The object of this study was to compare growth factor and cytokine profiles in canine autologous conditioned serum (ACS) to canine plasma. Blood collected from 16 medium to large breed dogs was used to produce ACS (Orthokine® vet irap 10 syringes) and citrated plasma (control). Canine-specific ELISA assays were run per manufacturers’ instructions for interleukin (IL)-10, IL-4, tumor necrosis factor (TNF)-α, insulin-like growth factor (IGF)-1, fibroblast growth factor (FGF)-2, transforming growth factor (TGF)-β1, IL-1β, and interleukin-1 receptor antagonist (IL-1ra). Serum, in addition to plasma and ACS, was collected from an additional 6 dogs for TNF-α, IL-1β, and IL-1ra analysis (total of 22 dogs). Data were analyzed for differences in cytokine concentrations between ACS, plasma, and serum using the Wilcoxon signed-rank test with significance set at P<.05.There was a large variability in growth factor and cytokine concentrations between individual dogs in both plasma and ACS. There were no significant differences in IL-10, TNF-α, IGF-1, FGF-2, and TGF-β1 concentrations between ACS, plasma, or serum. ACS concentrations of IL-1β (median, range; 46.3 pg/mL, 0-828.8) and IL-4 (0.0 pg/mL, 0-244.1) were significantly increased compared to plasma (36.6 pg/mL, 0-657.1 and 0.0 pg/mL, 0-0, respectively). IL-1ra concentrations in ACS (median, range; 3458.9 pg/mL, 1,243.1-12,089.0) were significantly higher than plasma (692.3 pg/mL, 422.5- 1,475.6), as was the IL-1ra:IL-1β ratio (39.9 and 7.2, respectively). / Master of Science

Intra-articular bupivacaine is a common analgesic used in dogs with orthopedic disease. Bupivacaine has been linked to chondrotoxicity. The mechanism for bupivacaine's chondrotoxicity is unknown, but may involve inflammation. Prostaglandin E2 (PGE2) is an inflammatory mediator and a marker of joint inflammation. The aim of this study was to compare synovial fluid PGE2 concentrations after a single intra-articular injection of bupivacaine with a saline control in normal canine stifles. We hypothesized that bupivacaine stifles would have a significantly elevated PGE2 concentration compared to controls. Stifles from eight healthy, adult Beagles were randomly selected as the treated stifle and infused with bupivacaine. The contralateral stifle was injected with saline. Synovial fluid was collected before and after injection. PGE2 was quantified using a commercial ELISA. Data were transformed and mixed model ANOVA was performed with significance set at p<0.05. There were no significant differences in PGE2 concentration between treatment groups or times. Samples acquired with one or two aspiration attempts had significantly lower PGE2 concentrations than samples with =3 aspiration attempts (p=0.001). When adjusted for number of attempts, PGE2 concentrations were significantly higher 24 (p=0.003) and 48 (p=0.041) hours after injection compared to baseline in the bupivacaine group, but not in the saline group. Intra-articular bupivacaine injection did not result in increased synovial fluid PGE2 concentrations compared to controls; however, multiple aspiration attempts did, suggesting that synovial fluid PGE2 concentration is sensitive to multiple fluid collection attempts. Future studies investigating synovial fluid inflammatory mediators should consider methods to minimize aspiration attempts. / M. S.

Objective – 1) To determine whether a graft of fascia lata and part of the patellar ligament, used in an extracapsular fashion from the tibial crest to the femorofabellar ligament, would eliminate abnormal cranial drawer motion in the cranial cruciate ligament (CrCL) deficient stifle 2) To determine if two new tibial suture anchor points would enhance biomechanical function of the lateral fabellar-tibial suture (FTS). Study Design – Experimental. Animals – 28 canine cadaver hind limbs. Methods – Stifles were mounted in a jig that allowed tibial rotation during loading and were tested between loads of â 65 to 80 N in caudal and cranial drawer respectively. Stifles were tested with the CrCL intact followed by one of four stabilization techniques after CrCL transection: lateral graft technique (LGT) and three FTS with different tibial anchor points. Results – Differences in cranial drawer motion (displacement) and stiffness between the LGT and standard FTS were not significant in two data sets, when compared to the intact CrCL. The FTS with the anchor point in the tibial crest showed the least displacement of all stabilization methods. Differences in stiffness were not significant between the stabilization techniques. Conclusions – Stability provided by the LGT is comparable to that of the standard FTS for the CrCL-deficient stifle in the cadaver. Altering the tibial anchor points for the FTS did not improve stiffness or result in a further decrease in cranial drawer motion. Clinical Relevance – The LGT could be used for the treatment of acute and chronic CrCL ruptures in the dog. A clinical study is recommended. / Master of Science

Objective: To evaluate pressor sensitivity to catecholamines in dogs after induction of iatrogenic hyperadrenocorticism (I-HAC) by serial arterial blood pressure measurements during infusions of increasing dose rates of norepinephrine. Animals: Eight dogs with I-HAC induced by administration of oral hydrocortisone at a mean dose of 3.3 mg/kg PO TID for 42-49 days and 8 control dogs which received empty gelatin capsules PO TID for 42-49 days. Procedure: Systolic, diastolic, mean blood pressure and heart rate measurements were recorded after sequential administration of increasing dose rates of norepinephrine (0.1, 0.125, 0.2, 0.3, 0.4, 0.6 and 0.8 mg/kg/min) for 10 minutes. The changes in systolic, diastolic, mean blood pressure and heart rate were compared between control dogs and dogs with I-HAC. Results: Dogs in the I-HAC group had a more pronounced pressor response to norepinephrine infusions than control dogs. The infusions were not completed in 7 of the 8 dogs in the I-HAC group versus 3 dogs in the control group due to severe elevations in systolic blood pressure. The mean change in systolic blood pressure was consistently higher in dogs in the I-HAC group. The difference was statistically significant at the 0.2 mg/kg/min norepinephrine dose rate. The mean change in heart rate was consistently lower in the I-HAC group, a difference that was significant at the 0.2 mg/kg/min norepinephrine dose rate. Conclusions and clinical relevance: Increased pressor sensitivity or decreased baroreceptor response to norepinephrine was seen in dogs with I-HAC suggesting that this mechanism is involved in the development of hypertension in canine hyperadrenocorticism. / Master of Science

Recent work in humans with protein losing nephropathies has revealed increased urine concentrations of sulfated glycosaminoglycans (GAGs). Differences exist between normal patients, those with glomerulonephritis (GN), and those with amyloidosis thus potentially allowing differentiation without a renal biopsy. Aims of this study were to validate a simple spectrophotometric assay used to measure canine urinary GAGs, establish a normal reference range, and determine optimal storage conditions. Urine GAG concentrations were measured in a limited number of dogs with glomerulonephritis or amyloidosis. Fourteen healthy dogs were placed in metabolic cages and all urine was collected for 24 hours. Serum and urine creatinine concentrations were measured at the beginning and end of the collection period. Urine collected at the beginning of the 24-hr period was centrifuged and the supernatant used to measure a spot GAG concentration and a spot glycosaminoglycan to creatinine ratio (GCR). A well mixed aliquot of the 24-hr sample was centrifuged, the supernatant used to measure the 24-hr total GAG, and stored at 4°C and -20°C for 1, 7, and 30 days. All dogs were used to determine effects of time and temperature (n=14), however, only dogs with an endogenous creatinine clearance > 2 ml/min/kg (n=10) were used to determine normal values. A standard absorption curve using a 1,9-dimethlymethylene blue dye and dilutions of chondroiton-4-sulfate was developed to estimate total GAG concentration. Repeated measures analysis of variance was used to test for effects of storage temperature and time on stability of urinary GAG. A p-value of < 0.05 was considered significant. Relationships between spot urinary GAG concentration, spot urinary GAG to creatinine ratio (GCR) and 24-hr total GAG excretion were estimated using simple linear regression. Single urine samples were collected by cystocentesis from dogs with GN or renal amyloidosis. The diagnosis was confirmed by clinical evaluation or by histologic analysis. Urine protein, creatinine and GAG concentrations were measured. There were no time or temperature effects on urine GAG concentrations for up to 1 day at 4°C and 30 days at -20°C. Mean 24-hr total GAG excretion ± standard deviation was 1.586 ± 0.461 mg/kg of body weight. Mean spot GAG concentration and spot GCR were 5.007 ± 1.588 mg/dl and 0.023 ± 0.01 respectively. Neither spot GAG concentration (R2=0.4216) nor GCR (R2= 0.0839) were adequate predictors of 24-hr total GAG. The GCR's from dogs with renal disease were not different from normal dogs. This study established normal total urinary GAG values in dogs. Contrary to findings in humans, there was no correlation between 24-hr total sulfated GAG and spot GCR in dogs, limiting clinical utility of this test. Further work is needed to determine if either total sulfated GAG or the spot GCR can be used to differentiate causes of protein-losing nephropathies in dogs. / Master of Science

This project is concerned with method development in mass spectrometry (MS)-based proteomics in order to find putative biomarkers for early pregnancy ofdomesticated dogs. It is of importance for dog breeders to know whether the dogsbecome pregnant post-mating. Unlike humans, dogs are not known to possess aspecific hormone that indicates fetal development; therefore other biomarkers mustbe investigated. The approach of choice in this project was to look at proteins throughMS-based proteomics. For this purpose, serum samples from 11 pregnant dogs (case,different breeds) and 7 non-pregnant dogs (control, all beagle dogs) were sampledbefore-hand at the Swedish University of Agricultural Sciences. Each dog wassampled Day 1, Day 8, Day 15, Day 22 and Day 29 after optimal mating. Twodifferent proteomics approaches were conducted: Bottom-up (“Shotgun”) proteomicsand targeted proteomics (“targeted analysis”). In this study, Label-free Quantification(LFQ) was employed, which is a relative quantitative technique. The massspectrometer of choice was the Quadrupole-Orbitrap QExactive plus massspectrometer coupled to a nano-Ultra Performance Liquid Chromatography (UPLC).Method optimization was done with respect to concentration of samples prior to MSanalysis, as well as different LC-gradients. From shotgun screening experiments, itwas possible to identify 252 proteins. Ultimately, 9 proteins were investigated usingtargeted final analysis: CRP, SERPINC1, CP, PROS1, SERPING1, A2M, AGP,SERPINA1 and HP. For targeted final analysis, 21 peptides were considered.Calibration curves were constructed using 8 of the 21 targeted peptides; 1 peptide perprotein, except for HP which had 2 peptides per protein. The SERPINA1 and CPproteins had no appropriate peptides for targeted final analysis and were thusexcluded. It was confirmed that CRP was up-regulated in case dogs compared tocontrol dogs. The other investigatedproteins showed no significant signs of regulation. In order to improve the results; itwould be desirable to include more dogs in the study which would benefit thestatistics of protein regulation. However, the use of isotopic labeled standards andemployment of a Parallel Reaction Monitoring (PRM) method should be prioritizedfor obtaining absolute quantitative data.

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The purpose of this investigation is to determine the pressure-flow relationships in the isolated canine cerebral vascular bed. These relationships in vessels which supply an organ surrounded by bone are theoretically influenced by limiting factors which are not present in those vessels surrounded by soft tissue. An evaluation of the extent of these limitations is essential to thorough understanding of the dynamics of the cerebral vascular bed.

Orientadora: Márcia Ferreira da Rosa Sobreira / Coorientador: Hélio José Montassier / Banca: Ketherson Rodrigues Silva / Banca: Ruben Pablo Schocken Iturrino / Banca: Marita Vedovelli Cardozo / Banca: Diane Meyre Rassi / Resumo: A cinomose canina é uma das enfermidades infectocontagiosas mais importantes que acomete os cães. É uma doença causada pelo vírus da Cinomose ("Canine Distemper Virus" - CDV), um Paramyxovirus, do gênero Morbillivirus, de ocorrência mundial, sem sazonalidade, sem predileção de sexo ou raça, apresenta maior incidência em animais jovens, podendo acometer todas as idades. No Brasil, pesquisas sobre o uso de técnicas que comparam os diferentes testes diagnósticos direto para o vírus da cinomose (CDV) associado a análise molecular e filogenética do mesmo ainda continuam escassas. Além disso, são poucos os estudos sobre epidemiologia molecular que relatam diferenças marcantes na análise filogenética do gene hemaglutinina (H) do CDV entre os isolados de campo e as cepas de referência do CDV, especialmente àquelas utilizadas na produção de vacinas. Dessa maneira, o presente trabalho tem como principal objetivo avaliar os principais métodos diagnósticos laboratoriais para identificação do CDV em cães com suspeita de cinomose associando à análise filogenética do gene H dos isolados de campo. O Ensaio imunocromatografico direto (IC), a RT-PCR e a Nested-PCR foram avaliados para detecção do CDV em diferentes amostras clínicas (urina, suabes retais e conjuntivais) de 62 animais suspeitos de cinomose, provenientes do atendimento ambulatorial do hospital veterinário da Unesp (Jaboticabal-SP) e de clínicas particulares da região. Após detecção viral as amostras foram submetidas a sequenciame... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Canine distemper is one of the most important infectious diseases that affects dogs. It is a Canine Distemper Virus (CDV), a Paramyxovirus, of the genus Morbillivirus, a global deforest, without seasonality, without predilection of sex or race, that is constituted by the majority of the animals, being able to affect all ages. In Brazil, research on the use of techniques that compare the different direct diagnostic tests for the distemper virus (CDV) associated with a molecular and phylogenetic analysis of the same are still scarce. In addition, there are some studies on molecular epidemiology that report differences in the phylogenetic analysis of the hemagglutinin (H) gene from CDV between the fields of action and as a reference for CDV, especially the practices used in the production of vaccines. Thus, the main objective of this study is to identify laboratory diagnoses for the identification of CDV in dogs with suspected pair association to the phylogenetic analysis of the H gene of the field isolates. The direct immunochromatographic (CI) assay, RT-PCR and nested-PCR were evaluated for the detection of CDV in different animal species (urine, swabs and conjunctival) of 62 suspected cases of distemper from the outpatient clinic of the Unesp (Jaboticabal-SP) and the private medical records of the region. After viral detection as sequential sequencing followed by phylogenetic analysis of the CDV H gene. A total of 42 animals were positive at least one of the experiment techni... (Complete abstract click electronic access below) / Doutor