Pharmacology and pore-forming domains of the cystic fibrosis transmembrane conductance regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel member of the ATP-binding cassette (ABC) superfamily of membrane proteins. CFTR has two homologous halves, each consisting of six transmembrane spanning domains (TM) followed by a nucleotide binding fold, connected by a regulatory (R) domain. This thesis addresses the question of which domains are responsible for Cl^- selectivity, i.e., which domains line the channel pore.

To address this question, novel blockers of CFTR were characterized. CFTR was heterologously expressed in Xenopus oocytes to study the mechanism of block by two closely related arylaminobenzoates, diphenylamine-2-carboxylic acid (DPC) and flufenamic acid (FFA). Block by both is voltage-dependent, with a binding site ≈ 40% through the electric field of the membrane. DPC and FFA can both reach their binding site from either side of the membrane to produce a flickering block of CFTR single channels. In addition, DPC block is influenced by Cl^- concentration, and DPC blocks with a bimolecular forward binding rate and a unimolecular dissociation rate. Therefore, DPC and FFA are open-channel blockers of CFTR, and a residue of CFTR whose mutation affects their binding must line the pore.

Screening of site-directed mutants for altered DPC binding affinity reveals that TM-6 and TM-12 line the pore. Mutation of residue 5341 in TM-6 abolishes most DPC block, greatly reduces single-channel conductance, and alters the direction of current rectification. Additional residues are found in TM-6 (K335) and TM-12 (T1134) whose mutations weaken or strengthen DPC block; other mutations move the DPC binding site from TM-6 to TM-12. The strengthened block and lower conductance due to mutation T1134F is quantitated at the single-channel level. The geometry of DPC and of the residues mutated suggest α-helical structures for TM-6 and TM-12. Evidence is presented that the effects of the mutations are due to direct side-chain interaction, and not to allosteric effects propagated through the protein. Mutations are also made in TM-11, including mutation S1118F, which gives voltage-dependent current relaxations. The results may guide future studies on permeation through ABC transporters and through other Cl^- channels.

MicroRNA-132 is a physiological regulator of hematopoietic stem cell function and B-cell development

MicroRNAs are a class of small non-coding RNAs that negatively regulate gene expression. Several microRNAs have been implicated in altering hematopoietic cell fate decisions. Importantly, deregulation of many microRNAs can lead to deleterious consequences in the hematopoietic system, including the onset of cancer, autoimmunity, or a failure to respond effectively to infection. As such, microRNAs fine-tune the balance between normal hematopoietic output and pathologic consequences. In this work, we explore the role of two microRNAs, miR-132 and miR-125b, in regulating hematopoietic stem cell (HSC) function and B cell development. In particular, we uncover the role of miR-132 in maintaining the appropriate balance between self-renewal, differentiation, and survival in aging HSCs by buffering the expression of a critical transcription factor, FOXO3. By maintain this balance, miR-132 may play a critical role in preventing aging-associated hematopoietic conditions such as autoimmune disease and cancer. We also find that miR-132 plays a critical role in B cell development by targeting a key transcription factor, Sox4, that is responsible for the differentiation of pro-B cells into pre-B cells. We find that miR-132 regulates B cell apoptosis, and by delivering miR-132 to mice that are predisposed to developing B cell cancers, we can inhibit the formation of these cancers and improve the survival of these mice. In addition to miR-132, we uncovered the role of another critical microRNA, miR-125b, that potentiates hematopoietic stem cell function. We found that enforced expression of miR-125b causes an aggressive myeloid leukemia by downregulation of its target Lin28a. Importantly, miR-125b also plays a critical role in inhibiting the formation of pro-B cells. Thus, we have discovered two microRNAs with important roles in regulating normal hematopoiesis, and whose dregulation can lead to deleterious consequences such as cancer in the aging hematopoietic system. Both miR-132 and miR-125b may therefore be targeted for therapeutics to inhibit age-related immune diseases associated with the loss of HSC function and cancer progression.

Some constructions, related to noncommutative tori; Fredholm modules and the Beilinson-Bloch regulator

A noncommutative 2-torus is one of the main toy models of noncommutative geometry, and a noncommutative n-torus is a straightforward generalization of it. In 1980, Pimsner and Voiculescu in [17] described a 6-term exact sequence, which allows for the computation of the K-theory of noncommutative tori. It follows that both even and odd K-groups of n-dimensional noncommutative tori are free abelian groups on 2^n-1 generators. In 1981, the Powers-Rieffel projector was described [19], which, together with the class of identity, generates the even K-theory of noncommutative 2-tori. In 1984, Elliott [10] computed trace and Chern character on these K-groups. According to Rieffel [20], the odd K-theory of a noncommutative n-torus coincides with the group of connected components of the elements of the algebra. In particular, generators of K-theory can be chosen to be invertible elements of the algebra. In Chapter 1, we derive an explicit formula for the First nontrivial generator of the odd K-theory of noncommutative tori. This gives the full set of generators for the odd K-theory of noncommutative 3-tori and 4-tori.

In Chapter 2, we apply the graded-commutative framework of differential geometry to the polynomial subalgebra of the noncommutative torus algebra. We use the framework of differential geometry described in [27], [14], [25], [26]. In order to apply this framework to noncommutative torus, the notion of the graded-commutative algebra has to be generalized: the "signs" should be allowed to take values in U(1), rather than just {-1,1}. Such generalization is well-known (see, e.g., [8] in the context of linear algebra). We reformulate relevant results of [27], [14], [25], [26] using this extended notion of sign. We show how this framework can be used to construct differential operators, differential forms, and jet spaces on noncommutative tori. Then, we compare the constructed differential forms to the ones, obtained from the spectral triple of the noncommutative torus. Sections 2.1-2.3 recall the basic notions from [27], [14], [25], [26], with the required change of the notion of "sign". In Section 2.4, we apply these notions to the polynomial subalgebra of the noncommutative torus algebra. This polynomial subalgebra is similar to a free graded-commutative algebra. We show that, when restricted to the polynomial subalgebra, Connes construction of differential forms gives the same answer as the one obtained from the graded-commutative differential geometry. One may try to extend these notions to the smooth noncommutative torus algebra, but this was not done in this work.

A reconstruction of the Beilinson-Bloch regulator (for curves) via Fredholm modules was given by Eugene Ha in [12]. However, the proof in [12] contains a critical gap; in Chapter 3, we close this gap. More specifically, we do this by obtaining some technical results, and by proving Property 4 of Section 3.7 (see Theorem 3.9.4), which implies that such reformulation is, indeed, possible. The main motivation for this reformulation is the longer-term goal of finding possible analogs of the second K-group (in the context of algebraic geometry and K-theory of rings) and of the regulators for noncommutative spaces. This work should be seen as a necessary preliminary step for that purpose.

For the convenience of the reader, we also give a short description of the results from [12], as well as some background material on central extensions and Connes-Karoubi character.