Ionized calcium (Ca2+) is a signaling messenger that controls numerous cellular processes essential for life. The fidelity of Ca2+ signaling depends on the mechanisms that dynamically regulate its cytosolic concentration and maintain it at a low level in a resting cell. Plasma Membrane Ca2+ ATPase (PMCA) is a high affinity Ca2+ extrusion pathway involved in Ca2+ homeostasis and signal transduction. PMCA are encoded by 4 genes (PMCA1-4), which are expressed in a tissue dependent manner. The diversity of PMCA isoforms is further increased by alternative splicing. Changes in PMCA activity occur in heart failure and hypertension. Specific inhibitors of other ion transporters such as thapsigargin and digoxin, have made their mark in cell biology, but the currently used inhibitors of PMCA (vanadate and eosin) are non-specific. Thus, selective inhibitors of PMCA are needed to discern its role in Ca2+ signaling in physiology and pathophysiology. We introduced the concept of caloxins - peptides that specifically inhibit the activity of PMCA by binding to one of its five extracellular domains (exdoms) 1 to 5. The earlier caloxins including 2a1 and 3a1 were obtained by screening a phage display random 12-amino acid peptide (Ph.D-12) library for binding to synthetic peptides based on the exdom sequences. However, they all had low affinity. The objective of this research was to develop caloxins with high affinity and PMCA4 isoform selectivity. A two-step screening method was developed to screen the Ph.D-12 library to first bind to the synthetic exdom of PMCA4, followed by affinity chromatography using PMCA protein purified from human erythrocyte ghosts (mainly PMCA4). This method was used to obtain caloxins 1b1 and 1b2 to bind to the N and C-terminal halves of the exdom 1 of PMCA4, respectively. Both caloxins 1b1 and 1b2 had a 10-fold higher affinity than the prototype caloxin 2a1 and showed slight PMCA4 isoform preference. To engineer inhibitors with greater affinity and PMCA4 isoform selectivity, Ph.D caloxin 1b1 like peptide library was constructed. Most of the peptides expressed in this library differed from caloxin 1b1 in 0, 1, 2 or 3 amino acid residues at random. The library was screened to obtain several peptides one of which was caloxin 1c2. Caloxin 1c2 had 200-fold higher affinity than caloxin 2a1 and was isoform selective, with greater than 10-fold affinity for PMCA4 than for PMCA isoforms 1, 2 or 3. Thus, caloxin 1c2 is the first high affinity PMCA inhibitor that also is selective for an individual PMCA isoform. The second aim of this research was to establish that caloxin 1c2 binds to PMCA protein in erythrocyte ghosts. Two photoreactive caloxin 1c2-derivatives containing the photoactivable residue benzoylphenylalanine (Bpa) and a C-terminal biotin tag were used. Bpa substituted tryptophan at position 3 (3Bpa1c2-biotin) and serine at position 16 (16Bpa1c2-biotin) in caloxin 1c2. Both the derivatives inhibited PMCA activity in the erythrocyte ghosts. The intensity of the biotin label in the photolabeled erythrocyte ghosts was much stronger with 3Bpa1c2-biotin, which was then used in the subsequent experiments. The photolabeled proteins in erythrocyte ghosts were detected as a 250-270 kDa doublet in Western blots using streptavidin and the PMCA specific antibody. The degree of photolabeling depended on the UV-crosslinking time, and on the concentrations of 3Bpa1c2-biotin and the ghost protein. The selectivity of the photolabeling site was confirmed by decreased photolabel incorporation at 250-270 kDa doublet in the presence of excess caloxin 1c2 and the synthetic exdom 1X peptide of PMCA4. The photolabeled erythrocyte ghosts were solubilized and analyzed by immunoprecipitation with the PMCA specific antibody. The immunoprecipitate showed a 250-270 kDa doublet in Western blots using streptavidin. This confirmed that PMCA protein was photolabeled by the photoreactive derivatives of caloxin 1c2. Thus, caloxin 1c2 inhibits PMCA activity by binding to the exdom 1X of PMCA4. My work in M.Sc. initiated the concept of caloxins in the literature. This research has taken it to the stage where we can obtain caloxins selective for individual PMCA isoforms. This contrasts with the relative paucity of inhibitors specific for individual isoforms of other ion pumps. The high affinity isoform selective caloxin 1c2 and previous caloxins are being used to study PMCA physiology in our lab and by other researchers. Since caloxins act when added extracellularly and it is possible to obtain PMCA isoform selective caloxins, it is anticipated that they will aid in understanding the role of PMCA in signal transduction and homeostasis in health and disease. / Thesis / Doctor of Philosophy (PhD)

A doença renal crônica (DRC) evolui de forma progressiva e o hiperparatiroidismo secundário renal (HPTSR) é uma das importantes alterações que causa a perda adicional de néfrons e o comprometimento de varios sistemas; o paratormônio (PTH) é considerado como uma importante toxina urêmica. A avaliação do PTH sérico em felinos com DRC pode trazer informações para o melhor entendimento da fisiopatologia do HPTSR, suscitando possíveis medidas terapêuticas. Observou-se que gatos com DRC (n=40) apresentaram aumento significante (p < 0,05) da concentração sérica de paratormônio intacto (PTHi) quando comparados a gatos clinicamente normais (n=21; grupo controle). Quanto a avaliação do PTHi nos subgrupos de DRC (ESTÁGIOS II, III e IV de evolução da afecção, classificados segundo o International Renal Interest Society IRIS), constatou-se diferença significante entre os gatos dos ESTÁGIOS III e IV com os gatos clinicamente normais; em relação ao ESTÁGIO II, apesar de não ter sido observada diferença significante, a maioria dos gatos apresentou aumento da concentração sérica de PTHi (> 60,2 pg/mL). Em relação aos valores das concentrações séricas de fósforo e de cálcio iônico, detectou-se a tendência de concentrações séricas progressivamente maiores de fósforo (hiperfosfatemia) nos estágios mais avançados da doença (frequências de 16,7%, 27,3% e 100% nos ESTÁGIOS II, III e IV, respectivamente); inversamente, as concentrações séricas de cálcio iônico foram progressivamente menores (respectivamente, frequência de hipocalcemia de 8,3%, 9,1% e 66,7%). Constatou-se diferença significante quanto as concentração de bicarbonato plasmático entre os gatos clinicamente normais e os gatos com DRC (acidose metabólica em 42,5% dos casos), como também entre os gatos clinicamente normais e os animais dos subgrupos DRC ESTÁGIOS II e III (bicarbonato plasmático < 16,8 mmol/L observado em 40,9% e 33,3%, respectivamente). Não foram detectadas diferenças significantes quanto as concentrações séricas de cálcio total ou de pH sanguíneo nas comparações múltiplas. Em relação aos valores da multiplicação entre as concentrações séricas de cálcio total e de fósforo, a análise estatística demonstrou resultados similares àqueles da análise das concentrações séricas de fósforo. Sugere-se que o estímulo para o aumento da concentração sérica de PTHi, e o consequente HPTSR, observado nos gatos com DRC no estágio IV, tenha decorrido da hipocalcemia iônica e da hiperfosfatemia apresentadas por estes animais, ativando a regulação pela paratireóide. Quanto aos gatos nos estágios II e III da DRC, outros fatores, além da hipocalcemia e da hiperfosfatemia, devem estar envolvidos no aumento da síntese de PTHi, sugerindo-se a necessidade de investigação, por exemplo, das concentrações séricas de calcitriol; ainda no subgrupo ESTÁGIO II, a hipercalcemia iônica observada em 50% dos gatos sugere a possibilidade do envolvimento da acidose metabólica, que poderia comprometer a fração ionizada do cálcio. A correlação positiva observada entre o fósforo e o PTHi sugere a possibilidade de avaliação indireta de HPTSR pela determinação do fósforo sérico; entretanto nos gatos nos estágios II e III da DRC, esta avaliação indireta não se mostrou adequada, pois observou-se concomitantemente normofosfatemia e aumento do PTHi, indicando-se a necessidade da determinação do PTHi para avaliação do HPTSR nesses estágios da DRC em gatos. / Chronic kidney disease (CKD) develops gradually, causing several changes and renal secondary hyperparathyroidism (RSHPTH) is one of those alterations, which, besides causing loss of additional nephrons, increases the morbidity and mortality due to the action of parathyroid hormone (PTH) as an important uremic toxin. Evaluation of serum PTH in cats with CKD may add information for the better understanding of RSHPTH pathophysiology, arising possible therapeutic procedures. Cats with CKD (n = 40) showed significant increase (p < 0.05) in serum intact parathyroid hormone (iPTH) as compared with clinically normal cats (n = 21, control group). In subgroups of CKD (STAGES II, III and IV of CKD, classified as recommended by International Renal Interest Society - IRIS), significant difference was observed between clinically normal cats and cats with CKD in STAGES III and IV; in reference to the STAGE II, although no significant difference was observed, most of these cats showed an increase in serum iPTH (> 60.2 pg / mL). In relation to values of phosphorus and ionized calcium serum concentrations, a trend of progressively higher serum concentrations of phosphorus (hyperphosphatemia) was detected in the late stages of the disease (16.7%, 27.3% and 100% in STAGES II, III and IV, respectively), in opposite, serum concentrations of ionized calcium progressively decreased (respectively, ionized hypocalcemia, 8.3%, 9.1% and 66.7%). Plasma bicarbonate concentrations were significant different between clinically normal cats and cats with CKD (metabolic acidosis observed in 42.5% of the cases), and between clinically normal cats and cats with CKD STAGES II and III (plasma bicarbonate < 16.8 mmol/L observed in 40.9% and 33.3% of the cases, respectively). No significant differences were detected for serum concentrations of total calcium and blood pH, in multiple comparisons analysis. In relation to the values of serum concentrations of calcium and phosphorus product, the results were similar to those obtained from phosphorus serum concentrations evaluation. The results suggested that the stimulus for the increase in serum iPTH, and follow development of RSHPTH, observed in cats with CKD STAGE IV, was in consequence of ionized hypocalcemia and hyperphosphatemia, enabling the regulation by the parathyroid. However, for the cats in STAGES II and III of CKD, other factors, beyond hypocalcemia and hyperphosphatemia, may be involved to cause the increase of iPTH synthesis, and calcitriol serum concentrations must be investigated; in cats with DRC STAGE II, 50% of the cases presented ionized hypercalcemia, suggesting the influence of metabolic acidosis in ionized calcium fraction. The correlation observed between serum phosphorus and iPTH may suggest the possibility of indirect evaluation of RSHPTH by means of phosphorus serum concentration; however in cats with DRC, STAGES II and III, this indirect assessment may not be adequate as normal serum levels of phosphorus as well as increased serum iPTH were observed in those cats, indicating the need for the determination of serum iPTH to evaluate RSHPTH.

A doença renal crônica (DRC) evolui de forma progressiva e o hiperparatiroidismo secundário renal (HPTSR) é uma das importantes alterações que causa a perda adicional de néfrons e o comprometimento de varios sistemas; o paratormônio (PTH) é considerado como uma importante toxina urêmica. A avaliação do PTH sérico em felinos com DRC pode trazer informações para o melhor entendimento da fisiopatologia do HPTSR, suscitando possíveis medidas terapêuticas. Observou-se que gatos com DRC (n=40) apresentaram aumento significante (p < 0,05) da concentração sérica de paratormônio intacto (PTHi) quando comparados a gatos clinicamente normais (n=21; grupo controle). Quanto a avaliação do PTHi nos subgrupos de DRC (ESTÁGIOS II, III e IV de evolução da afecção, classificados segundo o International Renal Interest Society IRIS), constatou-se diferença significante entre os gatos dos ESTÁGIOS III e IV com os gatos clinicamente normais; em relação ao ESTÁGIO II, apesar de não ter sido observada diferença significante, a maioria dos gatos apresentou aumento da concentração sérica de PTHi (> 60,2 pg/mL). Em relação aos valores das concentrações séricas de fósforo e de cálcio iônico, detectou-se a tendência de concentrações séricas progressivamente maiores de fósforo (hiperfosfatemia) nos estágios mais avançados da doença (frequências de 16,7%, 27,3% e 100% nos ESTÁGIOS II, III e IV, respectivamente); inversamente, as concentrações séricas de cálcio iônico foram progressivamente menores (respectivamente, frequência de hipocalcemia de 8,3%, 9,1% e 66,7%). Constatou-se diferença significante quanto as concentração de bicarbonato plasmático entre os gatos clinicamente normais e os gatos com DRC (acidose metabólica em 42,5% dos casos), como também entre os gatos clinicamente normais e os animais dos subgrupos DRC ESTÁGIOS II e III (bicarbonato plasmático < 16,8 mmol/L observado em 40,9% e 33,3%, respectivamente). Não foram detectadas diferenças significantes quanto as concentrações séricas de cálcio total ou de pH sanguíneo nas comparações múltiplas. Em relação aos valores da multiplicação entre as concentrações séricas de cálcio total e de fósforo, a análise estatística demonstrou resultados similares àqueles da análise das concentrações séricas de fósforo. Sugere-se que o estímulo para o aumento da concentração sérica de PTHi, e o consequente HPTSR, observado nos gatos com DRC no estágio IV, tenha decorrido da hipocalcemia iônica e da hiperfosfatemia apresentadas por estes animais, ativando a regulação pela paratireóide. Quanto aos gatos nos estágios II e III da DRC, outros fatores, além da hipocalcemia e da hiperfosfatemia, devem estar envolvidos no aumento da síntese de PTHi, sugerindo-se a necessidade de investigação, por exemplo, das concentrações séricas de calcitriol; ainda no subgrupo ESTÁGIO II, a hipercalcemia iônica observada em 50% dos gatos sugere a possibilidade do envolvimento da acidose metabólica, que poderia comprometer a fração ionizada do cálcio. A correlação positiva observada entre o fósforo e o PTHi sugere a possibilidade de avaliação indireta de HPTSR pela determinação do fósforo sérico; entretanto nos gatos nos estágios II e III da DRC, esta avaliação indireta não se mostrou adequada, pois observou-se concomitantemente normofosfatemia e aumento do PTHi, indicando-se a necessidade da determinação do PTHi para avaliação do HPTSR nesses estágios da DRC em gatos. / Chronic kidney disease (CKD) develops gradually, causing several changes and renal secondary hyperparathyroidism (RSHPTH) is one of those alterations, which, besides causing loss of additional nephrons, increases the morbidity and mortality due to the action of parathyroid hormone (PTH) as an important uremic toxin. Evaluation of serum PTH in cats with CKD may add information for the better understanding of RSHPTH pathophysiology, arising possible therapeutic procedures. Cats with CKD (n = 40) showed significant increase (p < 0.05) in serum intact parathyroid hormone (iPTH) as compared with clinically normal cats (n = 21, control group). In subgroups of CKD (STAGES II, III and IV of CKD, classified as recommended by International Renal Interest Society - IRIS), significant difference was observed between clinically normal cats and cats with CKD in STAGES III and IV; in reference to the STAGE II, although no significant difference was observed, most of these cats showed an increase in serum iPTH (> 60.2 pg / mL). In relation to values of phosphorus and ionized calcium serum concentrations, a trend of progressively higher serum concentrations of phosphorus (hyperphosphatemia) was detected in the late stages of the disease (16.7%, 27.3% and 100% in STAGES II, III and IV, respectively), in opposite, serum concentrations of ionized calcium progressively decreased (respectively, ionized hypocalcemia, 8.3%, 9.1% and 66.7%). Plasma bicarbonate concentrations were significant different between clinically normal cats and cats with CKD (metabolic acidosis observed in 42.5% of the cases), and between clinically normal cats and cats with CKD STAGES II and III (plasma bicarbonate < 16.8 mmol/L observed in 40.9% and 33.3% of the cases, respectively). No significant differences were detected for serum concentrations of total calcium and blood pH, in multiple comparisons analysis. In relation to the values of serum concentrations of calcium and phosphorus product, the results were similar to those obtained from phosphorus serum concentrations evaluation. The results suggested that the stimulus for the increase in serum iPTH, and follow development of RSHPTH, observed in cats with CKD STAGE IV, was in consequence of ionized hypocalcemia and hyperphosphatemia, enabling the regulation by the parathyroid. However, for the cats in STAGES II and III of CKD, other factors, beyond hypocalcemia and hyperphosphatemia, may be involved to cause the increase of iPTH synthesis, and calcitriol serum concentrations must be investigated; in cats with DRC STAGE II, 50% of the cases presented ionized hypercalcemia, suggesting the influence of metabolic acidosis in ionized calcium fraction. The correlation observed between serum phosphorus and iPTH may suggest the possibility of indirect evaluation of RSHPTH by means of phosphorus serum concentration; however in cats with DRC, STAGES II and III, this indirect assessment may not be adequate as normal serum levels of phosphorus as well as increased serum iPTH were observed in those cats, indicating the need for the determination of serum iPTH to evaluate RSHPTH.

Mild traumatic brain injury (mTBI), often referred to as concussion, has become increasingly recognized as a serious health issue in the general population. The prevalence of mTBI in athletes, particularly repeated injuries in young athletes, is of great concern as injuries to the developing brain can have long-term detrimental effects. In this study we used a novel awake closed-head injury (ACHI) model in rodents to examine repeated mTBI (rmTBI), to determine if repeated injuries produced the neurological and molecular changes evident with human concussion. Animals were administered 4, 8, and 16 rmTBIs and acute neurological assessments were performed after the injuries. Changes in glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (Iba-1) levels were assessed using Western blot analysis at one day following rmTBI in the ipsilateral dentate gyrus (DG) and the cornu ammonis (CA) regions of the hippocampus and the cortex (CX) indicative of astrocyte and microglial cell reactivity. Results indicated that the ACHI model produces neurological deficits immediately after the injuries, with the most deficits arising in the rmTBI16 group. Despite deficits in all injury groups, histological staining with cresyl violet revealed no significant morphological tissue damage to the brain. Western blot analysis, however, showed a significant increase in DG and CX GFAP expression in the rmTBI16 group with no changes in Iba-1 levels. This suggests an acute activation of astrocytes in response to injury, with a delay or absence of microglial activation. Our findings show that with repetitive concussions, we are able to detect acute neurological and molecular changes in the juvenile female brain. However, further investigation is necessary to determine if these are transient changes. / Graduate

Bakgrund: Provmaterial för joniserat kalcium är känsligt för pH-förändringar och med tanke på svårstuckna patienter är det betydelsefullt att undersöka lägre fyllnadsnivåers påverkan på analysresultatet. På grund av olika pre-analytiska faktorer kan tidsgränsen (4 timmar) för analys av standardbikarbonat överskridas. Förvaring av post-analytiska serumprover medför att kompletteringsanalyser kan beställas. Begränsad dokumentation finns om avkorkade serumprovers stabilitet i rumstemperatur. Syfte: Syftet var att undersöka hur lägre fyllnadsnivåer av serum påverkar analysresultatet för joniserat kalcium, om standardbikarbonatsprover på helblod kan analyseras senare än 4 timmar och hur länge serumprover kan stå i rumstemperatur utan kork för eventuella kompletteringsanalyser. Metod: Koncentrationen av analyten joniserat kalcium i serumprover med fyllnadsnivåerna 1 mL och 2 mL jämfördes med maximalt fyllda provrör. Kylskåpsförvarade helblodsprover analyserades för standardbikarbonat efter 4-7 timmar. Avkorkade serumprover analyserades för 10 biokemiska analyter efter att ha stått i rumstemperatur 2-8 timmar. Genomsnittlig procentuell avvikelse jämfördes med en analytisk och biologisk imprecisionsgräns för att bedöma analyters stabilitet. Resultat och slutsatser: Analysresultat av joniserat kalcium i lägre fyllnadsnivåer var tillförlitliga. Stabiliteten av standardbikarbonatsproverna kunde inte bedömas och därmed kunde inte en eventuell tidsgränsändring rekommenderas. De biokemiska analyterna var stabila upp till 8 timmar i rumstemperatur. / Background: Ionized calcium concentrations decrease when samples are exposed to air. Due to pre-analytical factors, the 4 hour time limit for analysis of standard bicarbonate, can sometimes be exceeded. There is limited documentation about additional analyses on post-analytic decapped serum samples stored at room temperature. Aim: The aim was to examine how lower sample volumes affect the concentration of ionized calcium, if the time limit for analysis of standard bicarbonate on whole blood can be prolonged and how long decapped serum samples can be stored at room temperature for eventual additional analyses. Methods: The concentration of ionized calcium was analyzed on serum samples filled with 1 mL and 2 mL and were compared to maximally filled samples. Refrigerated whole blood samples were analyzed for standard bicarbonate after 4-7 hours. Ten biochemical analytes were measured in decapped serum samples after 2-8 hours of storage at room temperature. The mean percentage deviation was compared to an analytical and biological imprecision limit to determine analyte stability. Results and conclusions: Ionized calcium concentrations in lower sample volumes were reliable. The stability of standard bicarbonate could not be determined, therefore a longer possible time limit could not be recommended. The biochemical analytes were stable for 8 hours.