The aim of this thesis was to reveal segments of the car customers in the Czech Republic. Firt section of this thesis contains theoretical informations about market segmentation. The second section is dedicated to an analysis and evaluation of the research, which has been conducted in terms of my thesis. At the and I described the discovered segments and suggested options for their approach.

The aim of my diploma thesis was to evaluate, whether the marketing strategy of the Nissan company is efficient and what is its position among the companies operating in the automotive industry in the Czech republic. The thesis is divided into 4 parts. First part deals with definition of basic marketing terms and their comparison with the practices implemented at Nissan. The second part is focused on the company itself, its postions on the European and Czech market and specific marketing methods, which Nissan uses. Situation analysis is the subject of the third part. This part is focused automotive market in Czech Republic and its development, product portfolio of Nissan and analysis of competitors and target group. The last part is based on a case study of new model launch on the market. It includes critical evaluation of new model launch and communication activities. In the end, the thesis evaluates success on new model launch on the market.

No description available.

The purpose of this diploma thesis „Parking Planning and Calculation„ is car parks at selected buildings. The goal of this thesis is to optimize the selected of targeted units for calculate the number of parking spaces. First part of the work is focused on the measurement itself and data analysis. The next part of the work consists of comparison of calculated parking places according to standards with real number of parking places.

Thesis is focused on the impact of the inside noise of the car on conditional driver safety, which is presented by reaction times on simulated stimulus. In introduction, there is a theoretical definition of the security of the vehicles, inside noise, and its methods of measurement. Practical part consists of description and realization of the measurements of the impact on the inside noise in the car on reaction time of drivers. Conclusion includes evaluations of the measurements and findings, which results from them.

archives@tulane.edu / Background: Human Immunodeficiency Virus-1 (HIV-1) has killed over 35 million and infects 1.8 million new people each year. Antiretroviral therapy (ART), although effective controlling plasma viremia and transmission, does not purge latent or persistent reservoirs necessary to eliminate infection, and must be maintained for life. It is thus imperative to discover therapeutics that provide both lifetime suppression of viral loads and depletion of viral reservoirs. Methods: To harness the immunosurveillance capacity of highly functional and persistent CMV-specific adaptive response, rhesus PBMCs were stimulated with rhCMV peptide pools (IE1, IE2, and pp65) to expand rhCMV-specific T cells. These cells were then genetically modified with retroviral vectors expressing a CD4 extracellular domain linked to T cell intracellular signaling domains that instruct CTL activity, converting them into HIV-specific effector cells. Vectors combine CD4 targeting with an maC46 fusion inhibitor to protect against viral entry. In a reversal of the critical step in the HIV viral lifecycle whereby virus targets new CD4+ host cells using its Env glycoprotein, these genetic modifications redirect host immune responses to target and kill Env expressing infected cells. We hypothesize that continuous stimulation of CD4-CAR T cells through their rhCMV-specific TCR will maintain activated T effector memory CTL capable of targeting HIV infected cells. Results: We find that autologous rhPBMCs can be expanded ex vivo with rhCMV peptides up to therapeutically relevant numbers for adoptive transfer. This rhCMV-specific T cell expansion enriches cells in a phenotype consistent with T effector memory differentiation. Following genetic modification and adoptive transfer, cells reach peak expansion at seven days post infusion into ART suppressed or unsuppressed SHIV infected Rhesus Macaques. We observe these cells capable of persisting in vivo for at least 2 years following reinfusion. Furthermore, these cells are maintained in vivo in an effector memory phenotype throughout the duration they were analyzed. Despite this, SHIV plasma viral loads remain unchanged. Conclusion: These studies establish use of rhCMV-specific T cells as an effective way to produce persistent genetically modified cells targeting SHIV. Future studies will need to further increase in vivo expansion, protection, and CTL activity as viral loads remain detectable. / 1 / Nathan Michel Johnson

Glioblastoma (GBM) remains the most aggressive primary brain tumor in adults. Since 2005, Standard of Care (SoC) consists of surgical resection followed by radiation and adjuvant chemotherapy with temozolomide. Treatment failure is attributed to intratumoral heterogeneity with populations capable of mechanisms to repair damaged DNA. Given the lack of progress to improve patient outcomes, the current work encompasses how multi-omic approaches can be utilized to uncover novel biology in GBM and develop precision medicines to exploit these cancer specific phenomena. Using patient derived GBM samples I first used the surface marker CD133 to interrogate glioblastoma stem cells, a subpopulation of cells identified to withstand conventional therapies and lead to tumor relapse. I used a genome-wide CRISPR-Cas9 library to conduct an unbiased loss-of-function phenotypic screen to identify regulators of CD133. I then validated SOX2 as a direct transcription factor to PROM1 encoding CD133. These findings further show the untapped potential of CRISPR to uncover novel biology to directly apply to broader fields of stem cells and cancer biology. Next, I combed GBM data sets at transcriptomic and proteomic levels to identify understudied proteins as potential targets for immunotherapies. Glycoprotein nonmetastatic melanoma protein B (GPNMB) has previously been identified as a clinically relevant target in GBM and shown to be active in the tumor immune microenvironment. I found GPNMB to be upregulated in recurrent GBM and macrophage populations which can be exploited in a more comprehensive manner to treat GBM. Through a series of models, I elucidated how GPNMB influences GBM biology, its effectiveness as a target for Chimeric Antigen Receptor T-cells, and how it can be paired with CD133 therapies to provide better coverage of tumor cells. Together, these studies highlight how advances in pre-clinical models and technologies can be leveraged to develop new therapies in a rational manner. / Thesis / Doctor of Science (PhD) / Glioblastoma (GBM) remains an aggressive and incurable brain cancer despite decades of intense research. Treatment failure is due to the untargeted approaches currently undertaken in the clinic. The current work uses multiples methods to interrogate how GBM grows and develops over time. Using GBM samples from consenting patients, I investigated an important population of the tumor using a surface marker CD133 and CRISPR to study which genes influenced it. I then successfully validated SOX2 as a direct regulator of CD133 expression. Next, I combed multiple data sets for a target to kill GBM cells without harming healthy tissue in patients. I found Glycoprotein Non-Metastatic Melanoma Protein B (GPNMB) to be exploitable and used several experimental methods to investigate its role in GBM progression. Finally, we used a novel immunotherapy to eliminate cells which express GPNMB. Together, these findings could apply to the broader field of stem cell biology and be used for a more targeted method to eliminate the cancer entirely.

Cross-centerline crashes occur rarely in the United States but are especially severe. This type of crash is characterized by one vehicle departing over a centerline and encountering a vehicle traveling in the opposite direction. In recent years, automakers have started developing and implementing lane departure warning (LDW) on newer vehicles. This system provides the potential to reduce or significantly impact the frequency of cross-centerline crashes. The objective of this thesis was to estimate the potential crash and injury benefits of a LDW system if installed on every vehicle in the US fleet. This research includes the following 1) a characterization of cross-centerline crashes in the United States today with current and future prevention methods, 2) a reconstruction methodology used for all crashes including rollovers and heavy vehicles, and 3) a simulation model and approach method used to estimate potential benefits of LDW systems on cross-centerline crashes. Cross over to left crashes account for only 4% of non-junction non-interchange crashes but account for 44% of serious injury crashes of the same type. As part of this research, 42 cross-centerline crashes were reconstructed and simulated as if they had a LDW system installed. Accounting for driver capability to react to a LDW alert, crash reduction benefits ranged from 22 – 30%.Using injury risk curves, the probability of experiencing a MAIS2+ injury in a cross-centerline crash was reduced by 29% when using a LDW system. / Master of Science

Adoptive immunotherapy using chimeric antigen receptor (CAR)-modified T cells is an effective treatment for hematological malignancies that are refractory to conventional chemotherapy. To address a wider variety of cancer entities, there is a need to identify and characterize additional target antigens for CAR-T cell therapy. The two members of the receptor tyrosine kinase-like orphan receptor family, ROR1 and ROR2, have been found to be overexpressed on cancer cells and to correlate with aggressive cancer phenotypes. Recently, ROR1-specific CAR-T cells have entered testing in phase I clinical trials, encouraging us to assess the suitability of ROR2 as a novel target for CAR-T cell therapy. To study the therapeutic potential of targeting ROR2 in solid and hematological malignancies, we selected two representative cancer entities with high unmet medical need: renal cell carcinoma and multiple myeloma. Our data show that ROR2 is commonly expressed on primary samples and cell lines of clear cell renal cell carcinoma and multiple myeloma. To study the efficacy of ROR2-specific CAR T cell therapy, we designed two CAR constructs with 10-fold binding affinity differences for the same epitope of ROR2. We found both cell products to exhibit antigen-specific anti-tumor reactivity in vitro, including tumor cell lysis, secretion of the effector cytokines interleukin-2 (IL-2) and interferon-gamma (IFNγ), and T cell proliferation. In vivo studies revealed ROR2 specific CAR-T cells to confer durable responses, significant survival benefits and long-term persistence of CAR-expressing T cells. Overall, there was a trend towards more potent anti-tumor efficacy upon treatment with T cells that expressed the CAR with higher affinity for ROR2, both in vitro and in vivo. We performed a preclinical safety and toxicology assessment comprising analyses of ROR2 expression in healthy human and murine tissues, cross-reactivity, and adoptive T cell transfer in immunodeficient mice. We found ROR2 expression to be conserved in mice, and low-level expression was detectable in the male and female reproductive system as well as parts of the gastrointestinal tract. CAR-T cells targeting human ROR2 were found to elicit similarly potent reactivity upon recognition of murine ROR2. In vivo analyses showed transient tissue-specific enrichment and activation of ROR2-specific CAR-T cells in organs with high blood circulation, such as lung, liver, or spleen, without evidence for clinical toxicity or tissue damage as determined by histological analyses. Furthermore, we humanized the CAR binding domain of ROR2-specific CAR-T cells to mitigate the risk of adverse immune reactions and concomitant CAR-T cell rejection. Functional analyses confirmed that humanized CARs retained their specificity and functionality against ROR2-positive tumor cells in vitro. In summary, we show that ROR2 is a prevalent target in RCC and MM, which can be addressed effectively with ROR2-specific CAR-T cells in preclinical models. Our preliminary toxicity studies suggest a favorable safety profile for ROR2-specific CAR-T cells. These findings support the potential to develop ROR2-specific CAR-T cells clinically to obtain cell products with broad utility. / Adoptive Immuntherapie mit T-Zellen, die chimäre Antigenrezeptoren (CAR) exprimieren, ist ein effektiver Behandlungsansatz für Chemotherapie-resistente Blutkrebserkrankungen. Die Übertragung dieses Konzepts auf weitere Krebsarten erfordert die Identifikation und Charakterisierung neuer Zielstrukturen für die CAR-T Zelltherapie. ROR1 und ROR2, die beiden Mitglieder der Familie der Rezeptortyrosinkinase-ähnlichen Orphan-Rezeptoren, werden auf einer Vielzahl von Tumoren überexprimiert und korrelieren mit einer schlechten Prognose und höherer Krebs-Invasivität. Kürzlich konnte ROR1 als Zielstruktur für die CAR-T Zelltherapie bestätigt werden und die Effektivität und Sicherheit ROR1 spezifischer CAR-T Zellen wird derzeit im Rahmen klinischer Phase-I Studien näher untersucht. Aus diesem Grund waren wir daran interessiert, das therapeutische Potenzial ROR2-spezifischer Zelltherapie zu untersuchen. Als Modellsysteme hierfür wählten wir das Nierenzellkarzinom und das Multiple Myelom als repräsentative hämatologische und solide Krebserkrankungen mit hohem medizinischem Bedarf aus. Unsere Daten zeigen, dass ROR2 häufig auf Zelllinien und primären Tumorproben des klarzelligen Nierenzellkarzinoms und des Multiplen Myeloms vorkommt. Um die Effektivität ROR2-spezifischer CAR-T Zellen zu untersuchen, wurden zwei CAR Konstrukte mit zehnfach unterschiedlichen Bindungsaffinitäten für dasselbe Epitop von ROR2 hergestellt. Beide Zellprodukte zeigten hohe, antigen-spezifische Antitumor-Reaktivität in vitro – insbesondere im Hinblick auf Tumorzell-Lyse, Sekretion der Zytokine Interleukin-2 (IL-2) und Interferon gamma (IFNγ) und T-Zell Proliferation. In vivo beobachteten wir langanhaltende Antitumor-Effektivität durch ROR2-spezifische CAR-T Zellen, sowie signifikante Überlebensvorteile und langfristige T-Zell Persistenz. Außerdem beobachteten wir, sowohl in vitro als auch in vivo, einen Trend zu stärkerer Antitumor-Effektivität von T-Zellen, die den CAR mit höherer Affinität für ROR2 exprimierten. Im Rahmen einer präklinischen Toxikologie-Studie analysierten wir die Expression von ROR2 im gesunden Gewebe, die Kreuz-Reaktivität ROR2-spezifischer CAR-T Zellen und deren Sicherheit durch adoptiven T-Zell Transfer in immun-defiziente Mäuse. Unsere Daten zeigen, dass ROR2 in H. sapiens und M. musculus gleichermaßen exprimiert wird und ROR2 Expression war insbesondere in den weiblichen und männlichen Reproduktionsorganen und Teilen des Gastrointestinaltrakts detektierbar. Wir konnten außerdem zeigen, dass CAR-T Zellen, die menschliches ROR2 erkennen, vergleichbare Antitumor-Reaktivität gegen Zellen, die murines ROR2 exprimieren, auslösen. Unsere in vivo Analysen zeigten temporäre Anreicherung und Aktivierung ROR2-spezifischer CAR-T Zellen in gut durchbluteten Geweben, wie Lunge, Leber und Milz, in der Abwesenheit klinischer Anzeichen für Toxizität oder histologisch nachweisbarer Gewebsschädigungen. Um die Risiken immunologischer Nebenwirkungen und die damit einhergehende Abstoßung ROR2-spezifischer CAR-T Zellen zu reduzieren, humanisierten wir die CAR Bindedomäne. Unsere Daten zeigen, dass humanisierte ROR2-spezifische CAR-T Zellen vergleichbare Spezifität und Funktionalität gegen ROR2-positive Tumorzellen in vitro aufweisen. Insgesamt zeigen unsere Daten, dass ROR2 eine häufig auftretende Zielstruktur auf der Oberfläche von RCC und MM Zellen ist und diese in präklinischen Modellen effektiv mittels ROR2-spezifischer CAR-T Zellen adressiert werden kann. Unsere vorläufigen Toxizitätsdaten deuten darauf hin, dass ROR2-spezifische CAR-T Zellen ein vorteilhaftes Sicherheitsprofil aufweisen. Alles in allem unterstützen diese Daten das Potenzial der klinischen Entwicklung ROR2-spezifischer CAR-T Zellen als Zellprodukte mit breit gefächerter Anwendbarkeit.

Glioblastoma is a deadly brain cancer with discouraging standard of care. New methods like convection enhanced delivery and chimeric antigen receptor T cells (CAR-T) are promising treatments that can be translated to glioblastoma. In this study, CAR-T cell flow through a hydrogel was explored in the context of in-vitro convection enhanced delivery. A culture method to create large spheroids mimicking tumors from preexisting glioblastoma stem cell lines was fabricated, a convection enhanced delivery system for in-vitro testing was designed, and characterization of the CAR-T cells using the in-vitro system took place. The spheroid culture method was successfully optimized to produce spheroids large enough to act as a sufficient tumor in little time, the in-vitro set-up successfully administered treatment, and CAR-T cells were found to increase their velocities through a medium as their injection velocity increased. It was discovered that the density of the spheroid plays a crucial role in treatment delivery, often times driving how treatment will move through the spheroid. This system can be used in the future studies to test the killing potential of CAR-T cells to a tumor in-vitro. / Master of Science / Glioblastoma is a deadly brain cancer with current treatments that are discouraging at best. New methods must be utilized to aid in patient recovery. Chimeric antigen receptor T-Cells (CAR-T) are a promising treatment that can be used in glioblastoma. In this study, CAR-T cell behavior is defined in the context of in-vitro convection enhanced delivery. A large spheroid, or sphere of cells, mimicking a tumor was created, a convection enhanced delivery system set-up for in-vitro testing was designed, and characterization of CAR-T cell behavior using the in-vitro system took place. The spheroids were successfully cultured to act as a sufficient tumor, the in-vitro set-up successfully administered treatment, and CAR-T cells were found to increase their velocities in a gel as their injection velocity increases. It was discovered that the density of the spheroid plays a crucial role in treatment delivery, often times driving how treatment will move through the spheroid. This system can be used in the future studies to test the killing potential of CAR-T cells to a tumor in-vitro.