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Next-Generation protein sequencing (NGPS) creates newfound ways of fully identifying every protein species in a single biological organism. It is an effort to use technology to determine proteomic data. The purpose of this research project is to use the current technology to sequence proteins and potentially find treatments for some diseases that are common today. Through NGPS, scientists can identify low abundant proteins including those that go through post-translational modifications (PTM) [1]. NGPS will allow us to fully determine protein sequences from protein samples using mass spectrometry with the ultimate goal of being able to determine the primary sequence of the protein in the given sample [1]. Antibodies are a specific class of proteins that aid our bodies in the immune response. Due to their variability in the complementary-determining region (CDR), NGPS will be used to determine the heavy and light chain sequences [2]. The goal of this technology is to fully determine the primary sequence of a protein in a given sample. The randomness of an antibody’s variable (V), diversity (D), and joining (J) genes (VDJ recombination) makes each protein unique. VDJ recombination refers to the process of T cells and B cells randomly assembling different gene segments. This process allows the antibody to make specific receptors that can recognize different molecules presented on the surface of antigens. Proteases are enzymes that break down proteins and peptides. By using different proteases with varying cutting rules, we can digest the antibody and run it through high mass accuracy determining instrument [1]. NGPS allows us to utilize mass spectrometry technology to measure proteins or polypeptides. Because of these measurements, post-translation modifications, including glycosylation, can be detected, unlike in DNA sequencing technology. Protein sequencing has the opportunity to play a major role in the fight against the COVID-19 outbreak and serve as curative measures for the treatment and Type 2 Diabetes [3]. Proteomics can serve as the basis of vaccine development as well as monitoring treatment. Utilizing techniques such as mass spectrometry could reveal the structure of the virus and ultimately allow for engineered tissues to produce the protein in large amounts in a lab setting. Currently, many companies are utilizing highly sensitized technology to carry out the goals of NGPS. The Oxford Nanopore is a company that uses technology to develop and explore more ways to undergo protein analysis. The methods used by this company involve using protein nanopores to mutate residues in pores to determine the overall sequence. The company utilizes modified aptamers that are drawn to the nanopore current. These aptamers can bind with some, but not all pores, allowing for the differentiation between target and non-target proteins. Nicoya Life Sciences is another company that uses Open Surface Plasmon Resonance (SPR) to detect molecular interactions. SPR uses an analyte (a mobile molecule) to bind to a ligand and observe changes in the refractive index. SPR allows researchers the ability to characterize the binding kinetics and affinities of monoclonal antibodies. SPR is an extremely promising technique to sequence proteins due to its flexibility in being able to work with a variety of molecules including lipids, nucleic acids, cells, viruses, nanoparticles, proteins, antibodies, carbohydrates, and more. The original goal behind NGPS was to establish a method to sequence proteins to aid in the early detection of common diseases such as Type 2 Diabetes. After significant research, it is now known that NGPS can be done in a variety of ways to accomplish a common goal—sequencing proteins and understanding how amino acids affect the human body. In the case of diseased states, NGPS can help researchers find ways to diagnose, treat, and cure diseases early on. Focusing on antibodies allows us to manipulate the body’s immune response to rid the host of pathogens. NGPS, however, is advancing at a much slower rate than anticipated by companies due to its many limitations including not being able to sequence large peptides, difficulties in material and composition of the sample, and needing to label small peptides to begin degradation. Ideally, finding a way to combine the high accuracy and specificity of certain techniques, the ability to detect low abundant proteins in others, and the flexibility of Open SPR would allow researchers and companies to create the standard for NGPS. Creating effective antibodies is precisely why NGPS has such great potential today. Ultimately, I found that as a standalone, Open SPR is the most effective method. However, as the research shows, there are limitations with each method, including Open SPR. The conclusion shows that it is necessary to find a combination of these techniques and create an accurate method, potentially using different technologies, to establish the most effective way to sequence proteins.

Lung cancer is one of the main causes of death worldwide, with most patients suffering from an advanced unresectable or metastatic non-small cell lung cancer. The mortality trends are mostly related to patterns of tobacco use, specifically from cigarettes. Tobacco is the basic etiological agent found as a consequence of the inhalation of tobacco smoke. Published data show the use of interferons (IFNs) in the treatment of lung tumours due to their potential in displaying antiproliferative, anti-angiogenic, immunoregulatory, and proapoptotic effects. Type1 IFNs have been employed as treatments for many types of cancer, both for haematological cancers and solid tumours. The IFN-γ (naked) functions as an anticancer agent against various forms of cancer. Hence, this study aimed to investigate the genoprotective and genotoxic effects of IFN-γ liposome (nano) on 42 blood samples from lung cancer patients, compared to the same sample size from healthy individuals. The effectiveness of IFN- γ liposome against oxidative stress was also evaluated in this study. A concentration of 100U/ml of IFN-γ liposome was used to treat the lymphocytes in: Comet and micronucleus assays, Comet repair, Western blotting and real-time polymerase chain reaction (qPCR) were based on a preliminary test for the optimal dose. The lymphocytes from lung cancer patients presented with higher DNA damage levels than those of healthy individuals. IFN-γ liposome was not found to induce any DNA damage in the lymphocytes. Also, it caused a significant reduction in DNA damage in the lymphocytes from lung cancer patients in; Comet, Comet repair and micronucleus assays. Furthermore, the 100U/ml of IFN-γ liposome significantly reduced the oxidative stress caused by H2O2 and appeared to be effective in both groups using the Comet and micronucleus assays. Results from; Comet, Comet repair and micronucleus assays were consistent. The data obtained indicated that IFN-γ in both forms (naked INF-γ and INF-γ nano-liposome) may potentially be effective for the treatment of lung cancer and showed the ability of IFN-γ liposome to reduce the DNA damage more than the naked form. The IFN-γ in both forms has also shown anti-cancer potential in the lymphocytes from lung cancer patients by regulating the expression of p53, p21, Bcl-2 at mRNA and protein levels by up-regulating the p53 and p21 to mediate cell cycle arrest and DNA repair in lung cancer patients. The findings of this study are consistent with the view that the naked IFN-γ and liposome could have a significant role in cancer treatment, including lung cancer. / Mutah University in Jordan

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Bakgrund: Runt 429 000 barn drabbas årligen av cancer världen över. Att leva med cancer och genomgå behandlingar är en svår utmaning för barn och närstående. Coping är ett verktyg som hjälper individer möta påfrestande situationer. Många studier belyser copingstrategier använda av föräldrar och andra personer nära cancerdrabbade barn, men underlaget är inte lika stort gällande barns egna beskrivningar av sin coping. Av denna anledning är det väsentligt att identifiera använda copingstrategier ur barns perspektiv. Syfte: Syftet med litteraturstudien var att beskriva vilka copingstrategier barn med cancer använder sig av för att hantera sjukdomen och dess behandling. Metod: Studien är en beskrivande litteraturstudie. Databassökningen genomfördes i PubMed. Sökningen resulterade i tio artiklar där åtta var kvalitativa, en var kvalitativ med mixad metod och en var kvantitativ. Framkomna data analyserades genom tematisk dataanalys. Huvudresultat: Barn med cancer använde olika copingstrategier som kategoriseras som problemfokuserade, emotionsfokuserade och meningsskapande copingstrategier. Till problemfokuserade copingstrategier hörde ”Symtomlindring och underlättande av behandling”, ”Information, kunskap och insikt” samt ”Anpassning och bibehållande av en normal vardag”. I emotionsfokuserade copingstrategier återfanns ”Stöd och känslohantering”, ”Distrahering och undvikande” samt ”Acceptans, motivation och framtidshopp”. Meningsskapande copingstrategier var ”Positiv omvärdering” och ”Personlig utveckling och att se sjukdomen som en utmaning”. Slutsats: Barn med cancer använder olika copingstrategier för att hantera sin sjukdom och dess behandling. Strategierna tillämpas växelvis beroende på situation och syftet med användningen av vald copingstrategi är individuell. Genom ökad kännedom kring ämnet kan cancerdrabbade barn erbjudas personcentrerad vård och lämpligt stöd. / Background: About 429 000 children are annually affected by cancer worldwide. Living with cancer and undergoing treatment is a difficult challenge for children and close relatives. Coping is a tool that helps individuals face stressful situations. Many studies shed light on coping strategies used by parents and other people close to children affected by cancer, but the basis is not as extensive regarding children's own descriptions of their coping. For this reason, it is essential to identify used coping strategies from children's perspective. Aim: The purpose of the literature study was to describe which coping strategies children with cancer use to manage the disease and its treatment. Method: The study is a descriptive literature study. The database search was conducted in PubMed. The search resulted in ten articles, eight of which were qualitative, one was qualitative with a mixed method and one was quantitative. The data were analyzed through thematic data analysis. Main results: Children with cancer used different coping strategies categorized as problem-focused, emotion-focused and meaning-making coping strategies. Problem-focused coping strategies included “Symptom relief and facilitation of treatment”, “Information, knowledge and insight” and “Adaptation and maintaining a normal everyday life”. In emotion-focused coping the strategies found were “Support and emotion management”, “Distraction and avoidance” and “Acceptance, motivation and hope for the future”. Meaning-focused coping strategies were “Positive reappraisal” and “Personal development and seeing the disease as a challenge”. Conclusion: Children with cancer use different coping strategies to deal with their illness and its treatment. The strategies are applied alternately depending on the situation and the purpose of using the chosen coping strategy is individual. Through increased awareness of the subject, children affected by cancer can be offered person-centred care and appropriate support.

This document reviews qualitative assessments used to explore the impact on art therapy interventions with patients in cancer treatment. The study explored the use of qualitative assessment in evaluating patient perspective on receiving art therapy adjunctly with cancer treatment. In addition, the research aimed to determine if art therapy interventions are perceived as effective in helping cancer patients reduce stress, cope, improve quality of life, express emotions, and reduce cancer-related symptoms during and after cancer treatment through qualitative assessment. Approximately 300 cancer patient experiences were reviewed through surveying qualitative studies that explored the effects of art making in cancer treatment through qualitative assessment such as interviews, questionnaires, observations, and open-ended questions. This archival research used a thematic approach to identify emergent themes in format, administration techniques, and impact in qualitative assessments to learn about the patient art therapy experience. The emergent themes were discovered while surveying information regarding types of formats and administration procedures used in qualitative cancer research. These findings suggest that qualitative assessments used in art therapy programs are a useful tool to determine how art interventions may help address patient's psychosocial needs, provide coping skills, and relieve cancer–related symptoms.

Bladder Cancer is the tenth most common malignancy globally, and is the thirteenth most common cause of tumor associate morbidity. Bladder cancer is largely stratified into two categories: Non-Muscle Invasive Bladder Cancer (NMIBC) and Muscle Invasive Bladder Cancer (MIBC). NMIBC represents disease localized to the urinary bladder, and can be stratified into low and high-grade disease. MIBC represents an aggressive class of bladder cancer, with invasion into the underlying muscle layers of the bladder. MIBC can be classified as either non-metastatic MIBC, with disease localized to the bladder corpus, or metastatic MIBC, with disease spreading to sites beyond the bladder corpus. High grade NMIBC presents significant risk for progression to MIBC, and collectively both high grade NMIBC and MIBC bladder cancers demonstrate poor prognostic outcomes in clinical settings in terms of responses to therapy, recurrence risks, and overall survival. Hexaminolevulinate is a precursor of Protoporphyrin IX (PpIX) in the heme biosynthetic pathway. Hexaminolevulinate has been FDA approved under the trade name Cysview for diagnostic usage in blue light cystoscopies for fluorescence mediated visualization of disease along the bladder wall. I demonstrate that in addition to its diagnostic utility, Cysview and blue light irradiation can be utilized clinical as a potential therapeutic modality. I demonstrate the significant selective cytotoxicity of Cysview in combination with blue light against patient derived organoids (PDOs) from primary bladder cancers. My results determine that Cysview and blue light induce a rapid cell death program mediated by an influx in Reactive Oxygen Species (ROS) production, resulting in less than 5% viability within 24 hrs of treatment. This massive loss in viability is observed in low and high grade NMIBC, as well as MIBC derived PDOs with diverse mutational profiles. The results of this work demonstrate that PDOs are a significant platform for assessing therapy responses for correlation with the large patient population. Furthermore, the work identifies photodynamic therapy with Cysview and blue light irradiation as a putative therapeutic modality for localized bladder cancers, with the potential for significant improvement in patient outcomes. The identification and characterization of the therapeutic effects of Cysview come at a critical time during a global shortage of conventional therapeutics for localized bladder cancer, and presents a pathway for patients affected by these shortages. Progression in bladder cancer has been understood to be driven by processes governing subpopulation and cell state and lineage transformation. Previous studies identify phenotypic plasticity within a subset of bladder cancers and have correlated this phenomenon with an increased risk for disease progression from NMIBC to MIBC. In previous work, a subset of PDOs derived from luminal primary tumors demonstrated significant degrees of luminal to basal plasticity in vitro. In my analysis of these PDOs using transcriptomic and chromatin accessibility data, I identified a transcriptomic and epigenetic signature unique to plastic PDOs. Furthermore, I identified HNF1B, GRHL2, GATA6, and SNAI2 as putative regulators of luminal to basal plasticity in bladder cancer. Using these molecular profiles, I correlated the plasticity phenotype with reduction in overall survival using data from published bladder cancer patient cohorts. Finally, I developed a novel transcriptomic subtypes classification scheme and an accompanying R package to classify epithelial heterogeneity in bladder cancer, based on the transcriptomic subtypes I identified in bladder cancer PDOs.

In 1867, Dr. Wilhelm Busch decisively exposed a cancer patient to erysipelas and notedtumor regression. The practice of inoculating tumors with bacteria became more widespread with the work of Dr. William Coley, beginning in 1891, who inoculated inoperable tumors and observed complete regressions though at times notable toxicity. These microbial manipulations of immunity now form the roots of cancer immunotherapy in modern history. Alongside the blossoming of cancer immunology since, the development of techniques to alter biological systems has given rise to synthetic biology. Together, these fields allow the programming of biological systems to precisely guide the cancer-immune interplay. As mammalian immunity targets bacterially-derived antigens due to the immunostimulatory nature of microbes, and tumors express various antigens, synthetic alteration of microbes to function as safe and effective anti-tumor vaccines is a natural proposition. In this work, I describe my development of such a microbial system comprised of a synthetic tumor-antigen construct optimized for expression in bacteria, the immunotoxin Listerolysin O, and a genetically edited probiotic E. coli chassis with multi-functional protease deletions. This platform encodes and delivers high levels of diverse tumor antigens sets, remodels the tumor microenvironment, and stimulates productive and durable anti-tumor immunity to control and eliminate primary and metastatic tumors. We show that this system induces tumor antigen-specific CD4+ and CD8+ T cells, activates NK cells, recruits and activates dendritic cells, and reduces immunosuppressive regulatory T cells, B cells, and myeloid cells within the tumor microenvironment. This work thus establishes a new class of anti-tumor vaccine which modulates all arms of immunity to achieve robust anti-tumor efficacy.

Thesis (M. Tech.) - Central University of Technology, Free State, 2009