Thesis (M.A.) PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / High dose chemotherapy followed by autologous stem cell transplantation (ASCT) remains the standard of care for patients with relapsed or refractory (R/R) diffuse large B cell lymphoma (DLBCL) who are chemosensitive to salvage therapy. There is now evidence that the achievement of complete remission by PET scan (PET-CR) after salvage therapy is a favorable determinant of ASCT outcome, implying that PET response should be part of the prognostic assessment for patients considering ASCT. However, it is unclear whether other prognostic factors are still relevant in patients getting post-salvage PET scanning. Moreover, while ASCT is often also used for patients with R/R transformed indolent lymphoma (TIL), there are no data on whether prognostic factors that are important for DLBCL patients, especially PET response to salvage, are similarly prognostic in this population. We conducted a retrospective study of 143 patients with R/R DLBCL and TIL who were transplanted in the last decade and had a post-salvage PET scan prior to ASCT. We examined prognostic factors in both groups, and constructed a prognostic score for DLBCL patients. For patients with DLBCL, post-salvage PET response was an important prognostic factor. Advanced age and symptomatic relapse were also significantly associated with inferior outcome. A simple score could stratify patients into 3 risk groups with 4-year post-ASCT overall survival of 84%, 59%, and 10%, and 4-year progression-free survival of 67%, 41% and 0% (p<0.0001 for both). However, none of those factors (including PET response to salvage) could be demonstrated for TIL, likely because of the limited sample size. Our novel prognostic score for DLBCL patients undergoing ASCT may be useful for prognostication, for stratification in clinical trials, and to motivate the design of new strategies for patients in the highrisk group, who may not derive benefit from standard ASCT. Those factors, however, do not apply to patients with TIL, which has important implications for their treatment and inclusion in ASCT clinical trials with larger sample sizes. / 2031-01-01

Thesis (MSc. (Biochemistry)) -- University of Limpopo, 2017 / The malignancy of cancer cells is responsible for the high death rate in patients diagnosed with metastatic cancers. Medicinal plants represent a reservoir of bioactive compounds that can be useful in the management of cancer. In this study, semi-purified extracts of Ricinus communis leaves were evaluated for their potential to serve as an anti-metastatic agent by using in vitro assays that tested their effects on a number of processes related to metastasis. The exhaustive extraction procedure was employed to generate the crude acetone extracts of R. communis leaves. The crude extracts were then subjected to solvent-solvent fractionation to yield six semi-purified extracts (n-butanol, Chloroform, Ethyl-acetate, n-hexane, Methanol + H2O and H2O). Thin layer chromatography (TLC) was done to determine the phytochemical composition of the semi-purified extracts as well as their antioxidant potential. Non-polar fractions showed to have a diverse mixture of phytochemicals with, however, very limited antioxidant activity. On the other hand, polar fractions showed to have phytochemical compounds with strong antioxidant potential. TLC guided the selection of n-hexane and n-butanol as fractions of great phytochemical diversity and antioxidant activity, respectively. The selected fractions were then assessed for their effect on the viability of normal fibroblasts (BUD-8) and breast (MCF-7) cancer cells using the MTT assay. The n-butanol fraction was shown to significantly decrease the viability of BUD-8 at concentrations above 200 µg/ml. The n-hexane fraction, however, showed to significantly affect the viability of the cells even at lower concentrations. On the positive side, the reduced viability of BUD-8 cells after exposure to both fractions was followed by an increase in cell proliferation after 24 hours suggesting that the extracts exhibited cytostatic rather than cytotoxic effects. Treatment of MCF-7 cells with different concentrations (100-500 µg/ml) of the fractions showed a dose- and time-dependant decrease in cell viability. Hoechst stain also confirmed the non-toxicity of the fractions to MCF-7 cells at 100 and 200 µg/ml. The fractions also showed to possess free radical scavenging activities by reducing the amount of intracellular ROS as demonstrated by the DCFH-DA fluorescent assay. Fluorescence intensity was strongly reduced in cells treated with the fractions and elevated in H2O2-treated and untreated MCF-7 cells. The effect of the fractions on metastasis was assessed by determining their effects on MCF-7 cell migration, attachment and invasiveness using wound healing assay, adhesion assay and Boyden chamber invasion assay, respectively. The wound healing assay showed the fractions to have strong inhibitory activities on the migration of MCF-7 cells. The xii ability of the cells to attach to cell culture treated plates was also greatly reduced in cells treated with the fractions. The n-butanol fraction was demonstrated to exhibit a time- and dose-dependent inhibition on MCF-7 cell invasion by reducing the cells’ capability to penetrate through the matrigel matrix to the bottom of the porous membrane. Gelatin-zymography was done to assess the effect of the n-butanol fraction on activity of MMP-2 and MMP-9. The fraction showed to completely inhibit the gelatinolytic activity of MMP-2 and no band corresponding to the molecular weight of MMP-9 was observed, suggesting that MCF-7 cells produce undetectable levels of MMP-9. The n-butanol fraction further showed to down-regulate the expression of a range of proteins such as MMP-9, uPA, VEGF, TGF-β1 implicated in metastasis and angiogenesis determined using the human angiogenesis antibody array kit. This study demonstrated that the fractions of R. communis extracts have the ability to inhibit major processes of the metastatic cascade by down-regulating the expression of proteins relevant to metastasis. Thus, the fractions can be considered as potential anti-metastatic agents functional in the regulation and/or treatment of malignant of cancers. / National Research Foundation (NRF)

Until now, the role of cyclin dependent kinase 5 (CDK5) in cardiac pathophysiology has not been explored. While CDK5 has been well studied in the neuroscience/Alzheimer’s field as a cyclin-independent kinase, there is currently no investigation into the cardiac-specific role of CDK5. Recently, it was established that inhibition of CDK5 in stem cell derived cardiomyocytes from individuals with Timothy Syndrome (TS) rescued the delayed inactivation phenotype; TS is a fatal genetic long QT syndrome (LQTS) caused by delayed inactivation of the L-type voltage gated Ca2+channel CaV1.2. While it is evident that CDK5 plays an important role in regulating CaV1.2 function, its role in cardiac tissue remains to be elucidated. To determine whether CDK5 is essential for cardiac function, two separate mouse models were established—a cardiac-deficient Cdk5 mouse model (Cdk5 flox x αMHC-MerCreMer+) and a Cdk5 activation mouse model via overexpression of Cdk5’s known activator, p35 (Cdk5r1/p35 OE x αMHC-MerCreMer+). Immediately after spatiotemporal induction of deficiency/activation of Cdk5 in adult mice, echocardiography, histology and proteomic analysis were performed to examine effects on cardiac structure and function. Analysis of cardiac function and morphology in Cdk5 deficient mice revealed severe systolic dysfunction and a dilated cardiomyopathy-like phenotype. These results were further validated by a pathway analysis of quantified global proteome changes. Conversely, mice with an activation of Cdk5 displayed only minor changes in cardiac function with a modest reduction in fractional shortening and ejection fraction. Notably, these mice did not have any significant changes in cardiac chamber morphology, nor any significant changes to their global proteome. Interestingly, however, phosphoproteomic analysis revealed over 3,000 differentially phosphorylated proteins. Pathway and gene ontology analysis of proteome changes revealed significant hits related to cell adhesion. Evidence for the extensively studied role of CDK5 in the brain has demonstrated a critical role for CDK5 kinase activity in the regulation of cell adhesion. Alterations in cell adhesion are observed in a number of cardiac pathologies including heart failure and dilated cardiomyopathy; it is therefore plausible that CDK5 potentially regulates cardiac function via cell adhesion mechanisms. A comparison of the phospho-proteome acutely after Cdk5 depletion vs the phospho-proteome acutely after Cdk5 activation, allowed for the identification of a novel cardiac-specific Cdk5 substrate, beta taxilin (Txlnb). Validation of this potential phospho-substrate with an in situ proximity ligation assay demonstrated the co-localization of Cdk5-Txlnb in wildtype mouse cardiac tissue sections. When looking at co-localization in Cdk5 deficient tissue sections, no signals were observed. Lastly, our lab obtained donor cardiac tissue samples from individuals who passed away due to either heart failure or non-cardiac causes (serving as control cardiac tissue). Analysis of cardiac tissue samples revealed a significant increase in both CDK5 and p35 expression in heart failure samples. Dysregulation of phosphorylation has been implicated in cardiac dysfunction, with known contribution to contractile failure and a number of cardiac pathologies including cardiomyopathies. These findings further support a role for CDK5 in cardiac function. In conclusion, it appears that CDK5 is imperative for the maintenance of healthy cardiac function. Cardiac-specific homozygous and heterozygous Cdk5 deficiency revealed severe systolic dysfunction along with a dilated cardiomyopathy-like phenotype. While the effects of Cdk5 activation in the heart need to be further investigated, initial findings report significant downstream effects on the phosphorylation of a number of proteins, including Txlnb. Moreover, Txlnb was identified as a potential novel cardiac-specific substrate of Cdk5. The importance of identifying a role for CDK5 in the heart extends beyond this study. CDK inhibitors have been at the forefront of drug development for cancer therapeutics and immunotherapy. While modulation of CDK5 activity may be beneficial in one physiological system, it may prove deleterious in another. It is therefore imperative that the full range of molecular and physiological roles of each CDK be fully elucidated prior to therapeutic application. Furthermore, outcomes from this study have the potential to be translational for drug discovery and the development of new therapeutic avenues for heart disease.

Bakgrund: I Sverige drabbas en av tre av cancersjukdom men två av tre överlever. Fler kvinnor än män drabbas av cancersjukdom där ärftlighet och ohälsosamma levnadsvanor är betydande faktorer. Oro, rädsla och ångest är vanliga reaktioner vid ett cancerbesked och under cancerbehandlingen. Det kan påverka individens lidande och livslust. Primärvården har en samordnande roll i sjukvården och i framtiden kommer primärvården få ett större ansvar för cancersjukvården. Distriktssköterskan behöver därför ha ökad kunskap kring patienter med cancersjukdom avseende oro, rädsla och ångest. Syfte: Syftet i denna studie var att belysa kvinnors erfarenheter under cancerbehandling.  Metod: Studien har en kvalitativ design där datainsamling utgjordes av åtta självbiografier författade av kvinnor. Narrativ tematisk analys användes för att analysera berättelserna. Analysen genomfördes på både manifest och latent nivå.  Resultat: De åtta kvinnornas upplevelser av oro, rädsla och ångest under cancerbehandlingen beskrevs genom tre teman med nio kategorier. De tema som framkom var Den ständiga pendlingen mellan trygghet och tuffa påfrestningar, betydelsen av att ta tillvara på tiden och förändringarna på ett personligt plan. Det framkom att kvinnorna hade känslor som pendlade mellan ensamheten under cancerbehandlingen gentemot tryggheten som fanns bland familj och vänner. De fann också strategier för att hantera sjukdomens biverkningar som påverkade deras identitet. Ett sjukdomslidande uppstod vilket bidrog till ett livslidande men trots det kunde kvinnorna finna livskraft.  Slutsats: Kvinnor som befann sig under cancerbehandlingen upplevde stor oro kring cancerbehandlingens effekt och risken för att återinsjukna. Återkommande upplevelser var ensamhet, rädsla, ångest och identitetsförändring. Distriktssköterskan behöver därför vara medveten om deras upplevelser vid bemötande och vårdande. Genom ett etiskt förhållningssätt kan en jämställd vård appliceras och stärka värdigheten hos kvinnor drabbade av cancersjukdom. / Background: In Sweden one of three suffer from cancer only two of three survive. More women than men suffer from cancer because of genetic and unhealthy determinations. Worry, fear and anxiety are common reactions notified of a cancer diagnosis and during the treatment of cancer. It can affect the persons suffering from the disease and their life. Primary care has a coordinating role and will have more responsibility in the future reagarding cancer care. Due to this, that the district nurse must have increased knowledge about patients with cancer in terms of worry, fear and anxiety.  Aim: The aim was to illuminate women's experiences under treatment of cancer. Method: The study has a qualitative design and the data collection was based on eight autobiographical written by women. The study has been analyzed using a narrative thematic analysis. The analysis was performed on a manifest and a latent level. Results: The eight women's experiences of worry, fear and anxiety under treatment of cancer was described in three themes and nine categories. The themes that emerged were: The constant oscillation between safety and rough distress, The importance of making the most of the time and The changes on personal levels. It appears that the feelings oscillated between loneliness under the treatment of cancer and safeness from loved ones. The women found strategies to handle the side effects under the treatment of cancer that affected their personal levels. The women could find vitality despite suffering from the disease and suffering from life.  Conclusion: Women who were under treatment of cancer experienced anxiety over the treatment's long term effects and the risk of relapses. Loneliness, worrying, anxiety and identity crises were common in the result. District nurses, therefore, need to be conscious about the patients' experiences when interacting and treating cancer patients. Through an ethical approach, equal care can be applied and strengthen the dignity for women suffering from cancer.

Colon cancer is one of the most common cancers in both the United States (US) and throughout the world. Over the last 30 years, despite the development of multiple classes of effective anti-tumor agents, colon cancer has consistently remained the second leading cause of mortality amongst all cancers and is today responsible for over 50,000 deaths a year in the US alone. Among the greatest challenges to the successful treatment of colon cancer is its heterogeneity in terms of drug-sensitivity, whereby it is often difficult to identify which patients will benefit from a specific class of anti-tumor agents before treatment has begun. It is therefore imperative to identify predictive biomarkers that can be leveraged to distinguish which colon tumors are most likely to respond to individual anti-cancer drugs. This will help develop new therapeutic algorithms that can maximize patient survival by rapidly matching individual patients with the specific treatment combinations that are most likely to benefit them as well as sparing them the toxicities from drugs that would be ineffective. Previous studies have reported that human colon carcinomas lacking expression of the caudal-type homeobox 2 (CDX2) transcription factor can be leveraged as a predictor of benefit from adjuvant chemotherapy containing 5-fluorouracil (5-FU). Lack of CDX2 expression associates with microsatellite instability (MSI), as well as several histopathological and molecular features that associate with exceptionally poor prognosis such as poor differentiation, lympho-vascular invasion, and BRAF mutation. However, the molecular mechanisms linking lack of CDX2 expression with increased drug sensitivity are currently unknown. In the first section of this study, we conducted a high throughput screen (HTS) aimed at identifying clinically approved anti-tumor drugs that display selective activity against colon carcinomas lacking CDX2 expression (CDX2-negative). The results of our screening, which compared an isogenic pair of CDX2+/+ and CDX2-/- cell lines generated by genetic inactivation of CDX2 using CRISPR/Cas9 constructs, revealed that CDX2-negative colon cancer cells display increased sensitivity to anti-tumor drugs that are substrates of the ATP binding cassette sub-family B member 1 (ABCB1) transporter. ABCB1 is a drug-efflux protein known for its capacity to extrude multiple classes of anti-tumor agents from the cytoplasm, therefore contributing to drug-resistance in cancer cells. Importantly, analysis of CDX2 and ABCB1 expression in two independent gene-expression databases (NCBI-GEO: n=2115; TCGA: n=478) revealed that a lack of CDX2 expression is invariably associated with lack of ABCB1 expression in human primary colon carcinomas. Furthermore, our molecular studies revealed that forced expression of CDX2 in human CDX2-negative colon cancer cells was capable of inducing expression of ABCB1, while genetic inactivation of CDX2 in human CDX2-positive cancer cells using CRISPR/Cas9 constructs resulted in loss of ABCB1 expression, thus establishing CDX2 as a direct mechanistic regulator of ABCB1 expression. Amongst all of the anti-tumor drugs identified as being ABCB1 substrates with preferential activity against CDX2-negative colon cancer cells, we observed that paclitaxel was the FDA-approved drug with the greatest degree of selectivity with a 10-fold difference in IC50. When tested in vivo against a collection of human patient derived xenograft (PDX) lines representative of both CDX2-negative and CDX2-positive colon carcinomas, paclitaxel displayed selective activity against CDX2-negative models, often inducing volumetric regression of established lesions. Our study, therefore, identified paclitaxel as a clinically approved anti-tumor agent that should be investigated for use in the treatment of CDX2-negative colon carcinomas. In the second portion of our study, we sought to conduct a preliminary evaluation of the possibility of using immune checkpoint inhibitors (ICIs) for the treatment of CDX2-negative colon carcinomas. ICIs have been shown to display substantial anti-tumor activity against colon carcinomas with microsatellite instability (MSI) and against epithelial malignancies over-expressing the immune-suppressive molecule PD-L1/CD274. Because CDX2-negative tumors are enriched for MSI and high levels of PD-L1/CD274, they are predicted to include a subgroup that is responsive to ICIs. However, not all MSI tumors respond to ICIs and, contrary to the majority of MSI tumors, the subgroup of MSI tumors characterized by a CDX2-negative phenotype is often associated with poor prognosis. Because the clinical activity of ICIs is dependent upon expression of class-I HLA molecules by tumor cells, we decided to evaluate whether CDX2-negative tumors were associated with inactivating mutations in class-I HLA genes. Our attention focused on a highly conserved poly-cytosine repeat region in the coding sequence of HLA-A (c.621_627) and HLA-B (c.621_626) genes. Because this sequence fulfilled the molecular definition of microsatellite, we predicted it to be highly susceptible to frameshift mutations (insertions or deletions) in MSI colon tumors. Indeed, a search across three independent genetic databases (TCGA, COSMIC, EBI) confirmed that this highly conserved poly-cytosine repeat region was targeted by recurrent and deleterious mutations in at least one HLA-A or HLA-B allele of at least 13% (n=21/156) of human MSI colon tumors, as compared to 0.3% (n=2/770) of human colon tumors with a microsatellite stable (MSS) phenotype (p<0.0001). Among tumors assessable for CDX2 expression, this specific type of class-I HLA mutations was more frequent among CDX2-negative (12%; n=6/49) as compared to CDX2-positive (1.5%; n=5/340) colon tumors (p<0.001), but was similar within MSI CDX2-negative (21%; n=6/28) and MSI CDX2-positive (17%; n=5/30) subgroups. In summary, this work achieved two main results: 1) it identified paclitaxel, a clinically approved anti-tumor drug, as a new treatment option for patients with CDX2-negative colon cancers, which represents an extremely aggressive subgroup of colorectal malignancies; 2) it revealed that, in human MSI colon tumors, class-I HLA genes are prone to recurrent frameshift mutations in a genomic hotspot, mutations that are likely to associate with tumor resistance to ICIs and that they are therefore likely to represent a new class of actionable predictive biomarkers for both MSI and CDX2-negative colon carcinomas. These findings will help advance our understanding of colon cancer biology, and hopefully improve treatment algorithms for the clinical management of colon cancer patients.

The PI3-kinase/AKT/mTOR pathway plays a central role in cancer signaling. While p110α is the catalytic α-subunit of PI3-kinase and a major drug target, PTEN is the main negative regulator of the PI3-kinase/AKT/mTOR pathway. PTEN and p110α protein expression in tumors is commonly analyzed by immunohistochemistry, which suffers from poor multiplexing capacity, poor standardization, and antibody cross-reactivity, and which provides only semi-quantitative data. Here, we present an automated, and standardized immuno-matrix-assisted laser desorption/ionization mass spectrometry (iMALDI) assay that allows precise and multiplexed quantitation of PTEN and p110α concentrations, without the limitations of immunohistochemistry. IMALDI, which combines immuno-enrichment with analysis using a benchtop MALDI-Time-of-Flight (TOF) mass spectrometer, is an especially well-suited method for translating mass-spectrometry based assays into the clinical lab. We systematically optimized the iMALDI workflow regarding sensitivity, robustness, and throughput while developing highly flexible automation protocols using a Bravo 96LT liquid handling robot. We further developed custom R scripts to improve data visualization and analysis. One hour digestion using a protein to trypsin ratio of 1:2, followed by direct immuno-enrichment for 1 h yielded high and consistent peptide recoveries. We demonstrated that the PTEN and p110α iMALDI assays can be multiplexed using both simultaneous and sequential enrichment, reducing the amount of required sample material as well as simplifying the workflow. The PTEN+p110α iMALDI assay was validated and demonstrated high accuracy for both target proteins (90-112% recovery of known spiked-in concentrations) as well as high precision and 5-day reproducibility (overall CVs of 9%) across the linear range of the assay (0.6 to 20 fmol). Lower limits of quantitation below 1 fmol were achieved. Endogenous PTEN and p110α were quantified in cell lines as well as fresh-frozen tumor tissue samples. A novel two-point internal calibration strategy (2-PIC) was developed, based on spiking two peptide isotopologues into the sample as internal standards, avoiding the need for an external calibration. We quantified endogenous PTEN in a Colo-205 cell line using the PTEN iMALDI assay, as well an orthogonal PTEN immuno-multiple reaction monitoring (immuno-MRM) method to demonstrate this technique. Excellent agreement was shown between both calibration approaches (residual standard deviation between 2-PIC and external calibration of 1.6-5.8%), as well as high correlation between PTEN iMALDI and PTEN immuno-MRM (R²= 0.9966) and good agreement between quantified amounts (0.48±0.01 and 0.29±0.02 fmol/µg of total protein). Finally, we analysed a set of patient samples from a AKT inhibitor AZD5363 drug trial using a multi-site workflow combining the developed PTEN+p110α assay with established AKT1+AKT2 iMALDI assays and untargeted proteomics. We demonstrated how the combination of targeted and untargeted proteomics approaches may be used to gain novel insights into the tumor biology of patient tissue samples. Further, we showed that the PTEN iMALDI assay has good correlation with a comparable immunohistochemistry method (R²=0.86), and that our assays can be further multiplexed, reducing the required amount sample material. Thus, we showed that iMALDI is promising tool for biomarker quantitation. / Graduate / 2022-08-12

Orientador: Fernando Lucas Primo / Resumo: Neste trabalho de mestrado profissional foi possível obter um sistema nanoestruturado polimérico contendo Quinizarina (QZ), um corante fotossensibilizador que emerge como nova alternativa para tratamento antitumoral, utilizando a terapia fotodinâmica (TFD). As nanopartículas foram produzidas em escala, utilizando um método inovador de Nano Spray Dryer as quais foram caracterizadas por diferentes métodos, tais como: Espectroscopia por Absorção e Emissao de Fluorescência na região do Ultra-Violeta Visível (UV-Vis), Espalhamento de Luz dinâmica (DLS) e Microscopia Eletrônica de Varredura (MEV). Através das caracterizações observou-se nanopartículas monodispersas com diâmetro hidrodinâmico reduzido, verificou-se também que as nanopartículas foram preparadas de forma eficiente, o qual obteve-se a encapsulação das partículas QZ camada por camada (layer by layer). As nanopartículas foram preparadas utilizando uma rota de baixo custo e com altissimo rendimento (>70%). Após a completa caracterização do sistema, as nanopartículas foram aplicadas em testes biológicos in vitro utilizando dois tipos de linhagens celulares: a fibroblastóide (NIH-3T3) e cancerígena de origem melanocítica (B16-F10). Realizou-se estudos de biocompatibilidade, internalização utilizando QZ livre e encapsulada (uptake) e microscopia confocal em ambas as linhagens utilizadas e testes de fototoxicidade na linhagem melanocítica. Observou-se através dos estudos de uptake que houve uma maior internalização da quiniza... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In this professional master's work it was possible to obtain a polymeric nanostructured system containing Quinizarin (QZ), a photosensitizing dye that appears as a new alternative for antitumor treatment, using a photodynamic therapy (PDT). The nanoparticles were produced in scale using an innovative Nano Spray Dryer method, and characterized by different methods such as: Absorption Spectroscopy and Fluorescence Emission in the Ultra-Violet and Visible Region (UV-Vis), Dynamic Light Scattering (DLS) and Scanning Electron Microscopy (SEM). Through caracterizations, was observed that the nanoparticles produced where monodispered and small hydrodynamic diameter, the particles where prepared in a efficient way, which encapsulated the QZ particles layer by layer. The nanoparticles were prepared using a low cost, high yield (> 70%) route. After a complete characterization of the system, nanoparticles were applied in in vitro biological tests using two types of cell line: a fibroblast (NIH-3T3) and a melanocytic cancer (B16-F10). Biocompatibility, uptake, using free and encapsulated QZ and confocal microscopy studies were performed in both cell lines, and phototoxicity tests were performed in melanocytic cell line. It was observed through uptake studies that there was a greater internalization of nanostructured quinizarin, due to its high biocompatibility, compared to free quinizarin. The internalization study by confocal microscopy demonstrates that the QZ nanostructured system fun... (Complete abstract click electronic access below) / Mestre

Bioengineered probiotics enable new opportunities to improve colorectal cancer (CRC) prevention, screening, and treatment strategies. With CRC incidence on the rise in younger populations, there is an increased need to engineer technologies that enhance patient access to diagnostic exams and disease management. This dissertation presents the development of an orally-delivered probiotic to screen for and treat early CRC lesions with a particular emphasis on translatability factors including: safety of probiotic use, exploration of oral delivery, and testing in clinically relevant models. At the interface of immunology, synthetic biology, and the microbiome fields is the overarching concept that microbes play a critical role in the tumor microenvironment (TME). The innate ability of bacteria to seek out tumor-specific signatures and proliferate within their necrotic cores due to reduced immune surveillance enables the precise immunoengineering of the local TME. Here, we will design, characterize, and test a probiotic encoded with a lysis mechanism to aid in biocontainment and maximize the release of recombinantly-produced diagnostic and immunotherapeutic cargo. In this lysis circuit, bacteria grow to a critical density within tumors and synchronously lyse, locally releasing their payload. A small fraction of bacteria remains to reseed the population and the cycle continues, resulting in repeated and sustained drug delivery. Drawing from advancements in immunology, we engineered bacteria to produce immune checkpoint inhibitors. Monoclonal antibodies targeting immune checkpoints have revolutionized cancer therapy, but only work in a subset of patients and can lead to a multitude of toxicities, suggesting the need for more targeted delivery systems. Due to their preferential colonization of tumors, bacteria are a natural chassis for the localized delivery of such therapeutics. Therefore, we engineered a commercially available probiotic, E.coli Nissle 1917 (EcN), for the controlled production and intratumoral release of nanobodies targeting programmed cell death protein – ligand 1 (PD-L1) and cytotoxic T- lymphocyte-associated protein-4 (CTLA-4) using the described lysing release mechanism. We demonstrate that a single injection of this engineered probiotic enhanced therapeutic response compared to analogous clinically-relevant antibodies, resulting in tumor regression in syngeneic mouse models. In an effort to create a more effective therapeutic for poorly immunogenic cancers, we utilized the modularity of our platform to slow tumor growth in mouse models of established CRC by combining it with a probiotically-produced cytokine, granulocyte-macrophage colony stimulating factor (GM-CSF). We sought to expand upon the relevance of this approach for early-stage CRC screening and treatment, by characterizing the platform in CRC precancerous lesions, or adenomas. When orally-delivered, EcN robustly colonized adenomas in genetically-engineered and orthotopic murine models of CRC, and human CRC patients. Leveraging adenoma-specific colonization, we probed for EcN presence in fecal matter, demonstrating its utility as a non-invasive screen for adenomas. For more accessible testing, we engineered EcN to produce salicylate and showed that it could be detected in the urine of tumor-bearing mice for days after oral delivery of the probiotic. Moreover, we demonstrated that the therapeutic effectiveness of our previously engineered therapeutic strain, producing PD-L1, CTLA-4 and GM-CSF, was maintained when delivered orally, ultimately resulting in significant adenoma reduction. Altogether, this dissertation aims to highlight the potential for engineered EcN to be used as a safe, orally-deliverable screening and therapeutic platform for early-stage CRC disease. While we have chosen to focus on CRC here, we will conclude by discussing efforts to adapt this platform to work in combination with other cellular therapies and therapeutic indications, ultimately engineering a platform to impact a broader patient population.

Introduktion: Munhälsan har stor betydelse för den allmänna hälsan och påverkar livskvalitén. God munhälsa innebär att munnen är fri från sjukdomar såsom cancer. Tidigare studier visar på att cancerbehandlingar kan ge orala biverkningar som påverka munhälsan negativt, vilket leder till ett ökat lidande för patienterna och ger en ökad belastning på vården.  Syfte: Syftet är att beskriva patienters upplevelser av sin munhälsa under och efter en cancerbehandling oavsett diagnos och behandling.  Metod: Allmän litteraturstudie baserad på kvalitativa studier. Resultaten från studierna som uppfyllde syftet analyserades med hjälp av en artikel skriven av Popenoe, Langius-Eklöf, Stenwall och Jervaeus, som är en praktisk guide för att analysera allmänna litteraturstudier i tre steg. Sammanlagt analyserades 22 artiklar.  Resultat: Analysen resulterade i fyra kategorier och elva subkategorier. Kategorierna var: fysiska förändringar, psykosociala upplevelser, behov av information och stöd och copingstrategier. Studierna visar på att orala biverkningar från olika cancerbehandlingar har en stor påverkan på patienternas upplevda munhälsa.  Slutsats: Studiens resultat indikerar på att patienterna påverkas av de orala biverkningarna i olika omfattningar. Bristande information, bemötande och stöd från sjukvården gällande deras orala biverkningar bidrog till ett ökat lidande för patienterna. Studiens resultat anses vara av klinisk relevans för sjuksköterskor som arbetar med patienter med cancer, för att kunna förebygga vårdlidande och ge en individuellt anpassad vård. / Introduction: Oral health is important for general health and affects the quality of life. Good oral health means that the mouth is free from diseases such as cancer. Previous studies show that cancer treatments can cause oral side effects that affect oral health negatively, which leads to increased suffering for patients and an increased burden on care. Aim: The aim is to describe patients` experiences of their oral health during and after cancer treatment regardless of diagnosis and treatment. Method: General literature study based on qualitative studies. The results of the studies that fulfilled the purpose were analyzed using an article by Popenoe, Langius-Eklöf, Stenwall, and Jervaeus, which is a practical guide for analyzing general literature reviews in three steps. In total 22 articles were analyzed.  Result: The analysis resulted in four categories and eleven subcategories. The categories were: physical changes, psychosocial experiences, need for information, and support and coping strategies. Studies show that oral side effects from various cancer treatments have a major impact on patients' perceived oral health. Conclusions: The result of the study indicates that the patients are affected by the oral side effects to varying degrees. Lack of information, response, and support from health care regarding their oral side effects contributed to increased suffering for the patients. The result is considered to be of clinical relevance for nurses who work with cancer patients, to be able to prevent suffering from care and provide individually tailored care.

Bakgrund: En av de mest förekommande sjukdomar hos kvinnor är bröstcancer. Bröstcancer uppstår genom cancer i bröstkörtel eller bröstkörtelgångarna. Behandling av bröstcancer innebär förändringar i vardagslivet. Kvinnor med bröstcancer lider av psykisk ohälsa där depression är vanligt. Sjuksköterskans roll är väsentlig för att upptäcka och förebygga ohälsa samt främja hälsa. Syfte: Syftet med studien var att belysa faktorer som är karaktäristiska för depression hos kvinnor under behandling för bröstcancer. Metod: En litteraturöversikt med deduktiv ansats valdes. Tolv kvantitativa artiklar sammanställdes efter litteratursökning och kvalitetsgranskning. Resultat: Fem kategorier av faktorer skapades: fysiska faktorer, psykiska faktorer, sociala faktorer, sociodemografiska faktorer och vårdrelaterade faktorer. Huvudfynd som framträdde var brist på socialt stöd, nedsatt sömnkvalitet och ökad oro samt ångest. Slutsats: Under behandling för bröstcancer finns det många faktorer som är karaktäristiska till depression. Dessa faktorer leder till nedsatt livskvalitet och många kan förebyggas genom omvårdnadsåtgärder. Förslag på omvårdnadsåtgärder är att sjuksköterskan ska bidra med socialt stöd, ge egenvårdsråd för förbättrad sömn och utveckla tillgängligheten för gruppterapi. Det anses finnas en vinst med att utföra screening för depression av alla patienter med bröstcancer för att effektivt identifiera och förebygga ohälsa hos denna patientgrupp.