Altered Amino Acid Sequence Affects Amyloid Formation In Aβ(25-35)

Many neurodegenerative diseases are associated with protein misfolding where the protein forms β-sheet rich polymers called amyloid. Alzheimer’s disease (AD) is an amyloid aggregation disease involving the Aβ(1-42) peptide fragment of the Amyloid Precursor Protein (APP) in the nervous tissue of the brain. Within the peptide there are regions thought to be more critical for amyloid formation; one of which is Aβ(25-35). This sequence has the ability to form amyloid at sufficient peptide concentrations. Within this region, residues 33-35 have been found by terminal deletions to be a core region (1), the residues necessary for amyloid formation. Using Cys-scanning mutagenesis the estimated core region (31-34) is slightly different. Previous papers have shown, the peptide loses the ability to form amyloid fibrils when the sequence of amino acids is changed (2). For this experiment, four different Aβ(25-35) sequences were tested, each having the same composition but a different sequence. FPLC was used in conjunction with Thioflavin-T fluorescence to monitor amyloid development. The results showed a definite effect on amyloid formation as compared to wild type. Sequence S-N showed nearly no change in behavior. Sequence S-M appeared to loose all amyloid forming ability. The remaining two sequences were unable to remain in solution long enough to be accurately tested

Prevention of HIV transmission

The human immunodeficiency virus (HIV) infects and replicates within individuals for the duration of their life. Initial infection results in little to no symptoms for years or even decades. These individuals are infectious and capable of further spreading HIV while completely unaware of their own status. This ability to transmit without detection is what lead to the unknown and thereby unopposed global spread of HIV type one (HIV-1). Once a test was developed to detect HIV, the virus was found in nearly all major countries around the world. Development of a cure has proven to be exceedingly difficult. So far, the only successful tactic to reduce the number of infected HIV individuals has been to prevent HIV transmission. Traditionally, the most effective way to prevent viral transmission is with a vaccine, but an effective HIV vaccine for wide spread use has not been developed. Therefore, alternative HIV transmission prevention strategies have been used. These strategies largely depend on behavioral modifications and include: 1) utilization of universal precautions in medical settings, 2) increased emphasis on HIV testing and self awareness of infection status, 3) encouraged use of protective measures such as condoms and male circumcision, 4) prophylactic use of antiretroviral drugs to limit mother to child transmission MTCT, and 5) the newly available oral pre-exposure prophylactic use of Truvada in HIV negative individuals. Together, these strategies have contributed to nearly eliminating HIV transmission in medical settings and greatly reduced MTCT. Unfortunately, HIV continues to spread globally largely due to sexual transmission. While condom usage is highly efficient to prevent transmission, their use is limited by acceptability and consent. In this dissertation, I evaluated the potential of topically applied interventions to be a novel, effective form of protection against HIV transmission as well as established a novel line of investigation evaluating microbial contributions to HIV transmission. Using humanized mice as a model of HIV transmission, I evaluated two antiretroviral drug based topical pre-exposure prophylaxis (PrEP) for efficacy; tenofovir and maraviroc. In both instances, these drugs were found to be protective when used prior to HIV exposure. Concerns regarding the dual use of tenofovir for treatment as well as PrEP prompted me to evaluate transmission of a tenofovir resistant strain of HIV. This study demonstrated a surprisingly large defect in transmission for tenofovir resistant HIV, which suggests that use of Tenofovir for PrEP may not result in a significant increase of circulating tenofovir resistant strains of HIV. I also utilized the humanized mouse model to start a completely novel line of investigation evaluating the effect of microbial populations on HIV transmission. While these studies are ongoing, preliminary results have shown a clear effect of microbiome composition on rectal HIV transmission. These results are significant in two ways. First, this is the first evidence that humanized mice are viable tools for microbiome based studies. Second, commensal microbiota does affect HIV transmission efficiency. Taken together, the following dissertation supports further efforts to curb the HIV epidemic by development of topical interventions (microbicides) and lends credence toward interventions based on commensal microbiome manipulations.Doctor of Philosoph

Effect of Amino Acid Subsititution in Set1 on Histone H3 Methylation and Gene Silencing in Saaccharomyces Cerevisiae

Chromosomal DNA in our cells is wrapped around a histone protein octomer like thread on a spool, forming a structure called a nucleosome. Series of nucleosomes form the nuclear chromosomes found in all eukaryotic organisms. Modifications to histone proteins can change how accessible chromosomal DNA is to protein complexes that act on DNA. DNA sequences that are inaccessible are called silent chromatin and regions that can’t be transcribed are subject to “gene silencing.” Proper gene silencing is necessary for normal cell development and regulation. Incorrect or missing histone modifications can cause the loss of gene silencing and uncontrolled gene expression similar to the situation in cells of patients with cancer or leukemia. My project focuses on a histone modifying complex COMPASS. COMPASS is composed of eight proteins, one of which is the histone H3 methyltransferase Set1. There are seven Set1 homologs in yeast and over 60 Set1-like proteins in humans, including MLL, which is known to be associated with human leukemia. Previous studies have shown that Set1 and most COMPASS proteins are essential for gene silencing at the ribosomal DNA locus (rDNA) in yeast. The SET domain is the active site of the Set1 histone methyltransferase, where methyl groups are covalently attached to the fourth lysine residue (K4) of histone H3. My goal is to investigate the effect of six individual amino acid substitutions in the SET domain of Set1; Y967A, I972A, Y993A, H1017L, Y967F, and G951A, on histone H3 methylation and gene silencing. These altered Set1 proteins are being expressed in the yeast Saccharomyces cerevisiae. Using Western blots and marker genes, the effect of these mutations are compared to wild type Set1. My data show that there are defects in histone H3 methylation in the amino acid substitution variants of Set1. In five of the mutants there is a complete loss of H3K4 methylation. In the future, we will determine if these altered Set1 proteins are assembled into the COMPASS complex. By characterizing the catalytic domain of Set1 using amino acid substitution variants, we will acquire a better understanding of the related proteins in humans

HIV-1 infection of microglial cells in a reconstituted humanized mouse model and identification of compounds that selectively reverse HIV latency.

Most studies of HIV latency focus on the peripheral population of resting memory T cells, but the brain also contains a distinct reservoir of HIV-infected cells in microglia, perivascular macrophages, and astrocytes. Studying HIV in the brain has been challenging, since live cells are difficult to recover from autopsy samples and primate models of SIV infection utilize viruses that are more myeloid-tropic than HIV due to the expression of Vpx. Development of a realistic small animal model would greatly advance studies of this important reservoir and permit definitive studies of HIV latency. When radiation or busulfan-conditioned, immune-deficient NSG mice are transplanted with human hematopoietic stem cells, human cells from the bone marrow enter the brain and differentiate to express microglia-specific markers. After infection with replication competent HIV, virus was detected in these bone marrow-derived human microglia. Studies of HIV latency in this model would be greatly enhanced by the development of compounds that can selectively reverse HIV latency in microglial cells. Our studies have identified members of the CoREST repression complex as key regulators of HIV latency in microglia in both rat and human microglial cell lines. The monoamine oxidase (MAO) and potential CoREST inhibitor, phenelzine, which is brain penetrant, was able to stimulate HIV production in human microglial cell lines and human glial cells recovered from the brains of HIV-infected humanized mice. The humanized mice we have developed therefore show great promise as a model system for the development of strategies aimed at defining and reducing the CNS reservoir

Inefficient Vaginal Transmission of Tenofovir-Resistant HIV-1

Transmission of drug-resistant HIV has been postulated to be a threat to current first-line antiretroviral therapy (ART) regimens and the efficacy of several antiretroviral-based preexposure prophylaxis (PrEP) strategies being tested. Here we evaluated the effect of the common tenofovir (TFV) resistance mutation K65R on vaginal HIV transmission. Our results demonstrate that despite no overt loss of overall replication competence in vivo, this mutation results in significantly reduced mucosal transmission. When transmitted, the mutant virus eventually reverted to the wild type in 2 of 3 animals examined

ART influences HIV persistence in the female reproductive tract and cervicovaginal secretions

The recently completed HIV prevention trials network study 052 is a landmark collaboration demonstrating that HIV transmission in discordant couples can be dramatically reduced by treating the infected individual with antiretroviral therapy (ART). However, the cellular and virological events that occur in the female reproductive tract (FRT) during ART that result in such a drastic decrease in transmission were not studied and remain unknown. Here, we implemented an in vivo model of ART in BM/liver/thymus (BLT) humanized mice in order to better understand the ability of ART to prevent secondary HIV transmission. We demonstrated that the entire FRT of BLT mice is reconstituted with human CD4+ cells that are shed into cervicovaginal secretions (CVS). A high percentage of the CD4+ T cells in the FRT and CVS expressed CCR5 and therefore are potential HIV target cells. Infection with HIV increased the numbers of CD4+ and CD8+ T cells in CVS of BLT mice. Furthermore, HIV was present in CVS during infection. Finally, we evaluated the effect of ART on HIV levels in the FRT and CVS and demonstrated that ART can efficiently suppress cell-free HIV-RNA in CVS, despite residual levels of HIV-RNA+ cells in both the FRT and CVS

Rectal Transmission of Transmitted/Founder HIV-1 Is Efficiently Prevented by Topical 1% Tenofovir in BLT Humanized Mice

Rectal microbicides are being developed to prevent new HIV infections in both men and women. We focused our in vivo preclinical efficacy study on rectally-applied tenofovir. BLT humanized mice (n = 43) were rectally inoculated with either the primary isolate HIV-1(JRCSF) or the MSM-derived transmitted/founder (T/F) virus HIV-1(THRO) within 30 minutes following treatment with topical 1% tenofovir or vehicle. Under our experimental conditions, in the absence of drug treatment we observed 50% and 60% rectal transmission by HIV-1(JRCSF) and HIV-1(THRO), respectively. Topical tenofovir reduced rectal transmission to 8% (1/12; log rank p = 0.03) for HIV-1(JRCSF) and 0% (0/6; log rank p = 0.02) for HIV-1(THRO). This is the first demonstration that any human T/F HIV-1 rectally infects humanized mice and that transmission of the T/F virus can be efficiently blocked by rectally applied 1% tenofovir. These results obtained in BLT mice, along with recent ex vivo, Phase 1 trial and non-human primate reports, provide a critically important step forward in the development of tenofovir-based rectal microbicides

Pathways to child and adolescent psychiatric clinics: a multilevel study of the significance of ethnicity and neighbourhood social characteristics on source of referral

<p>Abstract</p> <p>Background</p> <p>In the Swedish society, as in many other societies, many children and adolescents with mental health problems do not receive the help they need. As the Swedish society becomes increasingly multicultural, and as ethnic and economic residential segregation become more pronounced, this study utilises ethnicity and neighbourhood context to examine referral pathways to child and adolescent psychiatric (CAP) clinics.</p> <p>Methods</p> <p>The analysis examines four different sources of referrals: family referrals, social/legal agency referrals, school referrals and health/mental health referrals. The referrals of 2054 children aged 11-19 from the Stockholm Child-Psychiatric Database were studied using multilevel logistic regression analyses.</p> <p>Results</p> <p>Results indicate that ethnicity played an important role in how children and adolescents were referred to CAP-clinics. Family referrals were more common among children and adolescents with a Swedish background than among those with an immigrant background. Referrals by social/legal agencies were more common among children and adolescents with African and Asian backgrounds. Children with Asian or South American backgrounds were more likely to have been referred by schools or by the health/mental health care sector. A significant neighbourhood effect was found in relation to family referrals. Children and adolescents from neighbourhoods with low levels of socioeconomic deprivation were more likely to be referred to CAP-clinics by their families in comparison to children from other neighbourhoods. Such differences were not found in relation in relation to the other sources of referral.</p> <p>Conclusions</p> <p>This article reports findings that can be an important first step toward increasing knowledge on reasons behind differential referral rates and uptake of psychiatric care in an ethnically diverse Swedish sample. These findings have implications for the design and evaluation of community mental health outreach programs and should be considered when developing measures and strategies intended to reach and help children with mental health problems. This might involve providing information about the availability and accessibility of health care for children and adolescents with mental health problems to families in certain neighbourhoods and with different ethnic backgrounds.</p

Human Breast Milk and Antiretrovirals Dramatically Reduce Oral HIV-1 Transmission in BLT Humanized Mice

Currently, over 15% of new HIV infections occur in children. Breastfeeding is a major contributor to HIV infections in infants. This represents a major paradox in the field because in vitro, breast milk has been shown to have a strong inhibitory effect on HIV infectivity. However, this inhibitory effect has never been demonstrated in vivo. Here, we address this important paradox using the first humanized mouse model of oral HIV transmission. We established that reconstitution of the oral cavity and upper gastrointestinal (GI) tract of humanized bone marrow/liver/thymus (BLT) mice with human leukocytes, including the human cell types important for mucosal HIV transmission (i.e. dendritic cells, macrophages and CD4+ T cells), renders them susceptible to oral transmission of cell-free and cell-associated HIV. Oral transmission of HIV resulted in systemic infection of lymphoid and non-lymphoid tissues that is characterized by the presence of HIV RNA in plasma and a gradual decline of CD4+ T cells in peripheral blood. Consistent with infection of the oral cavity, we observed virus shedding into saliva. We then evaluated the role of human breast milk on oral HIV transmission. Our in vivo results demonstrate that breast milk has a strong inhibitory effect on oral transmission of both cell-free and cell-associated HIV. Finally, we evaluated the effect of antiretrovirals on oral transmission of HIV. Our results show that systemic antiretrovirals administered prior to exposure can efficiently prevent oral HIV transmission in BLT mice