Includes bibliographical references. / Biospecimens1 collected during routine oncology diagnostic and therapeutic interventions may be stored for future medical purposes. In accordance with legal and ethical principles, the patient provides informed consent for removal of the tissue for diagnostic or therapeutic reasons. Informed consent gives permission for the violation of bodily integrity that is inevitable with tissue removal.

Includes bibliographical references. / Public perceptions of vaccination have changed greatly since George Bernard Shaw unleashed his diatribe against the practice. Today it is recognised that, far from spreading disease, vaccination is one of the cheapest and most effective public health interventions. Immunization against infectious diseases has drastically reduced mortality and morbidity, particularly among children, and has diminished the disease burden caused by poliomyelitis, rubella, measles, tetanus, diphtheria and whooping cough, amongst others.

The report examines how the market, as well as purification conditions, forpeptide-oligonucleotide conjugates look like today. These conjugates are intended to be usedin drugs to treat otherwise incurable diseases but so far these types of molecules are still intest-phases and no finished conjugate is on the market yet. However, the drug SRP-5051 is inclinical phase II and it is expected to be seen on the market no earlier than May 2022. Thedrug consists of a phosphorodiamidate morpholino oligomer conjugated to a cationiccell-penetrating peptide. The aim of the project was to gain an understanding of what currentresearch looks like, what types of conjugates seem to be the most promising and whatchallenges there are with these. In addition, various purification possibilities for the mostpromising conjugates were also investigated. The report is a literature study and containsinterviews with experts in the field. The oligonucleotide most likely to occur in a futureconjugate consists of a charge-neutral phosphorodiamidate morpholino oligomer (PMO) orpeptide nucleic acid (PNA), which are spinal modifications that increase stability and affinity.The peptide in such a conjugate needs to perform endocytosis and is therefore expected to becationic. The amount of arginine in the peptide is correlated with both efficiency and toxicity,which is the major challenge of the conjugates today. The purification is performed with acation exchanger where the cationic charge of the peptide is utilized. Elution takes place witha salt gradient, if necessary a secondary purification step in the form of size exclusion is used.

The work presented in the thesis concerned theoretical study of light-matter nonlinear interactions. Two important aspects of such interactions have been examined, namely the nonlinear optical properties of a series of organic charge transfer molecules in solutions induced by the laser light and the propagation of the ultrafast high power laser through the nonlinear molecular medium. Special attention has been paid to understand the solvent effects on the two-photon absorption of a symmetrical diamino substituted distyrylbenzene chromophore, for which time-dependent density functional theory in combination with polarizable continuum model (PCM) have been employed. The dielectric medium alone has a rather small effect both on the bond length alternation and on the one-photon absorption spectrum, but noticeable effects on the two-photon absorption cross section. Both one- and two-photon absorptions are found to be extremely sensitive to the planarity of the molecule. Our calculations indicate that the experimentally observed anomalous solvent effect on the two-photon absorption of dialkylamino substituted distyrylbenzene chromophores can not be attributed to the intrinsic properties of a single molecule and its interaction with solvents. With the same theoretical approaches, two-photon absorption properties of interacting polar chromophores have been investigated to examine the validity of the widely used exciton model. Our first principles calculations have shown that the exciton model offers a conceptually simple interpretation for experimental observations, but is lack of predictability. The second part of the thesis is to investigate the propagation of ultrashort laser pulse through a one-dimensional asymmetric organic molecular medium by solving full Maxwell-Bloch equations using predictor-corrector finite-difference time-domain method. It focuses on the supercontinuum generation of spectra and the formation of attosecond pulses. It is shown that the supercontinuum generation is strongly modulated by both area and width of the pulse, which results from the interference between the splitting pulses in time-domain and is the implication of the time-energy uncertainty relation. The presence of permanent dipole moment in molecular medium has noticeable effects on the supercontinuum generation. Our calculations show that a well-shaped 132 attosecond pulse can be generated from a two femtosecond incoming pulse under certain conditions. Influences of carrier-envelope phase and time-dependent ionization on the spectral and temporal evolutions of the ultrashort pulses have also been discussed. / QC 20101122

The adrenergic hormones norepinephrine (NE) and epinephrine (EPI) are critical regulators of mammalian cardiovascular physiology. NE and EPI mediate stress responses to enhance cardiovascular function, however dysregulation of adrenergic signaling leads to heart failure, congenital heart malformations, and sudden cardiac death. Adrenergic hormone-expressing cells were found in the early embryonic heart, and NE has been determined essential for embryonic cardiac development. Despite extensive work in adults, the regulatory roles and adrenergic targets of these hormones during embryonic cardiac development have not yet been fully determined. Prior transcriptomic studies from our lab showed that expression of signal transduction and metabolic genes in embryos lacking adrenergic hormones were by far the most affected categories of genes. Thus, we hypothesized that adrenergic hormones stimulate early calcium signaling, and are required for sufficient supply of energy substrates for the metabolic shift from anaerobic glycolysis to aerobic respiration during heart development. We utilized the dopamine ?-hydroxylase knock-out (Dbh-/-) mouse model to examine effects of adrenergic-deficiency on calcium signaling and metabolism during heart development. Using calcium-imaging and patch-clamp techniques, we found that calcium transients, voltage-gated calcium channels, and L-type calcium currents in adrenergic-deficient embryonic hearts were not affected relative to controls indicating adrenergic stimulation did not influence early calcium signaling. Metabolomics analyses of adrenergic-deficient hearts revealed disruption in glycolytic and pentose-phosphate pathways as well as reduced activity of respective regulatory enzymes, glyceraldehyde 3-phosphate dehydrogenase and glucose 6-phosphate dehydrogenase indicating compromised glucose metabolism. Addition of pyruvate to embryonic hearts led to significant recovery of ATP concentrations and oxygen consumption rates, thereby supporting the hypothesis that adrenergic-deficient hearts are "starved" of metabolic substrates required for transitions from anaerobic glycolysis to aerobic metabolism. Overall, we showed that adrenergic hormones are not necessary for calcium signaling in the embryonic heart, but are essential regulators ensuring sufficient metabolic substrate and boosting enzymatic activities to fuel aerobic metabolism.

Nociceptive afferent axons of the stomach send signals centrally to the brain and locally to stomach tissues. Nociceptive axons can be detected with a variety of different markers. In particular, the neuropeptide substance P (SP) is one of the most commonly used markers for nociceptive nerves in the somatic and visceral organs. In addition, the Local release of SP contributes to tissue protection and repair in response to noxious stimuli. However, the topographical distribution and morphological structure of SP-immunoreactive (SP-IR) axons and terminals in the whole stomach have not yet been well determined. In this study, we labeled SP-IR axons and terminals in flat-mounts and serial sections of the antrum, pyloric sphincter, and duodenum (APD) of the mouse stomach. The flat-mount stomachs included the external muscles (longitudinal and circular layers) and the myenteric ganglionic plexus, whereas APD serial sections also included the submucosa and mucosa. Tissue samples were scanned using confocal and Zeiss M2 imager microscopy to acquire detailed high-resolution images of SP innervation and produce montages of the whole stomach flat-mount and APD serial sections. Additionally, we double-labeled the samples with vesicular acetylcholine transporter (VAChT) and tyrosine hydroxylase (TH), and injected the stomach walls with DiI-retrograde tracer to determine the connectivity map between SP-IR axons and their possible extrinsic origin. We found that: 1) SP-IR axons innervated all layers including the external muscles, myenteric ganglia, submucosa, submucosal ganglia, and mucosa. Many axons were immunoreactive for VAChT but not TH. 2) SP-IR axons and terminals formed an extensive network in the muscular layers and ran in parallel with the long axis of the external muscles. 3) SP-IR axons formed very dense terminal varicosities encircling individual neurons in the myenteric plexus 4) In the submucosa, SP-IR axons innervated blood vessels and submucosal ganglia and formed a nerve network around duodenal Brunner's glands. 5) In the mucosa, SP-IR varicose axons were organized into bundles in the muscularis mucosa and lamina propria at the base of the mucosa. Some SP-IR axons were found in the gastric subepithelial level and duodenal villi. 6) SP-IR axon density in the muscles and myenteric plexus was much higher than in the submucosa and mucosa. 7) The density of SP-IR axons and terminals in the flat-mounts varied in different stomach regions in the following order from high to low: antrum-pylorus, corpus, fundus, and cardia. 8) In the APD area, the muscular wall of the antrum and duodenum showed a higher SP-IR axon density than in the pyloric sphincter. 9) The extrinsic origin of SP-IR axons in the stomach derived mainly from the spinal T7-T11 dorsal root ganglia with a lesser contribution from the vagal nodose ganglia, but not from the celiac ganglia, the dorsal motor nucleus of vagus, or the nucleus tractus solitarius. Collectively, our data provide for the first time a map of the topographical distribution and morphology of SP-IR axons and terminals in the whole stomach with single-cell/axon/synapse resolution. This work will establish an anatomical foundation for functional mapping of the SP-IR nociceptive afferent innervation of the stomach and its pathological remodeling in gastrointestinal diseases.

Lung cancer is the leading cause of cancer death and the second most common cancer in the United States. Targeted therapies have led to an increase in survival gains; however, their use is dependent on presence of druggable mutations and hindered by emergence of drug resistance. 5-Aminoimidazole-4-carboxamide 1-ß-d-ribofuranoside (AICAR or Acadesine) is a natural metabolite of de novo purine synthesis pathway, catalyzed by adenylosuccinate lyase (ADSL), and it is a precursor to 5-amino-4-imidazole carboxamide ribonucleoside 5'-phosphate (ZMP). ZMP mimics adenosine monophosphate (AMP) and activates adenosine monophosphate-activated protein kinase (AMPK) and has been shown to specifically induce apoptosis in aneuploid cells. Cell viability assay was used to establish that AICAR can induce cytotoxicity in EGFR and KRAS mutated lung cancer cells irrespective of the mutational status.; thereby, making AICAR a potential anti-cancer drug candidate. However, AICAR's mechanism of cytotoxicity is not understood in lung cancer. FINDSITEcomb2.0 analysis, western blot and thermal shift binding assay identified the cytoplasmic tail of mucin 1 (MUC1-CT), a membrane glycoprotein overexpressed and aberrantly glycosylated in adenocarcinomas, is directly bound and degraded by AICAR. We also found that AICAR can disrupt Janus kinase 1 (JAK1) and MUC1-CT interaction and downregulate JAK1 and epidermal growth factor receptor (EGFR) signaling pathways. Moreover, EGFR mutant patient-derived xenograft tumor and mouse lung tumor tissue-derived organoids showed an enhanced reduction in size with co-treatment of AICAR, EGFR and JAK inhibitors. Moreover, AICAR significantly reduced xenograft tumor weight and size in nude mice without body weight loss Furthermore, MUC1 overexpression rescued AICAR-induced cytotoxicity in H1975 organoids. Therefore, this study is the first to discover MUC1 as the novel binding partner of AICAR and modulator of AICAR's toxicity and the first to show that AICAR can disrupt JAK1-MUC1-CT interaction.

Doxorubicin (Dox) has been one of the most effective chemotherapeutic drugs against a wide range of cancers. The use of Dox is restricted due to severe clinical side effects including Dox-induced skeletal muscle toxicity (DIMT). The exact molecular mechanisms in the progression and development of DIMT have not been established. We investigated the roles of inflammatory cell deaths pyroptosis and necroptosis in DIMT. Additionally, we explored the therapeutic potential of embryonic stem cell-derived exosomes (ES-Exos) in diminishing DIMT. Mice were used to develop DIMT and pre-diabetic animal models of muscle toxicity. Mice were subjected to muscle function and glucose tolerance testing. Soleus and gastrocnemius tissues were collected for protein expression assays including immunohistochemistry, western blot, ELISA, and RT-PCR to determine pyroptosis, inflammasome formation, M1 and M2 macrophages, necroptosis and necroinflammation levels. Histology staining was performed to determine atrophy and fibrosis. Muscle strength of mice were significantly (p < 0.05) diminished for Dox vs. controls whereas ES-Exos significantly (p < 0.05) mitigated this muscle dysfunction by enhancing muscle strength. In parallel, our results showed a significant (p < 0.05) increase in muscle atrophy, fibrosis, inflammasome formation leading to inflammation-induced cell death pyroptosis, necroinflammation, and pro-inflammatory M1 macrophage markers for Dox compared with controls. Treatment with ES-Exos significantly (p < 0.05) reduced atrophy, fibrosis, inflammation, inflammatory cytokines, and pyroptosis. Noticeably, ES-Exos treatment significantly enhanced anti-inflammatory M2 macrophages in the muscle. Necroptosis and necroinflammation markers, along with atrophy and fibrosis, were significantly (p < 0.05) increased in the combined model of Dox and pre-diabetes compared with pre-diabetes alone, suggesting that Dox administration in pre-diabetic conditions have enhanced detrimental effects. Overall, this novel study establishes the mechanistic roles of inflammatory cell deaths pyroptosis and necroptosis, inflammatory cytokines, and inflammatory macrophages in skeletal muscle toxicity. Moreover, we established the protective effects of Exos in muscle toxicity models involving Dox.

DCUN1D1 is an E3 ligase of the neddylation pathway. It mediates the posttranslational modification of majority of the cullin family of proteins with NEDD8. This activity is known to enhance ubiquitination of the cullin RING E3 ligases, however, the extent of the impact of DCUN1D1's activity is underexplored. Studies performed previously in our lab demonstrated the role of DCUN1D1 in prostate cancer in vitro and in vivo. We also identified potential inhibitors of DCUN1D1 which inhibited the proliferation of prostate cancer cells in a DCUN1D1-specific manner. This study seeks to determine the mechanism of action of DCUN1D1 in prostate cancer and to identify DCUN1D1 inhibitors using a proteomics approach. Immunoprecipitation-coupled mass spectrometry was performed to identify DCUN1D1 binding partners and we identified some known substrates of DCUN1D1 in the form of cullin 3, cullin 4B and cullin 5. We also observed that the DCUN1D1 pulldown products implicated the ubiquitin proteasome pathway, transcription, lipid metabolism and inflammatory pathways. SILAC quantitative proteomics analysis was also performed to determine the proteins that were differentially expressed in DU145 DCUN1D1 knockdown cells relative to DU145 control cells. Interestingly, we did not identify the cullin proteins or classical components of the neddylation pathway but identified the ubiquitin activating enzyme, UBA1. We also found that dysregulation of DCUN1D1 in prostate cancer led to a dysregulation in translation-related and protein processing activities such as dysregulation of eukaryotic protein translation, and protein processing in the endoplasmic reticulum. We also observed the recurrence of the WNT signalling pathway across the proteomics approaches. This culminated in the exploration of the mechanism of action of DCUN1D1 in prostate cancer using changes in protein expression as measured by western blot analysis. Significantly, we determined that DCUN1D1 mediates its mechanism of action in prostate cancer, through the neddylation pathway and preferential neddylation of cullin proteins. We also observed that knockdown of DCUN1D1 in prostate cancer led to the dysregulation of the ubiquitination and WNT/β-catenin pathways. Furthermore, advanced connectivity map analysis was performed to identify potential inhibitors of DCUN1D1 based on a proteomics approach. The drugs found to strongly connect with the DCUN1D1 knockdown signature included kinase inhibitors and anti-inflammatory agents. The above observations could lead to improved understanding of DCUN1D1 and its potential for molecular target based treatment of prostate cancer.

The development of computational models for predicting drug solubility has increased drastically during the last decades. Nevertheless these models still have diffculties to estimate the aqueous solubility as accurate as desired. In this thesis di erent aspects that are known to have a large impact on the aqueous solubility of a molecule have been studied in detail using various theoretical methods with intension to provide microscopic view on drug solubility. The rst aspect studied is the hydrogen bond energies. Eight drug molecules have been calculated using density functional theory and the validity of additive model that has often been used in solubility models is examined. The impact of hydrogen bonds in Infrared and Raman spectra of three commonly used drug molecules has also been demonstrated. The calculated spectra are found to be in good agreement with the experimental data. Another aspect that is important in solubility models is the volume that a molecule occupies when it is dissolved in water. The volume term and its impact on the solvation energy has therefore also been calculated using three di erent methods. It was shown that the calculated volume di ered signi cantly dependent on which method that had been used, especially for larger molecules. Most of the solubility models assume the solute molecule to be in the bulk of the solvent. The molecular behavior at the water/gas interface has been investigated to see how it di ers from bulk. It was seen that the concentration close to the interface was almost three times higher than in the bulk. The increase in concentration close to the surface depends on the larger gap between the interface energy and the gas phase energy than between the bulk energy and the gas phase energy. / QC 20101109