Indiana University-Purdue University Indianapolis (IUPUI) / Several pathological disorders are characterized by maladaptive decision-making (Dalley & Robbins, 2017). Decision-making tasks, such as Delay Discounting (DD), are used to assess the behavioral manifestations of maladaptive decision-making in both clinical and preclinical settings (de Wit, Flory, Acheson, Mccloskey, & Manuck, 2007). DD measures cognitive impulsivity and broadly refers to the inability to delay gratification (Hamilton et al., 2015). How decisions are made in tasks that measure DD can be understood by assessing patterns of behavior that are observable in the sequences of choices or the statistics that accompany each choice (e.g. response latency). These measures have led to insights that suggest strategies that are used by the agent to facilitate the decision (Linsenbardt, Smoker, Janetsian-Fritz, & Lapish, 2016). The current set of analyses aims to use individual trial data to identify the neural underpinnings associated with strategy transition during DD. A greater understanding of how strategy change occurs at a neural level will be useful for developing cognitive and behavioral strategies aimed at reducing impulsive choice. The rat dorso-medial prefrontal cortex (dmPFC) has been implicated as an important brain region for recognizing the need to change strategy during DD (Powell & Redish, 2016). Using advanced statistical techniques, such as demixed principal component analysis (dPCA), we can then begin to understand how decision representations evolve over the decision- making process to impact behaviors such as strategy change. This study was the first known attempt at using dPCA applied to individual sessions to accurately model how decision representations evolve across individual trials. Evidence exists that representations follow a breakdown and remapping at the individual trial level (Karlsson, Tervo, & Karpova, 2012; Powell & Redish, 2016). Furthermore, these representational changes across individual trials have previously been proposed to act as a signal to change strategies (Powell & Redish, 2016). This study aimed to test the hypothesis that a ‘breakdown’ followed by a ‘remapping’ of the decision representation would act as a signal to change strategy that is observable in the behavior of the animal. To investigate the relationship between trials surrounding the breakdown and/or subsequent remapping of the decision representation and trials surrounding strategy changes, sequences of trials surrounding the breakdown and/or remapping were compared to sequences of 9 trials surrounding the strategy-change trial. Strategy types consisted of either exploiting the immediate lever (IM-Exploit), delay lever (DEL-Exploit), or exploring between the two lever options (Explore). Contrary to the hypothesis, an overall relationship between breakdown and remapping trial sequences were not associated with change-trial sequences. In partial support of the hypothesis however, at the 4-sec delay when the subjective value of the immediate reward was high, a relationship between breakdown sequence and strategy change sequence was detected for when the animal was exploiting the delay lever (e.g. DEL-Exploit strategy). This result suggests that a breakdown in decision representation may act as a signal to prompt strategy change under certain contexts. One notable finding of this study was that the decision representation was much more robust at the 4-sec delay compared to the 8-sec delay, suggesting that decisions at the 4-sec delay contain more context that differentiate the two choice options (immediate or delay). In other words, the encoding of the two choice options was more dissociable at the 4-sec delay compared to the 8-sec delay, which was quantified by measuring the average distance between the two representations (immediate and delay) on a given trial. Given that Wistar rats are equally likely to choose between the immediate and delay choice alternatives at the 8-sec delay (Linsenbardt et al., 2016), this finding provides further support for current prevalent theories of how animals use a cognitive search process to mentally imagine choice alternatives during deliberation. If context which differentiates choice options at the 8-sec delay is less dissociable, it is likely that the cognitive search process would be equally likely to find either choice option. If the choice options are equally likely to be found, it would be assumed that the choice alternatives would also be equally likely to be chosen, which is what has been observed in Wistar rats at the 8-sec delay.

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Hepatitis B virus encoded X protein (HBx) contributes centrally to the pathogenesis of hepatocellular carcinoma (HCC). Aberrant activation of the Hedgehog (Hh) pathway has been linked to cancer. Thus, experiments were designed to test the hypothesis that HBx contributes to HCC via activation of Hh signaling. HBx expression correlated with up-regulation of Hh markers in human liver cancer cell lines, in HBx transgenic mice that developed HCC and in liver samples from HBV infected patients with HCC. The findings in human samples provide clinical validation of those in the HBx transgenic mice (HBxTg), and underscore the relevance of these transgenic mice to disease pathogenesis. Further, blockade of Hh signaling inhibited HBx stimulation of cell migration, anchorage independent growth, HCC tumorigenesis in HBx transgenic mice and tumor growth in xenograft model. These results suggest that the ability of HBx to promote cancer is at least partially dependent upon Hh activation and that activation of Hh signaling appears to be important for the development of HBx associated HCC. HBx also activates pathways that stimulate downstream Hh signaling, such as PI3K/AKT and Ras/Raf/MEK, also referred as non-canonical Hh signaling. Upon canonical Hh inhibition, compensatory activation of these pathways was seen in the presence of HBx in liver cancer cell lines and in HBxTg mice. Individual inhibition of these pathways also down-regulated Gli2 expression in HBx positive cell lines. These data suggests that in addition to canonical Hh signaling, activation of PI3K/AKT and ERK pathways by HBx leads to up-regulation of Gli2 expression in HBV-mediated HCC. This work identifies Hh pathway inhibition as a therapeutic strategy to slow tumor development and this work could lead to combination therapies that target Hh, AKT and ERK pathways, which may prevent or delay the appearance/progression of HCC. / Biology

ONC201 is a novel compound that upregulates endogenous TNF-Related Apoptosis-Inducing Ligand (TRAIL), in tumor and normal cells, restoring autocrine and paracrine anti-tumor activity within tumor cells, and upregulates the DR5 gene by activating the integrated stress response, inducing eIF2-alpha-dependent ATF4 and CHOP [1-3]. ONC201 also demonstrates potent anti-tumor effects on colorectal cancers [4, 5]. ONC201 presented a promising oral bioavailability, wide distribution throughout the body, and ability to cross the blood-brain barrier. Further, the unique ability of its TRAIL-and-DR5-based signaling to induce apoptosis in cancer cells and not normal cells adds to its appeal as an anti-cancer therapeutic and prompted clinical development [1-4, 6]. ONC201 has successfully completed an FDA advanced Phase I/II clinical trial in advanced aggressive refractory solid tumors. Results indicated that ONC201 is well-tolerated and recommended a phase II dose of 625 mg orally every 3 weeks. Several Phase I/II clinical trials are enrolling in multiple solid tumors and hematological malignancies [7, 8]. Chapter two of this study provides evidence that ONC201 dose intensification demonstrates an increased pharmacodynamic effect and an increasing anti-tumor efficacy in vivo while having a safe toxicity profile upon weekly dosing. This data influenced the Phase II clinical trials, which have now been adjusted to include weekly dosing. Given the potential anti-metastatic effects of TRAIL signaling and the role of TRAIL in the immune surveillance of cancer, we hypothesized that ONC201 would suppress metastatic tumor development and engage the immune system in its anti-cancer activity. We also establish that ONC201 provides an important anti-metastatic effect in CRC that should be pursued in both pre-clinical and clinical studies. Tail vein and surgical CRC models demonstrate that ONC201 inhibits the number and size of metastases. Evidence has shown that TRAIL can also inhibit cancer metastasis by possibly inducing cell death or TRAIL-sensitization in the primary tumor when cells undergo extravasation upon detachment from the primary tumor [9-11]. While we show that TRAIL plays a role in ONC201’s ability to inhibit migration/invasion in vitro, further investigation of the role of TRAIL in vivo is necessary. Our data indicates that ONC201 promotes a pro-immune response in CRC subcutaneous tumors with increased NK cells that play a role in ONC201’s efficacy in syngeneic models. Since activated natural killer cells express TRAIL, we established that ON201 can activate and induce TRAIL expression in NK cells [12, 13]. As we did not find any immune infiltrates in the metastases, we suggest that the effect of the micro-environment or in more clinically-relevant models with stromal environments should be pursued. Chapter 3 of this of thesis demonstrates the characterization of ONC201’s core structure and development of ONC201 analogues including their mechanistic differences and potential in vivo efficacy and safety. We have demonstrates the importance of the angular structure of ONC201 to ONC201’s anti-tumor efficacy [14]. The novel pharmacophore has now been called as imipridone and is essential for its anti-tumor activity, as the linear isomer had no anti-tumor effect. We leveraged this unique pharmacophore to synthesize ONC201 analogues with distinct therapeutic properties; namely, targeting ONC201-resistant tumor types or possessing distinct signaling properties. Imipridone R2 analogues have a lower IC50 and are more promising than their lead compound in certain tumor types. ONC212, a halide R2 analogue, demonstrates superior efficacy in vivo in melanoma xenografts, a large therapeutic window; but does have a rapid PK. Oncoceutics is currently developing ONC212 for a first-in human Phase I clinical trial. The fourth chapter of this study demonstrates the potential of a combinational therapy with ONC201 in colorectal cancer with bevacuzimab. Clinical trials in CRC and other tumor types have demonstrated that therapeutics targeting the vascular endothelial growth factor (VEGF) pathway, such as bevacizumab, are effective in combination with certain chemotherapeutic agents. ONC201 in combination with bevacuzimab led to superior results with almost no tumor growth. This result was re-capitulated in syngeneic models. Given that bevacuzimab is approved for metastatic CRC, we suggest that ONC201 in combination with bevacuzimab should be introduced into a combinatorial Phase II clinical trial. This thesis focuses on the importance of dose-intensification of ONC201 on its pre-clinical efficacy; establish an anti-metastatic effect; demonstrate an immune increase in subcutaneous models; and elucidate the role of the core angular structure in efficacy with the development novel ONC201 analogues. The importance of pre-clinical studies, ONC201’s analogues including the successful development of ONC212, and potentially advantageous combinational therapies with anti-angiogenics is explained in the chapters throughout. / Cancer Biology & Genetics

Alzheimer’s disease (AD), one of the major causes of disability and mortality in Western societies, is a progressive age-related neurodegenerative disorder. Increasing evidence suggests the etiology of AD may involve disruptions of zinc (Zn) homeostasis. We hypothesize that disruption of Zn homeostasis leads to alterations of Zn transporter (ZnT) proteins, resulting in increased production of neurotoxic amyloid beta (Aβ) peptide in AD brain. To address this hypothesis we carried out the following studies. 1. We characterized alterations of ZnT-1, ZnT-4 and ZnT-6 in the brain of preclinical AD (PCAD) subjects, who show no overt clinical manifestations of AD but demonstrate significant AD pathology at autopsy. 2. We identified the presence of ZnT-2 in human brain and compared protein levels in the brains of subjects with PCAD, mild cognitive impairment (MCI), early (EAD), and late-stage AD (LAD) to those in age matched normal control (NC) subjects. 3. We examined the relationship between protein levels of ZnT-1, ZnT-2, ZnT-4, ZnT-6 and Aβ produced by H4 human neuroglioma cells (H4-APP) transfected to overexpress amyloid precursor protein (APP), treated with short interfering RNA (siRNA) against each ZnT. Our data show a significant decrease (P < 0.05) of ZnT-1 and a significant increase of ZnT-6 in hippocampus/parahippo-campal gyrus (HPG) of PCAD subjects. In PCAD cerebellum (CER) the data show a significant increase of ZnT-4 and ZnT-6 compared to NC subjects. Levels of ZnT-2 were also significantly decreased in HPG of PCAD subjects compared to NC subjects. In addition, levels of ZnT-2 were significantly (P < 0.05) elevated in SMTG of PCAD and MCI subjects, compared to NC subjects. ZnT-2 was significantly (P < 0.05) elevated in HPG of EAD and LAD, and in SMTG of LAD brains, but was significantly (P < 0.05) decreased in LAD CER compared to NC subjects. siRNA mediated attenuation of each ZnT protein studied (ZnT-2, ZnT-4 and ZnT-6) led to significantly (P < 0.05) decreased production of Aβ compared to controls. Our results suggest alterations in Zn transport may play a role in Aβ processing and contribute to the neuropathology of AD.

This dissertation project aimed to demonstrate that pulmonary delivery of two anorectic gut secreted peptides, peptide YY (PYY) and oxyntomodulin (OXM) enabled food intake suppression and reduced body weight gain in rats via their systemic absorption from the lung and interaction with the brain. After PYY and OXM were administered to the lungs at varying doses, food intake and body weight gain were monitored in freely feeding rats. Significant 30-35 % food intake suppression was achieved for 4-6 h following pulmonary administration of endogenously active PYY3-36 and OXM1-37 at 0.80 and 0.50 mg/kg, respectively. Moreover, when administered daily for 7 days, these peptides enabled significant reduction of body weight gain by 39.4 and 62.3 %, respectively. However, neither of their active fragment peptides, PYY13-36, OXM30-37 and NAc-OXM30-37 was effective at doses equimolar to the effective doses of PYY3-36 and OXM1-37. For PYY3-36, its pulmonary administration caused c-Fos activation in the hypothalamus arcuate nucleus (ARC) only, which was concurrent to reduced orexigenic neuropeptide Y (NPY), suggesting its appetite suppression was mediated via the central nervous system (CNS). In contrast, OXM1-37 caused c-Fos activation in both the hypothalamus ARC and brainstem AP, which implied the involvement of the CNS control and vagal stimulation for this peptide. As it was clear that these effects resulted from their lung absorption and increased plasma levels, the pharmacokinetics of one of the peptides, PYY3-36 was characterized following pulmonary administration. The plasma profiles were dose-proportional and kinetically, non “flip-flop”, yielding the highest PYY3-36 concentrations (Cmax) of 75.0±9.3 and 726.3±69.0 ng/ml at 0.08 and 0.80 mg/kg, respectively, at 10 min. According to a new kinetic model developed in this project, the percent absolute bioavailability (% F) was estimated to be 12-14 %, as derived from the lung absorption (ka) and non-absorptive loss rate constant (knal) of 0.03 min-1 and 0.17-0.22 min-1, respectively. Overall, this research provided the first proof-of-concept for effective appetite suppression with pulmonary delivery of anorectic gut secreted peptides via systemic absorption.

A medicina nuclear é a área da ciência médica que usa radiofármacos, isto é um radionuclídeo ligado a uma molécula de interesse biomédico, para estudar o funcionamento do organismo. Com isso, essa ciência pode realizar diagnósticos de patologias ou tratamento oncológico. Os estudos pré-clínicos, estudo com pequenos animais, são requisitos de suma importância para o desenvolvimento dessa área. São esses estudos que permitem os testes de novos radiofármacos que possivelmente serão usados em humanos. Quando se trata de pequenos animais, os órgãos de estudo são muito pequenos, portanto é vital que o equipamento tenha uma ótima resolução espacial e boa sensibilidade. Na medicina nuclear, a tomografia por emissão de pósitron (Positron Emission Tomography, PET) tem as características necessárias para desenvolver esses estudos com pequenos animais. Os animais mais usados são ratos e camundongos, e como nem sempre esses animais representam modelo humano seria interessante ter um equipamento que também funcionasse em outros animais. Sendo esse tomógrafo caro, não é viável ter um equipamento para cada tipo de animal. O presente trabalho propõe justamente um único tomógrafo que possa ser usado em diferentes animais ou eventualmente em dois animais simultaneamente. Isso será alcançado variando o anel de detecção usado nesse tipo de tomógrafos. Isso é alcançado mais facilmente em geometrias retangulares, pois com apenas 4 hastes se consegue variar a distância máxima entre os detectores preservando a forma da geometria. O custo envolvido na construção física desse tomógrafo é elevado, consequentemente é interessante ter um teste preliminar que forneça dados que possam sustentar essa ideia. A melhor alternativa para esse teste é o uso de simulação computacional. GATE (Geant4 Application for Tomographic Emission) foi o programa escolhido para essa simulação, porque já é um software validado, isto quer dizer que é compatível com experimentos reais. Assim sendo, quatro simulações foram montadas, duas para geometrias circulares com diâmetros diferentes, e analogamente, duas quadradas. Para verificar qual das geometrias tem melhor performance, foram usados os métodos sugeridos pela norma NEMA NU 4-2008. Efetuados esses testes, pode-se observar que as geometrias quadradas tem resolução semelhante às circulares. A sensibilidade e a relação sinal-ruído são maiores nas geometrias quadradas. Portanto, conclui-se que no geral, as geometrias retangulares simuladas são melhores que as circulares. Esse resultado é motivador para dar início à construção física do tomógrafo, pois o mesmo permite desenvolver novos produtos de modo mais eficiente e com menos custo / Nuclear medicine is an area of medical science that uses radiopharmaceutical (a radionuclide bounded to a molecule of biomedical interest) to study human physiology by images. Thus, this science can perform diagnostics of diseases and eventually, in specific cases, cancer treatment. Before using a new radiopharmaceutical in humans it is necessary to test it in small animals. The organs in these animals are very small, consequently it is vital that the equipment has a great spatial resolution and high sensitivity. In nuclear medicine Positron Emission Tomography (PET) has this requirement. The most animal used to develop this kind of studies are rats and mice. However, they are not always representing human animal model, so equipment that works to other animals also it would be interesting. But it is not feasible to have equipment for each animal, because it will be very expensive. Therefore, the aim of this work is test one scanner that can be used in different animals or possibly in two animals at the same time. This will be achieved by varying the detection ring used in this type of scanners (PET). To do it, it is easier using rectangular geometries because moving only four stalks the distance between the detectors can be varied preserving the shape of the geometry. The cost involved in physical construction of this kind of tomograph is too high, therefore it is interesting have a preliminary test that provides some data which supports this idea. Computer simulation is a cheap alternative for this test and it is able to provide a reliable data. The software used to do the simulations was GATE (Geant4 Application for Tomographic Emission) because it has already validated, what means it is compatible with real experiments. Thus, four simulations were builded, two for circular geometries with different diameters and two for rectangular geometries. To check which kind of geometry has better performance, it used the methods suggested by NEMA NU 4-2008. At end of these tests, it is possible to observe that the spatial resolution in square geometries is similar to circular. The sensitivity and signal to noise ratio are higher in the square geometries. So, in general, it is concluded that the simulated rectangular geometries are better than circular certainly, this result can be motivating to begin physical construction of the scanner, as it allows developing new products more efficiently and with less cost.

New drugs are needed for better cancer management. Clinical trials are currently underway to assess the use of flavonoids (natural polyphenols) as anticancer agents. Among them, myricetin has been shown to induce cell cycle arrest and apoptosis in pre-clinical cancer models. We hypothesised that myricetin-derived novel flavonoids designed to enhance this natural potential and improve on the drug-likeness limitations of myricetin might have increased potential for their application in the management of breast and ovarian cancer. The effect of a library of novel flavonoids was screened on 3 panels of breast and ovarian cancer cell lines, representing different molecular subtypes and phenotypes, to assess their potency. The second-generation bi-methoxylated analogue AO-1530-OMe (Oncamex) was identified as the most effective candidate in the library, with sub-micromolar concentrations exerting a strong antiproliferative effect across almost all models studied. Results suggested that changes in the hydroxylation profile, the addition of methoxylations and a decyl alkyl chain were some of the structure-activity relationships contributing to this improved efficacy. Plate assays showed 8 h treatment with Oncamex reduced cell viability and induced cytotoxicity and apoptosis, concomitant with caspase activation and PARP cleavage. Pre-incubation with an antioxidant partially blocked these effects, suggesting the possible involvement of ROS modulation in the mechanism of action of Oncamex. Fluorescence microscopy reported the quick and stable delivery of Oncamex to the mitochondria. Fluorescent probes showed that Oncamex can induce mitochondrial superoxide production at concentrations associated with its antiproliferative effects. Study of the electrochemical properties of Oncamex by cyclic voltammetry supported this. Differential gene expression analysis following a microarray experiment showed Oncamex induces changes in the expression of genes controlling cell cycle and apoptosis. Together with previous results, the findings from this analysis led to the postulation of a model for the mechanism of action of Oncamex: due to its enhanced reactivity and mitochondrial targeting, Oncamex can generate mitochondrial superoxide, leading to mitochondrial dysfunction, membrane permeabilisation and the activation of the JNK pathway and the transcription factor FOXO3, which together contribute to the induction of intrinsic apoptosis and the inhibition of proliferation. Further proliferation assays on cell culture models also reported enhanced effect of Oncamex when administered in combination with paclitaxel and TRAIL. These improved responses were observed in breast and ovarian cancer models, including cells lines characterised by their treatment-resistant phenotype. Cotreatment with Oncamex also improved the effect of tamoxifen on anti-oestrogen resistant LCC9 breast cancer cells. Results from preliminary in vivo studies in mice implanted with the MDA-MB-231 breast cancer xenograft were consistent with an antiproliferative effect of Oncamex (25mg/kg/day) in vivo, as treatment inhibited tumour growth and reduced the expression of the marker of proliferation Ki-67 without signs of systemic toxicity. Tissues from this experiment also allowed for preliminary in vivo validation of the proposed mechanism of action of Oncamex by immunohistochemistry. The in vivo cytostatic effect of Oncamex was confirmed in a second in vivo experiment, which also investigated the effect of Oncamex at higher doses or in combination with paclitaxel. In conclusion, the novel flavonoid Oncamex has shown a promising antiproliferative effect in pre-clinical models of breast and ovarian cancer, including models of treatment-resistant cancers. Preliminary in vivo studies have demonstrated a partial recapitulation of the effect of Oncamex. A mechanistic model has been proposed by which Oncamex induces intrinsic apoptosis through its redox reactivity and mitochondrial targeting. These results support the potential of this prototypic candidate, although possible work in the structure and formulation of this candidate and further study and validation of its mechanism of action is needed for its continued development as an anticancer agent.

<p>Elderly committing suicide can be in a “preclinical phase” of dementia. Depressive symptoms may indicate a risk to develop a disease of dementia, for example Alzheimer’s Disease. Today almost 10% of the Swedish population older than 65 years suffer from a cognitive impairment diagnosed as dementia. Symptoms of dementia are associated with degenerative changes in the brain caused by a deposition of amyloid, leading among others things to a nerve cell death. A clinical diagnosis can be hard to set, and a definitive diagnose can only be set after a pathological examination, which only is possible after death. For this study we used Congo red staining of brains sections to find amyloid in autopsies from elderly people committing suicide. 35 cases (>60 year) were studied. Of the 35 cases 1/3 showed to be positive for amyloid deposition. This result in addition to other studies suggest that depressive symptoms is a “preclinical phase” of dementia, and therefore the suicide risk for this group must be consider to be elevated. However, more reliable prospective studies most be done to confirm this retrospective study.</p>