity adenosine kinase (TbAK), which phosphorylates adenosine, deoxyadenosine (dAdo), inosine and their analogs. RNAi experiments confirmed that TbAK is responsible for the salvage of dAdo and the toxicity of its substrate analogs. Cell growth assays with the dAdo analogs, Ara-A and F-Ara-A, suggested that TbAK could be exploited for drug development against the disease. It has previously been shown that when T. brucei cells were cultivated in the presence of 1 mM deoxyadenosine (dAdo), they showed accumulation of dATP and depletion of ATP nucleotides. The altered nucleotide levels were toxic to the trypanosomes. However the salvage of dAdo in trypanosomes was dramatically reduced below 0.5 mM dAdo. Radiolabeled dAdo experiments showed that it (especially at low concentrations) is cleaved to adenine and converted to ATP. The recombinant methylthioadenosine phosphorylase (TbMTAP) cleaved methylthioadenosine, dAdo and adenosine into adenine and sugar-1-P in a phosphate-dependent manner. The trypanosomes became more sensitive to dAdo when TbMTAP was down-regulated in RNAi experiments. The RNAi experiments confirmed that trypanosomes avoid dATP accumulation by cleaving dAdo. The TbMTAP cleavage-resistant nucleoside analogs, FANA-A and Ara-A, successfully cured T. brucei-infected mice. The DNA building block dTTP can be synthesized either via thymidylate synthase in the de novo pathway or via thymidine kinase (TK) by salvage synthesis. We found that T. brucei and three other parasites contain a tandem TK where the gene sequence was repeated twice or four times in a single open readi, Targeting the nucleotide metabolism of the mammalian pathogen Trypanosoma brucei.

Trypanosoma brucei causes African sleeping sickness in humans and Nagana in cattle. There are no vaccines available against the disease and the current treatment is also not satisfactory because of inefficacy and numerous side effects of the used drugs. T. brucei lacks de novo synthesis of purine nucleosides; hence it depends on the host to make its purine nucleotides. T. brucei has a high affinity adenosine kinase (TbAK), which phosphorylates adenosine, deoxyadenosine (dAdo), inosine and their analogs. RNAi experiments confirmed that TbAK is responsible for the salvage of dAdo and the toxicity of its substrate analogs. Cell growth assays with the dAdo analogs, Ara-A and F-Ara-A, suggested that TbAK could be exploited for drug development against the disease. It has previously been shown that when T. brucei cells were cultivated in the presence of 1 mM deoxyadenosine (dAdo), they showed accumulation of dATP and depletion of ATP nucleotides. The altered nucleotide levels were toxic to the trypanosomes. However the salvage of dAdo in trypanosomes was dramatically reduced below 0.5 mM dAdo. Radiolabeled dAdo experiments showed that it (especially at low concentrations) is cleaved to adenine and converted to ATP. The recombinant methylthioadenosine phosphorylase (TbMTAP) cleaved methylthioadenosine, dAdo and adenosine into adenine and sugar-1-P in a phosphate-dependent manner. The trypanosomes became more sensitive to dAdo when TbMTAP was down-regulated in RNAi experiments. The RNAi experiments confirmed that trypanosomes avoid dATP accumulation by cleaving dAdo. The TbMTAP cleavage-resistant nucleoside analogs, FANA-A and Ara-A, successfully cured T. brucei-infected mice. The DNA building block dTTP can be synthesized either via thymidylate synthase in the de novo pathway or via thymidine kinase (TK) by salvage synthesis. We found that T. brucei and three other parasites contain a tandem TK where the gene sequence was repeated twice or four times in a single open readi, Targeting the nucleotide metabolism of the mammalian pathogen Trypanosoma brucei.

Trypanosoma brucei causes African sleeping sickness in humans and Nagana in cattle. There are no vaccines available against the disease and the current treatment is also not satisfactory because of inefficacy and numerous side effects of the used drugs. T. brucei lacks de novo synthesis of purine nucleosides; hence it depends on the host to make its purine nucleotides. T. brucei has a high affinity adenosine kinase (TbAK), which phosphorylates adenosine, deoxyadenosine (dAdo), inosine and their analogs. RNAi experiments confirmed that TbAK is responsible for the salvage of dAdo and the toxicity of its substrate analogs. Cell growth assays with the dAdo analogs, Ara-A and F-Ara-A, suggested that TbAK could be exploited for drug development against the disease. It has previously been shown that when T. brucei cells were cultivated in the presence of 1 mM deoxyadenosine (dAdo), they showed accumulation of dATP and depletion of ATP nucleotides. The altered nucleotide levels were toxic to the trypanosomes. However the salvage of dAdo in trypanosomes was dramatically reduced below 0.5 mM dAdo. Radiolabeled dAdo experiments showed that it (especially at low concentrations) is cleaved to adenine and converted to ATP. The recombinant methylthioadenosine phosphorylase (TbMTAP) cleaved methylthioadenosine, dAdo and adenosine into adenine and sugar-1-P in a phosphate-dependent manner. The trypanosomes became more sensitive to dAdo when TbMTAP was down-regulated in RNAi experiments. The RNAi experiments confirmed that trypanosomes avoid dATP accumulation by cleaving dAdo. The TbMTAP cleavage-resistant nucleoside analogs, FANA-A and Ara-A, successfully cured T. brucei-infected mice. The DNA building block dTTP can be synthesized either via thymidylate synthase in the de novo pathway or via thymidine kinase (TK) by salvage synthesis. We found that T. brucei and three other parasites contain a tandem TK where the gene sequence was repeated twice or four times in a single open readi, Targeting the nucleotide metabolism of the mammalian pathogen Trypanosoma brucei.

Trypanosoma brucei causes African sleeping sickness in humans and Nagana in cattle. There are no vaccines available against the disease and the current treatment is also not satisfactory because of inefficacy and numerous side effects of the used drugs. T. brucei lacks de novo synthesis of purine nucleosides; hence it depends on the host to make its purine nucleotides. T. brucei has a high affinity adenosine kinase (TbAK), which phosphorylates adenosine, deoxyadenosine (dAdo), inosine and their analogs. RNAi experiments confirmed that TbAK is responsible for the salvage of dAdo and the toxicity of its substrate analogs. Cell growth assays with the dAdo analogs, Ara-A and F-Ara-A, suggested that TbAK could be exploited for drug development against the disease. It has previously been shown that when T. brucei cells were cultivated in the presence of 1 mM deoxyadenosine (dAdo), they showed accumulation of dATP and depletion of ATP nucleotides. The altered nucleotide levels were toxic to the trypanosomes. However the salvage of dAdo in trypanosomes was dramatically reduced below 0.5 mM dAdo. Radiolabeled dAdo experiments showed that it (especially at low concentrations) is cleaved to adenine and converted to ATP. The recombinant methylthioadenosine phosphorylase (TbMTAP) cleaved methylthioadenosine, dAdo and adenosine into adenine and sugar-1-P in a phosphate-dependent manner. The trypanosomes became more sensitive to dAdo when TbMTAP was down-regulated in RNAi experiments. The RNAi experiments confirmed that trypanosomes avoid dATP accumulation by cleaving dAdo. The TbMTAP cleavage-resistant nucleoside analogs, FANA-A and Ara-A, successfully cured T. brucei-infected mice. The DNA building block dTTP can be synthesized either via thymidylate synthase in the de novo pathway or via thymidine kinase (TK) by salvage synthesis. We found that T. brucei and three other parasites contain a tandem TK where the gene sequence was repeated twice or four times in a single open readi, Targeting the nucleotide metabolism of the mammalian pathogen Trypanosoma brucei.

Ribonucleotide reductase (RNR) is the only source for de novo production of the four deoxyribonucleoside triphosphate (dNTP) building blocks needed for DNA synthesis and repair. It is crucial that these dNTP pools are carefully balanced, since mutation rates increase when dNTP levels are either unbalanced or elevated. RNR is the major player in this homeostasis, and with its four different substrates, four different allosteric effectors and two different effector binding sites, it has one of the most sophisticated allosteric regulations known today. In the past few years, the structures of RNRs from several bacteria, yeast and man have been determined in the presence of allosteric effectors and substrates, revealing new information about the mechanisms behind the allosteric regulation. A common theme for all studied RNRs is a flexible loop that mediates modulatory effects from the allosteric specificity site (s-site) to the catalytic site for discrimination between the four substrates. Much less is known about the allosteric activity site (a-site), which functions as an on-off switch for the enzyme's overall activity by binding ATP (activator) or dATP (inhibitor). The two nucleotides induce formation of different enzyme oligomers, and a recent structure of a dATP-inhibited α(6)β(2) complex from yeast suggested how its subunits interacted non-productively. Interestingly, the oligomers formed and the details of their allosteric regulation differ between eukaryotes and Escherichia coli. Nevertheless, these differences serve a common purpose in an essential enzyme whose allosteric regulation might date back to the era when the molecular mechanisms behind the central dogma evolved.

La stabilité génomique, qui est essentielle à la vie, est possible grâce à la réplication et la réparation de l’ADN. Une des enzymes responsables de la réplication et de la réparation de l’ADN est la ribonucleotide reductase (RNR), qui est retrouvée chez la levure et chez l’humain. Cette enzyme catalyse la formation de déoxyribonucléotides et maintien le pool de dNTP requis pour la réparation et la réplication de l’ADN. L’enzyme RNR est un tétramère α2β2 constitué d’une grande (R1, α2) et d’une petite (R2, β2) sous-unité. Chez S. cerevisiae, les gènes RNR1 et RNR3 encodent la sous-unité α2 (R1). L’activité catalytique de RNR dépend d’une interaction avec le fer et de la formation d’un complexe entre R1 et R2. L’expression de toutes les sous-unités est inductible par les dommages causés à l’ADN. Dans cette étude, nous démontrons que des cellules qui n’expriment pas une des sous-unités, Rnr4, du complexe RNR sont sensibles à divers agents endommageant l’ADN, tels que le méthyl méthane sulfonate, la bléomycine, le péroxyde d’hydrogène et les rayons ultraviolets (UVC 254 nm). Au contraire, le mutant est résistant au 4-nitroquinoline-1- oxide (4-NQO), un composé qui engendre des lésions encombrantes. Par conséquent, le mutant rnr4Δ démontre une réduction marquée en mutations induites par le 4-NQO comparativement à la souche parentale. Nous voulions identifier la voie de réparation de l’ADN qui conférait cette résistance au 4-NQO ainsi que les protéines impliquées. Les voies BER, NER et MMR n’ont pas aboli la résistance au 4-NQO de la souche rnr4Δ. La protéine recombinante Rad51 ne joue pas un rôle critique dans la réparation de l’ADN et dans la résistance au 4-NQO. La délétion du gène REV3, qui encode une polymérase de contournement, impliquée dans la réparation post-réplication, a partiellement aboli la résistance au 4-NQO dans rnr4Δ. Ces résultats suggèrent que la polymérase Rev3 et possiblement d’autres polymérases translésion (Rev1, Rev7, Rad30) pourraient être impli, Genomic stability, critical for life, is controlled by DNA replication and repair. DNA replication and repair is mediated through many enzymes, one being ribonucleotide reductase (RNR), an enzyme found in both yeast and humans. RNR catalyzes the reaction involved in the formation of deoxyribonucleotides and is responsible for maintaining dNTP pools required for DNA repair and replication. RNR is an α2β2 tetramer consisting of a large (R1, α2) and small subunit (R2, β2). In S. cerevisiae RNR1 and RNR3 encode α2 (R1). RNR catalytic activity depends on its interaction with iron and on the formation of the complex between R1 and R2. All subunits are inducible by DNA damage. Here we show that cells lacking one subunit, Rnr4, of the RNR complex are sensitive to various DNA damaging agents such as methyl methane sulfonate, bleomycin, hydrogen peroxide, and ultraviolet radiation (UVC 254 nm). In contrast, the mutant is resistant to 4-nitroquinoline-1-oxide (4-NQO), an agent which produces bulky lesions. Consistent with this resistance, the rnr4Δ showed a sharp reduction in 4-NQO-induced mutations as compared to the parent. We wanted to determine which pathway was able to confer resistance to 4-NQO and thus targeted DNA repair proteins. The repair pathways BER, NER and MMR did not abolish 4-NQO resistance in rnr4Δ. Recombination protein Rad51 (NHEJ) was lethal in an rnr4Δ thus indicating no role in DNA repair and 4-NQO resistance. Deletion of the REV3 gene, encoding a DNA bypass polymerase involved in post replication repair, partially abolished 4-NQO resistance in rnr4Δ. These results suggest that Rev3 and possibly other translesion polymerases (Rev1, Rev7, Rad30) could play a role in the repair of bulky DNA lesions under low levels of dNTPs. DNA repair, a complex mechanism in yeast, involves a vast array of proteins, some yet to be discovered. Our results indicate that there is more than one protein involved in 4-NQO resistance and further investigation is required concern

Ribonucleotide reductase (RNR) is a key enzyme for the synthesis of the four DNA building blocks. Class Ia RNRs contain two subunits, denoted R1 (α) and R2 (β). These enzymes are regulated via two nucleotide-binding allosteric sites on the R1 subunit, termed the specificity and overall activity sites. The specificity site binds ATP, dATP, dTTP, or dGTP and determines the substrate to be reduced, whereas the overall activity site binds dATP (inhibitor) or ATP. By using gas-phase electrophoretic mobility macromolecule analysis and enzyme assays, we found that the Escherichia coli class Ia RNR formed an inhibited α4β4 complex in the presence of dATP and an active α2β2 complex in the presence of ATP (main substrate: CDP), dTTP (substrate: GDP) or dGTP (substrate: ADP). The R1-R2 interaction was 30–50 times stronger in the α4β4 complex than in the α2β2complex, which was in equilibrium with free α2 and β2 subunits. Studies of a known E. coli R1 mutant (H59A) showed that deficient dATP inhibition correlated with reduced ability to form α4β4 complexes. ATP could also induce the formation of a generally inhibited α4β4 complex in the E. coli RNR but only when used in combination with high concentrations of the specificity site effectors, dTTP/dGTP. Both allosteric sites are therefore important for α4β4 formation and overall activity regulation. The E. coli RNR differs from the mammalian enzyme, which is stimulated by ATP also in combination with dGTP/dTTP and forms active and inactive α6β2 complexes.