Houston Rescue and Restore Coalition human trafficking needs assessment

Background. Human trafficking, or "modern day slavery", is a complex public health issue that we must understand more fully before it can be effectively tackled. There have been very few empirical studies on human trafficking and estimates of global and national human trafficking victims vary widely. Free the Slaves, a non-profit organization, estimates that there are at least 27 million people in the world at any given time that can be classified as victims of human trafficking. Houston, Texas has been identified as a place where human trafficking may be more likely to exist due to its close proximity to Mexico and due to economic and population factors. Houston Rescue and Restore Coalition (HRRC) is a local organization that exists to raise awareness of human trafficking in Houston, Texas. To better serve victims of human trafficking, HRRC commissioned a community assessment of the services available to victims of human trafficking in the greater Houston metropolitan area.^ Purpose. The current study assessed the capacity of organizations and agencies within the greater Houston metropolitan area to deal with human trafficking issues; in particular, knowledge regarding human trafficking issues among these organizations and agencies was evaluated.^ Methods. A cross-sectional study design was used to conduct surveys with key informants/stakeholders from organizations and agencies within the greater Houston metropolitan area. The survey instrument included 41 items in three parts, and consisted of multiple choice questions, open-ended essay questions, and closed-ended 5 point Likert questions.^ Results. The findings from this study indicate that efforts must be made to increase comprehensive awareness of the issue of human trafficking, including the federal and state laws that have been enacted to combat this problem. The data also indicate that there are limited services provided to human trafficking victims within the greater Houston metropolitan area.^ Conclusion. The results of the survey will provide Houston Rescue and Restore Coalition with information that will assist them in targeting their efforts to combat human trafficking in Houston, Texas.^

Process to develop a State Model Law to reduce prescription drug trafficking while protecting pain management

This paper will discuss the intersection of pill mills and the under-treatment of pain, while addressing the unintended consequence that cracking down on pill mills actually has on medical professionals' treatment of legitimate pain in clinical settings. Moreover, the impact each issue has on the spectrum of related policy, regulatory issues and legislation will be analyzed while addressing the national impact on medical care. Lastly, this paper will outline a process to develop a State Model Law on this subject. This process will include suggestions for the future and how we can move forward to adequately address public safety needs and how we can attempt to mitigate the unintended impact prescription drug trafficking has had on a patient's right to appropriate pain management. This balance is achievable and this paper will address ways we can find this elusive balancing point through the development of a State Model Law. ^

Platelets and Anti-Angiogenic Resistance in Ovarian Carcinoma

Background: Resistance to targeted anti-angiogenic therapy is a growing clinical concern given the disappointing clinical impact of anti-angiogenic. Platelets represent a component of the tumor microenvironment that are implicated in metastasis and represent a significant reservoir of angiogenic regulators. Thrombocytosis has been shown to be caused by malignancy and associated with adverse clinical outcomes, however the causal connections between these associations remain to be identified. Materials and Methods: Following IRB approval, patient data were collected on patients from four U.S. centers and platelet levels through and after therapy were considered as indicators of recurrence of disease. In vitro effects of platelets on cancer cell proliferation, apoptosis, and migration were examined. RNA interference was used to query signaling pathways mediating these effects. The necessity of platelet activation for in vitro effect was analyzed. In vivo orthotopic models were used to query the impact of thrombocytosis and thrombocytopenia on the efficacy of cytotoxic chemotherapy, the effect of aspirin on thrombocytosis and cancer, and platelet effect on anti-angiogenic therapy. Results: Platelets were found to increase at the time of diagnosis of ovarian cancer recurrence in a pattern comparable to CA-125. Platelet co-culture increased proliferation, increased migration, and decreased apoptosis in all cell lines tested. RNA interference implicated platelet derived growth factor alpha (PDGFRA) and transforming growth factor beta-receptor 1 (TGFBR1) signaling. Biodistribution studies suggested minimal platelet sequestration of taxanes. Blockade of platelet activation blocked in vitro effects. In vivo, thrombocytosis blocked chemotherapeutic efficacy, thrombocytopenia increased chemotherapeutic efficacy, and aspirin therapy partially blocked the effects of thrombocytosis. In vivo, withdrawal of anti-angiogenic therapy caused loss of therapeutic benefit with evidence of accelerated disease growth. This effect was blocked by use of a small-molecule inhibitor of Focal Adhesion Kinase. Anti-angiogenic therapy was also associated with increased platelet infiltration into tumor that was not seen to the same degree in the control or FAK-inhibitor-treated mice. Conclusions: Platelets are active participants in the growth and metastasis of tumor, both directly and via facilitation of angiogenesis. Blocking platelets, blocking platelet activation, and blocking platelet trafficking into tumor are novel therapeutic avenues supported by this data. Copyright © 2012 Justin Neal Bottsford-Miller, all rights reserved.

Syntaxin 6- and microtubule- mediated intracellular trafficking contributes to Golgi and nuclear translocation of EGFR

Receptor-mediated endocytosis is well known for its degradation and recycling trafficking. Recent evidence shows that these cell surface receptors translocate from cell surface to different cellular compartments, including the Golgi, mitochondria, endoplasmic reticulum (ER), and the nucleus to regulate physiological and pathological functions. Although some trafficking mechanisms have been resolved, the mechanism of intracellular trafficking from cell surface to the Golgi is not yet completed understood. Here we report a mechanism of Golgi translocation of EGFR in which EGF-induced EGFR travels to the Golgi via microtubule (MT)-dependent movement by interacting with dynein and fuses with the Golgi through syntaxin 6 (Syn6)-mediated membrane fusion. We also demonstrate that the Golgi translocation of EGFR is necessary for its consequent nuclear translocation and transcriptional activity. Interestingly, foreign protein such as bacterial cholera toxin, which is known to activate its pathological function through the Golgi/ER retrograde pathway, also utilizes the MT/Syn6 pathway. Thus, the MT, and syntaxin 6 mediated trafficking pathway from cell surface to the Golgi and ER defines a comprehensive retrograde trafficking route for both cellular and foreign molecules to travel from cell surface to the Golgi and the nucleus.

Post-transcriptional Regulation of Mammalian Gene Expression in Non-coding Region of Target RNA

Tumor Suppressor Candidate 2 (TUSC2) is a novel tumor suppressor gene located in the human chromosome 3p21.3 region. TUSC2 mRNA transcripts could be detected on Northern blots in both normal lung and some lung cancer cell lines, but no endogenous TUSC2 protein could be detected in a majority of lung cancer cell lines. Mechanisms regulating TUSC2 protein expression and its inactivation in primary lung cancer cells are largely unknown. We investigated the role of the 5’- and 3’-untranslated regions (UTRs) of the TUSC2 gene in the regulation of TUSC2 protein expression. We found that two small upstream open-reading frames (uORFs) in the 5’UTR of TUSC2 could markedly inhibit the translational initiation of TUSC2 protein by interfering with the “scanning” of the ribosome initiation complexes. Site-specific stem-loop array reverse transcription-polymerase chain reaction (SLA-RT-PCR) verified several micoRNAs (miRNAs) targeted at 3’UTR and directed TUSC2 cleavage and degradation. In addition, we used the established let-7-targeted high mobility group A2 (Hmga2) mRNA as a model system to study the mechanism of regulation of target mRNA by miRNAs in mammalian cells under physiological conditions. There have been no evidence of direct link between mRNA downregulation and mRNA cleavages mediated by miRNAs. Here we showed that the endonucleolytic cleavages on mRNAs were initiated by mammalian miRNA in seed pairing style. Let-7 directed cleavage activities among the eight predicted potential target sites have varied efficiency, which are influenced by the positional and the structural contexts in the UTR. The 5’ cleaved RNA fragments were mostly oligouridylated at their 3’-termini and accumulated for delayed 5’–3’ degradation. RNA fragment oligouridylation played important roles in marking RNA fragments for delayed bulk degradation and in converting RNA degradation mode from 3’–5’ to 5’–3’ with cooperative efforts from both endonucleolytic and non-catalytic miRNA-induced silencing complex (miRISC). Our findings point to a mammalian miRNA-mediated mechanism for the regulation of mRNA that miRNA can decrease target mRNA through target mRNA cleavage and uridine addition

GENETIC INTERACTIONS OF FACTORS THAT REGULATE ALTERNATIVE RNA SPLICING IN THE MALE GERM LINE OF DROSOPHILA

Alternative RNA splicing is a critical process that contributes variety to protein functions, and further controls cell differentiation and normal development. Although it is known that most eukaryotic genes produce multiple transcripts in which splice site selection is regulated, how RNA binding proteins cooperate to activate and repress specific splice sites is still poorly understood. In addition how the regulation of alternative splicing affects germ cell development is also not well known. In this study, Drosophila Transformer 2 (Tra2) was used as a model to explore both the mechanism of its repressive function on its own pre-mRNA splicing, and the effect of the splicing regulation on spermatogenesis in testis. Half-pint (Hfp), a protein known as splicing activator, was identified in an S2 cell-based RNAi screen as a co-repressor that functions in combination with Tra2 in the splicing repression of the M1 intron. Its repressive splicing function is found to be sequence specific and is dependent on both the weak 3’ splice site and an intronic splicing silencer within the M1 intron. In addition we found that in vivo, two forms of Hfp are expressed in a cell type specific manner. These alternative forms differ at their amino terminus affecting the presence of a region with four RS dipeptides. Using assays in Drosophila S2 cells, we determined that the alternative N terminal domain is necessary in repression. This difference is probably due to differential localization of the two isoforms in the nucleus and cytoplasm. Our in vivo studies show that both Hfp and Tra2 are required for normal spermatogenesis and cooperate in repression of M1 splicing in spermatocytes. But interestingly, Tra2 and Hfp antagonize each other’s function in regulating germline specific alternative splicing of Taf1 (TBP associated factor 1). Genetic and cytological studies showed that mutants of Hfp and Taf1 both cause similar defects in meiosis and spermatogenesis. These results suggest Hfp regulates normal spermatogenesis partially through the regulation of taf1 splicing. These observations indicate that Hfp regulates tra2 and taf1 activity and play an important role in germ cell differentiation of male flies.

Cellular Trafficking of Single and Multistage Vectors

Nanomedicine is an innovative field of science which has recently generated many drug delivery platforms with exciting results. The great potential of these strategies rely on the unique characteristics of the devices at the nano-scale in terms of long time circulation in the blood stream, selective accumulation at the lesions sites, increased solubility in aqueous solutions, etc. Herein we report on a new drug delivery system known as a multistage system which is comprised of non-spherical, mesoporous silicon particles loaded with second stage nanoparticles. The rationally designed particle shape, the possibility to modulate the surface properties and the degree of porosity allow these carriers to be optimized for vascular targeting and to overcome the numerous biological barriers found in drug delivery. In this study we investigated the intra and inter cellular trafficking of the multistage system in endothelial cells bringing evidence of its bio-compatibility as well as its ability to perform multiple intra and inter cellular tasks. Once internalized in cells, the multi-particle construct is able to dissociate, localizing in different subcellular compartments which can be targeted for exocytosis. In particular the second stage nanoparticles were found to be secreted in microvesicles which can act as mediators of transfer of particles across the endothelium and between different endothelial and cancer cells.

RNA-SEQUENCING APPLICATIONS: GENE EXPRESSION QUANTIFICATION AND METHYLATOR PHENOTYPE IDENTIFICATION

My dissertation focuses on two aspects of RNA sequencing technology. The first is the methodology for modeling the overdispersion inherent in RNA-seq data for differential expression analysis. This aspect is addressed in three sections. The second aspect is the application of RNA-seq data to identify the CpG island methylator phenotype (CIMP) by integrating datasets of mRNA expression level and DNA methylation status. Section 1: The cost of DNA sequencing has reduced dramatically in the past decade. Consequently, genomic research increasingly depends on sequencing technology. However it remains elusive how the sequencing capacity influences the accuracy of mRNA expression measurement. We observe that accuracy improves along with the increasing sequencing depth. To model the overdispersion, we use the beta-binomial distribution with a new parameter indicating the dependency between overdispersion and sequencing depth. Our modified beta-binomial model performs better than the binomial or the pure beta-binomial model with a lower false discovery rate. Section 2: Although a number of methods have been proposed in order to accurately analyze differential RNA expression on the gene level, modeling on the base pair level is required. Here, we find that the overdispersion rate decreases as the sequencing depth increases on the base pair level. Also, we propose four models and compare them with each other. As expected, our beta binomial model with a dynamic overdispersion rate is shown to be superior. Section 3: We investigate biases in RNA-seq by exploring the measurement of the external control, spike-in RNA. This study is based on two datasets with spike-in controls obtained from a recent study. We observe an undiscovered bias in the measurement of the spike-in transcripts that arises from the influence of the sample transcripts in RNA-seq. Also, we find that this influence is related to the local sequence of the random hexamer that is used in priming. We suggest a model of the inequality between samples and to correct this type of bias. Section 4: The expression of a gene can be turned off when its promoter is highly methylated. Several studies have reported that a clear threshold effect exists in gene silencing that is mediated by DNA methylation. It is reasonable to assume the thresholds are specific for each gene. It is also intriguing to investigate genes that are largely controlled by DNA methylation. These genes are called “L-shaped” genes. We develop a method to determine the DNA methylation threshold and identify a new CIMP of BRCA. In conclusion, we provide a detailed understanding of the relationship between the overdispersion rate and sequencing depth. And we reveal a new bias in RNA-seq and provide a detailed understanding of the relationship between this new bias and the local sequence. Also we develop a powerful method to dichotomize methylation status and consequently we identify a new CIMP of breast cancer with a distinct classification of molecular characteristics and clinical features.

Estrogen induced desensitization of c -{\it fos\/} messenger RNA expression {\it in vivo\/}

In this thesis, we investigated the regulation of the nuclear proto-oncogene, c-fos by estrogen in vivo. In the uterus, estrogen causes a rapid, dramatic and transient induction of c-fos mRNA and this occurs by transcriptional activation. We have discovered a previously unrecognized regulatory mechanism by which fos becomes desensitized to estrogen following the transient induction. We investigated three aspects of this desensitization: (1) the kinetics and general characteristics of the phenomenon; (2) the molecular mechanism of the desensitization; and (3) the relationship of desensitization to estrogen stimulated DNA synthesis. The desensitization occurs between 3-24 hours after initial hormonal stimulation and is reversible within 72 hours. The desensitization is not species specific, in that it occurs in both the rat and mouse. The desensitization also occurs in at least two estrogen responsive tissues, the uterus and vagina. The desensitization is not unique to c-fos, since both c-myc and c-jun show similar patterns of desensitization. However, the desensitization is not observed with creatine kinase B (CKB), indicating that not all estrogen inducible genes become desensitized. In the second general area, we determined the desensitization is at the transcriptional level. The desensitization is homologous, but not heterologous, since estrogen induction does not desensitize c-fos to other agents. Other studies show that the desensitization is not due to the lack of functional estrogen receptors. Taken together, these findings suggest that the desensitization occurs at the level of the estrogen responsive element. In the third major area, we demonstrated that the desensitization appears to be related to estrogen induced DNA synthesis. Support for this suggestion comes from the observation that short acting estrogens which induce fos, but not DNA synthesis, do not produce desensitization. ^

Inhibitor of cytochrome c messenger RNA -protein interaction in stimulated rat skeletal muscle

The purpose of this work was to examine the possible mechanisms for the regulation of cytochrome c gene expression in response to increased contractile activity in rat skeletal muscle. The working hypothesis was that increased contractile activity enhances cytochrome c gene expression through a cis-element. A 110% increase in cytochrome c mRNA concentration was observed in tibialis anterior (TA) muscle after 9 days of chronic stimulation. Similar difference (120%) exists between soleus (SO) muscle of higher contractile activity and white vastus lateralis (WV) muscle of lower contractile activity. These results suggest that the endogenous cytochrome c gene expression is regulated by contractile activity. Cytochrome c-reporter genes were injected into skeletal muscles to identify the cis-element that is responsible for the regulation. Although the data was inconclusive, part of it suggested the importance of the 3$\sp\prime$-untranslated region (3$\sp\prime$-UTR) in mediating the response to increased contractile activity.^ RNA gel mobility shift (GMSA) and ultraviolet (UV) cross-linking assays revealed specific RNA-protein interaction in a 50-nucleotide region of the 3$\sp\prime$-UTR in unstimulated TA muscle. Computer analysis predicted a stem-loop structure of 17 nucleotides, which provides a structural basis for RNA-protein interaction. These 17 nucleotides are 100% conserved among rat, mouse and human cytochrome c genes and their 13 pseudogenes, suggesting a functional role for this region. The RNA-protein interaction was significantly less in highly active SO muscle than in inactive WV muscle and was dramatically decreased in stimulated TA muscle due to a protein inhibitor(s) associated with ribosome. It is possible that cytochrome c mRNAs undergoing translation are subject to a compartmentalized regulatory influence.^ The conclusion from these results is that increases in contractile activity induce or activate a protein inhibitor(s) associated with ribosome in rat skeletal muscle. The inhibitor decreases RNA-protein interaction in the 3$\sp\prime$-UTR of cytochrome c mRNA, which may result in increased mRNA stability and/or translation. ^

{\it cis\/} -acting determinants in the control of MuSVts110 RNA splicing

Like other simple retroviruses the murine sarcoma virus ts110 (MuSVts110) displays an inefficient mode of genome splicing. But, unlike the splicing phenotypic of other retroviruses, the splicing event effected upon the transcript of MuSVts110 is temperature sensitive. Previous work in this laboratory has established that the conditionally defective nature of MuSVts110 RNA splicing is mediated in cis by features in the viral transcript. Here we show that the 5$\sp\prime$ splice site of the MuSVts110 transcript acts as a point of control of the overall splicing efficiency at both permissive and nonpermissive temperatures for splicing. We strengthened and simultaneously weakened the nucleotide structure of the 5$\sp\prime$ splice site in an attempt to elucidate the differential effects each of the two known critical splicing components which interact with the 5$\sp\prime$ splice site have on the overall efficiency of intron excision. We found that a transversion of the sixth nucleotide, resulting in the formation of a near-consensus 5$\sp\prime$ splice site, dramatically increased the overall efficiency of MuSVts110 RNA splicing and abrogated the thermosensitive nature of this splicing event. Various secondary mutations within this original transversion mutant, designed to selectively decrease specific splicing component interactions, lead to recovery of inefficient and thermosensitive splicing. We have further shown that a sequence of 415 nucleotides lying in the downstream exon of the viral RNA and hypothesized to act as an element in the temperature-dependent inhibition of splicing displays a functional redundancy throughout its length; loss and/or replacement of any one sequence of 100 nucleotides within this sequence does not, with one exception detailed below, diminish the degree to which MuSVts110 RNA is inhibited to splice at the restrictive temperature. One specific deletion, though, fortuitously juxtaposed and activated cryptic consensus splicing signals for the excision of a cryptic intron within the downstream exon and markedly potentiated--across a newly defined cryptic exon--the splicing event effected upon the upstream, native intron. We have exploited this mutant of MuSVts110 to further an understanding of the process of exon definition and intron definition and show that the polypyrimidine tract and consensus 3$\sp\prime$ splice site, as well as the 5$\sp\prime$ splice site, within the intron at the 3$\sp\prime$ flank of the defined exon are required for the exon's definition; implying that definition of the downstream intron is required for the in vivo definition of the proximal, upstream exon. Finally; we have shown, through the construction of heterologous mutants of MuSVts110 employing a foreign 3$\sp\prime$ end-forming sequence, that efficiency of transcript splicing can be increased--to a degree which abrogates its thermosensitive nature--in direct proportion to increasing proximity of the 3$\sp\prime$ end-forming signal to the terminal 3$\sp\prime$ splice site. ^

Thermo -modulation of MuSVts110 splicing by inhibitory RNA sequences

Viral systems have contributed tremendously to the understanding of eukaryotic molecular biology. The proportional pattern of retroviral RNA expression offers many clues into the alternative splicing of cellular transcripts. The MuSVts110 virus presents an unusual expression system, where the mechanistic combination of RNA splicing and cellular transformation can be physiologically manipulated. Splicing of MuSVts110 pre-mRNA occurs inefficiently (30%-50%) at 33$\sp\circ$C or below and is subdued at 39$\sp\circ$C ($<$5%). Like most alternatively spliced cellular and retroviral transcripts, the MuSVts110 pre-mRNA contains cis-acting intron and exon sequences that attenuate splicing. These include a splicing inhibitory sequence at the 3$\prime$ end of the MuSVts110 v-mos exon, called the E2 Distal Element (E2DE), and a sub-optimal 3$\prime$ splice site. The E2DE directly inhibits MuSVts110 RNA splicing in a sequence-specific fashion at 39$\sp\circ$C but not at 28$\sp\circ$C, potentially through the association of cellular factors. Inefficient MuSVts110 splicing is pre-dominantly attributed to the utilization of multiple weak branchpoint sequences located between $-113$ and $-34$ nucleotides upstream of the 3$\prime$ splice site. The molecular control of MuSVts110 splicing, represented primarily by scattered multiple inefficient branchpoint sequences that are conditionally modulated by the E2DE at higher growth temperatures, is discussed. ^

Seawater carbonate chemistry, clearance rates, respiration rates, condition index and cellular turnover (RNA: DNA) of Juvenile King Scallop, Pecten maximus in a laboratory experiment

The decline in ocean water pH and changes in carbonate saturation states through anthropogenically mediated increases in atmospheric CO2 levels may pose a hazard to marine organisms. This may be particularly acute for those species reliant on calcareous structures like shells and exoskeletons. This is of particular concern in the case of valuable commercially exploited species such as the king scallop, Pecten maximus. In this study we investigated the effects on oxygen consumption, clearance rates and cellular turnover in juvenile P. maximus following 3 months laboratory exposure to four pCO2 treatments (290, 380, 750 and 1140 µatm). None of the exposure levels were found to have significant effect on the clearance rates, respiration rates, condition index or cellular turnover (RNA: DNA) of individuals. While it is clear that some life stages of marine bivalves appear susceptible to future levels of ocean acidification, particularly under food limiting conditions, the results from this study suggest that where food is in abundance, bivalves like juvenile P. maximus may display a tolerance to limited changes in seawater chemistry.