The emerging field of RNA nanotechnology necessitates creation of functional RNA nanoparticles, but has been limited by particle instability. Previously, it was found the three-way junction (3WJ) of the Phi29 DNA packaging motor pRNA was found to be ultra-stable and assemble in solution without the presence of metal ions. The three-way junction is composed of three short oligo RNA strands and proven to be thermodynamically stable. Here the assembly mechanism, thermodynamic and enzymatic stabilities, and kinetics are examined in order to understand the stability behind this unique motif. Thermodynamic and kinetics studies found that the pRNA 3WJ formed out of three components at a rapid rate creating a single-step three component collision with a lack of dimer intermediate formation while being governed by entropy, instead of the commonly seen enthalpy. Furthermore, the pRNA 3WJ proved to be stable at temperatures above 50 °C, concentrations below 100 pM, and produced a free energy of formation well below other studied RNA structures and motifs. With the high stability and folding efficiency of the pRNA 3WJ, it serves as an ideal platform for multi-branched RNA nanoparticles constructed through bottom-up techniques. RNA nanoparticles were constructed for the specific targeting of prostate cancer cells expressing Prostate Specific Membrane Antigen (PSMA) by receptor mediated endocytosis through the addition of an RNA aptamer; and the delivery of anti-miRNA sequences for gene regulation. The resulting nanoparticles remained stable while showing highly specific binding and entry in PSMA positive cells through cell surface receptor endocytosis. Furthermore, the entry of the nanoparticles allowed for the knockdown of against onco-miRNAs. Nanoparticles harboring antimiRNAs led to the upregulation of tumor suppressor genes, and signaling of apoptotic pathways. These findings display RNA nanotechnology can result in the production of stable nanoparticles and result in the specific treatment of cancers, specifically prostate cancer.

BAKGRUND: Prostatacancer (PCa) är den vanligaste cancerformen bland män i Sverige. Efter kirurgisk behandling, radikal prostatektomi (RP), drabbas många av urininkontinens och sexuella besvär. Studier på kvinnor visar att urininkontinens påverkar sexuell lust men det finns få liknande studier bland män. SYFTE: Syftet med studien är att jämföra självskattad sexuell lust bland män med och utan inkontinens, ett år efter RP. METOD: Denna kvantitativa tvärsnittstudie utgår från enkätmaterial insamlat i en pågående prospektiv studie. Data insamlat bland 1751 män, ett år efter RP, har analyserats och jämförts mellan två grupper (fall-kontrollstudie). RESULTAT: Resultatet visar att män med urininkontinens skattar lägre sexuell lust, ett år efter RP, än män utan urininkontinens samt att de i högre grad beskriver tanken på ett fortsatt liv med sina sexualproblem med negativa begrepp. KONKLUSION: Män som genomgått RP, och i synnerhet de som har urininkontinens, har nedsättningar i sexuallivet. Sjuksköterskan bör uppmärksamma och ge tid till samtal om påverkan på sexuallivet efter RP, med både patient och partner. Ökad kunskap inom området bidrar till ökad förståelse, och behövs för att sjuksköterskor ska kunna hjälpa dessa patienter. Nedsatt sexuell lust hos män är ett outforskat område och vidare forskning behövs. / BACKGROUND: Prostate cancer (PCa) is the most common form of cancer among Swedish men. Urinary incontinence and sexual dysfunction are two side effects that many men experience after surgery, radical prostatectomy (RP). Previous studies show that urinary incontinence has negative effects on women’s sexual desire. Few similar studies concern men. AIM: The aim of the study is to compare self-reported sexual desire among men with and without urinary incontinence, one year post-surgical treatment (RP). METHOD: This quantitative, cross-sectional study is based on a self-reported survey from an ongoing prospective study. Data collected from 1751 men, one year post-treatment, have been analyzed and compared between two groups (case-control study). RESULT: The result shows that men with urinary incontinence after RP have less sexual desire than men without urinary incontinence. These men are also less satisfied with the idea of spending the rest of their lives with their sexual problems. CONCLUSION: Men that have undergone RP, especially the ones who have urinary incontinence, experience sexual dysfunction. Nurses need to acknowledge and initiate dialogues with patients and their partners about effects on sexual life after RP. Research concerning men’s sexual desire will help nurses’ knowledge and ability to discuss sexual problems. More studies about men’s reduced sexual desire are necessary.

In recent years, the causal relationship between inflammation and cancer has gained wider acknowledgement and acceptance. While various types of immune cells are involved in the process of inflammation, macrophages represent the major inflammatory component of many tumors. Derived from circulating monocytes, these cells migrate to tumor sites in response to molecular cues present within the tumor microenvironment. Once there, interactions with neoplastic cells shape the differentiation and functional orientation of macrophages into two phenotypically distinct subsets: the “classically” activated M1 macrophages and the “alternatively” activated M2 macrophages. The preeminent paradigm in macrophage-related cancer research is that within the tumor stoma, macrophages acquire an M2 phenotype characterized by production of pro-angiogenic factors, ECM degrading enzymes and up-regulation of anti-inflammatory responses, thereby promoting tumor progression. M1 macrophages, on the other hand are thought to exert anti-tumorigenic effects due to their production of pro-inflammatory cytokines, and reactive oxygen species (ROS). While the generation of ROS during immune responses is an important aspect of immune regulation and host defense, excessive ROS production has been implicated in the pathogenesis of various degenerative diseases, including cancer. Yet, despite the well-established role of M1 macrophages in generating high levels of ROS via NADPH oxidase (NOX), M1 macrophages are still largely viewed as anti-tumorigenic. Hence, this study reevaluates the complex interaction between prostate cancer (PCa) cells and tumor-associated macrophages (TAMs), and operates on the premise that PCa cells promote a pro-tumor microenvironment, denoted by increased inflammation and oxidative stress, in part, through M1 macrophage-mediated, NOX-derived ROS production. Accordingly, immunofluorescent analysis of prostate tissue microarrays demonstrated an influx of M1 macrophages in prostate carcinoma. Immature monocytes co-cultured with the poorly tumorigenic prostate cell line, LNCaP, demonstrated changes in morphology and protein expression consistent with M1 macrophage polarization. PCa cells co-cultured with M1 macrophages displayed significantly higher intracellular ROS levels. Furthermore, M1-mediated ROS generation through NOXs increased prostate cell invasiveness and anchorage-independent growth. Taken together, results from this study suggest a potentially novel pro-tumorigenic function of M1 macrophages in early PCa progression, and aid in understanding the complex role of inflammation in cancer.

Ο καρκίνος του προστάτη, αποτελεί ένα από τα σπουδαιότερα προβλήματα υγείας και συγκεκριμένα την τρίτη πιο συχνή αιτία θανάτου από καρκίνο στους άνδρες. Ο υποδοχέας ανδρογόνων είναι ένας σημαντικός μοριακός στόχος στη θεραπευτική αντιμετώπιση του καρκίνου του προστάτη. Η επιβίωση και ο πολλαπλασιασμός των καρκινικών προστατικών κυττάρων εξαρτώνται από τη δράση των ανδρογόνων, τουλάχιστον κατά τα πρώιμα στάδια της νόσου. Λόγω αυτής της εξάρτησης, η αποστέρηση των ανδρογόνων αποτελεί την αρχική μέθοδο φαρμακολογικής παρέμβασης για την αντιμετώπιση του καρκίνου του προστάτη. Ωστόσο, παρά την αρχική αποτελεσματικότητα αυτής της προσέγγισης στην καταστολή του όγκου, εκείνος επανακάμπτει εντός δύο ή τριών ετών και εξακολουθεί να αναπτύσσεται ανεξάρτητα από την ύπαρξη ανδρογόνων. Οι υπάρχουσες θεραπευτικές προσεγγίσεις αδυνατούν να προλάβουν την επανεμφάνιση του όγκου. Προς αυτήν την κατεύθυνση, η ανάπτυξη νέων θεραπευτικών παραγόντων με αποτελεσματικότερη αντιανδρογόνο δράση, θα είχε σημαντική αξία στη θεραπεία του καρκίνου του προστάτη. Πρόσφατα, στεροειδικά παράγωγα, τα οποία έφεραν έναν εξαμελή λακταμικό δακτύλιο στη θέση-20 του στεροειδικού σκελετού εμφάνισαν ανασταλτική δράση έναντι του ανδρογονικού υποδοχέα καθώς και έναντι του πολλαπλασιασμού καρκινικών προστατικών κυττάρων. Επίσης, παράλληλες μελέτες της ερευνητικής ομάδας μας έχουν δείξει ότι τροποποιημένα στεροειδικά παράγωγα που έφεραν είτε ενδοκυκλική λακταμική ή εξωκυκλική αμιδική -NHCO- ομάδα, έχουν εμφανίσει εξαιρετική δράση έναντι αιματολογικών νεοπλασιών. Με βάση τα παραπάνω δεδομένα, στα πλαίσια της παρούσας μελέτης πραγματοποιήθηκε ο σχεδιασμός και η σύνθεση νέων 20-αμινοστεροειδών. Συγκεκριμένα επιτεύχθηκε η σύνθεση νέων μορίων που φέρουν ως κύρια δομικά χαρακτηριστικά έναν επταμελή λακταμικό Α-στεροειδικό δακτύλιο και ένα αμινο-υποκατεστημένο στερεογονικό κέντρο (C-20). Το συνθετικό σχήμα που ακολουθήθηκε, περιελάμβανε αρχικά τη διεύρυνση του Α-στεροειδικού δακτυλίου της πρεγνενολόνης μέσω μετάθεσης Beckmann της αντίστοιχης 3-κετοξίμης σε επταμελή λακταμικό δακτύλιο και μετέπειτα τη διαστερεοεκλεκτική μετατροπή της 20-κετο-ομάδας σε αμινο-υποκατεστημένο στερεογονικό κέντρο μέσω νουκλεόφιλης προσθήκης οργανομαγνησιακού αντιδραστηρίου στην αντίστοιχη ενδιάμεση Ν-[t-butyl-σουλφινυλ]-20-ιμίνη. Κατά την εξέλιξη της συνθετικής πορείας διερευνήθηκαν και βελτιστοποιήθηκαν οι πειραματικές συνθήκες των επιμέρους σταδίων. Η αποτίμηση της βιολογικής δράσης των νέων 20-αμινοστεροειδών αναμένεται να αποσαφηνίσει κατά πόσο οι παραπάνω δομικές τροποποιήσεις μπορούν να συμβάλλουν στην εκδήλωση αντιανδρογόνου και αντικαρκινικής δράσης. Παράλληλα, ο στεροειδικός σκελετός των νέων μορίων είναι εφικτό να οδηγήσει σε νέα τροποποιημένα παράγωγα για την εξαγωγή σχέσεων χημικής δομής-βιολογικής δραστικότητας. / Prostate cancer is one of the most important health problems and specifically the third most common cause of death because of cancer in men. The androgen receptor is an important molecular target for the treatment of prostate cancer. The survival and the proliferation of cancer prostatic cells depend on the action of androgens, at least at the early stages of the disease. Because of this dependency, androgen deprivation is the initial method in the pharmacological intervention for the treatment of prostate cancer. However, despite the initial effectiveness of this approach in tumor suppression, the tumor relapses within two or three years and continues to grow regardless the presence of androgens. The existing therapeutic approaches fail to prevent the revival of the tumor. Toward this direction, the development of new therapeutic agents with more effective antiandrogen activity, would have significant value in the treatment of prostate cancer. Recently, steroid derivatives, which carried a six membered lactam ring at C-20 of the steroid skeleton, exhibited inhibitory activity against the androgen receptor and also against the proliferation of prostate cancer cells. Furthermore, parallel studies of our research group have shown that modified steroid derivatives which carried either a lactam or an amide-NHCO-group, have shown excellent activity against haematological malignancies. Based on these data, in this study the design and synthesis of new 20-aminosteroid was achieved. Specifically we achieved the synthesis of new molecules bearing as main structural features one seven membered lactam A-steroid ring and an amino-substituted stereogenic center (C-20). The synthetic approach used, initially involved the pregnenolone A-steroidal ring expansion to a seven membered lactam ring via Beckmann rearrangement of the corresponding 3-ketoxime and then the diastereoselective conversion of the 20-keto group to an amino-substituted stereogenic center via nucleophilic addition of organometallic reagent to the corresponding intermediate N-[t-butyl-sulfinyl]-20-imine. During this process, the experimental conditions of the intermediate synthetic steps were investigated and optimized. The biological evaluation of the new 20-aminosteroids is expected to unravel whether these structural modifications may contribute to antiandrogenic and anticancer activity. Meanwhile, the steroidal skeleton of the new molecules may lead to the development of new modified derivatives for further structure-activity relationship studies.

published_or_final_version / abstract / Molecular Biology / Master / Master of Philosophy

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Pathology / Doctoral / Doctor of Philosophy

The immunoproteasome, known to play an important role in MHC class I antigen processing and presentation, have been linked to neurodegenerative diseases and hematological cancers. However, the pathophysiological functions of the immunoproteasome in these diseases are still not very well established. This can be attributed mainly to the lack of appropriate molecular probes that selectively target the immunoproteasome catalytic subunits. Herein, we report the development of a small molecular inhibitor (AM) that selectively targets the major catalytic subunit, LMP2, of the immunoproteasome. We show that the compound covalently modifies the LMP2 subunit with high specificity in human prostate cancer cell. AM was also shown to selectively inhibit the chymotrypsin-like activity of LMP2 subunit. More importantly, the anti-proliferative activity of AM is more pronounced in prostate cancer cells that highly express LMP2 without inducing toxicity in normal cells. These results implicate an important role of LMP2 in regulating cell growth of malignant tumors that highly express LMP2. Subsequently, the modes of action of AM were investigated. Prostate cancer cells that highly express LMP were shown to induce G2/M cell cycle arrest and apoptosis via PARP cleavage when treated with the compound. Similar to epoxomicin, the treatment of AM induced the accumulation of poly-ubiquitination in prostate cancer cells, which indicates the inhibition of proteolysis. However, unlike epoxomicin, the treatment of AM did not appear to inhibit the activation of inflammation. In conclusion, these results suggest that the LMP2 inhibitor, AM, may induce cytotoxicity prostate cancer cells that highly express LMP2 catalytic subunit in similar modes of action as epoxomicin but it does not involve the inflammatory pathway.

Prostate cancer (PCa) has been the leading cause of cancer death in men since1930. While studies pertaining to PCa have primarily focused on the disease and the subsequent side effects of treatment, psychological distress in this group has yet to be adequately addressed. The purpose of this dissertation was to: 1) conceptualize health related quality of life (HRQL) and health seeking behavior of men by describing lifestyle, cultural and health risks associated with being male, 2) evaluate the psychometric properties of the SF-12 Health Survey (SF-12) combined with the urinary and sexual portions of the UCLA PCa Index (UCLA-PCI), 3) investigate the psychological impact, coping styles and informational needs of a group of men diagnosed with PCa who have not yet undergone treatment. Men have been observed to underutilize health care services despite the fact that they are in poorer health, have higher mortality rates and lower life expectancies than women. Restricting emotions, being oriented toward success, having limited social networks and taking health risks are often associated with being male. Because incontinence and sexual dysfunction, the two most common side effects of PCa treatments impact men's quality of life, portions of the UCLA-PCI and the SF12 were analyzed. The psychometric analysis of the SF-12/UCLA-PCI and its three subcomponents confirmed the validity of the instrument. The SF-12 component had a Cronbach's alpha of 0.87, while the urinary and sexual subscales had a Cronbach's alpha of .86 and .91 respectively. All three scales were found to have good internal consistency. KEYWORDS: prostate cancer, health related quality of life, coping styles, psychological distress.

Background: Low-grade chronic inflammation has been implicated as a risk factor in prostate-related pathologies including benign hyperplasia and cancer. Sweet cherry containing the bioactive anthocyanin (ACN), has demonstrated tumor inhibitory action in model systems, specifically inhibition of inflammatory molecules and prostaglandin biosynthesis. Objective: To assess the urinary and plasma concentrations of ACN from the daily consumption of 3 cups of sweet cherries for 4 weeks and test the relationship of ACN levels and cherry consumption to inflammatory biomarkers in an at risk population. Results: Prostaglandin E2 Metabolite (PGEM) levels were reduced with cherry consumption in men with elevated baseline values. Conclusion: We conclude that 1c (142g) of sweet cherries 3 times daily for 4 weeks significantly reduced the COX-2 metabolite, PGEM, in men with elevated baseline levels. This was the first study to examine the chronic effects of daily sweet cherries on COX-2 inhibition in an at risk population.