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Bladder cancer (BlCa) is the second most common genitourinary cancer, affecting both men and women. Most (70%) cases present at the superficial stage; 20% of these recur with muscle-invasive disease. Major genetic alterations associated with BlCa include: loss/gain in expression or mutations in Retinoblastoma (RB) gene, human epidermal growth factor receptors (HERs), H-ras, p53 and FGFR3. Only p53 mutations are well correlated with invasive BlCa; other changes show variable correlations with disease status. To understand the progression of BlCa, a model of nine human BlCa cell sublines derived from a single parent but differing in in vivo characteristics, has been developed previously. These cells represent a heterogenous population from a single tumour and a model of different stages of BlCa progression, from non-tumourigenic to invasive. Two sublines were selected for further investigation: C3 (non-tumourigenic) and B8 (invasive). These were transfected with green (C3-GSP-2) and red fluorescent reporters (B8-RSP-gck) respectively to investigate the effects of their co-injection in vivo, specifically, promotion of C3 tumour growth by B8 cells. Surprisingly, B8 tumour growth was inhibited by C3 cells in vivo at different cell numbers and proportions of cells injected. Microarray analysis of C3 and B8 cells revealed differential expression of 1367 genes with dramatic differences in the transforming growth factor-?? and integrin-mediated pathways. Gene expression of BMP2, INHBB, FST, NOG, ID4 and TGF- ??1, in the TGF- ?? pathway was further analysed with qRT-PCR in all nine sublines. Expression of BMP2 was significantly related to tumourigenic potential (p=0.0238, Mann-Whitney) and INHBB to invasive ability (p=0.0476, Mann-Whitney). The BlCa model did not include a metastatic component. To broaden the model, cell lines were established from an invaded lymph-node (B8-RSP-LN) and a bone-metastasis (B8-RSP-BN) after subcutaneous and intra-cardiac injection of B8-RSP-gck cells. No significant differences were observed in the migratory capability and anchorage-independent colony formation of these metastatic cells compared with B8 cells. Evaluation of expression of the panel of TGF-beta genes (BMP2, INHBB, FST, NOG, ID4 and TGF- ??1) and metastasis-related genes (MMP9, MMP2 and KAI1) indicated that expression of BMP2, FST, ID4 and MMP9 was decreased or lost in the metastatic sublines.

There is much debate on the carcinogenic potential of disinfection by-products (DBP) in chlorinated water supplies. Until recently, epidemiological studies have been limited in their ability to examine accurately the risk of cancer with exposure to environmental carcinogens. This has largely been due to the long latency periods associated with cancer development, and the difficulties in accurately estimating chronic exposure. Although there is evidence, from predominantly case-control studies, of increased bladder cancer with exposure to chlorinated water supplies, the evidence is inconclusive. ¶ In an attempt to determine the carcinogenic potential of trihalomethanes (THMs) in chlorinated water, this study utilises DNA damage to bladder cells, evident as micronuclei, as a pre-clinical outcome measure. Using a pre-clinical marker helps overcome some of the limitations associated with long latency periods. The study improves on previous studies by estimating exposure to DBP at an individual level, and takes into consideration ingestion, inhalation and dermal exposure. ¶ A cohort study was undertaken in three Australian communities. The Bungendore (NSW) water supply was not chlorinated thereby providing a community unexposed to DBPs from chlorinated water. Canberra (ACT) and Adelaide (SA) had intermediate and relatively higher (but still within NHMRC guideline levels) of DBPs in the reticulation system. Trihalomethane levels in reticulated water (external dose) and in urine (internal dose) were used as exposure indices. As well, intake dose was computed by adjusting external dose for individual variations in ingestion and bathing. The primary outcome measure was the prevalence of micronuclei in bladder epithelial cells. A DNA index derived from flow cytometry was also used to estimate DNA damage in bladder cells. Associations between exposure and outcome were estimated using Poisson regression models, having identified and adjusted for interaction effects and confounders. ¶ A total of 529 participants were eligible to participate, of which 348 (65.8%) completed all aspects of the study. Analysis was limited to the 228 participants (65.53% of those who completed the study) who had slides suitable for micronuclei scoring. One hundred and forty three (63%) of the 228 participants were from the exposed communities, while 85 (37%) were from the unexposed community. This sample exceeded the estimated 50 per group required to detect a relative risk of 1.4, with a significance level of 0.05 and 80% power. ¶ External dose for total THM for the two chlorinated (exposed) communities ranged from 37.75 to 157.25 mg/l. Intake dose estimated by fluid intake diary ranged from 2.9 to 469.5 mg/l, while a retrospective questionnaire estimated intake dose to range from 0 to 409.4 mg/l. Internal dose (urine levels) of total THM for the same two communities ranged from 0 to 6.82 mg/l. Adjusted risk estimate for DNA damage to bladder cells (using the micronuclei assay) when total THM was assessed by available dose was 1.0002 (0.997 to 1.003), by intake dose estimated by fluid intake diary was 1.0001 (0.998 to 1.002), by intake dose estimated by questionnaire was 1.001 (0.999 to 1.003), and by internal dose was 1.05 (0.89 to 1.24). Using DNA index from flow cytometry as the outcome measure also did not identify significant associations, except when exposure was assessed as available dose of total THM (RR=1.0042; 1.0003 to 1.0081). ¶ The results suggest that THM levels are not significantly associated with DNA damage to bladder cell. This supports suggestions of THMs being non-genotoxic. Further work is required to assess the relationship between THM and the more mutagenic compounds, and to assess the carcinogenicity of the more mutagenic compounds at concentrations occurring in drinking water.

Arsenic is a human bladder carcinogen. Inorganic arsenic and methylated metabolites are excreted from the human body in urine. This study investigates the effects of arsenite [As(III)] and monomethylarsonous acid [MMA(III)] on human urothelial cells (UROtsa). Cytotoxicity studies found that MMA(III) was 20 times more toxic than As(III). In addition, UROtsa cells have the ability to biotransform As(III) to pentavalent and trivalent mono-methylated metabolites.To understand the mechanism of arsenic carcinogenesis, it is necessary to know which arsenicals are carcinogenic. Therefore, non-tumorigenic UROtsa cells were chronically exposed to 0.05 uM MMA(III) and monitored for signs of transformation. MMA(III)-treated cells (URO-MSC) became hyperproliferative after 12 weeks of exposure. Anchorage-independent growth was detected after 24 weeks of exposure to MMA(III). Gene array analysis conducted in URO-MSC cells after 52 weeks of exposure detected expression changes consistent with malignant transformation. Enhanced tumorigenicity in SCID mouse xenografts was also observed after 52 weeks of treatment.URO-MSC cells form squamous cell carcinoma, a histology associated with inflammation, when injected into SCID mice. Induction of cycolooxygenase-2 (COX-2) promotes proliferation, angiogenesis, and survival in cancer cells. To identify a potential mechanism of MMA(III) carcinogenesis, the effects of chronic and acute MMA(III) treatment on COX-2 expression were investigated. Western blot analysis revealed that COX-2 was induced in a time-dependent manner in URO-MSC cells. Acute MMA(III) exposure also increased COX-2 protein. To identify signal transduction pathways responsible for COX-2 induction, pharmacological inhibitors of various signaling pathways were co-administered with 0.05 uM MMA(III) and identified src and extracellular signal regulated protein kinase (ERK) activation to be responsible for COX-2 induction. Thus, MMA(III) causes ligand-independent activation of epidermal growth factor receptor (EGFR), which activates the signal cascade responsible for COX-2 expression. EGFR is elevated in URO-MSC cells. To determine if EGFR is a key mediator of URO-MSC cell tumorigenicity, inhibitors of downstream signal transduction (src, PI3K, and COX-1/-2) were found to reduce URO-MSC cell viability and growth in soft agar. Results from this work not only identify that MMA(III) can induce malignant transformation in human cells but also provides insight into the mechanism of arsenic-induced bladder cancer.

Bladder cancer (BlCa) is the second most common genitourinary cancer, affecting both men and women. Most (70%) cases present at the superficial stage; 20% of these recur with muscle-invasive disease. Major genetic alterations associated with BlCa include: loss/gain in expression or mutations in Retinoblastoma (RB) gene, human epidermal growth factor receptors (HERs), H-ras, p53 and FGFR3. Only p53 mutations are well correlated with invasive BlCa; other changes show variable correlations with disease status. To understand the progression of BlCa, a model of nine human BlCa cell sublines derived from a single parent but differing in in vivo characteristics, has been developed previously. These cells represent a heterogenous population from a single tumour and a model of different stages of BlCa progression, from non-tumourigenic to invasive. Two sublines were selected for further investigation: C3 (non-tumourigenic) and B8 (invasive). These were transfected with green (C3-GSP-2) and red fluorescent reporters (B8-RSP-gck) respectively to investigate the effects of their co-injection in vivo, specifically, promotion of C3 tumour growth by B8 cells. Surprisingly, B8 tumour growth was inhibited by C3 cells in vivo at different cell numbers and proportions of cells injected. Microarray analysis of C3 and B8 cells revealed differential expression of 1367 genes with dramatic differences in the transforming growth factor-?? and integrin-mediated pathways. Gene expression of BMP2, INHBB, FST, NOG, ID4 and TGF- ??1, in the TGF- ?? pathway was further analysed with qRT-PCR in all nine sublines. Expression of BMP2 was significantly related to tumourigenic potential (p=0.0238, Mann-Whitney) and INHBB to invasive ability (p=0.0476, Mann-Whitney). The BlCa model did not include a metastatic component. To broaden the model, cell lines were established from an invaded lymph-node (B8-RSP-LN) and a bone-metastasis (B8-RSP-BN) after subcutaneous and intra-cardiac injection of B8-RSP-gck cells. No significant differences were observed in the migratory capability and anchorage-independent colony formation of these metastatic cells compared with B8 cells. Evaluation of expression of the panel of TGF-beta genes (BMP2, INHBB, FST, NOG, ID4 and TGF- ??1) and metastasis-related genes (MMP9, MMP2 and KAI1) indicated that expression of BMP2, FST, ID4 and MMP9 was decreased or lost in the metastatic sublines.

Made available in DSpace on 2015-12-10T14:22:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-02-27. Added 1 bitstream(s) on 2015-12-10T14:29:05Z : No. of bitstreams: 1 000854691.pdf: 2953891 bytes, checksum: b9ca6d4c16761fe914f8188162cddefb (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O câncer de bexiga é a malignidade mais comum do trato urinário. O tratamento mais estipulado atualmente para os casos de câncer de bexiga não-músculo invasivo (CBNMI) é uma ressecção transuretral associada à administração de dose intravesical de manutenção de Bacillus de Calmette-Guerín (BCG). Entretanto, a imunoterapia com BCG também causa efeitos indesejáveis, além de aumentar a taxa de recidiva da doença secundária à interrupção do tratamento. Nesse contexto, o óxido de grafeno (GO) e/ou o óxido de grafeno reduzido (rGO) têm atraído significativo interesse no campo da detecção biológica, drug delivery e nas terapias anti-tumorais. Assim, o objetivo deste estudo foi caracterizar e comparar os efeitos antitumorais da Doxorrubicina (DOXO) e da Cisplatina (CIS) funcionalizadas na dispersão rGO com polímero Pluronic® F-68 frente ao tratamento do CBNMI. Inicialmente, foi realizada a caracterização do composto de rGO para conhecer suas propriedades químicas. Foram utilizadas 30 ratas da variedade Fischer 344, sendo constituído um grupo controle de cinco animais (Grupo 1), os quais receberam 0,3 mL de solução fisiológica à 0,9% por via intraperitoneal (i.p.). Nos demais animais foram administradas doses de 1,5 mg/kg de N-metil-N-nitrosouréia (MNU) intravesical (i.v.) e, após quatro doses, foram divididos em cinco grupos: Grupo Câncer (Grupo 2), Grupo Câncer+rGO (Grupo 3), Grupo Câncer+rGO+CIS (Grupo 4), Grupo Câncer+rGO+DOXO (Grupo 5) e Grupo Câncer+rGO+CIS+DOXO (Grupo 6), os quais receberam respectivamente: mesmo tratamento do Grupo 1; dose 0,2mL na concentração de 0,2mg/mL de rGO; 0,2mL da dispersão rGO com CIS a 0,05mg; 0,2mg de DOXO em 0,2mL da dispersão e 0,2mL da dispersão com as mesmas concentrações de quimioterápicos que os Grupos 4 e 5. Todos os tratamentos foram por via i.p., com administração semanal durante seis semanas consecutivas. Após 16 semanas de... / Bladder cancer is the most common malignancy of the urinary tract. The recommended first-line treatment for non-muscle invasive bladder cancer (NMIBC) following transurethral resection, is an induction course plus maintenance with intravesical Bacillus Calmette-Guerin (BCG). However, BCG immunotherapy causes undesirable effects, which contributes to treatment interruption, increasing cancer index recurrence. In this context, graphene oxide (GO) and/or reduced graphene oxide (rGO) has attracted increasing interest in the field of biological detection, drug delivery and cancer therapies. Thus, the aim of this study was to characterize and to compare the antitumor effects of Doxorubicin (DOXO) and Cisplatin (CIS) functionalized in rGO on dispersion with Pluronic F-68 polymer to the treatment of NMIBC. Initially, the characterization of the compound guarantee its chemical properties. It was used 30 female Fischer 344 rats, consisting a control group of five animals (Group 1), which received 0.3 ml of saline 0.9% intraperitoneally (i.p.). The remaining animals received 1.5 mg/kg of N-methyl-N-Nitrosourea (MNU) intravesical (i.v.) and after four doses, they were divided into five groups: Cancer Group (Group 2), Cancer + rGO Group (Group 3), Cancer + rGO + CIS Group (Group 4), Cancer + rGO + DOXO Group (Group 5), Cancer rGO + CIS + DOXO Group (Group 6), which received, respectively: the same treatment as group 1; received 0.2 mL dose at a concentration of 0.2mg/ml; received 0.2 mL of rGO dispersion with 0.05mg CIS; received 0.2 mg of DOXO in 0.2 mL of the dispersion; received 0.2 mL of rGO dispersion with the same chemotherapy combinations that groups 4 and 5. All treatments were i.p., with weekly administration during six consecutive weeks. After 16 weeks of treatment, urinary bladders were collected for histopathological, immunohistochemical and biochemical analysis, and still, blood collected for performing biochemical analyzes to obtain ...

Orientador: Wagner José Fávaro / Banca: Sérgio Pereira / Banca: Luiz Gustavo Chuffa / Resumo: O câncer de bexiga é a malignidade mais comum do trato urinário. O tratamento mais estipulado atualmente para os casos de câncer de bexiga não-músculo invasivo (CBNMI) é uma ressecção transuretral associada à administração de dose intravesical de manutenção de Bacillus de Calmette-Guerín (BCG). Entretanto, a imunoterapia com BCG também causa efeitos indesejáveis, além de aumentar a taxa de recidiva da doença secundária à interrupção do tratamento. Nesse contexto, o óxido de grafeno (GO) e/ou o óxido de grafeno reduzido (rGO) têm atraído significativo interesse no campo da detecção biológica, drug delivery e nas terapias anti-tumorais. Assim, o objetivo deste estudo foi caracterizar e comparar os efeitos antitumorais da Doxorrubicina (DOXO) e da Cisplatina (CIS) funcionalizadas na dispersão rGO com polímero Pluronic® F-68 frente ao tratamento do CBNMI. Inicialmente, foi realizada a caracterização do composto de rGO para conhecer suas propriedades químicas. Foram utilizadas 30 ratas da variedade Fischer 344, sendo constituído um grupo controle de cinco animais (Grupo 1), os quais receberam 0,3 mL de solução fisiológica à 0,9% por via intraperitoneal (i.p.). Nos demais animais foram administradas doses de 1,5 mg/kg de N-metil-N-nitrosouréia (MNU) intravesical (i.v.) e, após quatro doses, foram divididos em cinco grupos: Grupo Câncer (Grupo 2), Grupo Câncer+rGO (Grupo 3), Grupo Câncer+rGO+CIS (Grupo 4), Grupo Câncer+rGO+DOXO (Grupo 5) e Grupo Câncer+rGO+CIS+DOXO (Grupo 6), os quais receberam respectivamente: mesmo tratamento do Grupo 1; dose 0,2mL na concentração de 0,2mg/mL de rGO; 0,2mL da dispersão rGO com CIS a 0,05mg; 0,2mg de DOXO em 0,2mL da dispersão e 0,2mL da dispersão com as mesmas concentrações de quimioterápicos que os Grupos 4 e 5. Todos os tratamentos foram por via i.p., com administração semanal durante seis semanas consecutivas. Após 16 semanas de... / Abstract: Bladder cancer is the most common malignancy of the urinary tract. The recommended first-line treatment for non-muscle invasive bladder cancer (NMIBC) following transurethral resection, is an induction course plus maintenance with intravesical Bacillus Calmette-Guerin (BCG). However, BCG immunotherapy causes undesirable effects, which contributes to treatment interruption, increasing cancer index recurrence. In this context, graphene oxide (GO) and/or reduced graphene oxide (rGO) has attracted increasing interest in the field of biological detection, drug delivery and cancer therapies. Thus, the aim of this study was to characterize and to compare the antitumor effects of Doxorubicin (DOXO) and Cisplatin (CIS) functionalized in rGO on dispersion with Pluronic F-68 polymer to the treatment of NMIBC. Initially, the characterization of the compound guarantee its chemical properties. It was used 30 female Fischer 344 rats, consisting a control group of five animals (Group 1), which received 0.3 ml of saline 0.9% intraperitoneally (i.p.). The remaining animals received 1.5 mg/kg of N-methyl-N-Nitrosourea (MNU) intravesical (i.v.) and after four doses, they were divided into five groups: Cancer Group (Group 2), Cancer + rGO Group (Group 3), Cancer + rGO + CIS Group (Group 4), Cancer + rGO + DOXO Group (Group 5), Cancer rGO + CIS + DOXO Group (Group 6), which received, respectively: the same treatment as group 1; received 0.2 mL dose at a concentration of 0.2mg/ml; received 0.2 mL of rGO dispersion with 0.05mg CIS; received 0.2 mg of DOXO in 0.2 mL of the dispersion; received 0.2 mL of rGO dispersion with the same chemotherapy combinations that groups 4 and 5. All treatments were i.p., with weekly administration during six consecutive weeks. After 16 weeks of treatment, urinary bladders were collected for histopathological, immunohistochemical and biochemical analysis, and still, blood collected for performing biochemical analyzes to obtain ... / Mestre

INTRODUCTION: Bladder cancer (BC) is highly prevalent. It presents as either non-muscle invasive or muscle-invasive disease. The prognosis of muscle invasive disease is poor, with a 5-year survival rate of less than 50%. Treatment approaches for both types of BC have not advanced much in the last few years and new therapies are needed to overcome the large burden of BC. Recently, a large effort has been undertaken to classify BC into molecular subtypes. These analyses have revealed significant alterations in epigenetic modifiers in BC. A previous study from our group revealed that SH3GL2, a negative regulator of receptor tyrosine kinase (RTK) signaling, was lost with high frequency in BC, leading to increased growth of tumor cells in-vitro and in-vivo. Conversely, forced expression of SH3GL2 in BC cell lines attenuated oncogenic behaviors including growth and migration. In addition to genomic deletion, SH3GL2 is subject to methylation-induced silencing, a key epigenetic mechanism. OBJECTIVE: Epigenetic mechanisms of gene regulation are known to be perturbed in BC. The objectives of this study were to investigate methylation of the SH3GL2 promoter and to test whether agents that promote Deoxyribonucleic acid (DNA) demethylation could be used to re-express SH3GL2 thereby restoring regulation of RTK signaling. METHODS: Methylation of a specific CpG island in the SH3GL2 promoter was analyzed using methylation-specific Polymerase Chain Reaction (PCR) in a panel of BC cell lines with known SH3GL2 messenger Ribonucleic Acid (mRNA) status. Selected BC cell lines were treated with a variety of demethylating agents at different doses and for different times to evoke the re-expression of silenced SH3GL2. Demethylation inhibitors were combined with the histone deacetylase inhibitor, trichostatin A (TSA), to determine whether further re-expression could be achieved. RESULTS: The SH3GL2 promoter displayed differing extents of promoter methylation among cell lines examined. In RT4 cells, the only cell line with detectable expression of SH3GL2 mRNA and protein, the promoter was completely unmethylated. In contrast, T24 and 253J cells displayed significant promoter methylation with little to no SH3GL2 mRNA expressed, consistent with methylation-induced silencing. Treatment of T24 and 253J with 5-Aza-2’-deoxycytidine (5-Aza-dC, 20 M), a DNA methyltransferase (DNMT) inhibitor increased gene expression but this was not dose- or time-dependent. Two additional DNMT inhibitors, Zebularine and RG-108 were also tested. A much higher dosage of Zebularine was required to trigger activation (500 M) while RG-108 was unable to trigger gene reactivation at all. Combination treatment with 5-Aza-dC and TSA further increased SH3GL2 expression compared to either agent alone. These results suggest that DNA methyltransferase inhibition is an effective treatment to re-express SH3GL2 in cells with SH3GL2 promoter silencing. CONCLUSION: The present study shows silencing of SH3GL2 in a variety of BC cell lines as a consequence of DNA promoter hypermethylation. Treatment with demethylating agents was able to increase gene expression. Based on prior findings showing attenuation of tumor cell growth and migration with forced expression of SH3GL2, DNA methyltransferase inhibition represents an effective strategy to re-express SH3GL2 in BC and normalize tumor cell behavior. / 2020-07-03T00:00:00Z

Bladder cancer is currently the 6th most common cancer in the United States, resulting in 17,000 deaths annually. Clinically, bladder cancers are mostly urothelial carcinoma, classified as either non-muscle invasive bladder cancer (NMIBC) or muscle-invasive bladder cancer (MIBC), with the latter having a 5-year survival rate of merely 50%. With recent advances in next-generation sequencing, several international consortia have elucidated molecular subtypes of MIBC. The two major subtypes of MIBCs are basal and luminal; the basal subtype frequently exhibits hallmarks of squamous differentiation and highly expressed basal markers (CD44, KRT14, KRT6A, KRT6B). Tumors of the luminal subtype have papillary morphology and highly express differentiation-associated luminal markers (e.g., KRT20, PPARG, UPKs, and FOXA1). Notably, the transcription factor Peroxisome Proliferator Activated Receptor Gamma (PPARG) gene is frequently amplified in luminal MIBC. And recurrent activating mutations have been reported for its obligatory functional partner Retinoic X Receptor (RXR). In addition, the basal subtype is immune-infiltrated and is postulated as more likely to respond to immunotherapies. In contrast, the luminal subtype is immune-cold. Despite these advances in recent years, the molecular driver of subtype determination, specifically in luminal MIBC, remains poorly understood. Furthermore, subtype-specific targeted therapy for MIBCs is still in its infancy. Our previous work determined that Pparg activation can drive luminal tumor formation. We generated a novel Krt5CreERT2; VP16;Pparg transgenic mouse model, where Pparg expression is constitutively active in basal urothelial cells upon tamoxifen induction. During homeostasis, constitutive Pparg promoted luminal differentiation and cell cycle exit in basal cells but did not produce tumors. However, increased Pparg signaling in activated basal cells following 1-month exposure to bladder-specific carcinogen BBN produced luminal tumors. These tumors are similar both in morphology and molecular markers to human luminal MIBCs. The resulting VP16;Pparg luminal tumors have reduced Nf-kb expression and are immune cold compared to basal tumors. These findings suggest that Pparg is a driver of luminal tumor formation and a suppressor of immune infiltration in bladder cancer. In Chapter 2 of the thesis, I focus on the therapeutic potential of activating Pparg in basal MIBC. We treated mice bearing BBN-induced, Pparg-negative basal tumors with synthetic Pparg ligand - Rosiglitazone (Rosi) and Mek1/2 inhibitor Trametinib (Tram), both of which have been shown to induce Pparg signaling in vitro and in vivo. The combined RosiTram treatment induced apoptosis and significantly reduced tumor burden. The post-treatment urothelium appeared similar in morphology to a healthy urothelium. RosiTram treatment also restored normal urothelial differentiation and generated resident cell types (e.g., superficial cells, intermediate cells, Keratin5+ (K5), basal cells, and Keratin14+ (K14) basal cells) that are normally seen in a healthy urothelium. In contrast, basal tumors are almost entirely composed of K14-Basal cells. Mechanistically, RosiTram treatment partially restores differentiation through retinoic acid signaling and Ezh2 inhibition. Together, our study established targeted transcriptionally and epigenetically reprogramming as a promising differentiation therapeutic approach for basal bladder tumors.

Early detection and screening for urinary tract illnesses is a complex and widespread process which has implications for both preventative care, diagnosis, and treatment monitoring. In this paper, we investigate the use of Raman spectroscopy (RS) for the analysis of urine, a complex biological solution, for the detection of bladder cancer (BCa) and hematuria. Raman spectroscopy is a rapid, low cost, non-destructive analysis method with wide-ranging applicability due to the holistic data capturing nature of the scanning technique. Each Raman scan can be considered a 'snapshot' of the molecular makeup of the sample, and, through proper applications of algorithmic transformation and statistical analysis, many types of assessments can be performed on each sample. In this paper we address creating and utilizing a data pipeline for the purposes of analyzing and characterizing potential samples with hematuria and BCa. The algorithmic transformations utilized include baselining using either the Goldindec or ISREA methods, and intensity normalization. The statistical analysis methods utilized include principal component analysis (PCA), discriminant analysis of principal components (DAPC), analysis of variance (ANOVA), pairwise ANOVA, leave-one-out cross-validation (LOOCV), and partial least squares regression (PLSR). These components of the data pipeline serve to output qualitative or quantitative data, depending on the application. The Rametrix toolbox encompasses the tools required to transform and assess Raman spectra with PCA and DAPC. Using the Rametrix toolbox as well as ANOVA, pairwise ANOVA, and LOOCV, we were able to significantly detect the presence of bladder cancer in a specimen with 80% accuracy. Using the Rametrix toolbox, ANOVA, pairwise ANOVA, LOOCV, and PLSR, we were able to classify samples as pure urine, micro-, or macrohematuria with a greater than 91% accuracy, and quantify the amount of blood in the sample with a high correlation (R-squared value of 0.92). In combination, this style of data pipeline is shown to rapidly and accurately test for multiple symptoms or diseases using similar methodologies. / Master of Science / In the United States, over 37 million people live with chronic kidney disease, over 81 thousand new cases of bladder cancer will be diagnosed, and over 17 thousand people will die from bladder cancer. These serious renal and urinary tract illnesses require urinalysis as a major component of detection, diagnosis, and monitoring of the diseases. This level of required testing has significant costs, both in labor and financial impact. Reduction in both the labor and consumable reagent costs associated with urinalysis would serve to improve the ability for the healthcare system provide the necessary testing for these patients, and reduce the risk of shortages in both reagents and staff. We present a new analysis method, termed 'data pipeline', which would take data from a spectrographic data collection method, Raman Spectroscopy (RS), and generate useable output in the form of classification and quantification. These outputs are highly desired for urinalysis, as urine collection is largely the least invasive testing method related to the urinary tract. As we have shown, the RametrixTM toolbox, an algorithmic package of mathematical methods for assessing spectra, is the backbone of a data pipeline capable of detecting both hematuria, an early warning symptom of many urinary tract illness, and bladder cancer, a notably difficult to detect disease, with high accuracy. This method of analysis is non-destructive of the samples, requires no reagents or single use dipsticks, avoids subjective color assessment, and provides rapid results in a repeatable, potentially automatable manner. We investigate a critical component of this process, the baselining method, in order to further examine and refine the methodology by comparing the accuracy and statistical quality of the results with different baseline methods. It is our goal to implement this methodology with the best component processes, in order to achieve a highly robust, accurate tool for assisting in urinalysis testing.